PROBIOTIC BACTERIA FOR PREVENTION OF ATOPIC DISEASES DESIGN AND APPLICATION

PROBIOTIC BACTERIA FOR PREVENTION OF ATOPIC DISEASES DESIGN AND APPLICATION

Copyright 2005 by Clinical and Experimental Allergy for chapter 2 Copyright 2007 by Clinical and Experimental Immunology for chapter 3 Copyright 2009 by Allergy for chapter 4 Copyright 2009 by L. Niers for all other chapters All rights reserved. No part of this publication may be reproduced or transmitted in any form by any means without prior permission of the author. ISBN: 978-94-90122-64-5 Cover: Linde Ex. www.linde-ex.nl Lay-out and Printing: Gildeprint Drukkerijen, Enschede, the Netherlands Correspondence and reprint requests L.E.M. Niers, Wilhelmina Children s Hospital, University Medical Center Utrecht, room KE.04.133.1, PO BOX 85090, 3508 AB, Utrecht, the Netherlands L.E.M.Niers@umcutrecht.nl

PROBIOTIC BACTERIA FOR PREVENTION OF ATOPIC DISEASES DESIGN AND APPLICATION Probiotica voor de preventie van atopische aandoeningen Ontwikkeling en toepassing (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. J.C. Stoof, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 10 november 2009 des middags te 2.30 uur. door Laetitia Elisabeth Maria Niers geboren op 17 maart 1976 te Eindhoven

Promotor: Prof. dr. J.L.L. Kimpen Co-promotoren: Dr. M.O. Hoekstra Dr. ir. G.T. Rijkers The printing of this thesis was financially supported by Divisie Kindergeneeskunde Wilhelmina Kinderziekenhuis, Friesland foods, Winclove Bio Industries, Alk-Abello BV, Boehringer Ingelheim, Danone Research BV, Eijkman Graduate Fonds, Mead Johnson, Nestlé Nutrition, Nycomed, Phadia BV, Siemens Medical Solutions Diagnostics BV, Solvay Pharma BV, Stichting Gut flora in health and disease, Stichting Folia Orthica, Teva Pharma BV, VSM geneesmiddelen BV, Yakult Nederland BV, Zambon Nederland BV.

Voor mijn ouders

Contents Chapter 1 General introduction 9 Chapter 2 Identification of strong interleukin-10 inducing lactic acid bacteria which down-regulate T helper type 2 cytokines 27 Chapter 3 Selection of probiotic bacteria for prevention of allergic diseases: immunomodulation of neonatal dendritic cells 49 Chapter 4 The effects of selected probiotic strains on the development of eczema (The PandA study) 71 Chapter 5 Dynamic changes in intestinal microbiota in infants at risk of eczema during supplementation of probiotics 97 Chapter 6 Probiotic bacteria and modulation of the developing immune system 121 Chapter 7 General Discussion 143 Chapter 8 Summary en Nederlandse samenvatting 171 Dankwoord 197 Curriculum Vitae 201 List of publications 203 List of abbreviations 207

1 General introduction Parts of the introduction were published in: The hygiene hypothesis: The role of microbes in the prevention of atopy and atopic disease. In: Respiratory Tract Infections: Pathogenesis and emerging strategies for control. J.L.L. Kimpen and T. Ramilio, Horizon Scientific Press Ltd., Norfolk, UK, 2004

Chapter 1 10

Atopic diseases and allergy The term allergy, which is derived from the Greek allos meaning other and ergon meaning reaction, was first coined in 1906 by Clemens Peter Freiherr von Pirquet (1874-1929), an Austrian scientist and pediatrician, to describe a hypersensitivity reaction to smallpox vaccine. In allergic individuals immune responses to otherwise innocuous antigens produce allergic or hypersensitivity reactions upon re-exposure to the same antigen. The antibody IgE was found to be a major player in allergic immune responses 1. A familial or individual predisposition to develop sensitization and produce IgE antibodies upon exposure to allergens is called atopy 2. Atopy may contribute to manifest disease and symptoms such as eczema, food allergy, asthma and rhinoconjunctivitis, generalized as atopic diseases. Atopic diseases are common in children and add considerably to childhood morbidity. It is estimated that 30% of children suffer from atopic diseases during the first 18 years of life 3; 4. The incidence of (atopic) eczema and food allergy peaks during the first two years of life, while in schoolchildren asthma is the predominant atopic disease. Allergic rhinitis (hay fever) is the most important allergic manifestation during adolescence 5. In many cases, there is a sequence of early-onset eczema, followed by asthma and then rhinitis which is called the General introduction atopic march. The pathogenesis of atopic diseases is still unclear. Atopic diseases have a clear familial predisposition, they are multifactorial disorders in which alterations in many genes and 11 numerous environmental factors contribute to their pathogenesis 6; 7. The chance of developing atopic diseases increases from 15% to 50-80% in the offspring of families with a positive history of atopic diseases. In addition, many candidate susceptibility genes such as IL4-RA, ADAM33, TIM, PHF11, CD14 (most of which are involved in the immune system) have been identified in association with atopy and asthma 7. The treatment of allergic diseases is currently mainly based on avoidance of disease causing or disease aggravating factors. Pharmacological intervention with antihistamines and glucocorticoids offers considerable relief but no permanent cure is achieved. Furthermore, the development of atopic diseases can not be prevented, although breastfeeding and pre- and postnatal avoidance of passive smoking decrease risk and severity of later disease.

During the last decades, the prevalence of atopic sensitization and associated clinical phenotypes such as eczema, asthma and allergic rhinitis has increased in countries with a Western lifestyle 8; 9. Recent reports indicate that this global increase might be attenuated 10. It seems that the rise in asthma and allergic sensitization parallels decreased incidence of infectious diseases (in part due to the introduction of vaccination programs), improvement in living conditions, industrialization and urbanization 11. The exact cause of the increased prevalence of atopic diseases is still unknown. The time span is too short for genetic changes and therefore it seems more likely that one or more environmental changes operating on a genetically predisposed subset of the population drive the increasing incidence and prevalence of atopic diseases. Hygiene hypothesis The hygiene hypothesis states that the increase in the prevalence of atopic diseases may be due to a decreased exposure to microbes, or microbial products, early in life. The concept of the Chapter 1 hygiene hypothesis was first proposed by David Strachan, who reported an inverse association between hay fever and the number of children in the household. He found a similar association for eczema during the first year of life 12. From then, numerous epidemiological studies 12 supporting the hygiene hypothesis have been published. Specific infectious agents as well as overall microbial burden have been proposed as being protective against developing allergic disease. Measles 13, hepatitis A 14, mycobacterium tuberculosis infection and BCG vaccination 15;16, helminth infestation 17, other parasites 18 and Helicobacter pylori 19 have all been associated with protection against atopic diseases. Environmental factors which possibly reflect a change in microbial burden and exposure to microbial agents have been associated with the prevalence of atopic diseases as well. Both larger family size 20; 21 and frequency of day care visits showed an inverse relationship with the prevalence of atopic disease 21; 22. An increased exposure to infectious diseases due to more frequent child to child contacts is thought to be the underlying mechanism. The prevalence of atopic disease was in particular decreased in infants experiencing at least one upper airway infection before their first birthday 23. This emphasizes not only the importance of exposure to microbial products but also of timing. Furthermore, living on a farm

protects against developing atopic disease 24. Children who were taken into the stables or who drank non pasteurized milk on a regular basis have a substantially reduced risk of developing atopic diseases. Again, the aspect of timing is crucial: this protective effect was mainly present when the first year of life was spent on a farm. No protective effect was found if the child was only exposed after the age of 1 year 25. In addition, exposure of the mother to animal sheds during pregnancy had a protective effect on her offspring as well 25. Taken together, these studies indicate that early exposure to infectious agents or microbial products may play an important role in defining the developing immune response and the manifestation of atopic diseases (Figure 1). The window of opportunity to influence the manifestation of atopic diseases may be very early in life. General introduction 13 Figure 1: Genetic and environmental factors associated with atopic diseases

The intestinal microbiota and its role in the development of atopic diseases The gastrointestinal tract (GI-tract) colonized with the intestinal microbiota is the largest area of the body in direct contact with the microbial world outside. Therefore the GI-tract has recently received much attention regarding its potential role in the development of atopic diseases. The GI-tract contains 10 14 bacteria, ten times more than the number of cells in the body, and is composed of about 800 species and 7,000 strains 26. In utero, the gastro-intestinal tract is sterile, microbes start colonizing the gastro-intestinal tract during birth by exposure to the microbial flora present in the mother s birth canal. Early stages of colonization result from incidental microbial encounters with opportunistic early colonizers, i.e. Staphylococcus, Streptococcus, and Enterobacteria 27. Development of the infant intestinal microbiota is highly determined by interindividual variation, and is further influenced by numerous factors including time, mode of delivery, environment, microbial exposure, and nutrition 27; 28. At 1 year of age, the infants intestinal microbiota has developed into adult-like microbiota and is clearly different from the Chapter 1 microbiota at a younger age. Introduction of solid foods frequently precedes the shift to adult-like microbiota 27. This so-called commensal (derived from Latin together at the table ) microbiota have a symbiotic relationship with its host and is indispensable for digestion, synthesis of 14 micronutrients such as vitamins, and colonization resistance to potentially pathogenic bacteria. Furthermore, it is essential in postnatal maturation of the mucosal immune system and the development of immunological tolerance. The mucosal immune system is organized in Peyer s patches, mesenteric lymph nodes, isolated lymphoid follicles and further consists of the intestinal epithelium with lymphocytes scattered throughout the epithelium and lamina propria of the mucosa. Germ-free animals have smaller Peyer s patches, fewer intraepithelial T lymphocytes, and lower levels of secretory IgA. Furthermore, reconstitution of the intestinal microbiota of germ-free rodents with B. infantis restored their development of the otherwise defective oral tolerance 29. Remarkably, oral tolerance could only be restored when the intestinal microbiota were reconstituted in neonatal mice, not in older mice. This again underlines the aspect of timing and window of opportunity.

The presence or absence of certain bacteria has quite convincingly been associated with atopic diseases, indicating that the gut microbiota may indeed be involved in the etiology of atopic diseases. Analysis of the gut bacteria in industrialized countries indicates that the range of gut bacteria has narrowed compared to 40 years ago, and that the development of allergy in childhood is associated with having a more restricted gut flora 30. Furthermore, allergic children seem to harbor more Clostridium species and less Bifidobacterium species in their fecal microbiota 31; 32. In addition, the development of eczema has been associated with reduced diversity, less bifidobacteria, and the specific presence of E. coli and Bifidobacterium pseudocatenulatum 33-35. Differences in the intestinal microbiota between atopic and non-atopic children precede the development of atopic disease suggesting a potential causal relationship 31; 36. The microbiota hypothesis proposes that the increased prevalence of atopic diseases may be due to altered composition of the gastro-intestinal microbiota by factors associated with prosperity and improved hygiene, resulting in a disrupted development of immunological tolerance. Recent evidence suggests that the intestinal microbiota is required for proper development of regulatory T cells 37; 38. The immunology of atopic diseases General introduction Disrupted immunological tolerance may be of key importance in the pathogenesis of atopic 15 diseases. In allergic individuals, immune responses to otherwise innocuous antigens produce allergic reactions upon re-exposure to the same antigen. Allergic immune responses are mediated by type 2 CD4 + T helper (Th2) cells. Characteristic cytokines involved in allergic inflammation are interleukin (IL)-4 and IL-13 that drive IgE production, mucus secretion and airway hypersensitivity, and IL-5 that promotes eosinophil activation. In newborns, who develop atopy in later life postnatal maturation of cellular immune functions seems to be hampered already during the first year of life 39. High-risk children developing atopic disease by age 1 year seem to develop a Th2 cytokine profile characterized by high levels of IL-4, IL-5 and IL-13 early in life preceding clinical symptoms 40;41. Other reports have identified attenuated neonatal IFN-g responses and reduced capacity for production of IFN-g in infancy as a marker of the atopic

phenotype 42;43. Initially, the immunological explanation for the hygiene hypothesis was thought to be an imbalance between Th1 and Th2 cells. Insufficient infection-related Th1-responses during the neonatal period and infancy were considered to lead to unrestricted or unbalanced Th2-responses, resulting in atopic diseases. However, the Th2/Th1 paradigm could not be maintained since it has been shown that Th2-responses exist in the absence of allergic disease 18, diseases that are either Th1- or Th2 mediated are not mutually exclusive 44, and Th1 mediated autoimmune diseases are increasing in a similar rate as atopic diseases 11. Therefore, a role for recently rediscovered regulatory T cells and induction of tolerance has been postulated as factors in controlling the development of atopic disease 45. Figure 2 illustrates the development of T cell subsets. In children who have outgrown their cow s milk allergy, tolerance is associated with the presence of functional regulatory T cells 46. Recent studies indicate that allergenspecific regulatory T cell responses may be compromised in atopic diseases. So far, current data suggests that there may not be a numerical deficiency of regulatory T cells, but rather an imbalance between activation of regulatory T cells and effector cells following exposure to Chapter 1 allergens thereby hindering suppression to take place 47-50. Furthermore, it has been suggested that development of atopic diseases may be associated with impaired neonatal regulatory T cell function 51. Despite these associations of the immune system and atopic diseases, there is 16 currently no predictive biomarker for the development of atopic diseases. Probiotic bacteria Building on the hygiene hypothesis or more specific the microbiota hypothesis, modification of the intestinal microbiota by administration of microbes, in particular probiotic bacteria, may be a potential approach to prevent the development of atopic diseases in high-risk children (i.e. with a familial predisposition). Probiotic bacteria are lactic acid bacteria (Lactobacillus and Lactococcus spp), Bifidobacterium spp., Enterococcus spp. and Streptococcus spp., which are natural inhabitants in the intestines and are used for centuries in nutritional products because of their health promoting effect. The first documented scientific study on this issue appeared in 1905, performed by Elie Metchnikoff, who won the Nobel Prize 52 (Box 1). Scientific research into the mechanism of action of probiotics therefore started more than a century ago, and

Figure 2: Pathogens such as viruses, parasites, and bacteria and intestinal commensal bacteria such as probiotic bacteria interact with antigen presenting cells such as dendritic cells (DC) via pathogen associated molecular patterns (PAMPS), such as toll-like receptors (TLRs) and C-type lectins. DC play a pivotal role in polarizing Th1, Th2, Th17 and regulatory T cells. Co-stimulation between DC and T cells and the milieu of cytokines and tissue factors in which these complex interactions take place are involved in the nature of polarizing signals and outcome of T cell differentiation. General introduction the definition of what probiotic bacteria are has changed over the years. Lilly and Stilwell first introduced the term probiotics in 1965, which is derived from the Greek, meaning for life. They 17 defined probiotics as substances secreted by one micro-organism, which stimulate the growth of another 53. The World Health Organization (WHO) has adopted the definition of probiotic bacteria as Live micro organisms which when administered in adequate amounts confer a health benefit on the host. The most recent definition by the Finnish researcher Erika Isolauri is: probiotics are either living or inactivated microbiological cultures with a clear defined aim either to decrease risk of disease or to support the nutritional status 54. However, a good and unambiguous definition is not available, beneficial microbes may come close. The first study to assess the role of probiotics in the context of primary prevention of atopic diseases was published in 2001 by Kalliomaki et al. L. rhamnosus GG was administered prenatally during the last few weeks of pregnancy to the mothers and postnatally during the first 6 months of life to their offspring. The incidence of eczema at 2 years of age was reduced by around 50%

55. This effect was still evident at 4 years of age, but no reduction of IgE, sensitization, asthma of allergic rhinitis was observed 56. Currently, the results of five other studies have been published with conflicting results 57-61. It remains to be elucidated what exactly happens upon ingestion of probiotic bacteria and which effects or processes are initialized or stimulated. Probiotic bacteria may exert their effects at three levels 62 : intestinal microbiota modification, mucosal barrier fortification, and immunomodulation. Probiotic bacteria as part of the intestinal microbiota in the gut lumen assist in the digestion of food components, and may facilitate the colonization of commensal bacteria and prevent the colonization of pathogenic bacteria (level 1). Probiotic bacteria may strengthen the mucosal barrier by up regulation of mucous genes in intestinal goblet cells and prevention of bacterial adherence to the epithelium by competitive exclusion (level 2). Continuous cross talk via the intestinal epithelium, DCs and M cells (specialized epithelial cells) with the gut associated lymphoid tissues by probiotic bacteria may modulate the immune system (level 3) (illustrated in figure 3). The exact mechanisms of action of probiotic bacteria are unknown. Chapter 1 18 Figure 3: The potential mechanisms of action of probiotic bacteria. LF: lymphoid follicle

Aims and outline of the thesis The research described in this thesis was initiated after the first clinical trial addressing the possibilities of probiotic bacteria to prevent the development of atopic disease published by Kalliomaki M et al 63. Primary prevention of atopic disease by pre- and postnatal administration of probiotic bacteria was and is still promising. However, before probiotic bacteria can be recommended as preventive strategy for atopic diseases, results from previous clinical trials need to be confirmed. A gap in this field of research is the lack of studies that attempt to correlate results from in vitro screening assays with probiotics to outcomes in clinical trials. Furthermore, mechanisms of action of probiotic bacteria such as their influence on the intestinal microbiota and developing immune system are still largely unknown. This holds true as well for the relationship between various reported effects and clinical consequences. Research described in General introduction this thesis addresses these topics. In preparation of our clinical trial, the PANDA study, we aimed to make a rational choice from available strains based on their capacity to modulate immune responses. In chapter 2, the capacity of 13 different probiotic bacteria to induce or stimulate cytokine production of peripheral blood mononuclear cells (PBMCs) in healthy volunteers is described. Within the gastrointestinal tract professional antigen presenting cells such as dendritic cells (DCs) interact with the intestinal bacteria and play an essential role in the differentiation of naive T cells into Th1, 19 Th2 or regulatory T cells. In chapter 3 we describe the effects of four selected strains (out of the 13 thirteen tested in chapter 2) on cord blood monocyte-derived DCs and their impact on naive T cell differentiation. We selected three strains: B. bifidum, B. lactis, and Lc. lactis. This probiotic mixture was administered prenatally to pregnant women and postnatally to their offspring at high risk to develop atopic diseases in a randomized double blind, placebo-controlled trial. In this study, we primarily aimed to investigate the effect on the development of eczema and other atopic diseases during the first two years of life, and secondary the initial effects on early microbial colonization of supplemented strains and immune responses (chapter 4). Whether probiotic supplementation of these specific strains indeed results in a different composition of the intestinal microbiota beyond the supplemented strains (transiently or permanently), and if

specific alterations of the intestinal microbiota can be associated with protection against the development of atopic disease is described in chapter 5. The ontogeny of the immune system in high-risk children and the influence of probiotic supplementation on this process are described in chapter 6. A general discussion is presented in chapter 7, remaining questions are discussed, and suggestions for future research are described. A summary of this thesis in English and Dutch is provided in chapter 8. Chapter 1 20

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61 Wickens K, Black PN, Stanley TV, Mitchell E, Fitzharris P, Tannock GW, et al. A differential effect of 2 probiotics in the prevention of eczema and atopy: A double-blind, randomized, placebocontrolled trial. J Allergy Clin Immunol 2008 Aug 31. 62 van Santvoort HC, Besselink MG, Timmerman HM, van Minnen LP, Akkermans LM, Gooszen HG. Probiotics in surgery. Surgery 2008 Jan;143(1):1-7. 63 Kalliomaki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E. Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial. Lancet 2001 Apr 7;357(9262):1076-9. General introduction 25

2 Identification of strong interleukin-10 inducing lactic acid bacteria which down-regulate T helper type 2 cytokines Laetitia E M Niers 1, Harro M Timmerman 2, Ger T Rijkers 2, Grada M van Bleek 2, Nathalie O P van Uden 2, Edward F Knol 3, Martien L Kapsenberg 4, Jan L L Kimpen 1, Maarten O Hoekstra 1 1 Department of Pediatrics, University Medical Centre Utrecht, Wilhelmina Children s Hospital. 2 Laboratory of Pediatric Immunology, University Medical Centre Utrecht, Wilhelmina Children s Hospital. 3 Department of Dermatology/Allergology, University Medical Centre Utrecht. 4 Department of Cell Biology and Histology/Department of Dermatology, Academic Medical Centre, Amsterdam. Clinical and Experimental Allergy 2005;35:1481-1489

ABSTRACT Background Decreased exposure to microbial stimuli has been proposed to be involved in the increased prevalence of atopic disease. Such a relationship was indicated by enhanced presence of typical probiotic bacteria in the intestinal flora correlating with reduced prevalence of atopic disease. Recent clinical trials suggested that probiotic bacteria may decrease and prevent allergic symptoms, but which (different) species or strains may contribute is poorly understood. Objective We sought to select probiotic bacteria by their ability to modulate in vitro production of cytokines by peripheral blood mononuclear cells (PBMCs), to make a rational choice from available strains. Methods PBMCs, purified monocytes, and lymphocytes from healthy donors were co-cultured with 13 different strains of probiotic bacteria. The effect of lactic acid bacteria (LAB) on different cell Chapter 2 28 populations and effects on cytokine production induced by the polyclonal T cell stimulator phytohemagglutinin (PHA) was evaluated by measuring T helper type 1 (Th1), T helper type 2 (Th2) and regulatory cell cytokines in culture supernatants by multiplex assay. Results PBMCs cultured with different strains produced large amounts of IL-10 and low levels of IL-12p70, IL-5 and IL-13. In PHA stimulated PBMC cultures, the tested strains decreased the production of Th2 cytokines. Neutralizing IL-10 production resulted in partial to full restoration of Th2 cytokine production and concurred with an increase in pro-inflammatory cytokines such as IL-12p70 and TNF-a. Within the PBMCs, the CD14 + cell fraction was the main source of IL-10 production upon interaction with lactic acid bacteria. Conclusion Our results indicate that certain strains of lactobacilli and bifidobacteria modulate the production of cytokines by monocytes and lymphocytes, and may divert the immune system in a regulatory or tolerant mode. These specific strains may be favorable to use in prevention or treatment of atopic disease.

INTRODUCTION Modification of the intestinal flora early in life by administration of probiotic bacteria may be a potential approach to prevent atopic disease. Strachan DP et al 1989 1 suggested a decreased occurrence of Th1-associated infections to be a causative factor in the increased prevalence of allergy. More recently, modifications of the hygiene hypothesis have moved away from the direct impact of Th1-driving infections and towards the influence of the diversity of the microbial exposure in general during establishment of the intestinal flora 2;3. In industrial countries, having a high degree of hygiene, changed microbial exposure might cause an altered composition of the intestinal flora and thus, an alteration of the immunomodulatory microbial signaling posed during the critical stages of immune development in infancy 4. Atopic disease is the result of a T helper type 2 (Th2)-deviated immune response characterized by the production of the cytokines IL-4, IL-5 and IL-13. According to the hygiene hypothesis, insufficient infection-related Th1- responses during the neonatal period and infancy were considered to lead to unrestricted Th2- responses, resulting in allergic diseases. However, it has been shown that Th2-responses exist in the absence of allergic disease 3 and in the presence of strong Th1-responses 5. Therefore, a role for regulatory T cells and induction of tolerance has been postulated as factors in controlling the Identification of strong IL-10 inducing lactic acid bacteria development of atopic disease 6. At present, it is unclear how regulatory T cell responses that prevent atopic disease are initiated. 29 Because the gastrointestinal (GI) tract encompasses the largest surface of the human body where microbial products interact with the immune system, much attention has focused on the possibility that the intestinal flora may play a role in the development of atopic disease 2. Indeed, different bacterial colonization patterns have been observed in the intestinal flora of allergic children when compared to non-allergic children. Lactobacilli and bifidobacteria are more commonly found in the intestinal flora of non-allergic children, while allergic children seem to harbor higher numbers of clostridia and staphylococci 7; 8. Lactobacilli and bifidobacteria are most frequently investigated probiotic species for possible beneficial effects in prevention and treatment of atopic disease 9. Preliminary data indicate that probiotic bacteria can be effective in prevention and treatment of atopic disease 10-15. Perinatal administration of probiotic bacteria

(Lactobacillus rhamnosus GG) significantly reduced the development of eczema during the first 2-4 years of life in children with a family history of atopic disease 10; 11. In addition, specific probiotic strains have been beneficial in the treatment of infants suffering from atopic eczema and cow s milk allergy by significantly decreasing the SCORAD score 12; 13; 16. The mechanisms by which probiotic bacteria may modify immune-mediated diseases such as atopic disease, and which different species or strains may contribute are poorly understood. One suggested effective probiotic activity may be the ability to modulate a host s immunity 17; 18. To make a rational choice from available strains, we examined strains of the lactobacilli and bifidobacteria species for immunomodulatory activity. We sought to select probiotic bacteria based on their ability to modulate in vitro production of cytokines by mononuclear cells, and focused on suppression of Th2 cytokines and induction of regulatory cell-derived cytokines such as IL-10. MATERIALS AND METHODS Chapter 2 Cell preparation Sodium-heparinized blood was obtained by venepuncture from four healthy, adult donors, with 30 no history of atopic eczema, asthma or allergies. PBMCs were isolated by centrifugation (1000g for 20 minutes) over a Ficoll density gradient (Pharmacia, Uppsala, Sweden). After washing, the cells were counted and resuspended at a concentration of 5 x 10 6 cells/ml in RPMI 1640 tissue culture medium (Life Invitrogen), L-glutamin (2 mm), penicillin (100 U/ml) and streptomycin (100 mg/ml), and supplemented with 20 % heat-inactivated human AB serum (complete medium). Monocytes were purified from PBMCs by positive sorting using anti-cd14 conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD14 positive monocytes were recovered with a purity of > 90%. The negatively selected cells were used as source of peripheral blood lymphocytes (PBLs). Monocytes as well as PBLs were counted and resuspended at a concentration of 5 x 10 6 cells/ml in complete RPMI medium.

Bacterial strains and preparation of bacteria Thirteen strains out of a set of 75 probiotic strains were selected based on their resistance to acid, digestive enzymes and bile to allow their survival in the first part of gastrointestinal tract, their adherence to colonic cell lines like Caco-2, and general characteristics such as reproducible growth, antimicrobial activity (against microbes associated with atopy like St. aureus and clostridia), and shelf-life. The strains included in this study are Bifidobacterium (B) bifidum W23; B. breve W6; B. infantis W52; B. lactis W18 ; B. longum W51; Lactobacillus (Lb) brevis W63; Lb. casei W56; Lb. paracasei W72; Lb. plantarum W59; Lb. helveticus W60; Lb. rhamnosus W71; Lb. salivarius W24; Lactococcus (Lc) lactis W58. All strains were supplied and prepared by Winclove Bio Industries B.V., Amsterdam, the Netherlands. It should be noted that the term probiotic bacteria is defined as products containing a sufficient number of viable microorganisms to alter the host s microbiota to produce beneficial health effects. While all 75 strains are used in food products, not for all has a health promoting effect been demonstrated. Therefore, the selected bacteria will be referred to as lactic acid bacteria (LAB). From frozen stocks, pure strains were cultured in de Man, Rogosa and Sharpe broth (Merck, Darmstadt, Germany) at 37 C under anaerobic conditions for 22 hours. All bacteria were harvested by centrifugation (3000g for 15 minutes) during stationary growth phase. Pelleted bacteria were then washed three times in Identification of strong IL-10 inducing lactic acid bacteria PBS, concentration was determined by colony forming unit (CFU) counting, and diluted to a final working concentration of 10 8 CFU/ml in RPMI 1640. This stock suspension was aliquoted and 31 stored at 20 C. Survival of bacteria upon freezing and thawing was determined by amount of live and dead bacteria with staining for cfda (live) and Toto-1 (dead). For all strains tested, >80% was alive upon thawing. No significant differences were found in time. One fresh aliquot was thawed for every new experiment to avoid variability in the cultures between experiments. Stimulation of peripheral blood mononuclear cells The effect of probiotic bacteria directly on the different cell populations as well as possible interfering effects of bacteria on cytokine production induced by the polyclonal T cell stimulator phytohemagglutinin (PHA) were evaluated. Cell cultures were set up in duplicate or triplicate in 96-well-flat-bottom polystyrene microtitre plates (Nunc A/S, Roskilde, Denmark). All cultures

contained 0.5 x 10 6 PBMCs, monocytes or PBLs in complete medium. PBMCs were cultured in medium only or stimulated with PHA in a final concentration of 35 mg/ml, which was assessed as the optimal concentration in preliminary experiments. Live probiotic bacterial strains were added in a cell:bacteria ratio of 1:1. Negative control cultures contained unstimulated PBMCs, PHA stimulated PBMCs, monocytes, PBLs or probiotic bacteria. Where indicated, a neutralizing monoclonal antibody (mab) to human IL-10 (clone JES3-9D7, BD Pharmingen, San Diego, CA) was added in a final concentration of 5 mg/ml to PHA stimulated PBMCs cultures 10 minutes before addition of bacteria. The plates were incubated at 37 C in 5% CO 2. The culture supernatants were collected at 24 and 72 hrs and stored at 80 C until analysis of cytokines. Cytokine quantification in culture supernatants Cytokines were detected in supernatants by applying a multiplex assay using a procedure that has been described in detail elsewhere 19. This technique allows identifying 17 different cytokines within a single sample. Since Th2 cytokines are a cardinal feature of allergic immune responses, Chapter 2 for this application IL-4, IL-5 and IL-13 were measured. To address effects on the production of Th1 cytokines and regulatory cytokines, a set of pro-inflammatory cytokines (IL-1a, IL-1b, IL-2, IL-6, IL- 12p70, IFN-g and TNF-a) and IL-10 were measured. 32 Immunocytostaining and flowcytometry After co-culturing PBMCs and probiotic bacteria with and without PHA during 24 and 72 hrs, cells were collected per culture condition. Cells were resuspended in FACS buffer (PBS containing 0.02% azide, 2% FCS and 2 mm EDTA).To block non-specific binding of antibody reagents, cells were incubated with heat inactivated human serum (30 min at 4 C). Subsequently, cells were incubated in 50 ml of FACS buffer containing four appropriately diluted FITC-, PE-, PERCP-, or allophycocyanin (APC)-labeled mabs against human CD3, CD4, CD8, CD14, CD25, or CD69. All mabs were obtained from BD Biosciences. The stained cells were analyzed on a FACS-Calibur using CellQuest software (BD Bioscience, Mountain View, CA). Before intracellular staining, cells were incubated with different probiotic bacteria for 1 and 24 hrs at 37 C, where after 10 mg/ml Brefeldin A (BD PharMingen) was added for 5 additional hours. Cells were fixed in 2%

paraformaldehyde, permeabilized with saponin buffer 0.5 % (PBS containing 0.5% BSA, 0.05% azide and 0.5% saponin), and stained with anti-human IFN-g-FITC and anti-human IL-4-PE (both from BD PharMingen) to determine single-cell IL-4 and IFN-g content. Statistical analysis Statistical analyses were performed with the paired-samples T-test to reveal significant differences between cytokine production in response to different strains of bacteria. Differences were considered significant at P < 0.05. Statistical calculations were performed with SPSS 12.0.1 for windows. RESULTS Production of interleukin-10 by peripheral blood mononuclear cells is induced upon interaction with lactic acid bacteria To study the effect LAB may have in modifying cytokine production by immunopotent cells, we co-cultured PBMCs with 13 different strains of live LAB. Identification of strong IL-10 inducing lactic acid bacteria As shown in figure 1A, production of IL-10 by PBMCs was significantly induced by most of the LAB tested compared with unstimulated PBMCs. IL-10 responses were maximal at 24 hrs after 33 stimulation with the bacteria tested (Figure 1A) but remained significantly increased at 72 hrs (data not shown). Clear differences were observed between LAB regarding their capacity to induce IL-10 production. PBMCs produced minimal levels of IL-5 and IL-13 upon interaction with LAB after 24 hours and 72 hours of culture (Figure 1B shows the results for 72h of culture). No IL-4 production was detected (data not shown). PBMCs co-cultured with LAB showed significant enhancement the pro-inflammatory cytokines IL-1a and IL-1b. Similar results were obtained for production of IL-6, IFN-g, and TNF-a (Table I). Levels of IL-12p70 production were low with the exception of cultures stimulated with Lc. lactis, which stimulated the production of IL-12p70 by PBMCs (Table I). In the bacterial preparations themselves, none of the cytokines could be detected (data not shown).

IL-13 IL-5 Figure 1. Production of cytokines by PBMCs in response to probiotic bacteria Cytokines were analyzed by multiplex assay in supernatants collected from 24 hrs and 72 hrs cultures of human PBMCs with bacteria in a cell:bacteria ratio of 1:1. (a) Production of IL-10 by PBMCs in response to different bacterial species at 24 hrs of culture (optimal time point for IL-10). (b) Production of IL-13 and IL-5 by PBMCs at 72 hrs of culture with different bacterial species. Data are mean ± SEM values of four healthy donors and each culture condition per donor tested in Chapter 2 34 duplicate wells. (*, p < 0.05; **, p < 0.01). Different lactic acid bacteria modulated cytokine production of phytohaemagglutinin stimulated peripheral blood mononuclear cells To determine the effects of LAB interacting with stimulated mononuclear cells, PHA was added to the PBMCs during culture. Most of the bifidobacteria and lactobacilli significantly enhanced production of IL-10 by PHA stimulated PBMCs (Figure 2A shows results for 24h cultures), which was maximal at 24 hrs and remained high until 72 hrs. The capacity of probiotic strains to boost the production of IL-10 differed considerably between strains, with B. bifidum, B. infantis, B. lactis, Lb. casei, Lb. plantarum, Lb. helveticus, and Lc. lactis as the most potent inducers (Figure 2A) Upon interaction with LAB, the production of Th2 cytokines by PHA stimulated PBMCs was reduced. Production of IL-5 was significantly decreased with most of the tested strains (mean reduction 60 %, range 42-74%) compared to the negative control, i.e. PHA stimulated PBMCs only (Figure 2B). Levels of IL-13 were significantly decreased as well (mean reduction 65 %, range 56-71%) (Table II). In general, reduction of IL-5 and IL-13 tended to be more pronounced in cultures

with bifidobacteria when compared to the lactobacilli. In addition, probiotic strains stimulated the production of pro-inflammatory cytokines in PHA stimulated PBMCs. Similar to the results observed in unstimulated PBMCs, levels of IL-1a and IL-1b were increased by LAB. Comparable effects were observed for levels of IFN-g and TNF-a. Production of IL-12p70 was stimulated as well upon interaction with most of the LAB tested although levels of IL-12p70 were generally low. Production of IL-6 by PBMCs was highly induced by PHA. However, this effect of PHA was clearly less when PHA stimulated PBMCs were co-cultured with LAB. In contrast to unstimulated PBMCs, production of IL-6 when compared to the control PHA stimulated PBMCs was now decreased in co-culture with LAB (Table II). Interaction of PBMCs with LAB resulted in enhanced expression of CD25 and CD69 on CD4+ T-lymphocytes (Figure 3). Figure 2. Production of cytokines by PHA stimulated PBMCs in response to probiotic bacteria Cytokines were analyzed by multiplex assay in supernatants collected from 24 hrs and 72 hrs cultures of human PBMCs with bacteria in a cell:bacteria ratio of 1:1. A, Production of IL-10 by PHA stimulated PBMCs in response to different bacterial species at 24 hrs of culture (optimal time point for IL-10). B, Production of IL-5 by PHA stimulated PBMCs at 72 hrs of culture Identification of strong IL-10 inducing lactic acid bacteria 35 with different bacterial species. Data are mean ± SEM values of four healthy donors of two independent experiments and each culture condition per donor tested in duplicate wells. Significant differences between control and bacterial species were tested by the paired-samples T-test (*, p < 0.05; **, p < 0.01)

Chapter 2 36 Table I Cytokine levels of cultured unstimulated PBMCs in response to LAB (pg/ml) Cytokines IL-12 IL-13 IFN-gamma TNF-alfa Culture conditions IL-1alfa IL-1beta IL-2 IL-6 72 hrs 24 hrs 72 hrs 24 hrs 72 hrs 24 hrs 72 hrs 24 hrs 72 hrs 23 + 11 0.9 + 0.3 4.9 + 1.9 2.4 + 1.3 10.3 + 0.1 11.8 + 2.9 84.4 + 39.5 4.5 + 1.9 11.6 + 5.2 4996 + 2625 11 + 6.1 4.9 + 1.9 46 + 10 248 + 37 6627 + 1933 10027 + 3951 1749 + 630 544 + 181 2906 + 236 4.1 + 2.2 14.7 + 5.5 3.1 + 1.0 35 + 9.4 364 + 92 573 + 190 2917 + 474 970 + 266 3997 + 391 4.1 + 1.3 12.4 + 9.3 12.8 + 5.1 21.2 + 11 673 + 190 840 + 271 3750 + 761 861 + 224 3460 + 214 2.5 + 0.8 6.6 + 1.8 7.0 + 1.2 18.5 + 1.4 205 + 93 462 + 232 3307 + 577 841 + 188 3167 + 163 52 + 38 20.4 + 5.6 6.6 + 2.8 24.9 + 9.3 2836 + 1403 5488 + 3763 5431 + 810 2014 + 497 2947 + 198 8.8 + 4.4 13.9 + 7.7 4.1 + 1.5 35 + 8.4 1310 + 437 2972 + 1557 4289 + 1135 2137 + 610 7901 + 3215 8.0 + 2.9 8.3 + 5.2 11.1 + 4.4 30.6 + 12.3 376 + 137 917 + 348 2890 + 1045 910 + 393 3093 + 178 5.3 + 2.5 4.5 + 0.8 5.3 + 1.6 23.3 + 5.6 2339 + 794 4597 + 2517 4087 + 688 2144 + 523 3059 + 244 5.1 + 2.3 4.9 + 1.9 3.6 + 1.2 29 + 11.8 2696 + 847 2574 + 831 6457 + 1289 2660 + 300 3797 + 597 16.5 + 5.4 14.6 + 6.3 5.6 + 1.6 43 + 17 1827 + 645 3716 + 1861 5216 + 521 2419 + 506 3455 + 451 20.0 + 12.2 13.3 + 9.0 5.2 + 1.7 28.2 + 2.2 4120 + 1571 7330 + 3833 7982 + 2069 2694 + 542 5068 + 808 13.7 + 6.9 12.3 + 2.9 4.4 + 2.9 16.1 + 3.5 1551 + 706 2243 + 1533 2173 + 406 779 + 203 1364 + 1136 1.4 + 0.5 3.6 + 0.8 2.4 + 1.3 10.3 + 0.1 11.8 + 2.9 118 + 76 48 + 22 11.6 + 5.2 3371 + 328 210 + 89 107 + 31 4.9 + 2.1 29 + 11.8 11342 + 1648 15173 + 2275 6952 + 1395 4065 + 253 24 hrs 72 hrs 24 hrs 72 hrs 24 hrs 72 hrs 24 hrs Control no PHA 2.1 + 1.5 15 + 6 8 + 3 9 + 2 9.8 + 6.4 53 + 13 106 + 54 Control PHA 59 + 16 469 + 198 248 + 81 1572 + 1366 6722 + 4014 177 + 68 8356 + 5993 B. bifidum 857 + 83 483 + 18 6127 + 747 2946 + 282 7.2 + 3.2 89 + 20 1924 + 228 B. breve 502 + 51 251+ 62 5147 + 958 1641 + 407 29.6 + 10.4 39 + 13 2172 + 493 B. infantis 663 + 137 328 + 73 7028 + 2320 2089 + 651 13.4 + 10 35 + 12 9239 + 7693 B. lactis 1037 + 220 582 + 163 10710 + 2441 3763 + 1077 14.3 + 4.8 66 + 5 3973 + 1972 B. longum 750 + 146 446 + 60 6072 + 1265 2761 + 558 16.1 + 9.3 94 + 24 2192 + 583 Lb. brevis 252 + 82 160 + 45 2768 + 1164 1164 + 386 27.3 + 11.1 31 + 13 3554 + 656 Lb. casei 883 + 159 550 + 120 7515 + 1776 3239 + 688 16.9 + 5.4 56 + 13 2074 + 220 Lb. paracasei 1626 + 344 1003 + 182 12207 + 2861 5148 + 1034 7.2 + 4.2 51 + 3 8831 + 6754 Lb. plantarum 1136 + 100 730 + 76 8285 + 1560 4907 + 570 5.9 + 0.9 102 + 12 1903 + 176 Lb. helveticus 1317 + 168 747 + 99 20845 + 7389 4521 + 422 39.1 + 23.9 68 + 5 3184 + 1075 Lb. rhamnosus 524 + 80 295 + 58 4284 + 705 1857 + 286 11.4 + 5.5 57 + 18 2312 + 318 Lb. salivarius 9.1 + 7.8 17 + 7 91 + 65 52 + 37 12.8 + 5.2 49 + 15 1951 + 1472 Lc. lactis 3544 + 434 2014 + 267 13442 + 3546 11537 + 3487 11.4 + 4.6 54 + 0.1 2574 + 635 Values are means ± standard error of the mean (four healthy donors) Grey data differ significantly (P <0.05) from the control no PHA Bifidobacterium (B); LAB, lactic acid bacteria; Lactobacillus (Lb); Lactococcus (Lc); PBMCs, peripheral blood mononuclear cells; PHA, phytohaemagglutinin.

Table II Cytokine levels of PHA stimulated PBMCs in response to LAB (pg/ml) Cytokines IL-12 IL-13 IFN-gamma TNF-alfa Culture conditions IL-1alfa IL-1beta IL-2 IL-6 72 hrs 24 hrs 72 hrs 24 hrs 72 hrs 24 hrs 72 hrs 24 hrs 72 hrs 24 hrs 72 hrs 24 hrs 72 hrs 24 hrs 72 hrs 24 hrs 4996 + 2625 11 + 6 4.9 + 1.9 46 + 10 248 + 37 6627 + 1933 10027 + 3655 1749 + 630 544 + 181 2697 + 167 76 + 35 26.9 + 8.6 28 + 6.8 97 + 20 12514 + 1690 13284 + 5071 5404 + 1203 2697 + 522 3933 + 309 68 + 25 25.5 + 10.3 31 + 8.7 112 + 17 11875 + 1874 18164 + 6076 5284 + 2061 2647 + 432 3021 + 236 69 + 27 14.2 + 3.7 32 + 6.7 98 + 17 13820 + 1002 14233 + 4821 6634 + 1118 2643 + 514 3699 + 312 127 + 38 13.5 + 5.4 26 + 6.8 94 + 18 14326 + 1262 17168 + 5720 6602 + 1487 2819 + 392 3593 + 227 76 + 31 14.9 + 6.3 34 + 10 96 + 29 13210 + 1118 19385 + 6457 5582 + 629 3677 + 725 3856 + 435 36 + 15 17.1 + 8.6 37 + 12 138 + 29 12540 + 1463 17636 + 5906 3742 + 421 2020 + 326 3372 + 232 57 + 22 13.3 + 4.0 27 + 6.5 105 + 15 13054 + 1186 16956 + 5650 5489 + 692 2706 + 340 10078 + 3539 30 + 11 12.7 + 5.4 20 + 6.4 103 + 28 13408 + 1449 19872 + 6624 5488 + 1269 3071 + 517 4893 + 967 85 + 30 10.3 + 2.8 31 + 8.5 110 + 23 12737 + 1099 17420 + 5826 7971 + 1819 3652 + 545 4440 + 699 67 + 19 33.6 + 14.6 22 + 9 100 + 26 14393 + 1254 19325 + 6435 6669 + 1322 2914 + 332 6219 + 691 41 + 14 10.2 + 3.0 37 + 17 133 + 33 13000 + 1270 17606 + 5863 3335 + 221 1392 + 185 5057 + 1194 22 + 11 5.4 + 1.4 46 + 8.8 133 + 20 12821 + 1383 13816 + 5157 4574 + 716 1418 + 307 4241 + 736 211 + 55 34.3 + 7.8 16 + 6.6 78 + 17 14037 + 1502 16569 + 5570 5288 + 680 3627 + 451 Control PHA 59 + 16 469 + 198 248 + 81 1572 + 1366 6722 + 4014 177 + 68 8356 + 5993 B. bifidum 2044 + 162 2564 + 309 13016 + 2519 13722 + 5938 1695 + 880 78 + 11 3322 + 1785 B. breve 1115 + 234 2323+ 284 9499 + 5189 13804 + 5880 2444 + 1164 127 + 34 2021 + 529 B. infantis 1757 + 184 2385 + 219 19195 + 7001 8962 + 1369 2341 + 1054 108 + 18 1757 + 119 B. lactis 1484 + 153 2180 + 208 12298 + 2840 9020 + 1223 2927 + 1251 106 + 20 3154 + 769 B. longum 1402 + 227 2341 + 150 9482 + 2515 12546 + 2557 2319 + 967 128 + 23 2595 + 246 Lb. brevis 831 + 181 2086 + 274 9276 + 4626 7003 + 1712 2909 + 1386 148 + 35 3103 + 377 Lb. casei 1374 + 173 2340 + 153 12876 + 5044 10481 + 2421 2492 + 1298 110 + 30 2222 + 184 Lb. paracasei 2816 + 464 3438 + 315 15904 + 7886 12903 + 4981 1471 + 727 87 + 28 2647 + 156 Lb. plantarum 1737 + 323 2592 + 208 16354 + 7789 26742 + 13709 2464 + 1143 151 + 50 2460 + 170 Lb. helveticus 1620 + 290 2012 + 125 15529 + 3281 21273 + 10529 2176 + 955 95 + 15 3177 + 1001 Lb. rhamnosus 826 + 82 1433 + 186 7930 + 1916 5990 + 1566 3023 + 1202 125 + 37 3030 + 138 Lb. salivarius 502 + 114 1664 + 356 6824 + 3826 5014 + 2152 7882 + 5695 101 + 33 26025 + 13691 Lc. lactis 2959 + 343 2868 + 294 18645 + 7674 10519 + 3980 2130 + 945 72 + 28 2290 + 135 Values are means ± standard error of the mean (four healthy donors) Grey data differ significantly (P <0.05) from the controls Bifidobacterium (B); LAB, lactic acid bacteria; Lactobacillus (Lb); Lactococcus (Lc); PBMCs, peripheral blood mononuclear cells; PHA, phytohaemagglutinin. Identification of strong IL-10 inducing lactic acid bacteria 37

Lactic acid bacteria induce production of cytokines like IFN-g at the single cell level To confirm that LAB indeed stimulate production of cytokines by PBMCs intracellular production of IFN-g at the single cell level was measured. After 6 hours (1 hour of incubation with LAB followed by 5 hours incubation with BFA), no production IFN-g could be detected. After 24 hrs of culture, production of IFN-g by unstimulated PBMCs was induced by the bacterial species tested (0.11 % positive cells for control compared to 0.33-0.41% for tested strains). In PHA stimulated PBMCs, production of IFN-g was boosted progressively (0.75 % positive cells for control compare to 1.9-2.6% for tested strains) by LAB (data not shown). These data indicate that higher levels of cytokines in culture supernatants indeed reflect production induced by LAB and indicate an increased frequency of responding cells producing IFN-g. Chapter 2 38 Figure 3. Expression of activation markers by lymphocytes in response to probiotic bacteria. PBMCs were cultured with different bacterial species for 24 hrs and 72 hrs and evaluated on the expression of molecules associated with activated T cells. Plots are gated on CD3. A, Expression of CD25 on T-lymphocytes after 72 hrs of culture. Numbers in the upper right quadrant indicate the percentage of CD4 + CD25 total T cells and in the region the percentage of CD4 + CD25 bright T cells; B, CD69 expression on T-lymphocytes after 72 hrs of culture. Numbers indicate the percentage of positive cells in the corresponding quadrant. One representative experiment is shown of two different donors. From the six strains used in these experiments, three representative strains are shown.

Interleukin-10 regulated production of T helper type 2 and pro-inflammatory cytokines by peripheral blood mononuclear cells upon interaction with lactic acid bacteria. As the majority of the tested probiotic species enhanced the production of IL-10 by PBMCs, we next examined whether reduced Th2 cytokine levels measured in culture supernatants of PHA stimulated PBMCs in the presence of LAB could be attributed to their capacity to promote IL-10 production. Neutralization of IL-10 almost completely abrogated the ability of probiotic strains to reduce production of IL-13 by PHA stimulated PBMCs (Figure 4A). Levels of Th2 cytokines in culture supernatants were differently affected by neutralizing IL-10. IL-13 production was restored by almost all strains used, but the suppressive effect on IL-5 production sustained for several probiotic strains, like B. infantis, B. lactis, B. longum, Lb. salivarius, and Lc. lactis (Figure 4B). As production of IL-4 in a primary PBMC culture is low, regulation of IL-4 production by IL-10 was difficult to address. As a control, the neutralizing mab to IL-10 resulted in lower levels of IL-10 in culture supernatants compared to corresponding cultures without the antibody added (range of reduced IL-10 with mab as percentage: 61-90% after 24h of culture). Pro-inflammatory cytokines were significantly affected as well. Neutralizing IL-10 resulted in a robust boost of TNF-a production compared to the control, i.e. PHA stimulated PBMCs only (Figure 4C). Most pronounced effects were observed for IL-12p70. Both at 24 hrs and 72 hrs neutralizing IL-10 Identification of strong IL-10 inducing lactic acid bacteria dramatically increased levels of IL-12 in almost all different bacterial strains (Figure 4D shows results at 72 hrs). Not all strains had the capacity to increase production of IL-12p70 or TNF-a 39 when neutralizing IL-10. In cultures with Lb. paracasei, levels of IL-12p70 and TNF-a remained unaffected (Figure 4C and D).

Figure 4. Effects of neutralizing IL-10 on production of cytokines by PBMCs upon interaction with probiotic bacteria A neutralizing IL-10 mab (5 mg/ml) was added to PHA Chapter 2 40 stimulated PBMCs prior to addition of bacteria. IL-13 (A), IL-5 (B), TNF-a (C) and IL12p70 (D) were measured by multiplex assay in culture supernatants and results are shown for 72 hrs cultures. Data are mean ± SEM values of four healthy donors in which each culture condition was tested in duplicate wells. Significant differences between production of cytokines in culture conditions with or without the neutralizing IL- 10 mab added were tested by the paired-samples T-test (*, p < 0.05; **, p < 0.01). Production of IL-12p70 without mab IL-10 in response to the different bacteria tested was not significantly different from control PHA stimulated PBMCs.

Interleukin-10 is primarily produced by monocytes interacting with lactic acid bacteria To determine which cells are primarily stimulated to produce IL-10 upon interaction with LAB, PBMCs were depleted from CD14 positive monocytes and the negatively selected cells were designated as PBLs. LAB were able to induce IL-10 production directly in PBMCs and monocytes but not in PBLs (Figure 5A and 5C). Clear differences were observed between the capacities of different strains to induce IL-10 production in monocytes. Similar to our results in PBMC cultures, B. bifidum and Lc. lactis were the best inducers. Furthermore, production of IL-6 by monocytes was stimulated by LAB. IL-6 production seemed to be inversely correlated with production of IL- 10, i.e. B. bifidum and Lc. Lactis, which induced large amounts of IL-10, were the weakest inducers of IL-6, and vice versa for Lb. brevis and Lb. paracasei (Figure 5B and D). Consistent with our previous results, IL-12p70 production was low or absent. Identification of strong IL-10 inducing lactic acid bacteria 41 Figure 5. Production of IL-10 and IL-6 by PBMCs, monocytes and PBLs upon interaction with probiotic bacteria. PBMCs, CD14 + monocytes, and PBLs were cultured with different bacterial species. Cytokines were analyzed by multiplex assay in culture supernatants collected after 24 hrs and 72 hrs. Production of IL-10 in PBMCs, monocytes, and PBLs after 24 hrs (A) and 72 hrs (C) of culture. B, D, production of IL-6 by PBMCs, monocytes, and PBLs after 24 hrs and 72 hrs of culture, respectively. The results shown represent mean ± SEM of triplicate measurements in one healthy donor.

DISCUSSION In our study, we showed that live LAB from various species strain-specifically modulated immune responses by inducing the production of IL-10 by in-vitro cultured mononuclear cells. This coincided with reduced production of Th2 cytokines, minimal production of IL-12p70 but substantial induction of IFN-g and TNF-a. We have demonstrated potent effect of LAB on T cell activation in the presence of monocytes both in their resting state, as well as after PHA stimulation. One might argue that in vivo the most likely stimulus for T cells will be an antigendriven stimulation. Although we did not test this type of activation, we speculate that the effects of probiotics will be even more pronounced in an antigen-driven stimulation, because antigen is presented in close contact of the antigen-presenting cells with the T cells. Atopic diseases are characterized by predominant Th2 responses that involve IL-4, IL-5 and IL- 13. Our results confirm previous observations that LAB may have beneficial effects on atopic diseases by reducing the production of Th2 cytokines 20-22. Suppression of Th2 responses in Chapter 2 these studies 20 resulted from down-regulation of IL-4 and IgE production by pro-inflammatory cytokines such as IFN-g or IL-12. Consistent with previous studies in which PBMCs were exposed to various LAB 23; 24, we showed that the 13 strains tested induced pro-inflammatory cytokines 42 such as IL-1a, IL-1b, TNF-a, and IFN-g. However, our study suggests that the Th2 suppressive effect of probiotics may be mediated by immune regulatory functions not linked to an increase in IFN-g secretion or IL-12, but to the induction of regulatory cytokines such as IL-10. In this report, we suggest that LAB may divert the immune system into a regulatory mode by inducing IL-10. The importance of IL-10 in modulating immune responses by probiotic bacteria has recently been demonstrated. In a murine model, specific probiotic species ameliorated colitis by inducing the production of IL-10 by lamina propria mononuclear cells 25. Our results implicate as well a central role for IL-10 in the immunomodulatory properties of specific bacterial species, but clearly not for all strains tested. For specific strains, reduced production of Th2 cytokines by PBMCs in response to LAB resulted from concurrent IL-10 induction since neutralization of IL-10 restored levels of Th2 cytokines. Interestingly, reduction of IL-5 and IL-13 seemed differently regulated. Production of IL-13 was restored in almost all culture conditions to control levels

when neutralizing IL-10, but levels of IL-5 remained significantly reduced for probiotic strains like B. infantis, B. lactis, B. longum, Lb. salivarius, and Lc. lactis. Since IL-10 counteracts the production of IL-12, the poor IL-12 inducing capacity of the probiotic species tested in this study could be secondary to their capacity to promote IL-10. We therefore speculate that LAB can be potent inducers of IL-12 production in mononuclear cells when not inhibited by autocrine IL- 10 production. Alternatively, IL-12 production may also be underestimated due to juxtacrine signaling 26; 27. Of note, not all probiotic strains have the capacity to induce IL-12 production, for example Lb. paracasei in our study. Thus, besides IL-10 also other regulatory cytokines such as for instance TGF-b and other soluble factors may play a role in the regulatory capacities of LAB. In our experimental set-up, antigen-presenting cells (APC) (i.e. monocytes) were required to generate production of IL-10. It has previously been shown that L. plantarum and B. adolescentis induced the production of IL-10 and IL-6 in monocytes 28. However, we observed a reciprocal production of IL-6 and IL-10 by CD14 positive monocytes in response to LAB. IL-6 is produced by various cell types including monocytes and IL-6 is known as a pro-inflammatory cytokine with a role in the acute phase response. Recently, it was shown that production of IL-6 by antigen presenting cells upon microbial induction of the TLR pathway renders responder T cells refractory to the suppressive effect of CD4 + CD25 + Treg cells 29. Although rather speculative, our Identification of strong IL-10 inducing lactic acid bacteria results might indicate that specific LAB could contribute to an environment in which regulatory cells maintain tolerance. 43 To our knowledge, this is the first study showing that live LAB can modulate immune responses by inducing IL-10 in PBMCs, and these effects appeared to be strain-specific. In support of our results, it has been demonstrated that Lb. paracasei induced the development of IL-10 producing CD4 + T cells with low proliferative capacity 30. Moreover, genomic DNA released by exogenous bifidobacteria (B. breve and B. infantis) provided a stimulus for PBMCs to produce IL-10 31. Our results contrast with previous studies where IL-10 was not found to be induced by LAB 23; 24, but rather abundant production of IL-12 32; 33. The use of different strains might explain this discrepancy, since Christensen et al. reported great differences between six lactobacilli strains to induce production of key cytokines such as IL-12 and IL-10 34. Even when we studied six isolates of Lb. acidophilus, we found differences in their capacity to bind to immature dendritic cells (DC) (unpublished data).

We have chosen this approach to select specific probiotic strains for in-vivo purposes. Since live probiotic bacteria are applied in clinical trials, only live probiotic bacteria were tested in this study. Our data should be extrapolated with caution to what is presumably going on in the gutassociated immune system with regard to immunoregulation. Exactly how intestinal microbes interact with the mucosal immune system and what are the active components of LAB remains unclear. Since antigen presenting cells such as DC play a pivotal role in polarizing Th1, Th2 or regulatory T cells, we are currently investigating the effects of four selected strains (out of the 13 thirteen tested in this study) on cord blood monocyte-derived DC and their impact on naive T cells. Whether different bacteria have different clinical effects as suggested by in-vitro studies needs to be addressed further in clinical trials 9. To date, the crucial role of Th2 dominated immune responses in the pathogenesis of allergic diseases is well established. Recent studies indicated that regulatory T cells are likely to be involved in controlling expression of allergic diseases 35-37. Therapies, which enhance regulatory mechanisms and inhibit Th2 cell responses, might be of use in prevention and treatment of Chapter 2 atopic diseases 38. We speculate that specific strains of LAB may divert the immune system in a regulatory or tolerant mode and may therefore be beneficial in coping with or in the prevention of atopic disease. As probiotic strains differ in their capacity to do so, a careful selection for 44 therapeutic strains is necessary. Based on the present study, we have selected B. bifidum, B. infantis, and Lc. Lactis (because of their good IL-10 inducing capacity as well as efficient inhibition of IL-5 and IL-13) to be used as a multispecies probiotics in our clinical trial on primary prevention of allergic diseases by probiotic bacteria.

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3 Selection of probiotic bacteria for prevention of allergic diseases: Immunomodulation of neonatal dendritic cells Laetitia E M Niers 1, Maarten O Hoekstra 1, Harro M Timmerman 2, Nathalie O van Uden 2, Patricia M A de Graaf 2, Hermelijn H Smits 3 *, Jan L L Kimpen 1, Ger T Rijkers 2 1 Department of Pediatrics, Wilhelmina Children s Hospital, University Medical Centre Utrecht. 2 Laboratory of Pediatric Immunology, Wilhelmina Children s Hospital, University Medical Centre Utrecht. 3 Department of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, Netherlands. * Current affiliation: Leiden University Medical Centre, Parasitology, Cellular Immunology of Helminths, Leiden, the Netherlands Clinical and Experimental immunology 2007;149:344-352

ABSTRACT Modification of intestinal microbiota early in life by administration of probiotic bacteria may be a potential approach to prevent allergic disease. To select probiotic bacteria for in vivo purposes, we investigated the capacity of probiotic bacteria to interact with neonatal dendritic cells (DC) and studied the ensuing T cell polarizing effect. Immature DC were generated from cord blood derived monocytes and maturation was induced by maturation factors (MF), lipopolysaccharide (LPS) plus MF, and Bifidobacterium bifidum, Bifidobacterium infantis, Lactobacillus salivarius, Lactococcus lactis alone or combined with MF. After 12 days of co-culture with DC and Staphylococcus aureus enterotoxin B (SEB) as antigenic stimulus, cytokine production by autologous T cells was determined by intracellular cytokine staining. Additionally, cells were stimulated with CD3 and CD28 mabs and cytokines were measured in supernatants by multiplex assay. The probiotic strains induced partial maturation of DC. Full maturation of DC was induced for all strains tested when MF was added. The percentage of interleukin (IL)-4 producing T cells Chapter 3 was lower in T cell cultures stimulated with B. bifidum matured DC compared to MF and LPS matured DC, which coincided with a higher percentage of interferon (IFN)-g producing T cells. Furthermore, T cells stimulated by B. bifidum matured DC produced significantly more IL-10 50 compared to MF matured DC. These results indicate that selected species of the Bifidobacterium genus prime in vitro cultured neonatal DC polarize T cell responses and may therefore be candidates to use in primary prevention of atopic diseases.

INTRODUCTION The hygiene hypothesis states that decreased exposure to microbial stimuli early in life contributes to the increasing prevalence of atopic disease 1; 2. Much attention has focused on the intestinal microbiota and its potential role in the development of atopic disease 3. Enhanced presence of probiotic bacteria in the intestinal microbiota seems to correlate with protection against atopy since in the composition of the intestinal flora of non-allergic children bifidobacteria and lactobacilli are more commonly found 4-6. Intervention studies in which lactobacilli and bifidobacteria were studied for possible beneficial effects in prevention and treatment of atopic disease support the association between the intestinal microbiota and the development of atopic disease 7-10. It is not clear how intestinal microbes including probiotic bacteria interact with the intestinal mucosal immune system and which bacteria or bacterial products are beneficial. Probiotic bacteria may be capable of modulating the immune system 3; 11. For example, reconstitution of the intestinal microbiota of germ-free rodents restored their development of oral tolerance 12. In the gut, antigen-presenting cells (APC), in particular dendritic cells (DC) play a crucial role in innate as well as adaptive immune response against microbial antigens. DC are the main stimulators Probiotics modulate neonatal dendritic cell function of naive T cells and the nature of the T cell polarizing signals is largely determined by the type of microbial products encountered in the peripheral tissues 13. Previous studies have shown that 51 viable and killed probiotic bacteria demonstrate strain specific effects on the phenotype of human and murine DC 14-16, and on polarizing T helper cell responses via modulation of dendritic cell function 17-20. Therefore, we speculate that specific strains may have higher potential to target specific diseases such as atopic diseases. To this end, we chose to select probiotic bacteria based on their capacity to modulate immune responses in-vitro in preparation of our clinical trial on primary prevention of atopy and allergic disease (NCT00200954). Previously, we have examined the effects of thirteen strains of probiotic bacteria on their capacity to modulate cytokine production by adult peripheral blood mononuclear cells (PBMC) 21. We selected four strains to further investigate the effect of probiotic bacteria on neonatal immune cells. In this study, we investigated the effects of four selected probiotic strains on maturation of cord blood

monocyte-derived DC. Furthermore, the effect of DC matured in the presence of probiotic bacteria on polarization of the neonatal T cell response was examined. METHODS Bacterial strains and preparation of bacteria Four strains were selected for the present study based on their capacity to modify cytokine production of PBMC 21. These strains are Bifidobacterium (B) bifidum W23, B. infantis W52, Lactobacillus (Lb) salivarius W24, and Lactococcus (Lc) lactis W58. B. bifidum, B. infantis and Lc. lactis were selected based on their capacity to induce the production of interleukin (IL)-10 and reduction of IL-5 and IL-13 production. Lb. salivarius was included because of its contrasting effect, i.e. no induction of IL-10 production. All strains were supplied and prepared by Winclove Bio Industries, Amsterdam, the Netherlands. Pure strains were Chapter 3 cultured from frozen stocks as previously described 21. One fresh aliquot was thawed for every new experiment to avoid variability in the cultures. 52 Cell preparation Umbilical cord blood was obtained from deliveries of healthy children. The study was approved by the Medical Ethics Committee for human research of the University Medical Centre, Utrecht. Blood samples were collected in cord blood collection bags (MacoPharma, Utrecht, the Netherlands) and mononuclear cells were isolated by density gradient centrifugation over Ficoll- Hypaque (Pharmacia, Uppsala, Sweden). The cells were washed and resuspended in RPMI 1640 containing L-glutamine (2 mm) and penicillin (100 U/ml)/streptomycin (100 mg/ml) (all obtained from Invitrogen Life Technologies, Breda, the Netherlands) and supplemented with 2 % heatinactivated fetal calf serum (FCS). CD14 monocytes were purified by positive selection using anti-cd14 conjugated magnetic microbeads according to the manufacturer s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). Flow cytometric analysis showed that CD14 positive monocytes were recovered with a purity of > 90%. Subsequently, the negatively selected cells

were used to isolate naive T cells from cord blood mononuclear cells by positive selection with anti-cd4 conjugated magnetic microbeads (Miltenyi Biotec). In vitro generation and maturation of DC Immature DC (IDC) were generated by culturing cord blood CD14 + monocytes, as previously described 22. At day 6, maturation was induced by culturing the cells for 2 days with 50 ng/ ml IL-1b and 50 ng/ml tumor necrosis factor (TNF)-a (both Strathmann, Hamburg, Germany), subsequently referred to as maturation factors (MF), lipopolysaccharide (LPS) Escherichia coli (Sigma-Aldrich, St. Louis, MO, USA), plus MF (LPS-DC) and the different probiotic bacteria (20 x 10 6 CFU/ml; bacteria: cell ratio 10:1) in the presence or absence of MF. Expression of cell surface molecules and cytokine production of dendritic cells Expression of cell surface molecules and cytokine production was studied in in vitro generated DC as described above. The maturation status of DC was determined by cell surface analysis on day 8 of culture. DC were washed in fluorescence activated cell sorter (FACS) buffer (phosphatebuffered saline (PBS) containing 0.02% azide, 2% fetal calf serum (FCS) and 2 mm ethylenediamine tetraacetic acid (EDTA) and to block non-specific binding of Ab reagents incubated with heat Probiotics modulate neonatal dendritic cell function inactivated human serum (30 min at 4 C). Subsequently, cells were incubated in 50 ml of FACS buffer containing appropriately diluted fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, 53 peridinin chlorophyll protein (PERCP)-, or allophycocyanin (APC)-labeled mabs against human CD86, CD80, CD14, CD40, and HLA-DR (all from BD Biosciences, Mountain View, CA, USA). Cells were analyzed using FACS-Calibur and CellQuest software (BD Biosciences). Furthermore, at day 8 of the DC cultures, mature DC (2 x 10 4 cells) were stimulated with mouse CD40-ligand (CD40L)- expressing mouse plasmacytoma cells (J558, 2 x 10 4 cells; a gift from Dr. P. Lane, University of Birmingham, United Kingdom) overnight. Supernatants were collected and stored until further use.

Stimulation and culture of CD4 + T cells by mature DC Autologous CD4 + T cells (2 x 10 4 cells) were co-cultured with mature DC (5 x 10 3 cells) in the presence of the superantigen Staphylococcus aureus enterotoxin B (SEB) (100 pg/ml; Sigma- Aldrich) in 96 well flat-bottom culture plates (Nunc, Roskilde, Denmark). At day 5, 10 U/ml recombinant (r) IL-2 (Roche, Basel, Switzerland) was added to the cultures and the cultures were expanded for the next seven days. At day 5, 3 H thymidine incorporation was measured to investigate the capacity of the different matured DC to induce T cell proliferation. Cytokine production by CD4 + T cells On day 12, resting T cells were restimulated with 10 ng/ml phorbol myristate acetate (PMA) (Sigma-Aldrich) and 1 mg/ml ionomycin (Calbiochem, San Diego, CA, USA) for 6h, the last 5h in the presence of 10 mg/ml Brefeldin A (Sigma-Aldrich). Cells were fixed in 2% paraformaldehyde (PFA; Fluka, Deisenhofen, Germany), permeabilized with saponin buffer 0.5 % (PBS containing 0.5% bovine serum albumin (BSA), 0.05% azide and 0.5% saponin (Sigma-Aldrich), and stained Chapter 3 with anti-human IFN-g-FITC and anti-human IL-4-PE (both from BD Pharmingen) to measure intracellular IL-4 and IFN-g production by flow cytometry. In parallel, resting T cells were restimulated overnight with 0.4 mg/ml anti-cd3 mabs and 1 mg/ml anti-cd28 mabs (both 54 from BD Pharmingen). Supernatants were collected and cytokines were detected by multiplex immunoassay on a Luminex-100 system 23. Incubation of bacteria with transfected Chinese hamster ovary (CHO)-cell lines CHO cells transfected with human CD14 (CHO/CD14), human TLR-2 (CHO/CD14/TLR-2) and human TLR-4 (CHO/CD14/TLR-4) were kindly provided by Liana Steeghs (University Medical Centre Utrecht) and Douglas Golenbock (Division of Infectious Diseases and Immunology, University of Massachusetts Medical School). Upon activation via TLRs or CD14, expression of CD25 on the cell surface induced via a transfected nuclear factor kappa B (NF-kB) construct is used as a read out 24. PE-labeled anti-cd25 mab was purchased from Becton Dickinson. Cells were cultured and stimulated as described in detail elsewhere 25. In pilot experiments, viable, heat inactivated (incubated 30 minutes at 56 C) or PFA fixed bacteria were added in different

bacteria:cell ratios, i.e. 50:1, 20:1 and 10:1. Viable and heat inactivated probiotic bacteria in bacteria:cell ratio 50:1 was found to induce to the highest expression of CD25, which was used in further experiments. Heat inactivated Neisseria meningitides was used as a positive control for all three cell lines. CD25 expression was determined by flow cytometry. Statistical analysis All cultures were carried out in duplicate or triplicate. Values and error bars in the figures represent the mean ± standard error of the mean (SEM). Cytokine data were analyzed as continuous data. Non-parametric statistical analyses were performed with the Mann-Whitney U test to reveal significant differences in capacity to induce cytokine production. Differences were considered significant at P < 0.05. Statistical calculations were performed with SPSS 11.5 for windows. RESULTS Probiotic bacteria induce partial maturation of in-vitro cultured DC Probiotics modulate neonatal dendritic cell function We investigated the effect of four probiotic bacterial strains on the expression of CD40, CD80, CD86 and human leukocyte antigen D-related (HLA-DR) as maturation markers of DC. First, we 55 determined the optimal bacteria:dc ratio. A bacteria:dc ratio of 10:1 induced higher expression of co-stimulatory molecules compared to a ratio of 1:1. Higher bacterial doses had a cytotoxic effect on the DC (data not shown). Therefore, a 10:1 bacteria:dc ratio was selected for further experiments. Exposure of IDC to probiotic bacteria resulted in partial DC maturation (Figure 1a) and expression of CD86 and HLA-DR was enhanced significantly compared to IDC with B. bifidum, B. infantis and Lc. lactis (Figure 1b). No differences in expression of CD40 and CD80 were observed between IDC and DC cultured with the four tested strains. However, full maturation of DC comparable to MF-DC and LPS-DC was induced for all strains tested when MF were added (Figure 1a, b). In the presence of MF, no statistically significant differences were observed in the expression of CD86 and HLA-DR, whether bacteria were added or not. In subsequent

experiments, IDC were cultured with probiotic strains in the presence of MF to induce equal maturation in all groups. Chapter 3 56 Lc. Lactis no MF Lc. Lactis Lb. Salivarius no MF Lb. Salivarius B. Infantis no MF B. Infantis B. Bifidum no MF B. Bifidum LPS MF IDC Figure 1. Phenotype and maturation status of cord blood monocyte-derived dendritic cells (DC) upon exposure to selected probiotic bacteria. Immature DC were matured with maturation factors (MF), lipopolysaccharide (LPS) and probiotic bacteria (bacteria : cell ratio 10 : 1) in the presence or absence of MF. After 48 h expression of CD14, CD40, CD80, CD86 and human leucocyte antigen D-related (HLA- DR) was analysed by flow cytometry. A: Open histograms represent expression by immature DC, solid histograms show the level of expression as a result of treatment. One representative experiment of nine is shown. B: Mean expression of CD86 and HLA-DR in differently matured DC. The expression of CD86 and HLA-DR of immature DC was set at 100% and the relative expression in other culture conditions was compared to this value. Data represent mean expression ± standard error of the mean (n = 9). Statistical analysis was performed by Mann-Whitney U-test. * P < 0 05 compared to immature DC. Expression of CD86 and HLA-DR of all fully matured DC (MF, LPS, Bifidobacterium bifidum, B. infantis, Lactobacillus salivarius and Lactococcus lactis) was significantly higher (P < 0 01) compared to immature DC.

Probiotic bacteria do not modify production of cytokines by in-vitro cultured DC Next, we measured the capacity to produce cytokines by DC matured with different stimuli after ligation of CD40 by CD40-ligand (CD40L), mimicking the engagement by T cells. Compared to MF-DC and LPS-DC, production of IL-12 and TNF-α was not significantly (P > 0.05) affected by the presence of the four tested strains during maturation, although we did observe a trend towards increased production of IL-12 (Figure 2a, b). Furthermore, B. bifidum stimulated the production of IL-6 fourfold compared to MF and LPS DC, but not statistically significant (P = 0.909 and P = 0.277 respectively (Figure 2c). Other cytokines were also measured, but production of IL-10 was minimal. No production of IL-1a, IL-1b, IL-2, IL-4, IL-5 and IFN-g was observed (data not shown). Probiotics modulate neonatal dendritic cell function 57 Figure 2. Cytokine production by DC upon exposure to probiotic bacteria. Mature DC (day 8 of culture) were stimulated overnight with CD40-ligand transfected J558 cells to induce production of cytokines. IL-12 and TNF-a were measured in supernatants by multiplex assay. Data represent mean cytokine production ± SEM. Statistical analysis was performed by Mann-Whitney U test.

B. bifidum polarizes in-vitro-cultured DC to drive Th1 responses To investigate the T cell polarizing capacity of DC matured with probiotic bacteria, we performed co-cultures with autologous CD4 + T cells. CD4 + T cells proliferated to a similar extent, independently of a prior incubation of the DC with probiotic bacteria. In parallel, the expression of CD25 as cell surface marker of activation of CD4 + T cells was similar in all co-cultures, indicating equally activated T cells (data not shown). Next, the intracellular cytokine profile of the Th1 cytokine IFN-g and the Th2 cytokine IL-4 was determined. B. bifidum-stimulated DC reduced the number of IL-4 producing T cells and increased the number of IFN-g producing T cells (Figure 3a). The mean percentage of IL-4-producing T cells was decreased up to 50% in T cell cultures stimulated with B. bifidum compared to T cells activated by MF-DC and LPS-DC. In parallel, the mean percentage of IFN-g-producing T cells was increased (Figure 3b). The differences observed in percentages of IL-4 and IFN-g-positive T cells did not reach statistical significance (P > 0.05) due to large donor variability in the number of IL-4 and IFN-g-positive T cells, but consistent effects were observed in individual experiments. Chapter 3 Lc. lactis-stimulated DC seemed to have similar effects on CD4 + cells to B. bifidum but to a much lesser extent. The effect of B. infantis-dc on the number of IL-4 producing T cells was mainly donor dependent, but B. infantis-dc consistently increased the percentage of IFN-g producing 58 T cells (data not shown). Lb. salivarius-dc did not affect IL-4 or IFN-g production. Similar results were obtained when comparing the IFN-g/IL-4 ratio. For MF and LPS matured DCs, the IFN-g/IL-4 ratio was 3.44 and 3.70 respectively. B. bifidum matured DC induced the highest IFN-g/IL-4 ratio, 9.44. IFN-g/IL-4 ratio for B. infantis was 4.18, for Lb. salivarius 4.34 and for Lc. lactis 5.25. Besides IFN-γ and IL-4, we aimed to investigate the production of other cytokines by CD4 + T cells as well. To this end, cells were re-stimulated for 18 hrs with anti-cd3 mab and anti-cd28 mab and we measured levels of cytokines in the supernatants by multiplex assay. Indeed, in cultures of DCs co-cultured with B. bifidum, levels of IFN-g were significantly (Figure 4a). B. bifidum DC also stimulated production of TNF-α by CD4 + T cells as well (Figure 4b). A significant, although weaker, effect on IFN-g production was also observed for Lc. lactis co-cultured DCs (Figure 4a). Cytokine measurements in supernatants did not confirm reduced production of IL-4 for B. bifidum and Lc. lactis-dc as demonstrated with intracellular cytokine analysis (Figure

4c). Furthermore, production of IL-10 by T cells stimulated with B. bifidum-dc was significantly increased compared to MF-DC (Figure 4d). A Figure 3. Intracellular production of interferon (IFN)-γ and interleukin (IL)-4 by CD4 + T cells. Dendritic cells (DC) matured by different stimuli (5 10 3 cells) were co-cultured with CD4 + T cells B Probiotics modulate neonatal dendritic cell function (2 10 4 cells) and Staphylococcus aureus enterotoxin B (SEB) (100 pg/ml). After 12 days, CD4 + T cells were restimulated with phorbol myristate acetate (PMA) and ionomycin (last 5 h in the presence 59 of Brefeldin A) and IFN-γ and IL-4 production per cell was measured by intracellular staining and flow cytometry. (a) One representative experiment of nine is shown. The number in the dot plots are the percentages of cells in the corresponding quadrant of the representative experiment. (b) Data represent mean percentage ± standard error of the mean of IFN-γ and IL-4 producing T cells (n = 9) after co-culture with maturation factors (MF), lipopolysaccharide (LPS) and Bifidobacterium bifidum DC.

Chapter 3 Figure 4. Production of cytokines by CD4 + T cells. CD4 + T cells were stimulated as described in Fig. 3. 60 After 12 days of dendritic cell (DC) T cell co-culture, T cells were restimulated with anti-cd3 and anti- CD28 overnight. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 were measured in supernatants by means of multiplex assay. Data represent mean production ± standard error of the mean (n = 9). *P < 0 05. TLR activation by probiotic bacteria Next, we addressed the question whether cytokine production by probiotic primed DC may indicate the involvement of TLR binding. To this end, CHO-cell lines transfected with human CD14, human TLR-2 and human TLR-4 were incubated with live and heat inactivated probiotic strains. B. bifidum and B. infantis and to a lesser extent Lb. salivarius did activate TLR-2 as shown by increased expression of CD25, in contrast to Lc. lactis (Figure 5a). The capacity of these strains to activate TLRs was not different when live or heat-inactivated bacteria were used. None of the probiotic strains tested activated TLR-4 (Figure 5b) or CD14 (data not shown).

Heat inactivated Alive Probiotics modulate neonatal dendritic cell function 61 Figure 5. Activation of Toll-like receptors (TLRs) by probiotic bacteria. Chinese hamster ovary (CHO) cell lines transfected with human CD14, TLR-2 and TLR-4 were stimulated with live and heat-inactivated bacteria in a 50:1 bacteria:cell ratio. Expression of CD25 on the cell surface induced via a transfected nuclear factor kappa B construct was used via a read-out and determined by flow cytometric analysis. The negative control is the unstimulated transfected CHO-cell line, which has been left unstained. Heat-inactivated Neisseria meningitides was used as positive control in all cell lines. (a) Expression of CD25 indicated as mean fluorescence intensity (MFI) after activation of TLR-2. Negative control is CHO/ CD14/TLR-2 cell line. (b) Expression of CD25 indicated as MFI after activation of TLR-4. Negative control is CHO/CD14/TLR-4 cell line. Results are representative of two separate experiments.

DISCUSSION In the present study, we demonstrate that from the selected strains, B. bifidum had the most consistent effect in modulation of the immune response of neonatal cells. B. bifidum was most potent to polarize DC to drive Th1 cell responses involving increased IFN-g producing T cells concomitant with reduction of IL-4 producing T cells. Lc. lactis was much less effective in this respect. The phenotype of the DC or the cytokine production is not affected directly by any of the tested probiotic bacteria. Most studies investigating the in-vitro effects of probiotic bacteria on immune competent cells have used either murine models or cells from adult humans. Limited studies have addressed the in-vitro immune responses of newborns to bacteria of the intestinal flora. Results of previous studies indicate that different bacterial species and strains have differential effects on immune responses 14; 15; 17. Each strain seems to have its own unique immunomodulatory activity. This may hold true for their clinical application as well and may explain why beneficial effects have Chapter 3 been observed with perinatal administration of probiotic bacteria in high-risk children 8 or not 26. We chose the approach to select specific probiotic strains for in vivo purposes based on their capacity to modulate immune responses. Previously, we showed that B. bifidum, B. infantis, and 62 Lc. lactis reduced production of Th2 cytokines and were potent inducers of IL-10 production in PBMC 21. Subsequently, in this study we investigated the effects of these strains, with Lb. salivarius as control strain, on neonatal cord blood cells. Our results indicate that specific strains of probiotic bacteria are capable of modulating neonatal immune cells and their responses. Furthermore, it supports our approach to make a rational choice from available strains based on immunomodulatory activities 21. We showed that exposure of cord blood-derived IDC to selected probiotic bacteria lead to a moderate up-regulation of co-stimulatory molecules but did not induce full maturation independent from the presence of MF. After addition of MF, the maturation status of probiotic primed DC was not different from DC matured with MF or LPS plus MF. In support of our results, partial maturation of DC upon exposure to probiotic bacteria has been described in human and murine in-vitro cultured DC 14-16;18;27;28. Full maturation of DC with the tested strains

did not significantly affect production of IL-12 and TNF-α by DC, which is in line with previous observations 17. The presence of B. bifidum during maturation of DC stimulated IL-6 production by DC. IL-6 has been associated with the development of atopy 29; 30. In our experimental set-up, we did not observe Th2 skewing with B. bifidum matured DC. In previous studies other probiotic bacteria also inducted IL-6 production by DC, but in support of our results no Th2 skewing but rather Th1 skewing was observed 17; 18. Previously, it has been shown that CBMC and cord bloodderived monocytes produced IL-6, TNF-α, IL-12 (only by CBMC) as well as IL-10 in response to commensal gram-positive bacteria 31. DC mediated T cell activation is determined by expression of HLA-DR, co-stimulatory signals and cytokine production. In this study, the phenotype of DC as investigated by the expression of these signals does not seem to be affected by the presence of the tested probiotic bacteria during maturation of the DC. In particular, all the tested bacteria induced approximately the same level of IL-12. Various additional DC-derived molecules, inflammatory chemokines plus other members of the IL-12 family, i.e. IL-23 and IL-27, have the capacity to polarize T helper cells 13; 28; 32. Because the combination of these factors affects the fate of naive T helper cells, we investigated the effect of DC matured in the presence of probiotic bacteria on the polarization of naive T cell responses. Because of the presence of B. bifidum or Lc. lactis during maturation, Probiotics modulate neonatal dendritic cell function CD4 + T cells were skewed towards a Th1 response by increased production of IFN-γ and reduced production of IL-4. We therefore speculate that the tested strains indeed affect the phenotype 63 of mature DC, but may involve other DC-derived molecules such as ICAM1 (13), IL-23 and IL-27 28, and CXCL9/Mig (29). These results are, to our best knowledge, the first to show that neonatal naive T cells can be skewed towards a Th1 response upon exposure to probiotic bacteria and are in line with other recent publications 18;20. In contrast, human DC exposed to strains of lactobacilli used in other studies induced T cell hyporesponsiveness 19 or regulatory T cells 17. Because B. bifidum-dc polarized CD4 + T cells to produce significantly more IL-10 compared to MF-DC, we speculate that B. bifidum may favor the development of regulatory T cells as well. The exact mechanisms underlying the beneficial effects of probiotics are not understood completely, but may involve pattern recognition molecules such as TLRs. In our study, we

demonstrated that B. bifidum, B. infantis and Lb. salivarius were capable of activating TLR-2. Previous studies have indicated that gram-positive bacteria, such as bifidobacteria, are ligands for TLR-2 33. In support of our results, probiotic strains up-regulated TLR-2 transcripts in dendritic cells, suggesting that TLR signaling could be involved in dendritic cell maturation and activation 18. In addition, supernatant of B. breve induced DC maturation and activation through a TLR2 dependent pathway 33. Because we focus on the application of probiotic strains in atopic disease, we sought to select strains, which reduced neonatal Th2 responses and skewed T cell responses towards Th1 or regulatory T cells. An over-skewing towards Th1 might theoretically pose an enhanced risk for autoimmunity. The hygiene hypothesis also suggests that countries with lower sanitary status have a reduced frequency of autoimmune diseases. Furthermore, the last decades have shown not only a sharp increase in Th2-dominated allergic diseases but also a concomitant increase in Th1-mediated autoimmune diseases, such as multiple sclerosis and type I diabetes 34. These apparent controversies could be resolved if both development of autoimmune as well as allergic Chapter 3 diseases could be due to impaired function of regulatory T lymphocytes. Current interpretation of the hygiene hypothesis as well as accumulating experimental data point towards an important role of regulatory T lymphocytes 34. 64 It has been suggested that newborns that develop atopy in later life show a delayed postnatal maturation of cellular immune functions 35. High-risk children developing atopic disease in their first year of life developed a Th2 cytokine profile characterized by high levels of IL-4, IL-5 and IL-13 within the first 6 months 36. Other reports have identified weaker neonatal IFN-g responses and reduced capacity for production of IFN-g in infancy as a marker of the atopic phenotype 37;38. Our results indicate that specific probiotic strains are capable of driving in vitrocultured neonatal dendritic cells to induce Th1-cell responses. We suggest that selected strains of probiotic bacteria administered orally in the neonatal period and infancy may potentially modulate the immune responses that trigger disorders such as atopic eczema. We are currently investigating this issue in our clinical trial on primary prevention of atopy and allergic diseases by perinatal administration of probiotic bacteria (ISRCTN Register: ClinicalTrials.gov Identifier NCT00200954).

In conclusion, selected strains of Bifidobacteria species prime in vitro-cultured neonatal DC to drive Th1 responses. These strains may be good candidates to test whether probiotic strains can be applied in prevention or treatment of atopic disease. ACKNOWLEDGMENTS We thank Liana Steeghs and Douglas Golenbock for providing the CHO transfected cell lines with CD14, TLR2 and TLR4, and P. Lane and M. Kapsenberg for providing J558 CD40L expressing cell line. We like to thank E. Knol for critically reading the manuscript. This study was funded by the Wilhelmina Children s Hospital. None of the authors had financial relationship with a biotechnology and/or pharmaceutical manufacturer. Probiotics modulate neonatal dendritic cell function 65

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16 Drakes M, Blanchard T, Czinn S. Bacterial probiotic modulation of dendritic cells. Infect Immun 2004 Jun;72(6):3299-309. 17 Smits HH, Engering A, van der Kleij D, de Jong EC, Schipper K, van Capel TM, et al. Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin. J Allergy Clin Immunol 2005 Jun;115(6):1260-7. 18 Mohamadzadeh M, Olson S, Kalina WV, Ruthel G, Demmin GL, Warfield KL, et al. Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization. Proc Natl Acad Sci U S A 2005 Feb 14. 19 Braat H, van den Brande J, van Tol E, Hommes D, Peppelenbosch M, van Deventer S. Lactobacillus rhamnosus induces peripheral hyporesponsiveness in stimulated CD4+ T cells via modulation of dendritic cell function. Am J Clin Nutr 2004 Dec;80(6):1618-25. 20 Pochard P, Hammad H, Ratajczak C, Charbonnier-Hatzfeld AS, Just N, Tonnel AB, et al. Direct regulatory immune activity of lactic acid bacteria on Der p 1-pulsed dendritic cells from allergic patients. J Allergy Clin Immunol 2005 Jul;116(1):198-204. 21 Niers LE, Timmerman HM, Rijkers GT, van Bleek GM, van Uden NO, Knol EF, et al. Identification of strong interleukin-10 inducing lactic acid bacteria which down-regulate T helper type 2 cytokines. Clin Exp Allergy 2005 Nov;35(11):1481-9. 22 de Graaff PM, de Jong EC, van Capel TM, van Dijk ME, Roholl PJ, Boes J, et al. Respiratory syncytial virus infection of monocyte-derived dendritic cells decreases their capacity to activate CD4 T cells. J Immunol 2005 Nov 1;175(9):5904-11. 23 de Jager W, te Velthuis H, Prakken BJ, Kuis W, Rijkers GT. Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells. Clin Diagn Lab Immunol 2003 Jan;10(1):133-9. Probiotics modulate neonatal dendritic cell function 24 Lien E, Means TK, Heine H, Yoshimura A, Kusumoto S, Fukase K, et al. Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide. J Clin Invest 2000 Feb;105(4):497-504. 67 25 Lien E, Sellati TJ, Yoshimura A, Flo TH, Rawadi G, Finberg RW, et al. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem 1999 Nov 19;274(47):33419-25. 26 Taylor AL, Dunstan JA, Prescott SL. Probiotic supplementation for the first 6 months of life fails to reduce the risk of atopic dermatitis and increases the risk of allergen sensitization in highrisk children: a randomized controlled trial. J Allergy Clin Immunol 2007 Jan;119(1):184-91. 27 Veckman V, Miettinen M, Pirhonen J, Siren J, Matikainen S, Julkunen I. Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells. J Leukoc Biol 2004 May;75(5):764-71. 28 Smits HH, van Beelen AJ, Hessle C, Westland R, de Jong E, Soeteman E, et al. Commensal Gramnegative bacteria prime human dendritic cells for enhanced IL-23 and IL-27 expression and enhanced Th1 development. Eur J Immunol 2004 May;34(5):1371-80.

29 Heijink IH, Vellenga E, Borger P, Postma DS, de Monchy JG, Kauffman HF. Interleukin-6 promotes the production of interleukin-4 and interleukin-5 by interleukin-2-dependent and -independent mechanisms in freshly isolated human T cells. Immunology 2002 Nov;107(3):316-24. 30 Doganci A, Eigenbrod T, Krug N, De Sanctis GT, Hausding M, Erpenbeck VJ, et al. The IL-6R alpha chain controls lung CD4+CD25+ Treg development and function during allergic airway inflammation in vivo. J Clin Invest 2005 Feb;115(2):313-25. 31 Karlsson H, Hessle C, Rudin A. Innate immune responses of human neonatal cells to bacteria from the normal gastrointestinal flora. Infect Immun 2002 Dec;70(12):6688-96. 32 Lebre MC, Burwell T, Vieira PL, Lora J, Coyle AJ, Kapsenberg ML, et al. Differential expression of inflammatory chemokines by Th1- and Th2-cell promoting dendritic cells: a role for different mature dendritic cell populations in attracting appropriate effector cells to peripheral sites of inflammation. Immunol Cell Biol 2005 Oct;83(5):525-35. 33 Hoarau C, Lagaraine C, Martin L, Velge-Roussel F, Lebranchu Y. Supernatant of Bifidobacterium breve induces dendritic cell maturation, activation, and survival through a Toll-like receptor 2 pathway. J Allergy Clin Immunol 2006 Mar;117(3):696-702. 34 Bach JF. The effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med 2002 Sep 19;347(12):911-20. 35 Ngoc PL, Gold DR, Tzianabos AO, Weiss ST, Celedon JC. Cytokines, allergy, and asthma. Curr Opin Allergy Clin Immunol 2005 Apr;5(2):161-6. Chapter 3 36 van der Velden, V, Laan MP, Baert MR, de Waal MR, Neijens HJ, Savelkoul HF. Selective development of a strong Th2 cytokine profile in high-risk children who develop atopy: risk factors and regulatory role of IFN-gamma, IL-4 and IL-10. Clin Exp Allergy 2001 Jul;31(7):997-1006. 68 37 Prescott SL, Macaubas C, Smallacombe T, Holt BJ, Sly PD, Holt PG. Development of allergenspecific T-cell memory in atopic and normal children. Lancet 1999 Jan 16;353(9148):196-200. 38 Prescott SL, King B, Strong TL, Holt PG. The value of perinatal immune responses in predicting allergic disease at 6 years of age. Allergy 2003 Nov;58(11):1187-94.

Probiotics modulate neonatal dendritic cell function 69

4 The effects of selected probiotic strains on the development of eczema (The PandA study) Laetitia Niers 1, Rocío Martín 2, Ger Rijkers 1, 3, 4, Florien Sengers 1, Harro Timmerman 3, Nathalie van Uden 1, Hauke Smidt 2, Jan Kimpen 1, Maarten Hoekstra 1 1 Department of Pediatrics, Wilhelmina Children s Hospital, University Medical Center, Utrecht, the Netherlands. 2 Laboratory of Microbiology, Wageningen University, Wageningen, the Netherlands. 3 Department of Surgery, University Medical Center, Utrecht, the Netherlands. 4 Department of Medical Microbiology and Immunology, Sint Antonius Hospital, Nieuwegein, the Netherlands. Allergy 2009;64:1349-58

ABSTRACT Background Modification of the intestinal microbiota by administration of probiotic bacteria may be a potential approach to prevent allergic disease. We aimed to study primary prevention of allergic disease in high-risk children by pre- and postnatal supplementation of selected probiotic bacteria. Methods In a double blind, randomized, placebo-controlled trial, a mixture of probiotic bacteria selected by in-vitro experiments (B. bifidum, B. lactis and Lc. lactis; Ecologic Panda), was prenatally administered to mothers of high-risk children (i.e. positive family history of allergic disease) and to their offspring for the first 12 months of life. Results Parental-reported eczema during the first three months of life was significantly lower in the Chapter 4 intervention group compared to placebo, 6/50 vs. 15/52 (p = 0.035). After 3 months, the incidence of eczema was similar in both groups. Cumulative incidence of parental-reported eczema at 1 and 2 year was 23/50 (intervention) vs. 31/48 (placebo) and 27 (intervention) vs. 34 (placebo) 72 respectively. The number needed to treat was 5.9 at age 3 and 12 months and 6.7 at age 2 years. The intervention group was significantly more frequently colonized with higher numbers of Lc. lactis. Furthermore, at age 3 months in vitro production of IL-5 (146 pg/ml vs. 72 pg/ml; p= 0.04) was decreased in the probiotic-group compared to the placebo-group. Conclusions This particular combination of probiotic bacteria shows a preventive effect on the incidence of eczema in high-risk children, which seems to be sustained during the first two years of life. In addition to previous studies, the preventive effect appears to be established within the first three months of life.

INTRODUCTION Atopy and allergic diseases add considerably to childhood morbidity and the prevalence of allergic diseases has increased during the past few decades. This warrants the development of strategies to prevent allergic diseases. The hygiene hypothesis suggests that the increased prevalence of allergic diseases in children is associated with reduced exposure to microbial components early in life 1. Differences in the intestinal microbiota between allergic and nonallergic children have been described, which precede the development of allergic disease suggesting a potential causal relationship 2; 3. Furthermore, reconstitution of the intestinal microbiota of germ-free rodents with B. infantis restored their development of the otherwise defective oral tolerance 4. Building on this hypothesis, primary and tertiary prevention of allergic disease could be accomplished by administration of appropriate microbes. To date five clinical studies on primary prevention by probiotic bacteria of allergic diseases have been published with conflicting results regarding the effect on (atopic) eczema 5-9. No preventive effect on the development of other allergic diseases was reported so far. It has been questioned if, among other reasons, the use of different probiotic bacteria with assumingly different properties may have attributed to this disparity. Mechanisms by which probiotic bacteria The effects of selected probiotic strains on the development of eczema could induce a beneficial effect, in this context prevention of allergic disease, still remain to be elucidated. Probiotic bacteria may act at three different levels 10 : intestinal microbiota 73 modification, mucosal barrier fortification, and immunomodulation. Each probiotic strain has its unique immunomodulatory activity in vitro. This may hold true for their clinical application as well. We hypothesized that rationally selected strains might have more potential to target specific diseases such as allergic diseases. To study the possibilities of probiotic bacteria in primary prevention of allergic diseases in high-risk children, we selected three specific strains, B. bifidum, B. lactis and Lc. lactis. The strains were selected from a large collection of 69 potentially probiotic strains 11 on basis of their resistance to low gastric ph, pancreatic digestive enzymes and bile salts to allow their survival in the first part of the gastrointestinal tract, and general characteristics such as absence of production of D-lactate, reproducible growth and stable shelf-life. The selection of the specific bacteria was mainly based on their capacity to suppress

in vitro production of Th2 cytokines and to stimulate IL-10 production, both by PBMCs 12; 13. The three selected strains (Ecologic Panda) were administered prenatally to pregnant women and postnatally to their offspring at high risk to develop allergic diseases in a randomized double blind, placebo-controlled trial. In this study, we aimed to investigate the effect on the development of eczema during the first two years of life, and the initial effects on early microbial colonization and immune responses. METHODS Participants Families with a positive family history of allergic diseases, i.e. atopic eczema, food allergy, asthma or allergic rhinitis in either the mother, or the father plus an older sibling with a history of allergic diseases were recruited by an advertising campaign. Between March 2004 and July Chapter 4 2005, one hundred and fifty six pregnant women and their families were included at least two months before expected delivery. Children were excluded from the study if their mother received antibiotic treatment during the last two weeks of pregnancy, when the child was born 74 before 37 weeks of gestation, if the children received antibiotic treatment in the first two weeks of life, if ingestion of the study product was difficult due to vomiting or feeding problems in general for longer than 3 weeks from birth, or if the children had other major medical problems. Study design In a double blind, randomized, placebo-controlled clinical trial, the probiotic bacteria were prenatally administered to the pregnant mothers the last six weeks of pregnancy and postnatal for 12 months to their offspring. The intervention group received once daily 3 x 10 9 colony forming units (CFU) (1 x 10 9 CFU of each strain: B. bifidum W23, B. lactis W52 (previously classified as B. infantis), and Lc. lactis W58) of freeze dried powder of the probiotic mixture (Ecologic Panda, supplied by Winclove Bio Industries B.V., Amsterdam, the Netherlands). The individual probiotic cultures carry the European Union qualified presumption of safety (QPS). The control

group received placebo consisting of the carrier of the probiotic product, i.e. rice starch and maltodextran. Both supplements were dispensed as a stable powder in identical individually packed sachets containing 3 grams of material. The contents of each sachet was mixed with at least 10 ml of water, breast milk or infants formula, depending on the choice of feeding by the parents, and ingested as a suspension. Prior to the study, the stability and biological activity of the probiotic mixture in water, breast milk and infants formula was confirmed by measuring ph and CFU after suspending the product. During the study, the viability of the probiotic product was tested every 6 months. Block randomization with a block size of ten was used. Visits were scheduled at the age of 3 months, one year and two years. After birth, parents received at least one phone call to check on compliance and offer assistance if necessary to study related questions. Parental written informed consent was obtained. The study was approved by the Medical Ethics Committee of the University Medical Centre Utrecht, the Netherlands. Clinical outcome variables Parents were asked to complete a weekly diary of health status of their child including presence and complaints of eczema, infectious or atopy related complaints, visits to the family doctor, feeding habits including adverse events or difficulties with feeding, medication use, and The effects of selected probiotic strains on the development of eczema immunization status. Weekly completion of the diary was asked for to circumvent recall bias at the study visits. Furthermore, an adapted version of the British Medical Research Council 75 questionnaire 14;15 and the Dutch version of the European Community Respiratory Health Survey 16 was used to evaluate participants for symptoms indicative of food allergy, eczema, asthma, and allergic rhinitis at the age of 3, 12 and 24 months. In addition, parents were asked about their compliance and if they experienced problems with the daily administration of the study product. Parental-reported eczema was defined as eczema reported as complaint by the parents in the diaries. Physician-diagnosed eczema was defined as eczema reported by the parents and diagnosed as eczema by the family doctor or consulted physician. Eczema was defined as non-infectious dermatitis with typical features (redness, dryness, edema, oozing, and itching (scratching)) and distribution 17. Atopic eczema was defined as eczema plus sensitization, i.e. detectable (> 0.35 KU/ml) allergen specific IgE antibodies or a positive skin prick test (SPT).

A complete physical examination was performed at every visit. The presence and severity of eczema at the time of the visit were assessed by the basic clinical scoring system (BCSS) 18 and SCORAD 19. SPTs were performed at 24 months. Histamine dihydrochloride (10 mg/ml) was used as a positive control and the solvent (glycerine) as a negative control. SPTs were performed with allergen extracts for egg white, cow s milk, peanut, hazelnut, cat, dog, house dust mite (Dermatophagoides pteronyssinus), birch and grass (ALK-Abelló, Nieuwegein, the Netherlands). A wheal diameter at 15 minutes of > 3 mm larger than that of the negative control was considered positive. The physicians who were involved in the follow-up visits and physical examinations (LN, who performed all follow-up visits, and MH) were kept blinded with respect to group allocation until all children were seen at the age of two years (November 2007). Determination of serum total IgE and specific IgE Blood samples were collected at each follow-up visit to determine total IgE, a food panel (FP5, consisting of egg white, cow s milk, codfish, wheat, peanut and soybean), AlaTOP (multi allergen Chapter 4 screening test containing several inhalant allergens), and specific IgE for egg white, cow s milk, peanut, house dust mite and cat dander epithelium on the IMMULITE 2000 (Diagnostic Products Corporation, Los Angeles, CA, USA). For specific IgE tests and FP5, a positive result was defined 76 as a concentration of > 0.35 IU/ml. For AlaTOP a result was classified as positive at > 1.00 IU/ml. Molecular analysis of fecal microbiota Stool samples were collected according a prescribed schedule: four stool samples from birth during the first four weeks of life starting with the first stool of the newborn and with one-week intervals between each sample, one sample at 3 months of age. At 12 months of age, a stool sample 1 week and 1 day preceding the end of the administration period, and two stool samples after (1 and 2 weeks respectively) the end of intervention. In the analysis, samples 1 day before and 2 weeks after end of intervention were used. Microbial Community Profiling and Characterization (MCPC): This qualitative analysis is based on Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis, was performed by the Dr. van Haeringen Laboratories (Wageningen, the Netherlands).

DNA isolation for quantitative PCR and DGGE analysis: DNA was extracted from 200 mg of fecal samples taken from eight randomly chosen infants per group at seven different time points using the FAST DNA SPIN Kit (Q-biogene, Carlsbad, CA, USA). The DNA was eluted in 100 µl of autoclaved water and stored at -20 C. Quantitative PCR: SYBR Green PCR amplifications were performed in triplicate in an icycler iqreal-time Detection System (Bio-Rad Laboratories, Hercules, CA, USA) associated with the icycler Optical System Interface software (version 2, Bio-Rad). The number of Lc. lactis and bifidobacteria were determined in reactions of 25 µl with primers LcoLacF and LcoLacR 20, and Bif-recA-F and Bif-recA-R 21 respectively, both targeting a specific fragment of a single copy gene (reca). Reaction mixtures contained 1x SYBR Green PCR Mix (Bio-Rad), 0.5 µm of each Lc. lactis specific primers or 1 µm of each bifidobacteria specific primers, and either 5 µl of 10-fold diluted DNA extracts as template or water. PCR conditions for bacterial quantification were: initial denaturation at 95 ºC for 3 min followed by 40 cycles of 95 ºC for 15 s, annealing at 62 ºC (Lc. lactis) or 64 ºC (bifidobacteria) for 30 s, elongation at 72 ºC for 30 s and a final cycle at 95 ºC for 1 min. Following amplification, melting temperature analysis of PCR products was performed to determine the specificity of the PCR. Serially diluted reca PCR products obtained from genomic DNA of B. bifidum and Lc. lactis were used a real-time PCR control standard for quantification. The effects of selected probiotic strains on the development of eczema The amplicons were purified using DNA Clean and Concentrator-5 (ZymoResearch, Orange, CA, USA) according to the manufacturers instructions and the yield was quantified with the 77 Nanodrop-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA). The amount of gene copy numbers per ml was calculated from the spectrophotometric results using the average molecular weight of a nucleotide and a standard solution was serially diluted from 10 8 to 10 and used in each Q-PCR run. As reca occurs as a single copy gene in bacterial genomes, gene copy numbers are equivalent to cell numbers, assuming one genome per bacterial cell. Bifidobacterium-specific PCR amplification and DGGE: DNA isolated from infant feces at week 2, week 3 and week 12 was used as a template to perform the Bifidobacterium genus-specific PCR using 16S rrna gene targeting primers, Bif164-f and Bif662-GC-r 22. DGGE analysis of PCR amplicons was essentially performed as described previously 22 using the DCode System (Bio- Rad), only that a gradient of 45-50% denaturant was used.

Whole blood cultures and cytokine analysis Blood samples were collected at the age of 3 months. The blood was diluted 1:10 with RPMI 1640 containing L-glutamine (2 mm) and penicillin (100 U/ml)/streptomycin (100 mg/ml) (all from Invitrogen Life Technologies, Carlsbad, CA, USA). Whole blood cultures were carried out in quadruplicate. Cells were cultured in medium only or stimulated with a combination of anti- CD2 mab (CLB-T11.2/1 and CLB-T11.1/1, 1 μg/ml) and anti-cd28 mab (CLB-CD28/1, 4 μg/ml) (both obtained from Sanquin, Amsterdam, the Netherlands), or phytohemagglutinin (PHA; Murex Biotech Ltd, Kent, United Kingdom) at a final concentration of 25 μg/ml. Culture supernatants were collected at 48 and 72 hrs. Cytokines were detected in supernatants by multiplex assay (Luminex) as previously described in detail 23. 3 H thymidine incorporation was measured to determine lymphocyte proliferation. Statistical analysis To detect a 50% reduction in the frequency of (atopic) eczema (based on the data published by Chapter 4 Kalliomäki et al 9 ) at the 5% significance level with 80% power, 49 participants per group were required. A larger number was recruited to allow for an estimated 20% withdrawal rate. For baseline characteristics, χ 2 test was used to compare frequencies, and the independent samples 78 t test was used to compare continuous data. To study the effects of intervention, odds ratios with 95% CI were calculated by multiple logistic regression, correcting for potential confounders such as maternal atopy, birth weight, breastfeeding, and gender. Cytokine data were analyzed as continuous data. The cytokine data were normalized by logarithmic transformation and analyzed by the independent samples t test to determine differences between groups. QPCR data of the intestinal microbiota were analyzed by Anova/repeated measurements with Bonferroni post hoc test to correct for multiple comparisons. Differences were considered significant at 2-tailed P < 0.05. All calculations were performed with SPSS 14.0 for windows (Chicago, IL, USA).

RESULTS Baseline characteristics and participants A total of 156 participants were included in the study of which 102 completed the 3 months of follow-up. 98/156 participants were followed-up at 12 and 24 months of age (see Figure 1). 35 participants met the exclusion criteria after the mother was included. Participation was discontinued on parental request in 23 cases (see Figure 1). Motivational problems resulted in almost 15% of dropouts. Another reason for dropout was maternal (pregnancy related) health problems, which made further participation for these parents too demanding. The rate of dropouts was similar in both groups, as were the reasons for discontinuation. Baseline characteristics were similar in both groups (see Table I). The total number of pets at home was significantly higher in the placebo group compared to the intervention group (see Table I). Baseline characteristics of the dropouts did not differ from the participants who completed the study, which potentially rules out bias. Incidence of eczema and sensitization Eczema was reported by the parents in the weekly diaries during the first 3 months of life in The effects of selected probiotic strains on the development of eczema 15/52 (29%) children in the placebo group and 6/50 (12%) in the intervention group, odds ratio (OR) 0.322 (95% CI 0.108-0.960), p = 0.035 (see Figure 2A). Not all parents consulted their family 79 physician to evaluate their child s eczema. In 11/52 (21%) children in the placebo group and 3/50 (6%) children in the intervention group, eczema was diagnosed by the consulted physician, OR 0.217 (95% CI 0.053-0.891), p = 0.021 (see Figure 2A). The incidence of eczema increased during the second half of the first year of life. Between the age of 3-12 months and 12-24 months, the incidence of eczema increased but similar in both groups.

Chapter 4 80 Figure 1: Screening, enrollment and outcomes

Table I Baseline characteristics Placebo (n=52) Probiotics (n=50) Family characteristics Maternal atopic disease 44 (85%) 41 (84%) Age mother at time of birth 31.4 (30.4-32.5) 32.3 (31.1-33.5) Older sibblings - none 18 (35%) 17 (34%) - one or more 34 (65%) 33 (66%) Pets at home* 23 (44%) 11 (22%) - cat(s) 11 (21%) 8 (16%) - dog(s) 8 (15%) 3 (6%) - bird(s) 2 (4%) 0 (0%) - other 9 (17%) 2 (4%) Birth characteristics Male Gender 23 (44%) 18 (36%) Mode of delivery - vaginal 46 (89%) 46 (92%) - caesarean section 6 (11%) 4 (8%) Birth Weight (grams) 3658 (3520-3796) 3558 (3412-3705) Gestational age (weeks) 39.7 (39.3-40.1) 39,7 (39.2-40.2) Breastfeeding during the first year of life 41 (79%) 42 (84%) - mean duration of breastfeeding 7.2 (6.1-8.3) 7.0 (5.9-8.2) Duration intake study products mother (weeks) 6.0 (5.6-6.4) 6.0 (5.6-6.4) * p = 0.021 Data represent absolute numbers (percentage) or mean (95% CI) The effects of selected probiotic strains on the development of eczema Cumulative incidence of parental reported eczema at 1 and 2 years was 23/50 (intervention) vs. 30/48 (placebo), and 27 (intervention) vs. 33 (placebo), respectively (Figure 2B). At the age of 3 81 months, relative risk reduction was 58%, which decreased to 26 % and 22 % at respectively 1 year (OR 0.495 (95% CI 0.221-1.105) p 0.086) and 2 years of age (OR 0.518 (95% CI 0.227-1.180) p 0.117). To indicate the clinical significance of the observed effects on the development of eczema, the number needed to treat was calculated, which was 5.9 at the age of 3 and 12 months and 6.7 at the age of 2 year. The severity of eczema was similar in both groups and consisted of mild to moderate eczema in the majority of cases: mean (± SD) SCORAD score at 3 months was 21.0 (± 10.5) vs. 20.4 (± 10.9), at 1 year 19.6 (± 8.5) vs. 15.8 (± 6.2) (p 0.289), and at 2 year 18.7 (± 14.4) vs. 26 (± 13.8) (p 0.270), respectively for the placebo vs. intervention group.

A Proportion of 3 months old children with eczema (%) 50 Placebo Probiotics 40 30 20 10 B 0 40 Parental reported eczema Placebo Physician diagnosed eczema Probiotics Chapter 4 82 Cumulative incidence in absolute numbers 30 20 10 * 0 3 12 24 age in months Figure 2: A: Preventive effect of probiotic supplementation on eczema at age 3 months. Columns represent the percentage of affected children and bars are 95% CI. Univariate statistical analysis was performed with χ 2 test and multivariate analysis by multiple logistic regression analysis. B: Cumulative incidence in absolute numbers of parental reported eczema during the first two years of life. Univariate statistical analysis was performed with χ 2 test and multivariate analysis by multiple logistic regression analysis. * = P < 0.05.

No differences were observed in the cumulative incidence of atopic eczema. During the two year follow-up period, 8/48 (17%) of the placebo group vs. 10/50 (20 %) (p 0.876 by χ 2 test) of the intervention group suffered from eczema ánd were sensitized. The diagnosis of asthma or allergic rhinitis is difficult at this age, but no differences were observed in respiratory symptoms such as wheezing, coughing and rhinitis. Total IgE was measured in 89%, 94% and 92% of participants at the age of 3, 12 and 24 months respectively. No differences either were observed in median total IgE between groups at different ages or in the number of participants with increased total IgE at the different ages (data not shown). In the intervention group, a trend was observed towards more frequent sensitization, especially to food allergens, compared to the placebo group at the age of two years and during the whole study period (see Table II). However, this did not result in more food allergic participants in the probiotics group. Table II Sensitization: Total IgE, allergen specific IgE antibodies and skin-prick test reactions. Probiotics Placebo Total IgE (IU/ml)* 3 months 4.4 ± 1.3 3.8 ± 0.8 p = 0.665 12 months 18.5 ± 4.5 22.2 ± 4.2 p = 0.545 24 months 35.1 ± 7.7 54.6 ± 13.1 p = 0.210 one or more sige > 0,35 IU/ml 3 months 3/43 (6,9%) 1/51 (2%) p = 0.230 12 months 9/47 (19,1%) 4/45 (8,9%) p = 0.158 - food allergens 8/47 4/45 p = 0.247 - inhalation allergens 1/47 1/45 p = 0.975 24 months 9/44 (20%) 4/46 (9%) p = 0.113 - food allergens 8/44 (18%) 3/46 (6,5%) p = 0.091 - inhalation allergens 2/44 (5%) 3/46 (7%) p = 0.66 The effects of selected probiotic strains on the development of eczema 83 Positive SPT at 24 months 4/46 (9%) 4/47 (8,5 %) p = 0.975 - food allergens 2/46 (4%) 3/47 (6%) p = 0.66 - inhalation allergens 3/46 (7%) 1/47 (6%) p = 0.29 Sensitisation** - at 24 months 10/50 (20%) 7/48 (14,6%) p = 0.451 - ever (0-24 months) 17/50 (34%) 9/48 (18,8%) p = 0.087 - ever for foodallergens (0-24 months) 15/50 (30%) 7/48 (15%) p = 0.067 * data indicate mean ± SEM ** Either positive SPT or/and sige > 0,35 IU/ML

Composition of the gastrointestinal microbiota In a primary analysis, with randomly chosen fecal samples of 38/48 participants in the placebo group and 35/50 in the intervention group, the development of the intestinal microbiota was evaluated by MCPC which is a qualitative analysis based T-RFLP analysis. Participants in the probiotic group were significantly more frequently colonized with higher numbers of Lc. lactis compared to the placebo group during the first 3 months of life (data not shown). No differences were observed in the first four weeks of life in the number of children colonized by bifidobacteria. At the age of 3 months, all children in the probiotic group and 85% of the placebo were colonized with bifidobacteria (data not shown). Subsequently, to confirm and quantify these findings, the number of Lc. lactis and Bifidobacterium spp. was quantified using qpcr in fecal samples of eight randomly chosen participants in both groups. Lc. lactis was present in all fecal samples from the intervention group, but only in 2/8 from the placebo group. The number of Lc. lactis was significantly higher in the intervention group (see Figure 3A). Bifidobacterium spp. were present in all of the individuals in high numbers (see Figure 3B). To study qualitative Chapter 4 differences in the composition of Bifidobacterium spp., fecal samples were analyzed by DGGE. B. bifidum was present in all the samples analyzed from the intervention group (8/8) but only in half of the placebo group (4/8). Fecal colonization with B. lactis was more difficult to detect 84 (Figure 4). Cytokine production in whole blood cultures at 3 months of age In 46/48 participants of the placebo group and 43/50 in the intervention group whole blood cultures were performed and levels of cytokines at 48 and 72 hrs of culture were measured (Figure 5). Despite large individual variability, IL-5 was significantly reduced (p=0.04) in the intervention group at 72 hrs of cell cultures stimulated with anti CD2/CD28 mabs (Figure 5B). In the placebo group mean ± SEM levels of IL-5 were 145.7 ± 49.2 (pg/ml) vs. 72.2 ± 21.8 (pg/ml) in the probiotics group. For IL-13 a trend was observed towards decreased production in the intervention group at 48 and 72 hrs of cell cultures stimulated by anti CD2+CD28 mabs, 169.7 ± 63.9 (pg/ml) vs. 79.7 ± 22.8 (pg/ml) placebo vs. intervention group (p=0.08) and 442.1 ± 100.4 (pg/ml) vs. 299.2 ± 84.7 (pg/ml) placebo vs. intervention group (p=0.06) respectively (Figure 5A

The effects of selected probiotic strains on the development of eczema 85 Figure 3: Real time PCR quantification of Lc. lactis (A) and Bifidobacterium spp. (B). QPCR in stool samples of eight randomly chosen participants per placebo and intervention group at weeks 1, 2, 3, 4, 12, 52 and 54 using reca as target gene. Results are depicted as log gene copy numbers per gram feces. Each symbol represents data from an individual participant. Statistical analysis was performed with repeated measurements. The number of Lc. lactis is significantly (P < 0.0001) higher in the probiotic group during the first year of life. Bonferroni posttests indicated significant differences at week 2 (P < 0.01), week 3 and 4 (P < 0.001), and week 12 (P < 0.05).

PLACEBO PROBIOTIC M PB 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 PB M Chapter 4 Figure 4: DGGE of PCR-amplified bifidobacterial 16S rrna gene fragments from 8 randomly chosen participants per placebo group (numbered 1-8) and intervention group (9-16) after 3 weeks of 86 supplementation. The mobility of the PCR products obtained in DGGE was compared with the profile of the probiotic mixture Ecologic Panda obtained with the same primer set (lanes indicated by PB). Fragment A: Bifidobacterium bifidum; fragment B: Bifidobacterium lactis. and B). In a subgroup analysis, the mean production of IL-5 and IL-13 was consistently highest in the participants of the placebo group who developed eczema during the first three months of life: 2-3 fold increased compared to the participants in the placebo group who did not develop eczema. In the probiotics group, mean production of IL-5 and IL-13 was not different between the participants who did or did not develop eczema in the first 3 months of life, but was in general lower compared to the placebo group with no eczema. No differences of in vitro IL-10 production were found in CD2/CD28 stimulated cultures (see Figure 5A and B) or in PHA stimulated cultures

(data not shown). The in vitro lymphocyte proliferative response to either anti CD2/CD28 or PHA did not differ between the groups (data not shown). The effects of selected probiotic strains on the development of eczema 87 Figure 5: Production of cytokines in whole blood cultures at age 3 months. Whole blood cultures were left unstimulated (medium) or stimulated with anti CD2/anti CD28 mab, or PHA. Supernatants were collected at 48 hrs and 72 hrs and cytokines were measured by a multiplex assay (Luminex). A: anti CD2/CD28 48 hrs; B: anti CD2/CD28 72 hrs. Data represent mean ± SEM. * P < 0.05

DISCUSSION With perinatal administration of Ecologic Panda, we aimed at influencing early colonization with probiotic bacteria to provide immunoregulatory signals, which would result in primary prevention of allergic disease such as eczema. In this study, we have shown that administration of selected strains of probiotic bacteria prevented the development of eczema during the first two years of life in high-risk children. The children who received probiotic bacteria were more frequently colonized with higher numbers of Lc. lactis, and reduction of eczema at 3 months of age was paralleled by a decreased in vitro production of IL-5 and to a lesser extent IL-13. Thus far, five clinical trials on primary prevention of allergic disease by perinatal administration of probiotic bacteria have been published with inconsistent results 5-9. Two studies showed a preventive effect of probiotic supplementation on the development of eczema at the age of 2 years, with a relative risk reduction of 50% 9 and 26% 5. In a third study, the cumulative incidence of eczema was similar in intervention and placebo group, but less atopic eczema was observed Chapter 4 in the intervention group 8. In two studies supplementation of probiotic bacteria failed to reduce the risk of atopic dermatitis and resulted in increased sensitization to allergens in one study 6;7. A major reason for these discrepant clinical results may be the use of different probiotic strains or 88 preparations. In this respect however it is interesting that the results of Kopp et al. are in sharp contrast with the study of Kalliomaki et al. although an identical probiotic strain is used (LGG) in an also otherwise comparable study design 7;9. In contrast to most other studies, we have aimed to develop a target-specific combination of probiotic bacteria 12; 13. Our study indicates that the beneficial effects of probiotic bacteria are established within the first three months of life. We observed a relative risk reduction of 58% of parental reported eczema at 3 months of age. This effect seemed to be sustained until the age of two years although relative risk reduction decreased with age. At the age of 2 years, we found a similar relative risk reduction as Kukkonen et al 5. In high-risk children, the earliest signs of eczema can develop during the first three months of life, but the highest incidence rate occurs in the second half-year of life 24. In children older than 3 months, a similar incidence was observed in both groups. However, from a clinical perspective, reducing the burden of eczema certainly in the first year of life is very

valuable. We speculate that prenatal administration of probiotic bacteria to the mother might be essential in this process. Probiotic bacteria were administered during the last 6 weeks of pregnancy to the mother since this may cause temporary changes in the composition of the maternal intestinal microbiota and consequently may have influenced early colonization in their offspring. The vaginal and intestinal microbiota of the mother are one of the first colonizers of the neonatal gut 25. Very little is known about the immunological effects of maternal probiotic supplementation on the fetus. Modulation of fetal immune responses may be a mechanism of action although findings from recent studies are not consistent 26; 27. In line with previous observations, children in the probiotics group tended to be more frequently sensitized to food allergens, especially hen s egg, compared to the placebo group 6. Although this did not result in more food allergic participants in the probiotics group, sensitization to hen s egg at the age of 12 months has previously been found to be predictive of sensitization to inhalant allergens at a later age 28;29. However eczema, which was more prevalent in the placebo group, has been shown to be a predictor for the development of sensitization as well 30.No differences were observed in respiratory symptoms indicative of asthma or allergic rhinitis although diagnosis is difficult at this age. Since the participants of this study are all high-risk children who may develop allergy or atopic manifestations other than eczema later in childhood, all participants will be followed-up The effects of selected probiotic strains on the development of eczema at the age of 5-6 years old. Specifically asthma (including spirometry), allergic rhinitis and again allergic sensitization for food and inhalant allergens will be addressed. 89 Few studies have investigated the influence of probiotics on the intestinal microbiota of infants. In our study, administration of the probiotic mixture resulted in higher and more frequent colonization of Lc. lactis and more frequent colonization of B. bifidum. This indicates that the oral administration of the probiotic mixture was successful and compliance was good. The presence of Lc. lactis in a minority of participants in the placebo group could be explained by the fact that 30% of the lactating women have this microorganism in their breast milk 31 and most of the babies included in the study were breastfed. The apparent drop in mean carriage rate of Lc. lactis at 52 weeks may be caused by the expansion of the colonic anaerobic microbiota, which will dominate in the fecal samples. Perinatal administration of probiotic bacteria did not lead to an increase in carriage and total counts of bifidobacteria in the intestinal microbiota. The

supplied number of bifidobacteria was apparently not high enough to detect an elevation in the endogenous (high) numbers of bifidobacteria. Species-specific analysis however revealed that endogenous Bifidobacterium species have been replaced by B. bifidum since this microbe was more frequently located in the intestinal microbiota of the intervention group. In comparable intervention studies, supplemented strains were more frequently found in the intervention group at the end of the administration period, but no permanent colonization occurred 5; 32. Furthermore, LGG administration in the first months of life did not significantly interfere with the variation or quantity of the gut microbiota as measured by FISH analysis 33. Whether in our study permanent colonization will ensue and whether probiotic supplementation will result in modulation of the gut microbiota is subject of our current research. The results of this study suggest that primary prevention of eczema by perinatal administration of probiotic bacteria indeed involves modulation of the early colonization of the intestinal microbiota, which may result in modulating the development and maturation of the infants immune system. Modulation of the immune response via interaction with intestinal dendritic Chapter 4 cells with subsequent effects on T cell differentiation and induction of regulatory T cells has been suggested 34. Furthermore, recognition of commensal bacteria by TLRs on intestinal epithelial cells and cells of the mucosal immune system is essential for intestinal (immune) 90 homeostasis 35; 36. Probiotic signaling through TLRs may contribute to maintaining mucosal and intestinal homeostasis and thereby preventing eczema.in our study, we had a rather large number of dropouts, although this is not uncommon in this type of studies. Previous published studies also suffered from almost 25 % of dropouts 5; 6. Almost 40 % of the dropouts were based on parental request, mainly due to motivational problems, i.e. the study was too demanding. Trials may be prone to bias associated with post-randomization exclusion. However, we ensured that randomization was successful, i.e. placebo and intervention group were equal with respect to potential risk factors and confounders. In conclusion, this intervention study shows a preventive effect of early administration of selected probiotic bacteria on the incidence of eczema, but not atopic eczema in high-risk children. This preventive effect seems to be established within the first three months of life together with significant changes in the intestinal microbiota and decreased IL-5 production.

ACKNOWLEDGMENTS We would like to thank the children and their parents for their participation. We thank Nathalie van Uden for her technical assistance. We also like to thank Paul Westers, Centre for Biostatistics, University of Utrecht, the Netherlands, for assistance with statistical analysis. The effects of selected probiotic strains on the development of eczema 91

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29 Sherrill D, Stein R, Kurzius-Spencer M, Martinez F. On early sensitization to allergens and development of respiratory symptoms. Clin Exp Allergy 1999 Jul;29(7):905-11. 30 Almqvist C, Li Q, Britton WJ, Kemp AS, Xuan W, Tovey ER, et al. Early predictors for developing allergic disease and asthma: examining separate steps in the allergic march. Clin Exp Allergy 2007 Sep;37(9):1296-302. 31 Beasley SS, Saris PE. Nisin-producing Lactococcus lactis strains isolated from human milk. Appl Environ Microbiol 2004 Aug;70(8):5051-3. 32 Gueimonde M, Kalliomaki M, Isolauri E, Salminen S. Probiotic Intervention in Neonates-Will Permanent Colonization Ensue? J Pediatr Gastroenterol Nutr 2006 May;42(5):604-6. 33 Rinne M, Kalliomaki M, Salminen S, Isolauri E. Probiotic intervention in the first months of life: short-term effects on gastrointestinal symptoms and long-term effects on gut microbiota. J Pediatr Gastroenterol Nutr 2006 Aug;43(2):200-5. 34 Smits HH, Engering A, van der Kleij D, de Jong EC, Schipper K, van Capel TM, et al. Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin. J Allergy Clin Immunol 2005 Jun;115(6):1260-7. 35 Artis D. Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut. Nat Rev Immunol 2008 Jun;8(6):411-20. Chapter 4 36 Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 2004 Jul 23;118(2):229-41. 94

The effects of selected probiotic strains on the development of eczema 95

5 Dynamic changes in intestinal microbiota of infants at risk of eczema during supplementation of probiotics Laetitia E Niers 1, Rocío Martín 2, Ger T Rijkers 1,3,4, Susana Fuentes 2, Henk Panneman 5, Harro M Timmerman 6, Maarten O Hoekstra 1, Hauke Smidt 2 1 Department of Pediatrics, Wilhelmina Children s Hospital, University Medical Centre, Utrecht, the Netherlands. 2 Laboratory of Microbiology, Wageningen University, Wageningen, the Netherlands. 3 Department of Surgery, University Medical Centre, Utrecht, the Netherlands. 4 Department of Medical Microbiology and Immunology, Sint Antonius Hospital, Nieuwegein, the Netherlands. 5 Van Haeringen Laboratory, Wageningen, the Netherlands. 6 Nizo food research BV, Ede, the Netherlands. Submitted for publication

ABSTRACT Background Modification of the intestinal microbiota by administration of probiotic bacteria may be a potential approach to prevent atopic diseases. Objective This study aimed to evaluate the composition of the intestinal microbiota in the first year of life during probiotic supplementation, and its association with the development of atopic disease. Methods A mixture of probiotic bacteria (B. bifidum, B. lactis and Lc. lactis; Ecologic Panda) was prenatally administered to pregnant mothers of children at high-risk of developing atopic disease and their offspring for the first 12 months of life in a double-blind, placebo controlled, randomized trial. Stool samples were collected during the first 4 weeks of life, at 3 months and at 12 months of age before and after cessation of probiotic supplementation. The composition of the fecal Chapter 5 microbiota was studied by terminal restriction fragment length polymorphism analysis. Results Probiotic supplementation resulted in a significantly different composition of the fecal 98 microbiota (p 0.002). Especially during the first three months of life fecal samples of probiotic fed infants could be distinguished by the abundant presence of Lactococcus lactis, Streptococcus salivarius and members of the genera Lactobacillus, Clostridium and Staphylococcus. Successful intervention of probiotic bacteria, i.e. no development of eczema was associated with increased richness (mean difference 27.40 (95% CI 10.50-44.29) and diversity (mean difference 0.3430 (95% CI 0.10-0.58) as well as predominance of Enterococcus, Lactobacillus and Streptococcus species. Conclusions Probiotic supplementation resulted in a significantly different composition of the intestinal microbiota and thereby possible induced beneficial effects on the development of eczema.

INTRODUCTION Insufficient microbial exposure early in life has been hypothesized to be causally related to the development of allergic diseases 1-3. The human intestinal microbiota offers an extensive source of microbial exposure to the gut associated lymphoid system, thereby directing its maturation, including the development of tolerance 4-6. At birth, the gastrointestinal tract is sterile, but is rapidly colonized by various microorganisms. Primary colonization of the gastro-intestinal tract is largely dependent on the mode of delivery, level of exposure and type of nutrition. Disturbances in the intestinal microbiota have been linked to various diseases including allergic diseases 7; 8. The composition of the intestinal microbiota has indeed been shown to differ between healthy and allergic infants. Allergic children seem to harbor more Clostridium (C) species and less Bifidobacterium (B) species in their fecal microbiota 9-11. In addition, the development of eczema has been associated with reduced bacterial diversity 12 and the presence of Escherichia coli 13 and B. pseudocatenulatum 14. Prospective studies indicate that differences in intestinal microbiota composition precede the onset of clinical symptoms, suggesting a potential causal relationship. Consequently, the possibilities of primary prevention of allergic diseases by administration of probiotic bacteria have been explored. Results, however, were inconsistent regarding prevention Dynamic changes in intestinal microbiota by probiotic bacteria of (atopic) eczema, and no preventive effect on the development of other allergic diseases was reported so far 15-20. Very few studies have addressed the influence of probiotic supplementation on 99 early microbiota colonization of the intestine. Predominantly, the presence of the supplemented probiotic strains has been studied 17; 20; 21, but the effects of probiotic supplementation on early colonization are still largely unknown. In this study we investigated the influence of pre- and postnatal administration of selected probiotic bacteria, i.e. Ecologic Panda: B. bifidum, B. lactis and Lc. lactis, on the developing intestinal microbiota of neonates starting from birth during the first year of life, and prospectively correlated specific composition of the intestinal microbiota to the development of eczema during the first two years of life.

METHODS Subjects and study design A random selection of subjects were included in this study from a larger cohort of a randomized, double-blind, placebo controlled trial addressing the effect of pre- and postnatal administration of selected probiotic bacteria in primary prevention of allergic disease in high-risk infants, described in detail elsewhere 22. In brief, families in which the mother or the father plus an older sibling had a positive history or current symptoms of allergic disease, i.e. atopic eczema, food allergy, asthma or allergic rhinitis were included at least two months before expected delivery of the pregnant mother. Families were allocated randomly to either placebo or intervention group. The intervention group received once daily a probiotic mixture consisting of 3 x 10 9 colony forming units (CFU) of B. bifidum W23, B. lactis W52, and Lc. lactis W58 (1 x 10 9 CFU of each strain) in freeze dried powder (Ecologic Panda, Winclove Bio Industries B.V., Amsterdam, the Netherlands). The placebo group received the carrier of the probiotic mixture, i.e. rice starch Chapter 5 and maltodextrins. Placebo or probiotic mixture was administered prenatally to the pregnant mother during the last six weeks of gestation and postnatally to their offspring during the first year of life. 98 infants completed this study, 48 in the placebo group and 50 in the intervention 100 group. Viability of the freeze-dried probiotic bacteria preparation was guaranteed throughout the study by plate counting. Parental written informed consent was obtained. The study was approved by the Medical Ethics Committee of the University Medical Centre Utrecht, the Netherlands. Clinical outcomes variables Children were clinically examined at the age of 3, 12 and 24 months. Parents completed a weekly diary including the presence of eczema, visits to their family doctor or pediatrician and medication use. Parental reported eczema was defined as eczema reported by the parents in the diaries. The development of sensitization was assessed by collecting blood samples at the age of 3, 12 and 24 months for the determination of specific IgE and by performing skin prick tests (SPTs) at the age of 24 months. Specific IgE was measured for egg white, cow s milk, peanut, house

dust mite and cat dander epithelium on the IMMULITE 2000 (Diagnostic Products Corporation, Los Angeles, CA, USA). A positive result was defined as a concentration of specific IgE > 0.35 IU/ ml. SPTs were performed with allergen extracts for egg white, cow s milk, peanut, hazelnut, cat, dog, house dust mite (Dermatophagoides pteronyssinus), birch and grass, with histamine dihydrochloride (10 mg/ml) as a positive control and the solvent (glycerine) as a negative control (ALK-Abellò, Nieuwegein, the Netherlands). SPTs were regarded as positive at a wheal diameter at 15 minutes of > 3 mm. Stool sample collection After birth, stool samples were collected by the parents weekly during the first 4 weeks of life starting with the first stool of the newborn in the first week of life and subsequently with an interval of one week between each sample. Furthermore, parents collected one stool sample at 3 months of age. At the age of 12 months, administration of either the probiotic mixture or placebo was ended and parents collected four stool samples: 1 week and 1 day before ceasing treatment, and 1 and 2 weeks after discontinuing the intervention. For the current analysis, the samples 1 day before and two weeks after end of intervention were used. Stool samples were collected from diapers, placed in stool collection vials, and immediately frozen by the parents Dynamic changes in intestinal microbiota by probiotic bacteria in home freezers. At 3 and 12 months, i.e. during the visit to the outpatient clinic, frozen stool samples were transported on ice to the laboratory. Upon arrival, all samples were immediately 101 stored at - 20ºC until further analysis. 500 samples of 73/98 participants were analyzed. Microbial Community Profiling and Characterization (MCPC) Microbial Community Profiling and Characterization (MCPC) is a qualitative analysis based on Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis, and was performed by the Dr. van Haeringen Laboratories (Wageningen, the Netherlands). MCPC analysis was used to study the dynamics of the fecal microbial communities. DNA was isolated from fecal samples essentially as described by Carter and Milton 23. T-RFLP analysis was performed as described by Liu and co-workers 24. Bacterial 16S rrna genes were amplified by PCR using fluorescently labeled primers 8f and 926r, and amplicons were subsequently digested with MspI and Hinp1I

(New England Biolabs, Ipswich, MA, USA). The reaction products were then size separated by capillary electrophoresis (CE) using an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Lengths of fluorescently labeled T-RFs were determined using GeneScan software (Applied Biosystems) and compared with an MCPC Database which was built from sequence data that were obtained from the most recent release of EMBL sequence database available at the time of analysis (October 2008), and using the Patscan program 25. This method uses the length polymorphisms found in terminal restriction fragments of 16S rrna gene amplicons from different bacteria, and can therefore provide a sensitive method to assess microbial community diversity. The T-RFLP pattern of each sample is a composite of the number of fragments of unique lengths and the relative abundance of each fragment is reflected by the height of a peak in the electropherogram. Only peak heights of more than 25 fluorescence units were regarded as positive. In addition to the fecal samples, the T-RFLP patterns of the individual strains in the probiotic mixture were also determined. Chapter 5 DGGE, cloning and sequencing DGGE of 16S rrna gene fragments amplified with primers 357f-GC and 907r 26 was performed for selected samples with or without a high relative peak height of T-RF 559. PCR amplification 102 and DGGE analysis was done as described previously, but using a gradient of denaturants of 30 50% 27. Two isolates of Streptococcus salivarius were used as reference strains for DGGE analysis. The population corresponding to the predominant DGGE band shared by all samples with a high relative peak height of T-RF 559 was identified by cloning and sequencing of almost complete 16S rrna genes amplified with primers 27f and 1492r as described previously 27. The sequence was compared with those available in public databases, using the NCBI Blast server (www.ncbi. nlm.nih.gov) 28.

Statistical analysis To study the richness of the intestinal microbiota the number of T-RF peaks of each individual sample at a specific time point was calculated. Relative abundance of a specific T-RF in the total T-RFLP pattern was calculated by dividing the peak area of the particular T-RF with the total peak area of all T-RFs detected within a fragment length range of 50 to 620 bp. Only T-RFs with a relative abundance of at least 1% were included. Shannon-Wiener diversity index 29 was calculated as implemented in CANOCO 4.5 (Biometris, Wageningen, the Netherlands) 30 for each individual sample. Statistical analysis was performed by repeated measurements analysis with post-hoc Bonferroni test correcting for multiple comparisons. Statistical analysis was performed by SPSS 15.0 for windows (Chicago, IL, USA). A p-value of < 0.05 was considered statistically significant. Multivariate statistical analysis was performed using CANOCO 4.5 30; 31. The Principal Response Curves (PRC) analysis finds its origin in an ordination technique called partial redundancy analysis (partial RDA), which is a constrained form of principal component analysis. In this way, using PRC we focused on the differences with respect to microbiota composition between specified groups of samples (i.e. treatment, occurrence of eczema) at the different time points 32. The significance of the PRC was tested by performing an unrestricted Monte Carlo Permutation Procedure (MCPP) with 499 random permutations in the partial RDA Dynamic changes in intestinal microbiota by probiotic bacteria from which the PRC was obtained. 103

RESULTS High-throughput cultivation-independent 16S ribosomal RNA gene-targeted approaches, combined with multivariate statistical analyses, were used to assess the dynamics of fecal microbiota composition from birth onwards and during the first year of life in infants having a high risk of developing atopic symptoms. Infants participated in a clinical trial investigating the influence of pre- and postnatal administration of selected probiotic bacteria, namely a mixture (Ecologic Panda) of B. bifidum, B. lactis and Lc. lactis, on the developing intestinal microbiota of neonates, as well as on the onset of eczema 22. Richness and diversity of fecal microbiota Supplementation of probiotic bacteria did not affect richness or diversity of the fecal microbiota during the first year of life compared to the control group (Figure 1A and 1B). In both groups, the number of T-RFs observed in fecal profiles significantly increased between week 1 and week 2, Chapter 5 reflecting the physiological development of intestinal microbiota during the first weeks of life. The children s age had a significant effect on richness and diversity in both groups (Figure 1). In a subgroup analysis of eczema, children who received probiotic bacteria and did not develop 104 eczema during the first two years of life had a significantly richer and more diverse fecal microbiota during the first year of life compared to the children in the probiotics group who did develop eczema (Figure 2A and 2B). In fact, the children in the probiotic group without eczema had the highest richness and diversity scores. To the contrary, the children who developed eczema despite probiotic supplementation had the lowest richness and diversity of all subgroups. There was no significant difference in richness and diversity in the first year of life in the placebo group comparing children who did or did not develop eczema during the first two years of life (Figure 2A and 2B). There were no differences in richness and diversity in a subgroup analysis of sensitization during the first two years of life (p 0.54 and p 0.24 with RM analysis, probiotic and placebo group respectively) (data not shown).

Dynamic changes in intestinal microbiota by probiotic bacteria 105 Figure 1: Richness and diversity of the intestinal microbiota during the first year of life. Richness is indicated by the number of peaks in the total bacterial profile of the T-RFLP analysis of each individual sample at a specific time point. Diversity is indicated by Shannon-Wiener index. Data of both richness and diversity represent mean ± SEM. Statistical analysis was performed by repeated measurement (RM) analysis. A: Richness during the first year of life probiotic group vs. placebo group. Mean difference in richness placebo vs. probiotics -0.242 (-9.588 9.104 95% CI for difference), p 0.96. B: Shannon index of diversity during the first year of life probiotic group vs. placebo group. Mean difference in diversity placebo vs. probiotics 0.074 (-0.056 0.203 95% CI for difference), p 0.26.

Chapter 5 106 Figure 2: Richness and diversity in a subgroup analysis of eczema (infants that ever developed eczema during the first two years of life). Data of both richness and diversity represent mean ± SEM. A: Subgroup analysis of richness. Richness during the first year of life is significantly different between subgroups (p 0.0005). Differences between subgroups with Bonferroni post hoc test: Probiotics no eczema vs. probiotics eczema mean difference in richness 27.40 (10.50 44.29 95% CI for difference), p 0.0001; Placebo no eczema vs. placebo eczema mean difference in richness 10.84 (-7.11 28.79 95% CI for difference), p 0.629. B: Subgroup analysis of Shannon-Wiener index of diversity. Diversity during the first year of life is significantly different between subgroups (p 0.0013). Differences between subgroups with Bonferroni post hoc test: Probiotics no eczema vs. probiotics eczema mean difference in diversity 0.3430 (0.1000 0.5860 95% CI for difference), p 0.002; Placebo no eczema vs. placebo eczema mean difference in diversity 0.1086 (-0.1496 0.3668 95% CI for difference), p 1.000.

Composition of the intestinal microbiota during probiotic supplementation The changes of the intestinal microbiota over time during probiotic supplementation compared to that of infants in the placebo group were analyzed using PRC analysis of T-RFLP profiles. Probiotic supplementation resulted in a significantly different composition of the intestinal microbiota compared to the placebo group during the first year of life (Figure 3). The difference in composition was most apparent during the first 3 months of life. Several predominant T-RFs could be associated with the probiotic treatment. Specifically Streptococcus sp. (T-RF 559), Lactococcus lactis (T-RF 56-57), Lactobacillus sp. (T-RF 575), and Staphylococcus sp. (T-RF 153) were associated with probiotic supplementation (Figure 3). Predominant T-RFs in the placebo group were indicative of members of the Bacteroidetes sp. (T-RFs 82, 92) (Figure 3). Over time, the infants intestinal microbiota in both groups developed into adult-like microbiota and was at the age of one year clearly different from the microbiota at a younger age in both groups (Figure 4). At the age of one year no differences could be detected with PRC analysis between the placebo and probiotics groups, neither when probiotics were still administered (week 52) nor after cessation of probiotic supplementation (week 54) (Figure 3). Figure 3: The effect of perinatal administration Dynamic changes in intestinal microbiota by probiotic bacteria of probiotics on the infant fecal microbiota composition. 107 Principal Response Curve indicates the effects of perinatal administration of probiotics on the infant fecal microbiota composition. Values deviating from the reference value of zero ( ) indicate probiotic effect ( ). The left panel indicates T-RFs most strongly correlated with observed differences between groups. Partial redundancy analysis explains 2% of the total variance in the dataset. MCPP revealed that probiotic treatment correlated to the variation in the microbiota composition with a p-value of 0.002.

Figure 4: The effect of perinatal administration of probiotics on the infant fecal microbiota composition. Canonical Correspondence Analysis (CCA) indicates the effect of perinatal administration of probiotics on the infant fecal microbiota composition at different time points. Placebo is indicated by, probiotics is indicated by. The time variable of sampling is represented by vectors (weeks 1, 2, 3, 4, 12, 52 and 54). CCA analysis revealed that the infant fecal microbiota composition was significantly different at 52 and 54 weeks (p 0.002). Being the most consistently stimulated predominant T-RF peak during the first three months Chapter 5 of life by probiotic supplementation (Figure 3),T-RF peak 559 was further investigated by DGGE, followed by cloning and sequencing, and was identified to represent Streptococcus salivarius (Figure 5A). Whereas the prevalence of S. salivarius was not different between placebo and 108 probiotics group (Table I), probiotic supplementation significantly increased the relative abundance of S. salivarius during the first year of life (Figure 5B), specifically at week 2 and 3. In a subgroup analysis for eczema (occurrence of eczema during the first two years of life), the relative abundance of S. salivarius tended to be higher in children of the probiotic group who did not develop eczema compared to the participants who developed eczema despite probiotic supplementation, mean difference 2.67 (-0.321 5.656 95% CI for difference, p 0.078 (data not shown)). No such differences were detected in the placebo group (p 0.35). There was no association between the relative abundance of S. salivarius in the first year of life and the development of sensitization during the first two year of life in either the intervention group or the control group (data not shown).

Table I Prevalence of S. salivarius (T-RF 559) Placebo Probiotics p value* week 1 22/36 (61%) 24/34 (69%) 0.404 week 2 34/37 (92%) 33/34 (97%) 0.346 week 3 34/38 (90%) 33/34 (97%) 0.206 week 4 34/38 (90%) 32/33 (97%) 0.218 week 12 26/37 (70%) 30/34 (88%) 0.064 week 52 8/37 (22%) 8/35 (23%) 0.900 week 54 9/37 (24%) 7/35 (20%) 0.659 * Chi-square The composition of the intestinal microbiota in eczema vs. non-eczema PRC analysis of T-RFLP profiles was also used to compare the composition of the intestinal microbiota during the first year of life in children who did or did not develop eczema regardless of intervention by probiotic bacteria. The presence of predominant T-RFs corresponding to populations of Enterococcus sp., Bifidobacterium sp., Lactobacillus sp., Streptococcus sp. and Eubacterium sp. were inversely correlated with the development of eczema during the first two years of life (data not shown). To the contrary, T-RF peaks representing Clostridium sp. and Escherichia coli were predominant in the children who developed eczema during the first two years of life (data not shown). Next, the T-RFLP patterns of children who did or did not develop Dynamic changes in intestinal microbiota by probiotic bacteria eczema were analyzed separately for the probiotic group and the placebo group. There was no difference in the prevalence or relative abundance of the supplemented strain Lc. lactis 109 between the children who did or did not develop eczema in the probiotic group, indicating that overall compliance was good. During the first year of life, the microbiota composition of the children who developed eczema despite probiotic supplementation was significantly different from that of the children who did not (Figure 6A). Predominant T-RFs associated with eczema despite probiotic supplementation belonged mainly to Clostridium sp. (T-RFs 62, 187 and 214). In contrast, successful intervention with probiotics, i.e. no development of eczema up to the age of 2 years, was associated with T-RFs of Enterococcus sp. (T-RF 72), Lactobacillus sp. (T-RF 188), and Streptococcus sp. (T-RF 70). No significant difference in the composition of the intestinal microbiota was found by PRC analysis in the placebo group (Figure 6B).

Chapter 5 Figure 5: Presence and relative abundance of S. salivarius (T-RF 559). 110 A: DGGE analysis of V3-V5 region of 16S rrna gene fragments amplified by PCR from representative samples with high relative peak height of T-RF 559 (high 559) and randomly selected samples with low relative peak height (random). For reference, PCR products obtained from 2 strains of S. salivarius are included (S). The band representing S. salivarius (confirmed by cloning and sequence analysis) is indicated by the arrow. B: Relative abundance of T-RF 559 was calculated by dividing the peak height of the T-RF 559 by the total peak area of all T-RFs detected within a fragment length range of 50 to 620 bp. Data represent relative abundance in mean % ± SEM. Relative abundance of T-RF 559 (S. salivarius) during the first year of life is significantly higher in the probiotics group compared to placebo. Mean difference probiotics vs. placebo 2.793 (0.506 5.080 95% CI for difference), p 0.017 with repeated measurements analysis. Analysis of specific time points: week 2 mean difference probiotics vs. placebo 8.133 (2.791 13.47 95% CI), p < 0.001 and week 3 mean difference probiotics vs. placebo 5.974 (0.6326 11.32 95% CI), p < 0.05. Other time points p > 0.05 probiotics group vs. placebo group.

Figure 6: Eczema and the infant fecal microbiota composition. Principal Response Curve indicates the effects of eczema incidence on the infant fecal microbiota composition (A) in the probiotic group and (B) in the placebo group. A. The left panel indicates T-RFs most strongly correlated with observed differences between groups. Dynamic changes in intestinal microbiota by probiotic bacteria Values deviating from the reference value of zero ( ) indicate eczema occurrence (o). Partial redundancy analysis explains 3.9% of the total variance in the dataset. MCPP revealed that 111 eczema occurrence correlated to the variation in the microbiota composition with a p-value of 0.05. B. Values deviating from the reference value of zero ( ) indicate eczema occurrence ( ). Principal components analysis explains 2.6% of the total variance in the dataset. MCPP revealed no significant differences on the eczema occurrence for this group.

DISCUSSION In this study, we have shown that probiotic supplementation during the first year of life to children that have a high-risk of developing atopic disease resulted in a significantly different composition of the intestinal microbiota during the first three months of life. During probiotic supplementation, S. salivarius was significantly more abundant. In addition, Lc. lactis as well as members of the genera Lactobacillus, Clostridium and Staphylococcus were associated with probiotic supplementation. The development of eczema during the first two years of life in the probiotics group was associated with reduced richness and diversity, and significantly different intestinal microbiota especially during the first three months of life compared to children who did not develop eczema. These differences were already apparent in the first weeks of life, and are in line with previous reports 12; 33. A reduced diversity in microbial composition early in life was detected in British infants developing atopic eczema during the first 18 months of life 12. Even at the age of five years, a less diverse composition was found in Estonian Chapter 5 children suffering from allergy 34. This reduction in microbial diversity may be the consequence of increased relative abundance of a small number of specific key groups of microorganisms. In turn, high richness and diversity could be seen as the absence of overgrowth by opportunistic 112 microorganisms. In line with this reasoning several studies have shown that (increased) carriage of potential pathogens such as clostridia and enterobacteriaceae have been linked to an increased risk of developing atopic disease 9-11; 13; 35. Recently, bearing in mind that childhood antibiotic use has been associated with asthma and atopic disease, antibiotic use was found to result in decreased richness and diversity of the fecal microbiota 36. These results suggest that probiotic supplementation, which was previously found to prevent the development of eczema 22, exerts its beneficial effects at least to a significant extent by modulating the composition of the intestinal microbiota. This further seemed to be host-specific since the development of eczema despite probiotic supplementation could not be attributed to poor compliance. This finding may have implications for future studies. Early analysis of fecal microbiota of infants receiving a given mixture of probiotic bacteria could show increased richness and diversity of the intestinal microbiota. In the absence of such an effect, the window of opportunity may allow

to switch to another (combination of) probiotic and thereby providing a sort of second chance primary prevention. To our knowledge, our study is the first in which the composition of the intestinal microbiota in infants during probiotic supplementation has been investigated prospectively to this extent. Although potential modification of the intestinal microbiota as a rationale for applying probiotic bacteria in prevention of allergic diseases is generally acknowledged, thus far only few studies have reported on this issue: supplemented strains were more frequently found in the intervention group at the end of the administration period, but no permanent colonization occurred 17;21. Furthermore, LGG administration during the first six months of life did not significantly influence variation or quantity of the intestinal microbiota as measured by 16S rrna-targeted FISH analysis 37, but may have resulted in differences in the initial communities of bifidobacteria in favor of B. breve 38. In line with these previous findings, we have shown that administration of Ecologic Panda (B. bifidum, B. lactis, and Lc. lactis) results in significantly higher numbers of intestinal Lc. lactis and B. bifidum 22. The observed differences in colonization between placebo and intervention group already seemed to be present during the first weeks of life. It is tempting to speculate that these differences are due to the fact that the mothers were treated with probiotic bacteria during Dynamic changes in intestinal microbiota by probiotic bacteria the last 6 weeks of pregnancy. This may have caused temporary changes in the composition of the maternal intestinal microbiota and consequently may have influenced early colonization in 113 their offspring. Alternatively, these differences may be caused by postnatal supplementation of probiotic bacteria, which per protocol was started within 24 hours after birth. Stool samples obtained during the first week of life were taken ideally immediately after birth (first stool sample), but not all parents succeeded in doing this. Given our experimental setup, it is impossible to differentiate between these two possibilities. The effects of probiotic supplementation were most apparent during the first 3 months of life but appeared to fade away by the age of 1 year, despite continuing administration until the first birthday. The effect of probiotic supplementation may be overruled by more powerful factors determining colonization of the intestine. Development of the infant intestinal microbiota is highly determined by inter-individual variation, and is further influenced by numerous factors

including time, microbial exposure, and nutrition 39; 40. As time progresses the infants intestinal microbiota at 1 year of age develops into adult-like microbiota and is clearly different from the microbiota at a younger age 41. This trend was also clearly visible in the data obtained in the present study. Weaning has a major influence on the profiles of bacterial communities 42. Furthermore, introduction of solid foods frequently precedes the shift to adult-like microbiota 40. In our study, solid foods were introduced at a mean age of 5.5 months, with no differences between placebo and intervention group, but no stool samples were collected during this period, as this was not the focus of this study. We speculate that weaning and the introduction of solid foods may have restrained the influence of probiotic supplementation, but we do not know when exactly the effect of probiotic treatment is no longer apparent. Furthermore, with increasing intestinal microbial complexity during the first year of life, colonization resistance may influence the effects of probiotic supplementation. In addition, the expansion of the colonic anaerobic microbiota, which will dominate the fecal microbiota, may hamper identification of subtle differences and microbial changes in the small intestine. Chapter 5 The development of eczema during the first two years of life regardless of intervention by probiotics was associated with the predominance of Clostridium sp. and E. coli during the first year of life. To the contrary, Bifidobacterium sp., Lactobacillus sp. and Enterococcus sp. 114 were predominant in the children who did not develop eczema during the first two years of life. Both observations are in line with previous findings 9-11. Interestingly, Clostridium sp. were predominant in children who did develop eczema during the first year of life despite probiotic supplementation. Non-toxigenic C. difficile has previously been associated with parentally reported and clinically diagnosed eczema 35. Nevertheless, although T-RFLP is a qualitative method, identification at the strain level of Clostridium sp. was not possible. Mechanistically probiotic bacteria may alter the composition of the microbiota by competing for nutrients or via the inhibition of pathogens or colonization resistance 43. Furthermore, metabolic products produced from one bacterial species may provide substrates to support the growth of other populations. For example, production of exopolysaccharides synthesized by bifidobacterial strains may act as fermentable substrates for other intestinal bacteria 44. In addition, evidence is emerging from in vivo studies that probiotics can affect cellular processes of the host,

thereby activating its immune system 45.In conclusion, probiotic supplementation resulted in a significantly altered composition of the intestinal microbiota beyond the supplemented strains. The exact mechanism of action of probiotic bacteria is still largely unknown. This study, however, indicates for the first time that probiotic bacteria may prevent the development of eczema in high-risk children by inducing the development of a richer and more diverse microbiota, and stimulate or inhibit the presence of specific bacterial species, such as shown here with respect to the stimulation of S. salivarius. Future research will capitalize on findings reported here, providing a more detailed view on the dynamics of microbiota colonization associated with probiotic treatment as well as the prevention or onset of atopic disease, using the Human Intestinal Tract Chip (HITChip), a high-throughput 16S rrna gene-targeted DNA oligonucleotide microarray platform for the comprehensive profiling of human intestinal microbiota 46-49. ACKNOWLEDGMENTS We would like to thank the children and their parents for their participation. Dynamic changes in intestinal microbiota by probiotic bacteria 115

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6 Probiotic bacteria and modulation of the developing immune system Laetitia E Niers 1, Nathalie O van Uden 1, Jan L Kimpen 1, Maarten O Hoekstra 1, 1, 2, 3 and Ger T Rijkers 1 Department of Pediatrics, Wilhelmina Children s Hospital, University Medical Centre, Utrecht, the Netherlands. 2 Department of Surgery, University Medical Centre, Utrecht, the Netherlands. 3 Department of Medical Microbiology and Immunology, Sint Antonius Hospital, Nieuwegein, the Netherlands. Manuscript in preparation

ABSTRACT Background Modulation of the immune system is a potential mechanism of action by probiotic bacteria, but the effects of probiotic supplementation on the developing immune system of infants are still largely unknown. Methods A mixture of probiotic bacteria (B. bifidum, B. lactis and Lc. lactis; Ecologic Panda), was prenatally administered to mothers of high-risk children (i.e. with a positive family history of atopic diseases) and their offspring for the first 12 months of life in a double-blind, randomized, placebo-controlled study. Blood samples were collected at age 3, 12 and 24 months. At these moments, lymphocyte phenotyping was performed and cytokine profiles were measured in ex vivo (anti CD2/CD28 monoclonal antibodies or phytohemagglutinin) stimulated whole blood cultures and in plasma. Chapter 6 Results Patterns of Th2 cytokine production during the first two years of life differed between placebo and intervention group: IL-4 p 0.085, IL-5 p 0.004 and IL-13 p 0.054 (whole blood cultures after 122 48h stimulated with anti CD2/CD28 mab). At 3 months, IL-5 and IL-13 were reduced in the intervention group compared to the placebo group, which was no longer apparent one year. Th2 cytokine production was higher in the intervention group at 24 months: IL-4 20.4 vs. 9.0 pg/ml (p 0.030), IL-5 175 vs. 83 pg/ml (p 0.008) and IL-13 238 vs. 140 pg/ml (p 0.016). Probiotics did not affect the in vitro production of IL-10, IFN-g, or TNF-a during the first two years of life, nor did it affect the development of lymphocyte populations and subsets. Conclusions Probiotic supplementation resulted in decreased production of Th2 cytokines at the age of 3 months but significant increased production by the age of 2 years. This indicates that the beneficial effect of probiotics cannot be directly related to systemic cytokine profiles or ex vivo induced cytokines.

INTRODUCTION Atopic diseases are immunologically characterized by a predominant type 2 T-helper cell response to allergens. Under physiological conditions, the neonatal immune system seems to be Th2-skewed in line with the intrauterine environment (to prevent the fetus from stillbirth). Postnatal development of the immune system involves maturation of Th1 function and the development of tolerance. Children at high risk of atopic diseases have a reduced capacity and delayed development of Th1 interferon gamma (IFN-g) responses 1; 2. Other studies have identified a Th2 dominated cytokine profile characterized by high levels of IL-4, IL-5 and IL-13 within the first 6 months of life in high-risk children developing atopic disease in their first year of life 3, or a diminished capacity to respond to microbial stimuli 4. More recent studies indicate that regulatory T cells are likely to be involved in controlling expression of atopic diseases 5-7. Thus, induction or expansion of regulatory T cells has been suggested as an attractive strategy for prevention and management of atopic diseases 8. Atopic diseases have been associated with a persistent Th2 skewing as a result of insufficient exposure to environmental microbial stimuli. In order to provide the developing immune system with additional stimuli, probiotic bacteria are being studied for their capacity to prevent Immunomodulation by probiotic bacteria atopic diseases. The desired immunomodulatory effect of probiotic bacteria would therefore be reduction of Th2 function, and induction or augmentation of Th1 function, tolerance and 123 regulatory immune responses. Indeed, a great number of in vitro studies have addressed the immunological effects of different probiotic bacteria on mononuclear cells, which involved suppression of Th2 cytokines, stimulation of IFN-g, IL-12 and other pro-inflammatory cytokines, and the induction of regulatory T cells 9-11. Furthermore, different species and strains of lactic acid bacteria have been shown in vitro to modulate the expression of co-stimulatory molecules and cytokine production of murine and human dendritic cells 12;13. Despite the relative abundance of in vitro data, the in vivo effects of probiotic supplementation on the developing immune system of infants are still largely unknown. From clinical trials with a primary prevention as well as a tertiary prevention (treatment) design, probiotic supplementation has been associated with low-grade inflammation (i.e. higher plasma levels of

CRP) 14 and increased production of IFN-g upon ex vivo mitogen and superantigen stimulation of mononuclear cells 15. We have conducted a randomized, double blind, placebo controlled clinical trial in high-risk children on the effect of selected probiotic strains on eczema. Selected probiotic bacteria showed a preventive effect on the incidence of eczema in high-risk children established within the first three months of life and which seemed to be sustained during the first two years of life 16. This clinical effect coincided with decreased production of Th2 cytokines at the age of 3 months in the intervention group. In this study, we aimed to further investigate the effects of supplementation of selected probiotic strains 17; 18 prenatally and postnatally during the first year of life on the developing immune system during the first two years of life. METHODS Study design and participants Chapter 6 156 pregnant women and their families were recruited and included as described in detail previously 16. In brief, families were eligible if the mother or the father plus an older sibling had a positive history of allergic disease. In a double-blind, randomized, placebo-controlled clinical 124 trial 3 x 10 9 colony forming units (CFU) of the probiotic mixture Ecologic Panda (Bifidobacterium (B) bifidum W23, B. lactis W52 (previously classified as B. infantis) and Lactococcus (Lc.) lactis W28) (Winclove Bio Industries B.V., Amsterdam, the Netherlands) was administered daily during the last 6 weeks of pregnancy to the mothers and postnatally during the first year of life to their offspring. 98 infants completed the supplementation period and the two years of follow-up. Parental written informed consent was obtained and ethical permission was granted by the Medical Ethics Committee of the University Medical Centre Utrecht, the Netherlands. Clinical outcome variables and blood samples Follow-up visits were scheduled at the age of 3, 12 and 24 months. Children were clinically examined, eczema was evaluated by SCORAD score and a questionnaire (adapted version of the British Medical Research Council questionnaire 19; 20 and the Dutch version of the European

Community Respiratory Health Survey 21 ) was used to evaluate participants for symptoms or complaints of atopic diseases. Furthermore, parents completed a weekly diary including the presence of eczema, visits to their family doctor or pediatrician and medication use. Parental reported eczema was defined as eczema reported by the parents in the diaries. Atopic eczema was defined as eczema plus sensitization. Sensitization was defined as either elevated specific IgE (> 0.35 IU/ml) for one or more allergens and/or a positive SPT. Total and specific IgE was measured in sera collected at the age of 3, 12 and 24 months and SPTs were performed at 2 years of age as described previously 16. Blood samples were collected at each follow-up visit. Sodiumheparinized blood was used to collect plasma samples and to perform lymphocyte phenotyping and whole blood cultures. The numbers of samples (plasma and supernatants of whole blood cultures) available are provided in table I. Table I Number of samples available of whole blood cultures and plasma Placebo Probiotics 3m 12m 24m 3m 12m 24m Plasma 39 41 39 36 38 38 Supernatants whole blood cultures acd2/cd28 48H 45 39 44 43 32 36 acd2/cd28 72H 46 41 44 42 39 39 PHA 48H 45 39 44 43 32 37 PHA 72 H 46 47 44 42 42 39 Immunomodulation by probiotic bacteria 125 Lymphocyte phenotyping Whole blood (50 ml) was incubated with 10 ml of FACS buffer (PBS containing 0.02% azide) containing four appropriately diluted FITC-, PE-, PERCP-, or allophycocyanin (APC)-labeled mabs against human CD3, CD4, CD8, CD20, CD45RA, CD45RO and chemoattractant-homologous receptor expressed on Th2 cells (CRTH2). All mabs were obtained from BD Biosciences (Mountain View, CA, USA). After incubation during 10 minutes, red blood cells were lysed by the addition of FACS lysing buffer and incubation during 5 minutes. Cells were washed three times in FACS buffer and subsequently resuspended in 1 % paraformaldehyde (PFA) in phosphate buffered saline (PBS). The stained cells were analyzed on FACS-Calibur using CellQuest software (BD Biosciences, Mountain View, CA, USA). The different cell populations were counted and analyzed within the total lymphocyte population (i.e. the lymphocyte gate as determined on the basis of

forward scatter and side scatter characteristics). Within the lymphocyte population a total of 10 000 cells were counted. Data are expressed as percentage ± SEM of positive cells within the lymphocyte gate. For naive and mature CD4 cells, the absolute number of CD4/CD45RA positive cells and CD4/CD45RO positive cells respectively was divided by the absolute number of CD4 cells within the lymphocyte gate to calculate the percentage of naive and mature cells of CD4 positive lymphocytes. Whole blood cultures Whole blood cultures were carried out as described in detail elsewhere 16. In brief, cells were cultured in medium only or stimulated by anti-cd2 / anti-cd28 monoclonal antibody (mab) (CLB-T11.2/1, CLB-T11.1/1 (1 μg/ml) and CLB-CD28/1 (4 μg/ml); Sanquin, Amsterdam, the Netherlands) or phytohemagglutinin (PHA) (final concentration of 25 μg/ml); Murex Biotech Ltd, Kent, UK) and supernatants were collected at 48 and 72 h. To determine lymphocyte proliferation 3 H thymidine incorporation during the last 16 hrs of a 48 or 72 hrs culture period was measured. Chapter 6 Cytokine analysis Cytokines (IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, TNF-a and IFN-g) were determined in supernatants 126 of whole blood cultures by multiplex assay (Luminex) as described in detail previously 22. Furthermore, cytokines (IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-23, IL-25, TNF-a and IFN-g) were measured in plasma/serum samples collected at the age of 3, 12 and 24 months. For statistical reasons, cytokine concentrations below the limit of detection were assigned a value of 50% of the lowest measured value of that specific cytokine. Statistical analysis Power calculations to determine the size of our clinical study were based on the primary outcome variable, which was the incidence of eczema as described in detail previously 16. Outcome variables concerning the development of the immune system, i.e. lymphocyte phenotype and cytokine levels in supernatants of whole blood cultures and plasma samples were secondary outcome variables. Cytokine data were analyzed as continuous data and dichotomous data (detectable or non-detectable). Cytokine production in unstimulated (medium) whole blood cultures and

production of IL-12 after polyclonal stimulation were generally below detection limits and were therefore not included in the analysis. To determine differences between groups at distinct ages, the independent samples t-test was used. Differences between intervention group and placebo group for dichotomous data were determined by χ 2. To evaluate the development of cytokine production during the first two years of life in the intervention vs. placebo group, and subgroups, repeated measurements analysis was used. Pearson s correlation test was used to assess the correlations between total IgE vs. cytokine production, as well as cytokine production in whole blood cultures and plasma samples in corresponding samples. The risk of a type 1 error with multiple comparisons was generally acknowledged and therefore a difference at 2-tailed P < 0.01 was considered significant. All calculations were performed with SPSS 15.0 for Windows (Chicago, IL, USA). RESULTS The development of lymphocyte populations and subsets in high-risk infants The relative sizes of lymphocyte subpopulations in blood collected at the age of 3 months, 1 Immunomodulation by probiotic bacteria year and 2 years are shown in Table II. Within the CD3 + T lymphocyte population including CD4 + and CD8 + T cell subsets, fluctuations in the relative frequencies were limited. The percentage 127 of CD20 + B lymphocytes doubled within the first three months of life and thereafter remained within mean limits of 20-27% until the age of 2 years. At birth, a large part of the CD4 + T lymphocyte population consisted of naive (CD45RA + ) T cells, which further increased during the first year of life. During the first two years of life CD45RO + memory T lymphocytes increased at least a twofold. The percentage of CRTH2 positive cells was highest in cord blood, and then clearly decreased three fold during the first 3 months of life with a subsequent gradual increase until the age of two years (Figure 1). This pool of CRTH2 positive cells largely consisted of non T cells, probably basophils, and the number of CD3 + and CD4 + T cells, which were CRTH2 positive, was small and relatively constant during the first two years of life. Probiotic supplementation did not influence the development of lymphocyte populations during the first two years of life (Table II).

Figure 1: CRTH2 expression during the first two years of life. Data indicate percentage ± SEM of positive cells within the lymphocyte gate. Chapter 6 128 Table II Immunophenotyping of peripheral blood lymphocytes Cell population Age Placebo Probiotics χ2 CD3 positive cells CB 54.4 ± 3.4 52.3 ± 2.6 ns 3m 65.7 ± 1.6 65.7 ± 1.8 ns 12m 63.4 ± 1.3 64.5 ± 1.3 ns 24m 63.5 ± 1.2 61.6 ± 1.1 ns CD4 positive cells CB 38.9 ± 2.7 39.0 ± 2.3 ns 3m 47.7 ± 1.5 45.3 ± 2.3 ns 12m 45.1 ± 1.3 43.8 ± 1.5 ns 24m 42.0 ± 0.9 40.0 ± 1.0 ns CD8 positive cells CB 14.3 ± 1.1 13.4 ± 1.0 ns 3m 15.6 ± 0.8 18.7 ± 1.9 ns 12m 14.6 ± -.6 17.7 ± 1.4 ns 24m 18.1 ± 0.8 17.9 ± 0.9 ns CD20 positive cells CB 10.3 ± 0.9 12.3 ± 0.9 ns 3m 20.8 ± 1.5 21.3 ± 1.6 ns 12m 27.3 ± 1.3 25.4 ± 1.4 ns 24m 24.8 ± 1.1 25.3 ± 1.0 ns CRTH2 positive cells CB 3.3 ± 0.3 3.4 ± 0.4 ns 3m 1.6 ± 0.1 1.3 ± 0.1 ns 12m 2.1 ± 0.1 1.9 ± 0.1 ns 24m 2.3 ± 0.1 1.9 ± 0.2 ns Naive CD4 cells CB 76.6 ± 4.3 71.2 ± 2.9 ns (CD45RA/CD4 positive ) 3m 78.3 ± 3.2 78.9 ± 2.3 ns 12m 78.3 ± 2.2 80.7 ± 1.6 ns 24m 71.0 ± 1.6 73.4 ± 1.4 ns Memory CD4 cells CB 7.7 ± 1.3 9.9 ± 1.5 ns (CD45RO/CD4 positive) 3m 10.3 ± 0.6 13.3 ± 1.7 ns 12m 16.9 ± 1.2 16.3 ± 1.0 ns 24m 19.2 ± 1.5 19.5 ± 1.4 ns Data indicate mean percentage ± SEM. CB: Cordblood; m: months. NS: not significant

Cytokine production during the first two years of life The in vitro capacity to produce cytokines was studied in whole blood cultures from children at 3 months, 1 year, and 2 years. The data, expressed as dichotomous data depending on type of stimulation and duration of culture is shown in Table III. IFN-g was difficult to detect at the age of 3 months with maximal 56% of samples showing detectable levels depending on stimulus and duration of the culture. The capacity to produce IL-4 increased with age as well, but was certainly at the age of 3 months more pronounced than the capacity to produce IFN-g. Other Th2 cytokines, notably IL-5 and IL-13 were detected in the majority of samples at the age of 3 months and further during the first two years of life. These results suggest a Th2 polarization pattern in the whole study population during the first months of life. A similar pattern in capacity to produce different cytokines during the first two years of life was observed in plasma samples, although cytokines in general and specifically IL-4 and IL-5 were more difficult to detect which is a well-known phenomenon in plasma samples (data not shown). The capacity to produce cytokines (expressed in dichotomous data) in stimulated whole blood cultures or in plasma samples was not significantly different in the intervention group compared to the placebo group (Table III and data not shown, respectively). In whole blood cultures stimulated with acd2/cd28 mab the pattern of cytokine responses Immunomodulation by probiotic bacteria changed significantly with age (Figure 2 and Figure 3). IFN-g responses changed most markedly. At 3 months of age, IFN-g responses were generally low or undetectable as typically seen in very 129 young children. Between 3 and 24 months, IFN-g responses increased 14 fold (Figure 3) compared to an almost 4-fold increase for IL-5 responses (Figure 2b). Similar patterns of cytokine secretion, although less pronounced, were observed in plasma samples (Table IV). However, IL-4 and IL-5 did not change significantly with age in the whole study population (p 0.197 and p 0.153 respectively), and for IL-17 and IL-23 only a trend was observed (p 0.088 and p 0.021 respectively). Again, IFN-g changed most markedly during the first two years.

Chapter 6 130 Table III Percentage of samples of whole blood cultures with detectable levels of cytokines Placebo Probiotics Cytokine Culture condition 3m 12m 24m 3m 12m 24m IL-4 anti CD2/CD28 48 H 49 85 100 40 97 100 anti CD2/CD28 72 H 76 76 77 71 82 74 PHA 48H 58 95 89 67 100 95 PHA 72 H 83 100 50 83 100 64 IL-5 anti CD2/CD28 48 H 84 82 93 72 94 100 anti CD2/CD28 72 H 96 100 100 93 100 100 PHA 48H 93 97 89 98 100 95 PHA 72 H 96 98 100 95 100 100 IL-13 anti CD2/CD28 48 H 96 100 98 91 100 100 anti CD2/CD28 72 H 96 97 93 100 100 100 PHA 48H 98 100 100 100 100 95 PHA 72 H 98 98 98 100 100 100 IL-10 anti CD2/CD28 48 H 96 97 100 91 100 100 anti CD2/CD28 72 H 96 100 100 95 100 100 PHA 48H 100 100 100 100 100 97 PHA 72 H 98 100 100 100 100 100 IL-17 anti CD2/CD28 48 H n.a. 33 86 n.a. 50 92 anti CD2/CD28 72 H n.a. n.a. 86 n.a. n.a. 95 PHA 48H n.a. 39 95 n.a. 38 81 PHA 72 H n.a. 100 95 n.a. 100 95 IFN-γ anti CD2/CD28 48 H 31 59 93 35 63 94 anti CD2/CD28 72 H 50 68 98 43 82 97 PHA 48H 56 85 98 51 84 95 PHA 72 H 39 100 95 26 100 95 TNF-α anti CD2/CD28 48 H n.a. 85 100 n.a. 94 100 anti CD2/CD28 72 H 96 100 100 98 100 97 PHA 48H 100 97 98 100 100 95 PHA 72 H 98 98 100 100 100 100 Data indicate percentage of positive, i.e. detectable levels of cytokines, supernatants of whole bloodcultures. H = hours; M = months; N.a. = not available

Figure 2: Production of Th2 cytokines in whole blood cultures stimulated by acd2/cd28 antibodies at 3, 12, and 24 months of age. A: 48H of culture. B: 72H of culture. Immunomodulation by probiotic bacteria 131 Figure 3: Production of pro-inflammatory cytokines and IL-10 in whole blood cultures at 3, 12, and 24 months of age stimulated by acd2/cd28 antibodies after 72H of culture.

Table IV Cytokine levels in plasma Placebo Probiotics Cytokine 3m 12m 24m 3m 12m 24m IL-4 0.025 ± 0.013 0.32 ± 0.055 0.56 ± 0.20 0.43 ± 0.28 0.80 ± 0.35 1.96 ± 1.38 IL-5 0.68 ± 0.21 0.97 ± 0.54 1.8 ± 0.58 0.40 ± 0.140 1.40 ± 0.53 14.5 ± 10.6 IL-13 0.89 ± 0.34 3.81 ± 0.90 8.57 ± 1.70 2.01 ± 1.01 4.84 ± 1.38 12 ± 4.8 IL-25 415 ± 84 1787 ± 291 4576 ± 1155 2052 ± 972 2367 ± 729 4954 ± 1637 IL-10 10.7 ± 2.7 42.0 ± 9.9 30.3 ± 7.3 5.97 ± 0.95 45.2 ± 13.7 45.8 ± 21.0 IL-6 11.9 ± 4.8 98.6 ± 31.5 54.8 ± 12.6 6.9 ± 2.2 106 ± 38 58.8 ± 18.8 IL-17 0.07 ± 0.002 1.54 ± 0.71 3.46 ± 2.19 0.13 ± 0.043 2.28 ± 0.80 9.13 ± 6.47 IL-23 504 ± 242 635 ± 286 1141 ± 262 208 ± 67 663 ± 319 1190 ± 374 IFN-γ 0.72 ± 0.28 11.1 ± 2.9 41.4 ± 8.4 5.89 ± 4.65 15.3 ± 4.7 44.3 ± 12.4 TNF-α 0.41 ± 0.12 4.75 ± 1.45 4.35 ± 1.53 0.80 ± 0.35 8.29 ± 2.94 5.90 ± 2.23 Data are represented as mean ± SEM (pg/ml) Effects of probiotic supplementation on cytokine production Patterns of Th2 cytokine production in whole blood cultures were significantly different in the intervention group compared to the placebo group (Figure 2 A and B). Repeated measurement analysis indicated significant changes in Th2 cytokine production over time (Table V). More Chapter 6 importantly the interaction of age and group was consistently significant (p < 0.01) or showed a robust trend (p < 0.100) in both IL-5 and IL-13 (and to a lesser extent IL-4 as well) which means that cytokine production in both placebo and probiotics group were changing over time but were 132 changing in different ways (Table V). As we have published previously for the age of 3 months 16, IL-5 was reduced (p=0.04) in the intervention group at 72 hrs of cell cultures stimulated with anti CD2/CD28 mabs (Figure 2B), and for IL-13 a similar trend towards decreased production in the intervention group was observed at 48 (p=0.08) and 72 hrs (p=0.06) of cell cultures (Figure 2A and 2B) 16. No significant differences in cytokine production between intervention and placebo group were observed at the age of 12 months. At two years of age, the situation has changed in that the production of Th2 cytokines was consistently higher in the intervention group compared to the placebo group (Table V). Probiotic supplementation did not affect the production of IL- 10, IFN-g, and TNF-a during the first two years of life. The only exception was IL-10 after 48h of culture with PHA, which tended to develop differently between groups. In addition to the in vitro induced cytokine responses presented above, we also measured levels of cytokines in plasma samples. Probiotic supplementation did not significantly affect cytokine patterns in

plasma (Table IV). However, IL-25 was a five fold higher in the intervention group at 3 months, 2052 pg/ml vs. 415 pg/ml (p 0.077 independent samples t-test). Similar to whole blood cultures, IL-4 and IL-5 at the age of two years were higher in the intervention group. Furthermore, Th2 cytokine production in whole blood cultures at 24 months was significantly positively correlated with levels of Th2 cytokines in plasma samples at 24 months (data not shown). Thus, high in vitro producers of Th2 cytokines (primarily participants of the intervention group) also displayed high levels of Th2 cytokines in plasma. Finally, we have studied whether supplementation with probiotics bacteria would influence the in vitro lymphocyte proliferative responses. Neither the anti CD2/CD28 nor the PHA induced response differed between both groups (data not shown). Table V The influence of probiotic supplementation on cytokine responses in whole blood cultures Cytokine Polyclonal stimulation Repeated measurements analysis Cytokine production 24 months** Age* Interaction age x group* Placebo vs probiotic group IL-4 acd2/cd28 48h p 0.012 p 0.085 p 0.030 acd2/cd28 72h p 0.000 p 0.051 p 0.073 PHA 48h p 0.097 p 0.768 p 0.111 PHA 72 h p 0.080 p 0.744 p 0.076 IL-5 acd2/cd28 48h p < 0.001 p 0.004 p 0.008 acd2/cd28 72h p < 0.001 p 0.018 p 0.044 PHA 48h p 0.010 p 0.033 p 0.015 PHA 72 h p < 0.001 p 0.003 p 0.002 IL-13 acd2/cd28 48h p 0.653 p 0.054 p 0.016 acd2/cd28 72h p < 0.001 p 0.064 p 0.137 PHA 48h p < 0.001 p 0.068 p 0.006 PHA 72 h p 0.192 p 0.003 p 0.001 IL-10 acd2/cd28 48h p 0.009 p 0.318 p 0.467 acd2/cd28 72h p 0.114 p 0.358 p 0.726 PHA 48h p < 0.001 p 0.015 p 0.936 PHA 72 h p < 0.001 p 0.138 p 0.727 IFN-γ acd2/cd28 48h p < 0.001 p 0.231 p 0.512 acd2/cd28 72h p < 0.001 p 0.613 p 0.610 PHA 48h p < 0.001 p 0.239 p 0.815 PHA 72 h p < 0.001 p 0.546 p 0.936 TNF-α acd2/cd28 48h p < 0.001 p 0.900 p 0.856 acd2/cd28 72h p < 0.001 p 0.619 p 0.219 PHA 48h p 0.406 p 0.852 p 0.121 PHA 72 h p 0.012 p 0.414 p 0.506 Immunomodulation by probiotic bacteria 133 * Greenhouse-Geisser ** Independent samples t-test

The association of eczema, sensitization and total IgE with cytokine production To further investigate why Th2 cytokine production developed differently in the participants of the intervention group subgroup analyses for eczema (ever eczema during the first two years of life) and sensitization (ever sensitization (elevated sige > 0.035 IU/ml or a positive SPT) during the first two years of life) were performed. With repeated measurements analysis there was no consistent difference in the development of Th2 cytokines in either whole blood cultures or plasma between subgroups of eczema, specifically not between the subgroups probiotics no eczema vs. probiotics with eczema. Only patterns of IL-4 (p 0.047) and IL-5 (p 0.026) of whole blood cultures stimulated by PHA during 48h changed differently with age when comparing the subgroups probiotic no eczema vs. probiotic eczema: the participants who developed eczema despite probiotic supplementation produced the highest levels of IL-4 and IL-5 at 24 months. No such differences were observed in the placebo group. Th2 cytokine production in whole blood cultures at 24 months tended to be higher in the children who suffered from eczema between 12 and 24 months of age, but this was the case in both groups. Cytokine levels in plasma samples Chapter 6 were not different between subgroups of eczema (data not shown). Children in the probiotics group tended to be more frequently sensitized in general (34% vs. 18.8%, p 0.087, and specifically to food allergens (especially hen s egg) (30% vs. 15%, p 0.067) compared to the placebo group. 134 However, sensitization was not associated with increased production of Th2 cytokines in either whole blood cultures or plasma (data not shown). Furthermore, only at 12 months of age weak associations with Th2 cytokine production and total IgE were observed. Th2 cytokine production in plasma samples (IL-4, IL15, IL-13 and IL-25) was not correlated with the level total IgE (data not shown). DISCUSSION In this study, we have shown that the immune system in high-risk children is characterized by a high percentage of CRTH2 positive cells in cord blood, which may reflect the Th2 bias of the neonatal immune system. CRTH2 is expressed on Th2 lymphocytes, eosinophils and basophils

and promotes the production of Th2 cytokines. CRTH2 is associated with the development and maintenance of the allergic response 23-25. Postnatal development of the immune system is most markedly characterized by an extensive increase in the capacity to produce IFN-g, reflecting the maturation of Th1 function. Probiotic supplementation during the first year of life resulted in a significant decreased Th2 cytokine production at the age of 3 months but a clearly increased production of these cytokines at 24 months of age. Probiotic supplementation neither positively nor negatively affected the postnatal maturation of Th1 function, as well as the production of IL-10 as potential marker for the development of regulatory T cells. One of the potential mechanisms of action of probiotic bacteria is modulation of the immune system. It has been hypothesized that probiotic bacteria stimulate the gut associated lymphoid tissue (GALT) thereby modulating the developing immune system to reverse the prenatal Th2 bias into a more balanced immune response including tolerance and regulatory T cells to nondangerous antigens 26. Previously, probiotic supplementation in primary prevention of atopic diseases has been associated with low-grade inflammation, i.e. higher plasma levels of CRP, increased plasma levels of total IgE, IL-10, and total IgA 14. Probiotic supplementation did not affect ex vivo cytokine production of allergen-specific immune responses, responses to vaccine antigens (tetanus toxoid) nor early innate immune responses 27;28. In children with established Immunomodulation by probiotic bacteria atopic dermatitis or cow s milk allergy, in vivo probiotic treatment was associated with increased in vitro IFN-g responses 15;29. Of note, the effects on the immune system depended on the specific 135 species and strain of the probiotic bacteria used. In the latter study, Lactobacillus GG increased the level of IFN-g production in stimulated PBMCs but did not affect IL-4 production, while to the contrary a mixture of probiotic bacteria (LGG, L. rhamnosus, B. breve, and Propionibacterium freudenreichii spp shermanii) increased IL-4 production after treatment 29. Because the disturbances in Th2 and Th1 cytokines may be due to functional inactivity of regulatory T cells, induction or expansion of regulatory T cells has been suggested as an attractive preventive and therapeutic solution for atopic diseases 7;8. In many (review) articles, probiotic supplementation has been associated with induction of regulatory T-cell populations. In vitro studies and murine models lent support to this matter 30-33, but from clinical trials supplementation of probiotic bacteria did not affect Foxp3 mrna expression or the frequency of circulating regulatory T

cells 34, and has not convincingly been shown to be associated with increased production of IL-10 14;35. In line with these results, we also did not observe significant differences in plasma levels of IL-10 during the first two years of life nor in the ex vivo stimulated production of IL- 10 by mononuclear cells between placebo and probiotic group. This does however not exclude regulatory T cell activity at mucosal sites such as the gastro-intestinal tract, which is undetected in the peripheral circulation. In the present study, increased Th2 cytokine production in the probiotic group could not be clearly associated with the development of disease, i.e. eczema, or sensitization during the first two years of life. Furthermore, it is unclear at present if increased ex vivo production of Th2 cytokines at two years of age will have clinical consequences in the future. However, these findings possible indicate adverse effects of probiotic supplementation on Th2 pathways. A Th2 cytokine profile characterized by high levels of IL-4, IL-5 and IL-13 within the first 6 months of life in high-risk children has been associated with the development of atopic disease in the first year of life 3. In addition, children with atopic disease at age 6 years showed increased Th2 Chapter 6 cytokine responses at age 1 and 2 year to house dust mite 2. Despite these associations, ex vivo cytokine production is by no means an established or validated biomarker for atopic disease 2. Although atopic diseases are immunologically characterized by allergen-specific T cell memory 136 dominated by Th2 cell cytokines, increased presence of Th2 cytokines and IgE does not necessary implicate the development of atopic diseases. In chronic helminth and parasite infections, Th2 responses dominate in the absence of clinical allergy due to the presence of anti-inflammatory responses 36. The results of our study suggest that the potential of probiotic bacteria to modulate immune response in vitro seems to be different from the in vivo and/or ex vivo response. The probiotic bacteria used in this clinical trial were selected based on their in vitro capacity to suppress the production of Th2 cytokines and stimulation of IL-10 production in cells of healthy individuals 17;18. The capacity of the individual strains to modulate cytokine responses might be different from the probiotic mixture, since it has been shown that specific lactic acid bacteria neutralize or inhibit the cytokine production induced by other lactic acid bacteria 12. However, the probiotic mixture used by us in the clinical trial had a similar profile of suppressing Th2 cytokines and

stimulating the production of IL-10 and pro-inflammatory cytokines in PBMCs. In contrast to the individual strains, the probiotic mixture clearly stimulated the production of IL-12 in vitro, which potentially would lead to a stronger induction of Th1 responses. It is however more likely that results from in vitro cultured cells may not accurately represent in vivo responses to intestinal microbiota, due to complex interactions in the intestinal tract between its microbiota, intestinal epithelium and immune system. The intestinal epithelium forms a barrier between the intestinal microbiota and the underlying immune cells such as dendritic cells (DCs). The potential crosstalk between microbiota and cells of the mucosal immune system including DC has been studied in transwell models. Upon direct interaction with lactic acid bacteria (LAB), DCs were differentially activated and matured, but this effect was lost when evaluating indirect interactions with DCs through a monolayer of intestinal epithelial cells (IECs) 37. LAB did however reduce maturation of DCs through IECs by E. coli in a TLR2 dependent mechanism 38. IECs seemed to be largely unresponsive to gram-positive bacteria such as LAB but the presence of LAB interfered with the pro-inflammatory immune response of IECs to pathogens such as Salmonella typhimurium 39. Recently it was shown that signals from commensal bacteria might be acting directly on IECs to induce expression of cytokines such as IL-25 40. For obvious ethical, but also practical reasons, investigating the effects of probiotic bacteria on the mucosal immune system of the gut in Immunomodulation by probiotic bacteria children is impossible. As a surrogate, we measured IL-25 in plasma samples. During the first three months of life we observed a clearly different composition of the intestinal microbiota in 137 the probiotics group compared to the placebo group (Niers et al, submitted) which coincided with plasma levels of IL-25 at the age of 3 months being a 5 fold higher in the probiotics group compared to the placebo group (p 0.075). Higher levels of IL-25 were however not associated with richness, diversity, and relative abundance of Lc. lactis or Streptococcus salivarius. Previously it has been shown in children that timing and the type of bacteria colonizing the intestine of newborns may determine the modulation of the immune system 41; 42. The exact sequence of events between intestinal microbiota and systemic cytokine levels however is unknown, and statistically we could no find consistent and clearly significant correlations between parameters of intestinal colonization (richness, diversity, relative abundance of Lc. lactis or Streptococcus salivarius) and production of different cytokines.

In conclusion, supplementation of probiotic bacteria during the first year of life modulates the development of the immune system during the first two years of life by reducing Th2 cell cytokine responses at the age of 3 months but increasing Th2 cell cytokine responses at age 2 years. The clinical implications of increased presence and production of Th2 cytokines at two years of age are currently unclear. Long time follow-up at the age of 6 years when specifically asthma (including lung function (spirometry)), allergic rhinitis and again allergic sensitization for food and inhalant allergens will be assessed, will show the long-term clinical consequences of the administration of probiotic bacteria. ACKNOWLEDGMENTS We would like to thank the children and their parents for their participation. We thank Paul Westers, Center for Biostatistics, Julius Centre, University of Utrecht, the Netherlands for Chapter 6 assistance with statistical analysis. 138

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General discussion 7

Chapter 7 144

The scientific basis for the use of probiotic bacteria early in life Primary prevention of atopic diseases in young children by administration of probiotic bacteria early in life has a solid scientific basis (see Introduction). Various reproducible observations from numerous studies offer valid arguments supportive of a role of probiotic bacteria in preventing the development of atopic diseases in high-risk children, with the hygiene hypothesis as the main initiator. However, the hygiene hypothesis and associated observations have been questioned and should be put into perspective to appreciate clinical intervention trials such as described in this thesis. The hygiene hypothesis involves a theory on how the environment, in particular the exposure to microbes in early childhood, influences immune responses and the development of atopic diseases. More than circumstantial evidence supporting this theory is lacking since viral upper airway infections, bacterial cell wall products (LPS), oro-faecal transmitted infections, parasitic infections, and specific gastro-intestinal microbes all have been associated with the protection against atopic diseases. Thus, no specific causative factor can be pinpointed. There rather seems to be a myriad of environmental factors operating in a complex interplay with each other and the host with its complex immune system and genetic susceptibility. Because the pathogenesis of atopic diseases is largely unknown, the exact mechanism of action of specific General discussion environmental factors on the development of these diseases is unclear. Of note, the majority of these factors may merely reflect poorer levels of hygiene and may not be directly protective. 145 Furthermore, the epidemiological studies, which lend support to the hygiene hypothesis, have a cross sectional study design or are prospective cohort studies. With a cross sectional study design, it is difficult to differentiate between chance (indicating a possible causative relationship between two variables) and change of behavior (indicating a consequence rather than a cause). Cross sectional studies do not offer proof of causative relationships, but merely generate hypotheses. Data generated from prospective cohort studies have a higher level of evidence regarding causative relationships. If associated variables or factors precede the onset of disease, a potential causal relationship can be suggested, but conclusive evidence for causality from prospective studies is lacking. Conclusively, the nature of most supporting studies implicate that the hygiene hypothesis is truly a hypothesis, which must be verified by intervention

studies. The microflora hypothesis, which is a spin off of the hygiene hypothesis, proposes that prosperity and improved hygiene have altered the composition of the intestinal microbiota and have disrupted the maturation of the immune system and the development of immunological tolerance. It should be emphasized that the host s immune system may modify the colonization of the intestinal tract, and that these findings may reflect the characteristics of the immune system associated with atopic diseases rather than a causal relationship. Thus, mechanistic and clinical studies are needed to demonstrate the functional effect of microbes on the intestinal microbiota, the immune system and immune mediated diseases such as atopic diseases. For mechanistic studies, animal models are particularly useful. Germ-free animals have smaller Peyer s patches, fewer intraepithelial T lymphocytes, and lower levels of secretory IgA compared to conventional pathogen free animals, which emphasizes the pivotal role of the intestinal microbiota on the development of the immune system. Colonization of germ free mice with Bacteroides fragilis restores normal development of the immune system. Subsequently it was shown in a series of elegant experiments that the polysaccharide A from the capsule of B. fragilis Chapter 7 is responsible for this effect 1. The main question remains what exactly happens upon ingestion of probiotic bacteria. 146 The research described in this thesis adds the following aspects to our current knowledge and ongoing discussions on the effects of probiotic bacteria on the intestinal microbiota, the immune system and the development of atopic diseases: Probiotic bacteria may only be capable to exert their clinical beneficial effects if they induce a richer and more diverse microbiota, and stimulate or inhibit the presence of specific bacterial species. Probiotic supplementation during the first year of life is associated with decreased ex vivo production of Th2 cytokines at 3 months of age, but increased ex vivo production of Th2 cytokines at the age of two years. The potential of probiotic bacteria to modulate immune response in vitro seems to be different from ex vivo. The preventive effect of probiotic bacteria on eczema seems to be established early in life.

Effects of probiotics on the gastro-intestinal tract The intestinal commensal microbiota has a complex relationship with the host. Interaction between bacteria and the host can take place in the form of symbiosis, commensalism and pathogenicity. Symbiosis involves a relationship between two different species in which one or both species benefit from the presence of the other without harming each other. Commensalism involves the co-existence of species without beneficial or disadvantageous effects. Pathogenicity indicates damage to the host 2. Furthermore, the intestinal microbial species and strains have a complex relationship with each other. One possible mechanism of action of probiotic bacteria in the gastro-intestinal tract is influencing the composition of the intestinal microbiota and exerting metabolic activities in the gastro-intestinal tract. In chapter 5, we have shown that probiotic supplementation during the first year of life results in profound changes in the early colonization and composition of the intestinal microbiota, especially during the first three months of life. The changes were related to the supplemented strains but the presence and abundance of other species or specific bacteria such as Streptococcus salivarius were stimulated as well. On the other hand, the presence of the supplemented strains in the gastro-intestinal tract may also inhibit the presence or outgrowth of other bacteria. General discussion The underlying mechanisms by which the supplemented strains modulate the composition of the intestinal microbiota remain to be elucidated. Intestinal bacteria may positively or 147 negatively influence each other in various ways. The early colonizers of the newborn s intestinal tract can modulate the expression of genes of the intestinal epithelial cells, thereby creating a favorable niche for themselves and stimulating or inhibiting the presence of other bacteria 2. In addition, intestinal bacteria differ in their adhesion capacity, metabolic capacities and antibacterial properties. Furthermore, intestinal bacteria communicate and influence each other by means of quorum sensing 3. Quorum sensing involves chemical communication via small hormone-like molecules called auto-inducers. Another very important function of the intestinal microbiota is antimicrobial activity to potential pathogens called colonization resistance by competing for nutrients, adhesion sites and the production of antibacterial substances 4-6. From the supplemented strains B. bifidum, B. lactis (formerly classified as B. infantis), and Lc. Lactis, B.

lactis has been shown to display antimicrobial activity, especially to Enterococcus faecalis and a coagulase negative Staphylococcus (CNS) 7. It remains unknown if the clinical beneficial effects of probiotic bacteria as described in chapter 4 are a direct effect of the supplemented strains or are mediated by the overall composition (including richness and diversity) or specific species induced by probiotic supplementation. In experimental animal model of acute pancreatitis it has been found that induction of a commensal rat ileal bacterium (CRIB) is associated with the health promoting effects of a multispecies mixture of probiotic bacteria (H. Timmerman et al, manuscript in preparation). Whether this is a causal relation, meaning that the beneficial effects of the probiotic bacteria are mediated by CRIB, is currently being studied. The children in the probiotics group in our study had a significantly higher prevalence and abundance of Lc. lactis compared to the placebo group. There was no difference in the prevalence or the relative abundance of Lc. lactis between the children who did or did not develop eczema in the probiotic group. Due to the high physiological prevalence of bifidobacteria and the limited discriminating power of microbial Chapter 7 community profiling and characterization (MCPC) analysis regarding specific bifidobacterial strains, clinical beneficial effects could not be associated with the levels of supplemented B. bifidum and B. lactis. However, within the probiotic group successful intervention i.e. absence 148 of eczema during the first two years of life was clearly associated with more richness and diversity of the intestinal microbiota, several predominant terminal restriction fragments (T-RFs) (identified as members of the genera Lactobacillus, Clostridium and Staphylococcus) and tended to be associated with the relative abundance of Streptococcus salivarius. These results (chapter 5) justify the conclusion that probiotic bacteria may only be capable to exert their clinical beneficial effects if they induce a richer and more diverse microbiota, and stimulate or inhibit the presence of specific bacterial species. Furthermore, this seems to be a host specific process, which is not associated with compliance as measured by the relative abundance of the supplemented strain Lc. lactis. Probiotic supplementation during the first year of life does not seem to induce permanent effects on the composition of the intestinal microbiota, which is in line with other studies 8-10. The effect of probiotic supplementation seems to be overruled by more powerful factors determining colonization of the intestine, such as weaning, nutrition and

other microbial exposure. With increasing intestinal microbial complexity during the first year of life, factors like colonization resistance may limit the effects of probiotic supplementation. The lack of beneficial effects of probiotic supplementation in tertiary prevention or treatment of older children with (atopic) eczema could therefore be due to the inability of the probiotic bacteria to influence the composition of intestinal microbiota. The window of opportunity to, temporarily, modulate the intestinal microbiota by probiotic bacteria clearly lies within the first three months of life and appears to be closed at an older age. Effects of probiotics on the immune system Apart from the influence on composition of the intestinal microbiota, as discussed above, modulation of the immune system is another potential mechanism of action by probiotic bacteria. Although clinical and experimental evidence exists to support this concept, general claims of modulation of the immune system by probiotic bacteria overstate our current knowledge. The effects of ingested probiotic bacteria on the intestinal and systemic immune system involve complex host-microbe interactions which are just beginning to be thoroughly explored and understood 11. Many in vitro studies have addressed the effects of different probiotic General discussion bacteria on mononuclear cells, which involved suppression of Th2 cytokines, stimulation of IFN-g, IL-12 and other pro-inflammatory cytokines, and the induction of regulatory T cells (discussed in 149 more detail in chapter 2). We have shown that probiotic bacteria inhibit the in vitro production of IL-5 and IL-13 and stimulate the production of IL-10 by PBMCs. The capacity to suppress these Th2 cytokines differed greatly among species and strains and was partly mediated by IL-10. Furthermore, different species and strains of lactic acid bacteria have been shown to modulate the expression of co-stimulatory molecules and cytokine production of murine and human dendritic cells (discussed in more detail in chapter 3). We showed that B. bifidum and Lc. lactis in vitro prime neonatal dendritic cells to polarize naive T cells into Th1 cells and for B. bifidum possibly Tregs as well since the production of IL-10 of these T cells was significantly increased. It is clear from these and many other studies that considerable variation exists in the capacity for immune modulation in vitro among different lactic acid bacterial species and strains. The effect

of a probiotic bacterium is strain specific and cannot be extrapolated even to other strains of the same species. This seems to hold true for their clinical application as well. We have used this knowledge to make a rational choice of available probiotic bacteria. It is still largely unknown if probiotic bacteria may influence the human systemic immune system. From murine studies, it has been suggested that commensal microbiota may regulate immune responses outside the gut. If the endogenous microbiota is altered at time of first allergen exposure, mice can develop allergic airway responses to allergens 12-14. Furthermore, airway inflammation and allergic sensitization have been shown to be suppressed by orally administered probiotic bacteria in murine models associated with a shift to Th1 allergenspecific responses as well as T regulatory-dependent mechanisms 15;16. Previously it has been shown in children that timing and the type of bacteria colonizing the intestine of newborns may determine the modulation of the immune system 17;18. In healthy adults, administration of living L. plantarum bacteria induced mucosal gene expression patterns and cellular pathways indicative of immune tolerance in the duodenum 19. This is the first demonstration that probiotic Chapter 7 bacteria have an effect on the human (intestinal) immune system in vivo. Can all these data be integrated to explain the mechanisms of influence of probiotics on the immune system of infants? We showed that probiotic supplementation during the first year 150 of life is associated with decreased ex vivo production of Th2 cytokines at 3 months of age but with increased ex vivo production of Th2 cytokines at the age of 2 years. These results seem to conflict with the conclusions of chapter 2, in which we described the selection of the probiotic bacteria used in the clinical trial based on their in vitro capacity to suppress the production of Th2 cytokines and stimulation of IL-10 production. However, in vitro cultured cells may not accurately represent in vivo responses to intestinal microbiota, since the intestinal tract is characterized by complex interaction between its microbiota, host epithelial cells and mucosal immune system. The intestinal immune system can be divided into the intestinal epithelium and the gut associated lymphoid tissue, which functions in close contact and interaction with the systemic immune system. The intestinal epithelium forms a barrier between the intestinal microbiota and the underlying immune cells. Intestinal epithelial cells (IECs) seem to be largely unresponsive to

the intestinal microbiota but the presence of lactic acid bacteria interferes with the immune response of IECs to pathogens such as Salmonella typhimurium 20. Recently it was shown that signals from commensal bacteria may be acting directly on IECs to induce expression of cytokines such as IL-25 21. To study the effects of the specific probiotic bacteria used in our clinical trial on immune cells with IECs has a natural barrier, we used a transwell system in which a monolayer of IECs separates the bacteria from the basolaterally located PBMCs. Preliminary results indicate that the presence of IECs enhances production of IL-10 and IL-13 by stimulated PBMCs from high risk children. In the presence of IECs, the mixture of probiotic bacteria used in the clinical trial is not capable of suppressing the production of IL-13, but is still capable of enhancing the production of IL-10 and IFN-g. The production of TNF-a is only stimulated in the absence of IECs (personal communication L. Willemsen). These results indicate that the presence of IECs profoundly influence the production of cytokines of mononuclear cells upon exposure to probiotic bacteria. This provides a possible explanation for the conflicting results of the capacity of probiotic bacteria to modulate immune responses in vitro compared to ex vivo immune responses after probiotic supplementation. Another level at which probiotic bacteria may interact with the gastro-intestinal immune system is by translocation to mesenteric lymph nodes, sampling of luminal bacteria by dendritic General discussion cells or adhesion to M cells covering Peyer s patches. Dendritic cells (DC) in the gastro-intestinal immune system play a very important role in the immune response to intestinal bacteria 151 including commensal bacteria. DC are conditioned by epithelial-cell-derived factors and polarize naive T cells into Th1, Th2 or T regulatory cells (Tregs) 22. Furthermore, DC have an important role in atopic diseases 23. By controlling the development of tolerance to allergens, DC may regulate the development of atopic diseases. We showed that B. bifidum and Lc. lactis prime neonatal DC to polarize naive T cells into Th1 cells and for B. bifidum possibly Tregs as well since the production of IL-10 of these T cells was significantly increased. Probiotic bacteria presumably should interact directly with intestinal epithelial cells and cells of the immune system to be effective in modulation of immunity. This interaction is mediated by pattern recognition receptors such as the Toll-like receptor family (TLRs), C-type lectins (such as DC-sign) and NOD receptors, which recognize microbe-associated molecular patterns. We showed that B. bifidum

and B. lactis (formerly classified as B. infantis) are capable of binding to TLR2 but not to TLR4. Others have demonstrated that next to TLR2 also TLR9 can interact with probiotic bacteria or specific probiotic ligands 24-27. We further investigated the interaction of probiotic bacteria with DC by studying the binding of FITC labeled bacteria on immature DC (figure 1). B. bifidum and Lc. lactis showed substantial binding to DC compared to other strains. Preliminary results further indicated that B. bifidum binds to DC via DC-Sign (figure 2). Binding of probiotic bacteria to DCsign has previously been shown to prime DC to induce Tregs 28. Chapter 7 152 Figure 1: Interaction of probiotic bacteria with dendritic cells. Dendritic cells (DC) were cultured and differentiated from human peripheral monocytes with rgm- CSF and ril-4 for 6 days. Immature DC were incubated with FITC labeled bacteria. A: Confocal fluorescence microscopy shows individual DCs with attached and/or fagocytosed bacteria. B: The percentage of FITC positive DC was measured by FACS analysis (n= 10).

Figure 2: Interaction of B. bifidum with DC specific ICAM-3-grabbing non-integrin (DC-SIGN) receptor. Peripheral blood monocytes were differentiated in vitro in immature dendritic cells. A. The adherence of FITC-labeled B. bifidum to dendritic cells. Fluorescence intensity of the dendritic cells was measured by FACS analysis. B. Dendritic cells were pre-incubated with blocking DC-SIGN antibody (AZN-D1, a kind gift of Prof. Dr. Y. van Kooyk, Free University, Amsterdam). Binding of B. bifidum General discussion to dendritic cells was reduced as assessed by confocal fluorescence microscopy and fluorescence intensity by FACS analysis. 153

Chapter 7 154 Follow up Results Reference(s) / Location Strain(s) / Dosage a n b Duration of Supplementation Prenatal Postnatal incidence of eczema No difference in severity (SCORAD) Trend towards prevalence of allergic rhinitis and asthma 159 2-4 weeks 6 months c 2, 4, and 7 years L. rhamnosus GG (ATCC 53103) / 1 x 10 10 CFU/d Kalliomaki 2001; 2003; 2007 / Finland 232 4 weeks 12 months 2 years No difference in incidence of eczema IgE-associated eczema (IgE or positive SPT) No difference in severity (SCORAD) Trend toward sensitization, significant only in children with atopic mothers (positive SPT) L. reuteri (ATCC 55730) / 1 x 10 8 CFU/d Abrahamsson 2007 / Sweden 226 N/A d 6 months 1 year No difference in incidence of eczema incidence of atopic eczema (positive SPT) rate of sensitization (positive SPT) overall incidence of wheezing (26% vs. 13%) Lactobacillus colonization confirmed Taylor 2007 / Australia L. acidophilus (LAVRI-A1) / 3 x 10 9 CFU/d No difference in cumulative incidence of allergic diseases incidence of eczema (2 yrs) incidence of atopic eczema (IgE or positive SPT) (2 yrs) incidence of atopic eczema (IgE or positive SPT) in cesarean-delivered children (5 yrs) No difference in severity (SCORAD) Lactobacillus and Bifidobacterium colonization confirmed 1223 2-4 weeks 6 months 2 and 5 years Probiotic mixture e + 0.8 g galacto-oligosaccharides Kukkonen 2007; Kuitunen 2009 / Finland 6 months f 2 yeaers No difference in incidence of eczema No difference in severity (SCORAD) rate of recurrent episodes of wheezing bronchitis (26% vs. 9.1%) 105 4-6 weeks Kopp 2008 / Germany L. rhamnosus GG (ATCC 53103) / 5 x 10 9 CFU/d

2 years incidence of eczema (L. rhamnosus) IgE-associated eczema (positive SPT) (L. rhamnosus) severity (SCORAD 10) (L. rhamnosus) No difference in incidence of eczema or severity (B. lactis) Lactobacillus and Bifidobacterium colonization confirmed 474 5 weeks 6 months (mother) 2 yrs (infant) L. rhamnosus HN001 / 6 x 10 9 CFU/d or B. animalis ssp. lactis (HN019) / 9 x 10 9 CFU/d Wickens 2008 / New Zealand 253 N/A d 6 months 1 year No difference in incidence of eczema No difference in IgE-associated eczema (IgE or positive SPT) No difference in severity (SCORAD) 156 6 weeks 12 months 2 years incidence of eczema (both parent-reported and physician-diagnosed) No difference in IgE-associated eczema (IgE or positive SPT) No difference in severity (SCORAD) Trend toward sensitization, specifically food allergens Lactococcus and Bifidobacterium colonization confirmed Soh 2009 / Singapore B. longum (BL999) L. rhamnosus (LPR) ~2.8 x 10 8 CFU/d combined B. bifidum (W23) B. lactis (W52) Lc. lactis (W58) / 3 x10 9 CFU/d combined Niers 2009 / the Netherlands a Provided as a powder mixed in water, breast milk, or formula unless otherwise indicated. b At time of randomization. c Mothers could choose to administer the probiotic directly to the infant (56%) or to consume the probiotic and deliver via breast milk. d Supplementation was begun within 12 hours in infant formula (Soh 2009) or 48 hours of birth (Taylor 2007). N/A: not administered. e L. rhamnosus GG (ATCC 53103) / 5 x 10 9 CFU/d, L. rhamnosus LC705 (DSM 7061) / 5 x 10 9 CFU/d, B. breve Bb99 (DSM 13692) /2 x 10 8 CFU/d, Propionibacterium freudenreichii ssp. shermanii JS (DSM 7076) / 2 x 10 9 CFU/d f Mothers supplemented with the probiotic for 3 months, exposing infant via breast milk, followed by 3 months of direct supplementation of the infant. General discussion 155

Effects of probiotics on the development of atopic diseases To date, eight primary prevention studies including the PandA study using eight different probiotic bacteria or mixtures of probiotic bacteria have been published with conflicting results (see table). No adverse events have been found in the pregnant mothers or their children. The longest follow-up period now is 7 years 29. Many studies are still on the way 30. In five studies specific probiotic bacteria administered prenatally to the pregnant mothers and postnatally to their high-risk children reduced the incidence or prevalence of (atopic) eczema compared to infants receiving placebo. Other studies have failed to show a preventive effect. In addition, probiotic supplementation has been associated with increased prevalence of sensitization, and no preventive effect on the development of food allergies, asthma, and allergic rhinitis has been reported. This large heterogeneity in results might be explained by several factors. First, it is apparent that different probiotic strains or combinations of probiotics would be expected to elicit different effects. This explanation is insufficient, however, based on the observation that studies Chapter 7 using the same single strain of Lactobacillus rhamnosus GG (LGG) have reported dramatically different results 31;32. Next, differences in outcomes may be due, in part, to differences in routes of administration. While most studies of primary prevention have administered probiotics to 156 both the pregnant mother and infant, some supplemented only the infant and others relied on supplementation of the mother and the administration of protective factors to the infant via breast milk. The following issues will be further discussed: Only studies in which supplementation started during pregnancy have observed a preventive effect. Both studies, which did not supplement the pregnant women, reported no change in the incidence of eczema. Low rates of colonization of strains which failed to show a preventive effect on the development of eczema. Only preventive effect on eczema and no preventive effect on sensitization or any atopic disease other than eczema. The possibility of gene by environment interaction.

Only studies in which supplementation started during pregnancy observed a preventive effect on the development of eczema. This indicates that prenatal administration of probiotic bacteria to the mother might be essential in this process. The vaginal and intestinal microbiota of the mother are one of the first colonizers of the neonatal gut 33. Prenatal administration of probiotic bacteria may cause temporary changes in the composition of the maternal intestinal microbiota and consequently may influence early colonization in their offspring 34-36. This involves temporary infantile colonization of the supplemented strain to the mother, but also alterations in the microbiota not reflective of the administered bacteria have been reported. Prenatal administration with LGG, for example, did not result in infant colonization with LGG, but rather an increase in the percentage of infants with detectable B. longum 37. In most cases, the mothers suffered from an atopic disease. This may be related to an unfavorable balance of gut microbiota. The administration of probiotic bacteria may improve this balance, which consequently may be transferred to the offspring. Furthermore, probiotic supplementation to the mother might result in immune modulation in the mother, for instance in immunomodulatory factors in breast milk. Immune modulation via the mother may provide an unknown immune stimulation to the child. Supplementing pregnant and breastfeeding mothers with probiotics has been reported to alter immunological General discussion factors in cord blood and breast milk, although findings from recent studies are not consistent. The use of probiotics (L. rhamnosus strain GG and B. lactis Bb12) during pregnancy and lactation 157 reduced the risk of sensitization at the age of 12 months in infants of atopic mothers, potentially mediated by higher concentrations of TGF-b2 in colostrum 38. In addition, supplementation of L. rhamnosus HN001 during the last 2-5 weeks of pregnancy resulted in higher cord blood IFN-g levels and higher IgA levels in breast milk 39. However, supplementation of L. reuteri during pregnancy seemed to reduce levels of TGF-b2 in breast milk, and less sensitization was associated with low levels of this cytokine 40. Furthermore, LGG treatment of pregnant women failed to influence fetal antigen-specific immune responses 41;42. Conclusively, prenatal administration of probiotic bacteria may be essential in preventing the development of eczema in the offspring by influencing the developing intestinal microbiota and immunological factors in breast milk and cord blood, but the exact mechanism of action remains to be elucidated.

Low rates of colonization of strains which failed to show a preventive effect on the development of eczema. An effective probiotic strain or mixture of probiotic bacteria should be able to survive the host s digestive system to maintain sufficient viable microorganisms to reside in the intestinal tract and induce a beneficial clinical response. In line with this statement, we showed that reduced incidence of eczema at 3 months of age coincided with increased prevalence and abundance of the supplemented strains, i.e. Lc. lactis in particular (chapter 4). Not all primary prevention studies have confirmed the colonization of the supplemented probiotic bacteria in the intestine. Other studies which did show a preventive effect of specific probiotic strains or probiotic mixtures on the development of eczema ánd investigated the fecal microbiota showed increased prevalence and abundance of the supplemented strains as well 8;9. The probiotic strains, which failed to reduce the risk of (atopic) eczema, were detected at relatively low frequency. B. animalis subsp lactis was present in 22.6% of the fecal samples at 3 months of age, which increased to 53.1% at 24 months of age (background exposure in the placebo group reached to 15%). In the same Chapter 7 study supplementation of L rhamnosus HN001 substantially reduced the risk of eczema ánd was detected in 71.5% of the fecal samples in the intervention group at the age of 3 months (background exposure in the placebo group almost 30%) 43. Administration of L. acidophilus 158 during the first 6 months of life resulted in culturable levels of Lactobacillus species in maximal 36% of samples in the probiotic group compared to 21.6% in the placebo group at 6 months 44. However, high prevalence of the supplemented strains per se is not sufficient to prevent the development of (atopic) eczema. Although 72 to 85% of infants had positive fecal samples (determined by the detection of PCR amplicons) for the supplemented strains LGG and B. longum in the intervention group compared to 12% in the placebo group during the first three months of life 10, this probiotic combination failed to reduce the risk of (atopic) eczema 45. We showed that probiotic bacteria may only be capable to exert their clinical beneficial effects if they induce a richer and more diverse microbiota, and stimulate or inhibit the presence of specific bacterial species. In addition, specific probiotic strains or combinations that lack effect on eczema may be less effective in modulating the immune system.

Only preventive effect on eczema and no preventive effect on sensitization or any atopic disease other than eczema. The studies published thus far provide evidence for a protective role of at least some lactic acid bacteria species or combinations in the development of eczema, atopic eczema, or both (see table). There is little evidence that this is mediated through effects on allergic sensitization and no protective effect on asthma and/or allergic rhinitis has been reported. The clinical phenotype of eczema can been divided into atopic and non atopic eczema, i.e. eczema which coincides with allergic sensitization or not, respectively 46. The association between atopic eczema and sensitization varies widely between studies indicating that sensitization is neither a causal factor nor a requirement for the development of eczema 47. Specifically in young children, eczema precedes the development of sensitization. The majority of the studies did not observe an effect of probiotic supplementation on the incidence or prevalence of sensitization (either at least one positive skin prick test or elevated specific IgE during the follow-up period). Of note, we observed at trend towards more sensitization, specifically food allergens (hen s egg) at the age of two years. Others have found significantly more sensitization to aeroallergens at the age of 1 year after administration of L. acidophilus during the first 6 months of life 44. Strong IgE antibody responses to food proteins in infants must be considered as markers for General discussion atopic reactivity in general and are predictors of subsequent sensitization to aeroallergens at an older age 48. Sensitization to aeroallergens at a young age is a risk factor for the development 159 of asthma. Therefore, long-term follow-up is warranted to address the development of asthma after supplementation with probiotic bacteria. Eczema, asthma and allergic rhinitis are associated clinical phenotypes, which tend to cluster in families and individuals. Eczema may precede the development of asthma and allergic rhinitis. This sequence of events is called the atopic march of which the exact pathogenesis is unclear. It has been questioned if eczema, asthma, and allergic rhinitis are separate disease entities, which may coincide in an individual. However, loss-of-function mutations in the filaggrin gene which results in impairment of the epidermal barrier function were recently found to predispose to both atopic and non atopic eczema, and in the presence of eczema were associated with the development of asthma and allergic rhinitis 49. Although the concept that early prevention of

eczema might halt the progression of other atopic diseases such as asthma is highly intriguing, it remains to be demonstrated. This holds true as well for the assumption that probiotic supplementation may prevent atopic diseases in general since this has only been demonstrated for (atopic) eczema. For asthma prevention as well as allergic rhinitis longer observation periods are necessary. Most studies had a follow-up until the age of one or two years while asthma and allergic rhinitis develop during school age and adolescence. The longest follow-up period is currently 7 years but Kalliomaki et al only used asthma medication use (derived from the records of insurance companies) to assess the effect of LGG on the development of asthma. Other studies did not observe a difference in asthma like symptoms, mostly wheezing. However, pre-school wheezing is not equivalent to asthma. Therefore, long-term follow-up including a thorough diagnosis of asthma including lung function (spirometry and exhaled bronchial nitric oxide) is warranted. This accounts as well for allergic rhinitis. The possibility of gene by environment interaction Chapter 7 As indicated previously, a major reason for inconsistent results of clinical trials as summarized in table I may be the use of different probiotic strains or preparations. In this respect however it is interesting that the results of Kopp et al. are in sharp contrast with the study of Kalliomaki et 160 al. although an identical probiotic strain is used (LGG) in an also otherwise highly comparable study design 32;50. Of note, during the first 3 months exposure of the infants to LGG was only through breast milk, followed by 3 months of direct supplementation in the study by Kopp et al. Supplementation via breast milk may be less effective than direct supplementation to modulate the composition of the intestinal microbiota and the developing immune system during the critical window of opportunity. The studies were conducted in Germany and Finland respectively, hence differences in the study populations specifically genotype differences may contribute to disparate results as well. Both populations originated largely from different ancestors. For example, the predominant Y-chromosome haplogroup in Germans is R1b, compared to I (http://www.scs.uiuc.edu/~mcdonald/worldhaplogroupsmaps.pdf) in Finland. The predominant mitochondrial haplogroups are similar in Germany and Finland, expect for the Sámi people (also known as laps). In addition, not everyone benefits from

probiotic supplementation. Children do develop eczema despite probiotic supplementation. Is this lack of an effect the result of gene by environment interaction? Within the pathogenesis of atopic diseases in which alterations in many genes and numerous environmental factors are involved, gene by environment interactions will certainly contribute to the susceptibility and manifestation of disease. For instance, in children with the CC genotype at -159 of the CD14 gene, increasing exposure to endotoxin (LPS) is associated with reduced risk of eczema and allergic sensitization 51. It is tempting to speculate that the effect of probiotic bacteria will in part be determined by particularly the genotype of the (gastro-intestinal) immune system. Polymorphisms in CD14 and TLRs are potential candidates to affect the efficacy of probiotic bacteria. In our study population, we determined single-nucleotide polymorphisms (SNPs) in the promoter region of CD14 (CD14/-159 (C/T)), three SNPs in TLR-2 (TLR2/-16934 (A/T)) promotor polymorphism, TLR2/C1982A, and TLR2/G2258A), and one in TLR-4 (TRL4/C8851T). Preliminary results showed a significant interaction with randomization and the genotype of TLR2/-16934 promotor polymorphism with respect to the development of eczema at the age of 3 months. This indicates that TLR2 genotype influences the effect of probiotic bacteria. In children who are homozygous for the T allele, which involved 34.4% of the study population, probiotic intervention did not have a significant effect on the development of eczema at 3 months of age General discussion (OR 1.6 (95% CI 0.3-7.9), p 0.689). Probiotic supplementation resulted in significantly less eczema in children who are heterozygous or homozygous for the A allele (OR 0.113 (95% CI 0.022-0.579), 161 p 0.007). Since we previously showed that B. bifidum and B. lactis are capable of binding to TLR2 and this particular SNP in TLR2 is know to be functional, it may be concluded that the ability to benefit from probiotic supplementation may depend on TLR2 genotype. Our data do not have enough power to demonstrate an unequivocal role for TLR polymorphisms in determining the outcome of probiotic intervention.

Conclusions and future perspectives The PandA study is the first study of perinatal administration of probiotic bacteria for the primary prevention of atopic diseases in which strains were selected based on in vitro cytokine production. The PandA study thereby crossed the gap between in vitro effects of probiotic bacteria and outcomes in clinical trials. Furthermore, the PandA study tried to identify the immune modulating activities of probiotics in vivo in the context of clinical outcomes. Certain immunomodulatory effects demonstrated in vitro, such as the reduction in the Th2-associated cytokines IL-5 and IL-13, were subsequently observed ex vivo in whole blood cells obtained from probiotic-supplemented infants. Subsequently, the PandA study provided evidence that probiotic bacteria may induce beneficial clinical effects by modulating the early composition of the intestinal microbiota such as enhancing richness and diversity. In future studies the window of opportunity may allow to switch to another (combination of) probiotic in the absence of modulation of the intestinal microbiota and thereby a sort of second chance primary Chapter 7 prevention. Furthermore, future studies need to continue identifying the immune activities of probiotics in vivo in the context of clinical outcomes. Figure 3 summarizes the potential mechanisms of action and clinical effects of perinatal administration of probiotic bacteria. 162 There is a high public enthusiasm for probiotic use although conclusive evidence for health benefits is still required, and health claims from commercially available products overstate our current knowledge. Probiotics may be used to prevent and to treat a number of different diseases. The efficacy of treatment is proven in randomized double-blind placebo-controlled studies for a number of diseases of the gastrointestinal tract, like pouchitis, antibioticsassociated diarrhea, and Clostridium difficile-induced enterocolitis 52-54. Some studies suggest that probiotics may have beneficial effects in patients with lactose intolerance, rotavirus diarrhea, and irritable bowel disease. For other claims, proper supporting evidence is lacking. Detrimental 55 and potentially negative effects 29;44 have been reported as well which emphasizes the issue of safety of probiotic bacteria. This calls for a critical and cautious approach in developing future studies with probiotic bacteria. The Dutch Food and Consumer Product Safety Authority (VWA) recently conducted a literature survey concerning the safety of the consumption of probiotic

bacteria (www.vwa.nl). They concluded that there is no evidence for adverse effects in healthy individuals. Long term, possibly negative, effects in children should however be investigated. Worth mentioning is that different probiotics (even within a species) may differ with respect to effect. Therefore, it seems logical to hypothesize that not all probiotics will be beneficial in all diseases. In addition, probiotic bacteria are clearly not beneficial in every individual in the population, but probably only to those with a particular genetic susceptibility, maybe even within certain environmental co-factors. This challenges future studies how to choose monostrain or multispecies probiotics. The concept of strain selection based on in vitro cytokine profiles and potential interactions with the immune system could be used in combination with qualities concerning safety, survival of the first part of the gastro-intestinal tract, i.e. resistance to acid and bile, the capacity to reach high prevalence and abundance in fecal microbiota, and the capacity to modulate the resident intestinal microbiota. However, thorough strain selection does not guarantee probiotic effectiveness in vivo due to the complexity of the host, necessitating randomized clinical intervention. Future research should focus on the long-term effects of perinatal administration of probiotic bacteria regarding the development of sensitization, asthma and allergic rhinitis. Furthermore, the effects of prenatal administration of probiotic bacteria to pregnant women on their intestinal General discussion microbiota and immune system and subsequently on their offspring should be more thoroughly addressed. Mechanistic studies are needed to unravel which component(s) of probiotic bacteria 163 are crucial in determining their effects, what is the gastrointestinal niche of probiotic bacteria and how do they interact with the commensal microbiota and the gastrointestinal immune system. This should include well defined animal models but preferably human studies including intestinal biopsies (to study the intestinal microbiota and the first lining of the mucosal immune system of the gut) as well. Perinatal supplementation with probiotics holds promise for the prevention of childhood eczema. However, the clinical effects of single probiotic strains or combinations on the development of eczema should be confirmed in similar trials. The large heterogeneity in clinical studies and used probiotic preparations currently undermines solid scientific evidence and hampers the general recommendation to use probiotic bacteria in primary prevention of atopic diseases in high-risk children.

Chapter 7 164

Figure 3: Summary of potential effects of probiotic bacteria. a) After surviving the digestive process of the stomach and first part of the small intestine, probiotic bacteria may influence the composition of the developing intestinal microbiota by inducing more richness and diversity and by stimulating or inhibiting the presence of commensal bacterial communities. b) Recognition of probiotic bacteria by intestinal epithelial cells (IECs) may influence the secretion of cytokines and other tissue factors by IEC that direct the expression of cytokines and polarizing capacity of underlying dendritic cells (DC). c) Pattern recognition receptors including Toll-like receptors and C-type lectins are expressed by IECs and underlying intestinal dendritic cells and may recognize probiotic bacteria. Dendritic cells may interact with probiotic bacteria by extending their dendrites between tight junctions to directly sample the luminal microbiota, or bacteria may pass the intestinal epithelial barrier via M cells. d) In mesenteric lymphnodes and Peyer s patches, DC may promote the differentiation of regulatory T cells and IgA-secreting B cells (not shown) or polarize T helper cells into Th1 or Th2 cells. Probiotic bacteria may thereby modulate the intestinal as well as the systemic immune system. e) How probiotic bacteria eventually influence the development of atopic diseases is not fully understood. f) Prenatal administration of probiotic bacteria to the mother may influence the composition of the intestinal microbiota of her newborn and may modulate the neonatal immune system. General discussion 165

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30 Prescott SL, Bjorksten B. Probiotics for the prevention or treatment of allergic diseases. J Allergy Clin Immunol 2007 Aug;120(2):255-62. 31 Kalliomaki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E. Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial. Lancet 2001 Apr 7;357(9262):1076-9. 32 Kopp MV, Hennemuth I, Heinzmann A, Urbanek R. Randomized, double-blind, placebocontrolled trial of probiotics for primary prevention: no clinical effects of Lactobacillus GG supplementation. Pediatrics 2008 Apr;121(4):e850-e856. 33 Palmer C, Bik EM, Digiulio DB, Relman DA, Brown PO. Development of the Human Infant Intestinal Microbiota. PLoS Biol 2007 Jun 26;5(7):e177. 34 Gueimonde M, Sakata S, Kalliomaki M, Isolauri E, Benno Y, Salminen S. Effect of maternal consumption of lactobacillus GG on transfer and establishment of fecal bifidobacterial microbiota in neonates. J Pediatr Gastroenterol Nutr 2006 Feb;42(2):166-70. 35 Schultz M, Gottl C, Young RJ, Iwen P, Vanderhoof JA. Administration of oral probiotic bacteria to pregnant women causes temporary infantile colonization. J Pediatr Gastroenterol Nutr 2004 Mar;38(3):293-7. 36 Gronlund MM, Gueimonde M, Laitinen K, Kociubinski G, Gronroos T, Salminen S, et al. Maternal breast-milk and intestinal bifidobacteria guide the compositional development of the Bifidobacterium microbiota in infants at risk of allergic disease. Clin Exp Allergy 2007 Dec;37(12):1764-72. Chapter 7 168 37 Lahtinen SJ, Boyle RJ, Kivivuori S, Oppedisano F, Smith KR, Robins-Browne R, et al. Prenatal probiotic administration can influence Bifidobacterium microbiota development in infants at high risk of allergy. J Allergy Clin Immunol 2009 Feb;123(2):499-501. 38 Huurre A, Laitinen K, Rautava S, Korkeamaki M, Isolauri E. Impact of maternal atopy and probiotic supplementation during pregnancy on infant sensitization: a double-blind placebocontrolled study. Clin Exp Allergy 2008 Aug;38(8):1342-8. 39 Prescott SL, Wickens K, Westcott L, Jung W, Currie H, Black PN, et al. Supplementation with Lactobacillus rhamnosus or Bifidobacterium lactis probiotics in pregnancy increases cord blood interferon-gamma and breast milk transforming growth factor-beta and immunoglobin A detection. Clin Exp Allergy 2008 Jul 2. 40 Bottcher MF, Abrahamsson TR, Fredriksson M, Jakobsson T, Bjorksten B. Low breast milk TGFbeta2 is induced by Lactobacillus reuteri supplementation and associates with reduced risk of sensitization during infancy. Pediatr Allergy Immunol 2008 Sep;19(6):497-504. 41 Kopp MV, Goldstein M, Dietschek A, Sofke J, Heinzmann A, Urbanek R. Lactobacillus GG has in vitro effects on enhanced interleukin-10 and interferon-gamma release of mononuclear cells but no in vivo effects in supplemented mothers and their neonates. Clin Exp Allergy 2008 Apr;38(4):602-10. 42 Boyle RJ, Mah LJ, Chen A, Kivivuori S, Robins-Browne RM, Tang ML. Effects of Lactobacillus GG treatment during pregnancy on the development of fetal antigen-specific immune responses. Clin Exp Allergy 2008 Dec;38(12):1882-90.

43 Wickens K, Black PN, Stanley TV, Mitchell E, Fitzharris P, Tannock GW, et al. A differential effect of 2 probiotics in the prevention of eczema and atopy: A double-blind, randomized, placebocontrolled trial. J Allergy Clin Immunol 2008 Aug 31. 44 Taylor AL, Dunstan JA, Prescott SL. Probiotic supplementation for the first 6 months of life fails to reduce the risk of atopic dermatitis and increases the risk of allergen sensitization in highrisk children: a randomized controlled trial. J Allergy Clin Immunol 2007 Jan;119(1):184-91. 45 Soh SE, Aw M, Gerez I, Chong YS, Rauff M, Ng YP, et al. Probiotic supplementation in the first 6 months of life in at risk Asian infants--effects on eczema and atopic sensitization at the age of 1 year. Clin Exp Allergy 2009 Apr;39(4):571-8. 46 Johansson SG, Bieber T, Dahl R, Friedmann PS, Lanier BQ, Lockey RF, et al. Revised nomenclature for allergy for global use: Report of the Nomenclature Review Committee of the World Allergy Organization, October 2003. J Allergy Clin Immunol 2004 May;113(5):832-6. 47 Williams H, Flohr C. How epidemiology has challenged 3 prevailing concepts about atopic dermatitis. J Allergy Clin Immunol 2006 Jul;118(1):209-13. 48 Nickel R, Kulig M, Forster J, Bergmann R, Bauer CP, Lau S, et al. Sensitization to hen s egg at the age of twelve months is predictive for allergic sensitization to common indoor and outdoor allergens at the age of three years. J Allergy Clin Immunol 1997 May;99(5):613-7. 49 Marenholz I, Nickel R, Ruschendorf F, Schulz F, Esparza-Gordillo J, Kerscher T, et al. Filaggrin lossof-function mutations predispose to phenotypes involved in the atopic march. J Allergy Clin Immunol 2006 Oct;118(4):866-71. 50 Kalliomaki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E. Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial. Lancet 2001 Apr 7;357(9262):1076-9. 51 Simpson A, John SL, Jury F, Niven R, Woodcock A, Ollier WE, et al. Endotoxin exposure, CD14, and allergic disease: an interaction between genes and the environment. Am J Respir Crit Care Med 2006 Aug 15;174(4):386-92. General discussion 169 52 Szajewska H, Ruszczynski M, Radzikowski A. Probiotics in the prevention of antibioticassociated diarrhea in children: a meta-analysis of randomized controlled trials. J Pediatr 2006 Sep;149(3):367-72. 53 McFarland LV. Meta-analysis of probiotics for the prevention of antibiotic associated diarrhea and the treatment of Clostridium difficile disease. Am J Gastroenterol 2006 Apr;101(4):812-22. 54 Mimura T, Rizzello F, Helwig U, Poggioli G, Schreiber S, Talbot IC, et al. Once daily high dose probiotic therapy (VSL#3) for maintaining remission in recurrent or refractory pouchitis. Gut 2004 Jan;53(1):108-14. 55 Besselink MG, van Santvoort HC, Buskens E, Boermeester MA, van GH, Timmerman HM, et al. Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial. Lancet 2008 Feb 23;371(9613):651-9.

Summary en Nederlandse samenvatting 8

Chapter 8 172

Summary Atopic diseases such as (atopic) eczema, food allergy, asthma, and allergic rhinitis are common diseases. The cumulative incidence during childhood is estimated to be between 20 to 30%. Atopic diseases are immune-mediated diseases that are determined by interplay between genetic and environmental factors. Immunologically, atopic diseases are characterized by a predominance of T-helper type 2 (Th2) immune responses. This imbalance of the immune system is probably caused by an insufficient function of regulatory T cells (Tregs), which play a pivotal role in maintaining homeostasis of the immune system. In countries with a so called Western lifestyle an increase in the prevalence of atopic diseases has been observed during the last decades. This increase may be due to a reduced exposure to microbial antigens, which is supposed to be a prerequisite for a normal development of the immune system (the hygiene hypothesis). The development of atopic diseases may thus be the consequence of insufficient microbial stimulation of the immune system and insufficient development or activation of Tregs. The gastrointestinal tract is the largest surface in direct contact with the outer microbial environment and the intestines harbor the intestinal microbiota with approximately 10 14 Summary microbes, consisting of around 800 species and 7000 subspecies. At birth, the gastrointestinal tract is almost sterile but is rapidly colonized by microbes. The role of the intestinal microbiota 173 in the development of atopic diseases is illustrated by differences found in the intestinal microbiota between allergic and non-allergic children. Enhanced presence of typical probiotic bacteria in the intestinal flora correlated with reduced prevalence of atopic diseases. These differences are already present before the development of atopic diseases, which supports a causal relationship. Lactic acid bacteria such as Lactobacillus and Lactococcus spp., Bifidobacterium spp., Enterococcus spp., and Streptococcus spp. are natural inhabitants of the intestinal tract and are used for centuries as probiotic bacteria in nutritional products because of their suggested health promoting effects. Previously in a double-blind randomized placebo-controlled study Marko Kalliomäki showed that administration of Lactobacillus GG to pregnant women during the last

6 weeks of pregnancy and subsequently to their high-risk babies (i.e. with a positive family history of atopic diseases) during the first 6 months of life reduced the incidence of atopic eczema at the age of two years with approximately 50%. There was no preventive effect on the development of sensitization or symptoms of asthma or allergic rhinitis. Furthermore, this study did not provide new insights on the mechanisms of action of probiotic bacteria, which are still largely unknown. With the PandA (Probiotics and Allergy) study, we aimed to further investigate the possibilities of primary prevention of atopic diseases by perinatal administration of probiotic bacteria and their mechanisms of action. We aimed to answer the following questions: Primary study question : What is the effect of perinatal administration of probiotic bacteria on the incidence atopic diseases (i.e. atopic eczema, food allergy, asthma and allergic rhinitis) in high-risk children? Secondary study questions : 1. Does early administration of probiotics result in a (temporary or permanent) Chapter 8 different composition of the intestinal microbiota, which is compatible with beneficial clinical effects? 2. Is the immune system of the child modulated by early administration of probiotics? 174 - Does supplementation of probiotic bacteria prevent or reduce sensitization? - Are type-2 T helper cell responses suppressed and/or are regulatory T cells induced? It is clear that probiotics have specific targets of action, of which the immune system is one. Worth mentioning is that different probiotics (even within a species) may differ with respect to their effect. Therefore, it seems logical to hypothesize that not all probiotics will be beneficial in all diseases. In preparation of our clinical trial, we therefore aimed to select probiotic bacteria for primary prevention of atopic diseases in children by a rational approach. To our opinion, insight into the pathogenesis of atopic diseases is the key to success. The desired immunomodulatory effect of probiotic bacteria would therefore be reduction of Th2 function, and stimulation of Th1 function, tolerance and regulatory immune responses.

In vitro selection of probiotic bacteria We sought to select probiotic bacteria by their ability to modulate in vitro production of cytokines by peripheral blood mononuclear cells, to make a rational choice from available strains. This in vitro study is described in chapter 2. To this end thirteen strains out of a set of 75 lactic acid bacteria were selected based on their resistance to acid, digestive enzymes and bile to allow their survival in the first part of gastrointestinal tract, their adherence to colonic cell lines like Caco-2, antimicrobial activity (against microbes associated with atopy like Staphylococcus aureus and clostridia), and general characteristics such as reproducible growth and shelf-life. These thirteen strains consisted of five bifidobacteria strains, seven lactobacilli strains and one lactococcus strain (Lc. lactis). Peripheral blood mononuclear cells from healthy donors were cocultured with these thirteen different strains of lactic acid bacteria and T helper type 1 (Th1), T helper type 2 (Th2) and regulatory cell cytokines were measured in culture supernatants by multiplex assay. We showed that specific strains induced the production of large amounts of IL-10, which coincided with low levels of IL-12p70. Furthermore, lactic acid bacteria decreased the production of Th2 cytokines IL-5 and IL-13, which was IL-10 dependent for specific strains since neutralizing IL-10 production resulted in partial to full restoration of Th2 cytokine production and concurred with an increase in pro-inflammatory cytokines such as IL-12p70 and TNF-a. These Summary results indicated that specific strains of lactobacilli and bifidobacteria modulate the in vitro production of cytokines, and may divert the immune system in a regulatory or tolerant mode. 175 These specific strains may be favorable to use in prevention or treatment of atopic disease. Upon administration, probiotic bacteria may interact with the intestinal mucosal immune system, although exact mechanisms and active components are not fully understood. In the intestinal immune system, antigen-presenting cells such as dendritic cells (DCs) play a pivotal role in polarizing naive T cells into Th1, Th2 or regulatory T cells. In addition, we aimed to investigate the effect of lactic acid bacteria on neonatal immune cells. To this end, we investigated the effects of four selected strains, Bifidobacterium (B) bifidum, B. lactis (previously classified as B. infantis), Lactobacillus (Lb) salivarius, and Lactococcus (Lc) lactis (out of the 13 thirteen tested in chapter 2) on cord blood monocyte-derived DCs and their impact on naive T cells. In chapter

3, we showed that B. bifidum had the most consistent effect in modulation of the immune response of neonatal cells. B. bifidum was most potent to polarize DC to drive Th1 cell responses involving increased IFN-g producing T cells concomitant with reduction of IL-4 producing T cells. Lc. lactis was much less effective in this respect. Furthermore, T cells stimulated by B. bifidum matured DC produced significantly more IL-10 compared to control DC. The phenotype or the cytokine production of DC was not affected directly by any of the tested probiotic bacteria. In addition, we demonstrated that B. bifidum, B. infantis and Lb. salivarius were capable of binding to TLR-2. These results indicate that selected species of the Bifidobacterium genus prime in vitro cultured neonatal DC to polarize T cell responses and may therefore be candidates to use in primary prevention of allergic diseases. The question exactly how probiotics influence DC function (i.e. which parts of probiotic bacteria and which receptors on DC are involved) remains to be answered. As probiotic strains clearly differ in their capacity to modulate in vitro immune responses, a careful selection of strains for clinical use is necessary. Based on the in vitro studies described in Chapter 8 chapter 2 and 3 we selected B. bifidum, B. lactis (previously classified as B. infantis), and Lc. Lactis. As our main selection criteria, we applied IL-10 inducing capacity as well as efficient inhibition of IL-5 and IL-13. This multi-species probiotics was used in our clinical trial on primary prevention 176 of atopic diseases by probiotic bacteria, the PandA study. Primary prevention of atopic diseases by selected probiotic bacteria: the PandA study In a double-blind, randomized, placebo-controlled trial, the mixture of probiotic bacteria (3 x 10 9 CFU of B. bifidum, B. lactis, and Lc. Lactis) was administered prenatally to mothers of high-risk children (i.e. positive family history of atopic diseases) and to their offspring during the first 12 months of life. Primary outcome variables and the early effects of probiotic supplementation on the intestinal microbiota and developing immune system are described in chapter 4. Parentalreported eczema during the first three months of life was significantly lower in the intervention group compared to placebo. After 3 months, the incidence of eczema was similar in both groups. Cumulative incidence of parental-reported eczema at 1 and 2 year was 23/50 (intervention) vs. 31/48 (placebo) and 27 (intervention) vs. 34 (placebo) respectively. The number needed to

treat was 5.9 at age 3 and 12 months and 6.7 at age 2 years. Children in the probiotics group tended to be more frequently sensitized to food allergens especially hen s egg, compared to the placebo group but this did not result in more food allergic participants in the probiotics group. The intervention group was significantly more frequently colonized with higher numbers of Lc. lactis. Furthermore, at age 3 months in vitro production of IL-5 was decreased in the probioticgroup compared to the placebo-group. For IL-13 we observed a similar trend. We concluded that this particular combination of probiotic bacteria showed a preventive effect on the incidence of eczema in high-risk children, which seems to be sustained during the first two years of life. In addition to previous studies, the preventive effect appeared to be established within the first three months of life. The influence of probiotic supplementation on the infantile intestinal microbiota Modification of the intestinal microbiota by administration of probiotic bacteria may be a potential part of the mechanism of action of probiotic bacteria. We aimed to evaluate the composition of the intestinal microbiota in the first year of life during probiotic supplementation, and its association with the development of atopic diseases. We described our findings in chapter 5. During the course of the PandA study, participants collected stool Summary samples during the first 4 weeks of life, at 3 months, and at 12 months of age before and after cessation of probiotic supplementation. High-throughput cultivation-independent 16S 177 ribosomal RNA gene-targeted approaches (terminal restriction fragment length polymorphism analysis), combined with multivariate statistical analyses, were used to assess the dynamics of fecal microbiota composition from birth onwards and during the first year of life. Altogether, about 500 fecal samples were analyzed. Probiotic supplementation resulted in a significantly different composition of the fecal microbiota. Especially during the first three months of life fecal samples of probiotic fed infants could be distinguished by the abundant presence of Lactococcus lactis, Streptococcus salivarius and members of the genera Lactobacillus, Clostridium and Staphylococcus. Successful intervention of probiotic bacteria, i.e. the absence of eczema was associated with increased richness and diversity of the intestinal microbiota as well as predominance of Enterococcus, Lactobacillus and Streptococcus species. In conclusion, probiotic

supplementation resulted in a significantly different composition of the intestinal microbiota compared to placebo. In addition, probiotic bacteria may only be capable to exert their clinical beneficial effects if they induced a richer and more diverse microbiota, and stimulated or inhibited the presence of specific bacterial species. The effect of probiotic bacteria on the developing intestinal microbiota seemed to be a host specific process, which was not associated with compliance. Modulation of the developing immune system by probiotic bacteria Modulation of the immune system is a potential mechanism of action by probiotic bacteria, but the effects of probiotic supplementation on the developing immune system of infants are still largely unknown. We studied the effects of probiotic supplementation on the development of lymphocyte populations and ex vivo cytokine responses, which is described in chapter 6. In the PandA study, blood samples were collected at age 3, 12 and 24 months. At these moments, lymphocyte phenotyping was performed and cytokine profiles were measured in ex vivo (anti Chapter 8 CD2/CD28 monoclonal antibodies or phytohemagglutinin) stimulated whole blood cultures and in plasma. Patterns of Th2 cytokine production during the first two years of life differed between placebo and intervention group. At 3 months, IL-5 and IL-13 were reduced in the intervention 178 group compared to the placebo group, which coincided with reduced incidence of eczema in the intervention group. Differences in cytokine production were no longer apparent at one year of age. Th2 cytokine production was higher in the intervention group at 24 months, but this could not be associated with the development of atopic diseases. Probiotic supplementation neither positively nor negatively affected the postnatal maturation of Th1 function, as well as the production of IL-10 as potential marker for the development of regulatory T cells. Probiotic supplementation did not affect the development of lymphocyte populations and subsets. These results indicated that the beneficial effect of probiotics cannot be directly related to systemic cytokine profiles or ex vivo induced cytokines. The clinical implications of increased presence and production of Th2 cytokines at two years of age are currently unclear. Follow-up at the age of 6 years will show the long-term clinical consequences of the administration of probiotic bacteria.

Conclusions and discussion Reflecting on the research described in this thesis within the context of the current related literature, our main findings are that probiotic bacteria may induce beneficial clinical effects by modulating the early composition of the intestinal microbiota such as enhancing richness and diversity. Clearly, probiotic bacteria do not prevent the development of atopic diseases in every supplemented individual but probably only in those with a genetic susceptibility, suggesting complex gene by environment interactions. In the PandA study, the preventive effect of selected probiotic bacteria on eczema seems to be established early in life (within the first three months) and may in part be determined by TLR-2 genotype. Furthermore, probiotic bacteria seem to modulate the developing immune system, but beneficial effect of probiotics cannot be directly related to systemic cytokine profiles or ex vivo induced cytokines. Complex interactions between the host s intestinal immune system and intestinal microbiota are poorly understood, but for instance, intestinal epithelial cells will condition the response of dendritic cells and subsequent T cell responses upon interaction with probiotic bacteria. Future research should focus on the long-term effects of perinatal administration of probiotic bacteria regarding the development of sensitization, asthma and allergic rhinitis. Furthermore, the effects of prenatal administration of probiotic bacteria to pregnant women on their intestinal Summary microbiota and immune system and subsequently on their offspring should be more thoroughly addressed. Mechanistic studies are needed to unravel which component(s) of probiotic bacteria 179 are crucial in determining their effects, what is the gastrointestinal niche of probiotic bacteria and how do they interact with the commensal microbiota and the gastrointestinal immune system. This should include well defined animal models but preferably human studies including intestinal biopsies (to study the intestinal microbiota and the first lining of the intestinal immune system) as well.

Chapter 8 180

Nederlandse samenvatting Allergie is een overmatige reactie van het immuunsysteem op een overigens onschadelijke en ongevaarlijke prikkel (allergenen), zich uitend in ziekte. Allergische aandoeningen zijn vaak voorkomende ziekten, de cumulatieve incidentie op de kinderleeftijd is 20-30%. Bij kinderen onder de 3 jaar zijn de meest voorkomende allergische aandoeningen eczeem en voedselallergie terwijl tussen de 4 tot 12 jaar vooral astma op de voorgrond staat. Allergische rhinitis (hooikoorts) is de belangrijkste allergische manifestatie gedurende de adolescentie. Allergische aandoeningen worden veroorzaakt door genetische én omgevingsfactoren. Preventie van allergische aandoeningen is tot nu toe niet mogelijk. De behandeling bestaat uit het gebruik van medicijnen zoals antihistaminica en corticosteroïden, en het vermijden van allergenen en aspecifieke prikkels zoals passief roken. In landen met een westerse levensstijl is het voorkomen van allergische aandoeningen gedurende de laatste decennia schrikbarend toegenomen. De oorzaak voor deze toename is onbekend. Een mogelijke reden is verminderde blootstelling aan microbiële antigenen (van bijvoorbeeld virussen, bacteriën, parasieten) op jonge leeftijd, waarvan wordt verondersteld dat het nodig is voor een gebalanceerde ontwikkeling van het aangeboren en het verworven afweersysteem (de hygiëne hypothese). Nederlandse samenvatting Het afweersysteem, ook wel immuunsysteem genoemd, wordt onder andere gereguleerd door kleine eiwitten, cytokinen genaamd. Cytokinen worden geproduceerd door witte bloedcellen 181 waaronder T lymfocyten. Er bestaan een aantal belangrijke categorieën van cytokinen, met verschillende functies en welke worden geproduceerd door verschillende subsets van T helper (Th) lymfocyten. De Th1 (type 1) lymfocyten produceren vooral de cytokinen interleukine 2 (IL- 2) en g-interferon. Th2 (type 2) lymfocyten produceren IL-4, IL-5 en IL-13. Deze laatst genoemde cytokinen zijn vooral betrokken bij allergische reacties waaronder de productie van IgE antistoffen. Onder normale fysiologische condities is er een evenwichtige verdeling tussen Th1 en Th2 reacties, en dit evenwicht wordt in stand gehouden door de zogenaamde regulatoire T lymfocyten en andere cytokinen (IL-10). Allergische aandoeningen zijn immunologisch gemedieerde ziekten die worden gekarakteriseerd door een dominante Th2 cytokinen respons. Kinderen die allergische aandoeningen ontwikkelen in hun eerste levensjaar vertonen al dit

dominante Th2 cytokinen profiel rond de leeftijd van 6 maanden. Daarnaast hebben kinderen met een hoog risico op het ontwikkelen van allergische aandoeningen een verminderde capaciteit en een vertraagde ontwikkeling van hun Th1 (productie van g-interferon) functie. Deze disbalans wordt mogelijk veroorzaakt door een tekortschietende werking van de regulatoire T cellen, die belangrijk zijn voor het in stand houden of brengen van homeostase in het cellulaire immuunsysteem en het cytokine IL-10 speelt daarbij een rol. Tijdens de intra-uteriene ontwikkeling helt de immunologische balans van het nog ongeboren kind sterk over naar Th2, gedacht wordt om te voorkomen dat het kind door het immuunsysteem van moeder als lichaamsvreemd wordt afgestoten. Vanaf de geboorte wordt het immuunsysteem door prikkels van buiten waaronder expositie aan microben en microbiële antigenen zodanig gestimuleerd dat de Th1 cellen zich kunnen ontwikkelen en het systeem in balans komt door ontwikkeling en activatie van regulatoire T cellen. Naast genetische predispositie zou het krijgen van een allergische aandoening dus het gevolg kunnen zijn van onvoldoende blootstelling aan microbiële antigenen en dientengevolge onvoldoende ontwikkeling en activatie van regulatoire T cellen. Chapter 8 De darm en de bacteriën in de darm spelen een belangrijke rol in blootstelling aan microbiële antigenen. Het spijsverteringskanaal is het grootste lichaamsoppervlak dat in direct contact staat met de microbiële buitenwereld en huisvest ongeveer 10 14 bacteriën, bestaande uit 182 ongeveer 800 soorten en zo n 7000 stammen. Voor de geboorte is de darm steriel maar raakt direct na de geboorte gekoloniseerd met bacteriën. De rol van de bacteriën in de darm, de darmmicrobiota, in de ontwikkeling van allergische aandoeningen wordt geïllustreerd met de verschillen in darmflora tussen allergische en niet allergische kinderen. Niet allergische kinderen hebben op jonge leeftijd meer bifidobacteriën in hun darmflora, in tegenstelling tot allergische kinderen waar vaker Escherichia coli, Staphylococcus aureus en Clostridium species voorkomen. Deze verschillen zijn al aanwezig vóór het ontwikkelen van allergische ziekten, wat een causaal verband suggereert. Modulatie van de darmmicrobiota door bijvoorbeeld toediening van probiotica zou daarom een potentiële manier kunnen zijn om de ontwikkeling van allergische aandoeningen in hoogrisico (i.e. met een positieve familie anamnese voor allergische aandoeningen) te voorkomen. Probiotica zijn melkzuur bacteriën (Lactobacillus en Lactococcus soorten), bifidobacteriën,

enterococcen en streptococcen species die van nature voorkomen in de darm en al sinds eeuwen worden gebruikt in voeding vanwege hun gezondheidsbevorderende effecten. Probiotica worden door de World Health Organisation (WHO) gedefinieerd als Levende micro-organismen die, indien toegediend in voldoende hoeveelheid, een gezondheidsbevorderend effect hebben in de gastheer. Een echt goede, sluitende definitie van probiotica is niet zo gemakkelijk te geven, misschien is een kort en krachtig gezonde bacteriën nog wel het beste. In een dubbelblind gerandomiseerde en placebo-gecontroleerde studie toonde Kalliomaki voor het eerst aan dat toediening van Lactobacillus GG aan zwangere vrouwen gedurende de laatste 6 weken van de zwangerschap en vervolgens aan hun hoog-risico baby s gedurende de eerste 6 maanden van het leven leidde tot een reductie van de incidentie van (atopisch) eczeem op de leeftijd van 2 jaar met ongeveer 50%. Er werd geen verschil gevonden in de incidentie van astma en allergische rhinitis en in de ontwikkeling van sensibilisatie. Het exacte werkingsmechanisme van probiotica is nog steeds onbekend en bovengenoemde studie leverde geen nieuwe aangrijpingspunten op. De mogelijke werkingsmechanismen van probiotica kunnen liggen op 3 niveaus. Ten eerste zouden probiotica de ontwikkeling en samenstelling van de darmmicrobiota kunnen beïnvloeden. Ten tweede zouden probiotica van invloed kunnen zijn op het darmepitheel en daardoor de barrière functie van de darm gunstig kunnen beïnvloeden. Ten derde zouden Nederlandse samenvatting probiotica het mucosale immuunsysteem van de darm kunnen beïnvloeden en indirect het systemische immuunsysteem. Een groot gemis in het onderzoeksgebied van probiotica is de 183 vertaalslag van in vitro effecten van probiotische bacteriën naar de klinische toepasbaarheid en klinische effecten. Het doel van het onderzoek beschreven in dit proefschrift, waaronder de PandA (Probiotics and Allergy) studie, was de mogelijkheden van primaire preventie van allergische aandoeningen door perinatale toediening van probiotica en het onderliggende werkingsmechanisme nader te onderzoeken. Hierbij stelden wij ons de volgende onderzoeksvragen: Hoofdvraagstelling: Wat is het effect van perinatale toediening van probiotica op de incidentie van allergische aandoeningen (zoals eczeem en voedselallergie) in hoog risico kinderen?

Afgeleide vraagstellingen: 1. Leidt perinatale toediening van probiotica tot een (tijdelijk of blijvende) andere samenstelling van de darmmicrobiota, wat geassocieerd kan worden met gunstige klinische effecten? 2. Geeft vroege toediening van probiotica in-vivo modulatie van het immuunsysteem? - Voorkomt of vermindert perinatale toediening van probiotische bacteriën de ontwikkeling van sensibilisatie, in andere woorden atopie? - Worden type 2 T helper cel responsen onderdrukt en/of regulatoire T cellen gestimuleerd? Het is duidelijk dat probiotica verschillende specifieke aangrijpingspunten hebben. Eén daarvan is het immuunsysteem: het mucosale immuunsysteem in de darm en mogelijk ook het systemische immuunsysteem. Niet alle probiotica zijn gelijk en hoeven dus ook niet een gelijksoortig effect te sorteren. Het inzetten van een willekeurige probiotische bacterie tegen Chapter 8 een willekeurige ziekte leidt dan ook zelden tot het gewenste resultaat. Wij hebben een rationele aanpak gekozen om een specifieke combinatie van probiotische bacteriën te ontwikkelen voor primaire preventie van allergische aandoeningen bij kinderen. Voorwaarde voor kans op succes 184 is inzicht in de pathogenese of veronderstelde pathogenese van allergische aandoeningen. Versterking van regulatoire T cel functie en van IL-10 productie zou kunnen bijdragen aan het voorkomen of onderdrukken van allergische ziekten bij hoog-risico kinderen en dit is dan ook mogelijk het gewenste immunomodulerende effect van probiotica ter preventie van allergische aandoeningen. In vitro selectie van probiotische bacteriën Om probiotische bacteriën te selecteren vergeleken wij hun in vitro capaciteit om cytokinen productie van mononucleaire cellen uit perifeer bloed (PBMCs) van gezonde vrijwilligers te stimuleren en te beïnvloeden. Dit onderzoek wordt beschreven in hoofdstuk 2. Uit een verzameling van 75 verschillende melkzuurbacteriën werden 13 bacterie stammen voor deze experimenten geselecteerd. Allereerst zijn er een aantal algemene kenmerken waaraan de

te selecteren probiotica moeten voldoen: veiligheid, stabiliteit, antimicrobiële activiteit, en het bestand zijn tegen passage door de maag en het eerste gedeelte van de dunne darm. Dat laatste is belangrijk omdat er hiervoor een aanzienlijke variatie bestaat tussen stammen en het belangrijk is dat de probiotica levend de darm bereiken. De op basis van bovengenoemde selectie criteria 13 geselecteerde bacterie stammen bestonden uit 5 verschillende bifidobacteria, 7 lactobacillus stammen en 1 lactococcus (Lc. lactis). In een in vitro model werden PBMCs gekweekt met individuele probiotische bacteriën en werd cytokinen productie gemeten. Wij vonden dat specifieke bacteriën in staat waren de productie van het regulatoire cytokine IL-10 te stimuleren en dit ging gepaard met een lage productie van IL-12. Daarnaast vonden we dat Bifidobacterium (B.) bifidum en andere probiotische bacteriën in staat waren om Th2 cytokine productie (zoals IL-5 en IL-13) te onderdrukken. Bij de meeste bacteriën, maar niet allen, berustte dit effect op de versterking van IL-10 productie aangezien door blokkade van IL-10 productie het onderdrukkende effect op Th2 cytokinen teniet werd gedaan. Deze resultaten tonen aan dat specifieke stammen van bifidobacteria en lactobacillen de in vitro productie van cytokinen door cellen van het afweersysteem kunnen moduleren, waarbij ze mogelijk de regulatoire functie van het afweersysteem ondersteunen. Deze specifieke probiotische bacteriën zouden daarom bij voorkeur gebruikt kunnen worden in preventie van allergische aandoeningen. Nederlandse samenvatting Probiotica komen na toediening terecht in de darm, alwaar zij mogelijk een interactie aangaan 185 met het mucosale immuunsysteem van de darm. Hoe dit precies werkt en welke bacteriële antigenen hierbij een rol spelen is onbekend. In de in vivo situatie zijn gespecialiseerde dendritische cellen de zogenaamde professionele antigeen presenterende cellen die contact maken met de micro-organismen in het lumen van de darm, en vervolgens de richting van de immuunrespons bepalen. De dendritische cel bepaald, afhankelijk van het micro-organisme waarmee hij in contact is gekomen, of een naïeve T cel zich ontwikkelt tot een Th1, Th2 of een regulatoire T cel. We hebben daarom in vitro eveneens naar de aansturing van de cellulaire immuunrespons (T lymfocyten) door dendritische cellen gekeken. Wij kozen voor het gebruik van navelstrengbloed omdat we graag de effecten van probiotische bacteriën op het neonatale immuunsysteem wilden onderzoeken. In hoofdstuk 3 bestudeerden wij het effect van 4

geselecteerde probiotische bacteriën, B. bifidum, B. lactis (voorheen geclassificeerd als B. infantis), Lactobacillus (Lb) salivarius, en Lactococcus (Lc) lactis op dendritische cellen gedifferentieerd uit monocyten en hun invloed op naïeve T cellen. Ook hierbij vonden we dat specifieke probiotische bacteriën, in het bijzonder B. bifidum in staat waren dendritische cellen te sturen om de Th2 respons te onderdrukken en de Th1 respons te stimuleren. Mogelijk stimuleert B. bifidum ook regulatoire T cellen omdat de dendritische cellen gekweekt onder invloed van B. bifidum de IL- 10 productie van de T cellen stimuleerden. Hoe precies probiotica de functie van dendritische cellen kunnen beïnvloeden (dus welk onderdeel van de bacterie reageert met welke receptor op de dendritische cellen) is nog grotendeels onbekend. Wij toonden in hoofdstuk 3 wel aan dat toll-like receptor 2 een mogelijke receptor is voor B. bifidum, B. lactis en Lb. Salivarius. Wij konden niet aantonen dat het fenotype en de cytokinen productie van de dendritische cellen beïnvloed werden door de probiotische bacteriën maar er spelen zeer waarschijnlijk andere oppervlakte moleculen en cytokinen/chemokinen een rol. Concluderend laten onze resultaten zien dat specifieke stammen van bifidobacteria in staat zijn in vitro gekweekte dendritische Chapter 8 cellen te moduleren in hun sturing van de T cel respons. Deze specifieke stammen zouden goede kandidaten kunnen zijn voor primaire preventie van allergische aandoeningen. Aangezien probiotische bacteriën duidelijk verschillen in hun capaciteit immuunresponsen te moduleren 186 is een zorgvuldige selectie voor klinische toepassingen noodzakelijk. Op basis van bovenstaande gevonden eigenschappen hebben we een combinatie van 3 probiotica stammen samengesteld (B. bifidum, B. lactis en Lc.lactis) voor een klinische studie naar primaire preventie van allergie en astma in een groep hoog-risico kinderen, de PandA studie (Probiotics AND Allergy). Primaire preventie van allergische aandoeningen door geselecteerde probiotische bacteriën: de Panda studie. In een dubbelblinde, gerandomiseerde, placebo gecontroleerde studie werd de mix van geselecteerde probiotische bacteriën (3 x 10 9 colony forming units (CFU) van B. bifidum, B. lactis en Lc.lactis) dagelijks toegediend aan moeders tijdens de laatste 6 weken van de zwangerschap en aan hun hoog-risico (i.e. met een positieve familie anamnese voor allergische aandoeningen) kind gedurende het eerste levensjaar. De kinderen werden vervolgd tot de leeftijd van 2 jaar

met follow-up bezoeken op de leeftijd van 3, 12 en 24 maanden. In hoofdstuk 4 beschrijven wij de effecten van perinatale toediening van deze probiotica op de ontwikkeling van eczeem en voedselallergie. De ouders in de probiotica groep rapporteerden significant minder eczeem vergeleken met de placebo groep gedurende de eerste 3 levensmaanden. Daarnaast was er significant minder eczeem gediagnosticeerd door een (huis)arts in de probiotica groep op deze leeftijd. Na de leeftijd van 3 maanden was de incidentie van eczeem gelijk in beide groepen. De number needed to treat (het aantal kinderen dat je moet behandelen om bij 1 kind de ontwikkeling van eczeem te voorkomen) was 5,9 op de leeftijd van 3 en 12 maanden en 6,7 op de leeftijd van 2 jaar. De kinderen in de probiotica groep waren wel vaker gesensibiliseerd voor voedselallergenen, voornamelijk kippenei, maar dit leidde niet tot meer kinderen met daadwerkelijk voedselallergie in de probiotica groep. In hoofdstuk 4 toonden we ook aan dat onze interventie met probiotische bacteriën succesvol was: Lc. lactis was significant vaker en significant meer aanwezig in de darmmicrobiota van de kinderen in de probiotica groep vergeleken met de placebo groep. Immunologisch was er op de leeftijd van 3 maanden minder productie van Th2 cytokinen (IL-5 en IL-13) in de probiotica groep. Tot nu toe zijn zeven andere klinische studies gepubliceerd betreffende primaire preventie van allergische aandoeningen door toediening van probiotica met zeer wisselende resultaten. Drie studies lieten een Nederlandse samenvatting preventief effect zien op de ontwikkeling van eczeem met een relatieve risico reductie van 26-50%. In een vierde studie werd alleen een preventief effect geconstateerd wat betreft atopisch 187 eczeem (eczeem plus sensibilisatie). Een belangrijke reden voor de wisselende resultaten is het gebruik van verschillende bacterie stammen. Tevens is het opmerkelijk dat alléén studies waarin de probiotica prentaal zijn toegediend aan de moeder een preventief effect laten zien op de ontwikkeling van eczeem. De Panda studie verschilt van de andere studies door de in vitro selectie van de toegepaste melkzuurbacteriën. Wij concludeerden dat perinatale toediening van deze specifieke combinatie van geselecteerde probiotische bacteriën de ontwikkeling van eczeem op jonge leeftijd voorkomt en dat dit effect mogelijk gedurende de eerste 2 levensjaren. Het grootste effect wordt duidelijk gevonden in de eerste 3 levensmaanden en lijkt gepaard te gaan met veranderingen van de darmmicrobiota en modulatie van het immuunsysteem.

De invloed van perinatale toediening van probiotica op de ontwikkeling en samenstelling van de darmmicrobiota Het werkingsmechanisme van probiotica is grotendeels onbekend, maar toediening van probiotica zou mogelijk de ontwikkeling en samenstelling van de darmmicrobiota kunnen beïnvloeden. Wij bestudeerden de samenstelling van de darmmicrobiota gedurende het eerste levensjaar en onderzochten of de samenstelling van de darmmicrobiota geassocieerd is met de ontwikkeling van eczeem gedurende de eerste 2 levensjaren (hoofdstuk 5). Tijdens de panda studie verzamelden de ouders ontlastingmonsters van hun kind gedurende de eerste 4 weken na de geboorte, op de leeftijd van 3 maanden en rondom het stoppen van de probiotica op de leeftijd van 12 maanden. 500 ontlastingmonsters werden onderzocht met 16S ribosomaal RNA terminale restrictie fragment lengte polymorfismen (elke bacterie kan geïdentificeerd worden op basis van een specifiek stukje DNA). Perinatale toediening van probiotica leidde tot een significant andere samenstelling van de darmmicrobiota vergeleken met de placebo groep. Vooral gedurende de eerste 3 levensmaanden onderscheidde de probiotica groep zich van de Chapter 8 placebo groep door toegenomen aanwezigheid van Lactococcus lactis, Streptococcus salivarius en verschillende soorten Lactobacillen, Clostridia en Staphylococcen. Succesvolle interventie met probiotica, i.e. uitblijven van eczeem, was geassocieerd met grotere aantallen bacteriën 188 en grotere diversiteit in de samenstelling van de microbiota, en Enterococcen, Lactobacillen en Streptococcen als dominante species. Concluderend leidde perinatale toediening van probiotica tot een significant andere samenstelling van de darmmicrobiota. Bovendien leken gunstige klinische effecten van probiotica geassocieerd te zijn met een meer diverse darmmicrobiota en de aan- (of af-)wezigheid van specifieke bacterie stammen. Indien deze effecten in de gastheer uitbleven ontwikkelde de kinderen wel eczeem ondanks probiotica. Het uitblijven van een effect van probiotica op de darmmicrobiota hing niet samen met slechte therapietrouw, maar zou te maken kunnen met de gastheer zelf (bijvoorbeeld zijn genotype en zijn afweersysteem).

De invloed van perinatale toediening van probiotica op de ontwikkeling van het afweersysteem De effecten van probiotica op het intestinale en systemische immuunsysteem zijn grotendeels onbekend. In gezonde vrijwilligers (volwassenen) werd recentelijk aangetoond dat toediening van Lb. plantarum in het duodenum expressie van genen induceert die betrokken zijn bij cellulaire processen geassocieerd met de ontwikkeling van tolerantie. Het invasieve karakter van dergelijk onderzoek maakt het medisch ethisch onmogelijk dit bij kinderen uit te voeren. Wij onderzochten (daarom) de effecten van probiotica op het systemische immuunsysteem. Bloedmonsters werden afgenomen op de leeftijd van 3, 12 en 24 maanden. Zowel het fenotype als de cytokinenproductie van lymfocyten werden onderzocht. De capaciteit om Th2 cytokinen te produceren bleek zich significant anders te ontwikkelen gedurende de eerste twee levensjaren in de probiotica groep vergeleken met de placebo groep. Zoals eerder vermeld was de productie van de Th2 cytokinen IL-5 en IL-13 op de leeftijd van 3 maanden lager in de probiotica groep. Op de leeftijd van 1 jaar vonden wij geen verschillen. Echter op de leeftijd van 2 jaar produceerden de lymfocyten significant meer Th2 cytokinen (IL-4, IL-5 en IL-13) in de probiotica groep. De betekenis hiervan is onduidelijk, er komen niet meer allergische aandoeningen voor in de probiotica groep. Toediening van probiotica had geen effect op de ontwikkeling van Th1 cellen (g-interferon Nederlandse samenvatting productie) of regulatoire T cellen (IL-10 productie). Toediening van probiotica had geen effect op het fenotype en het voorkomen van de verschillende soorten lymfocyten. Concluderend 189 kunnen de klinische effecten van probiotica in de Panda studie niet direct geassocieerd worden met blijvende veranderingen in patronen van cytokinen productie. Het is onduidelijk of wat de klinische betekenis is van de toegenomen productie van Th2 cytokinen in de probiotica groep. Klinische follow-up rond de leeftijd van 6 jaar zal meer inzicht geven in de lange termijn effecten, waaronder de ontwikkeling van astma, van perinatale toediening van probiotica.

Conclusies en discussie De Panda studie is de eerste studie waarin probiotische bacteriën werden geselecteerd op basis van in vitro productie van cytokinen voor toepassing in een interventie studie naar de mogelijkheden van primaire preventie door perinatale toediening van probiotica. De Panda studie is hiermee een elegant voorbeeld van translationeel onderzoek, dat de vertaalslag probeert te maken tussen in vitro effecten van probiotica en hun daadwerkelijk klinische toepasbaarheid en effecten. Bepaalde immunomodulatoire effecten van de geselecteerde probiotische bacteriën die in vitro werden waargenomen, zoals de verminderde productie van Th2 cytokinen IL-5 en IL-13 werden vervolgens ook waargenomen in ex vivo volbloedkweken van deelnemers in de interventie groep. Daarnaast werd in de Panda studie aangetoond dat gunstige klinische effecten (i.e. het voorkomen van eczeem) van geselecteerde probiotische bacteriën geassocieerd waren met modulatie van de intestinale microbiota, zoals toegenomen variatie en diversiteit, in de eerste 3 levensmaanden. De periode waarin het mogelijk lijkt de samenstelling van de intestinale microbiota te beïnvloeden is wel beperkt tot de eerste 3 Chapter 8 levensmaanden (the window of opportunity). Dit is een belangrijk gegeven om de potentiële werkzaamheid van probiotische bacteriën te bepalen en biedt de mogelijkheid in toekomstige studies bij het uitblijven van modulatie van de intestinale microbiota binnen the window of 190 opportunity over te stappen op andere probiotische bacteriën. Dit onderzoek levert een belangrijke bijdrage aan de kennis van de mogelijkheden van probiotica in de primaire preventie van allergische ziekten bij hoog-risico kinderen en de mogelijke werkingsmechanismen van probiotica op de ontwikkeling van de darmmicrobiota en de ontwikkeling van het afweersysteem. Onze bevinding dat probiotica alleen in staat zijn gunstige klinische effecten te induceren als zij de diversiteit en samenstelling van de darmmicrobiota veranderen ligt conceptueel gezien misschien voor de hand maar is echter nooit dusdanig beschreven in de literatuur en is daarom één van de belangrijkste resultaten. Bovendien is het duidelijk dat probiotica niet in elk individu de ontwikkeling van allergische ziekten kunnen voorkomen. Het effect van probiotica is mogelijk (deels) afhankelijk is van het genotype van het individu, en wordt dus bepaald door complexe interacties tussen genotype en omgevingsfactoren. In de Panda studie is het preventieve effect van probiotica op de ontwikkeling

van eczeem het meest uitgesproken in de eerste drie levensmaanden en mogelijk deels bepaald door toll-like receptor 2 genotype. Daarnaast kunnen probiotica de ontwikkeling van het afweersysteem beïnvloeden, echter gunstige klinische effecten van probiotica bijvoorbeeld het voorkomen van eczeem kunnen niet duidelijk gerelateerd worden aan systemische cytokinen profielen dan wel ex vivo geïnduceerde cytokinen productie. Complexe interacties tussen de intestinale microbiota waaronder probiotica en het intestinale en systemische afweersysteem van de gastheer liggen ten grondslag aan het werkingsmechanisme van probiotica maar zijn tot op heden nog grotendeels onbekend. Zo gaan intestinale epitheelcellen een interactie aan met intestinale bacteriën, zowel commensalen als pathogenen. Intestinale epitheelcellen spelen zo een zeer belangrijke rol in de effecten van intestinale bacteriën en dus ook probiotische bacteriën op het intestinale immuunsysteem. In toekomstig onderzoek moet de nadruk liggen op de lange termijn effecten van perinatale toediening van probiotica waaronder de ontwikkeling van sensibilisatie voor inhalatie allergenen, astma en allergische rhinitis. Bovendien dienen de effecten van prenatale toediening van probiotica aan de zwangere vrouw op haar intestinale microbiota en afweersysteem (en vervolgens hun pasgeborenen) grondig onderzocht te worden, aangezien alléén in de studies waarin de ontwikkeling van eczeem werd voorkomen door toediening van probiotica de Nederlandse samenvatting zwangere moeder ook werd behandeld. Diermodellen lenen zich goed voor mechanistische studies naar probiotica (welke receptoren zijn betrokken, welke antigenen zijn van belang) 191 echter humane studies waarbij darmbiopten worden genomen maken het mogelijk zowel de effecten van toediening van probiotica op de intestinale microbiota evenals op onderdelen van het intestinale immuunsysteem in de mens te onderzoeken. Het beschreven onderzoek is van belang voor de kindergeneeskunde. Allergische aandoeningen zijn zeer prevalent op de kinderleeftijd en de ontwikkeling ervan is tot op heden niet te voorkomen. Perinatale toediening van probiotica is een veelbelovende mogelijkheid, in ieder geval ter voorkoming van eczeem. Echter door de wisselende resultaten van primaire preventie studies met probiotica is het nog te vroeg om perinatale toediening van probiotica op grote schaal toe te gaan passen in hoog-risico kinderen. Bovendien dient door de grote verschillen in effecten van individuele en combinaties van probiotische bacteriën klinische effectiviteit

gedegen te worden vastgesteld. Voorzichtigheid blijft echter ook geboden. Toegenomen sensibilisatie door toediening van probiotica zou ongunstig kunnen zijn aangezien sensibilisatie op jonge leeftijd een risicofactor is voor astma. Gunstige of negatieve effecten op de ontwikkeling van astma en allergische rhinitis zijn tot op heden niet beschreven maar dienen ook nog grondig onderzocht te worden. Daarnaast worden probiotica al in meerdere zuigelingenvoedingen en andere commercieel verkrijgbare producten toegepast. Gezondheidsbevorderende effecten toegeschreven aan deze producten overtreffen de huidige kennis over de effecten van probiotica. Inzicht in de mogelijkheden en onmogelijkheden van probiotica is noodzakelijk voor een adequate voorlichting voor ouders en dit onderzoek biedt hier een evidente bijdrage aan. Chapter 8 192

Nederlandse samenvatting 193

Dankwoord Curriculum vitae List of publications List of abbreviations

Dankwoord 196

DANKWOORD Promoveren is meer dan alleen onderzoek (leren) doen. Het is ook heel veel relaties en interacties aangaan met verschillende personen. De relaties en interacties in de darm tussen de bacteriën en de gastheer (wij dus), maar ook de bacteriën onderling zijn een voorbeeld van symbiose en kun je onderverdelen in mutualisme, commensalisme en pathogeniciteit. Symbiose in enge betekenis kan je omschrijven als een samenleving van twee levensvormen die gunstig of noodzakelijk is voor beide levensvormen (ook wel mutualisme genoemd). Commensalisme is een interactie waarbij de één voordeel heeft zonder de ander te beïnvloeden. Bij pathogeniciteit wordt schade berokkend. Dit soort interacties/relaties vind je niet alleen in de darm, maar overal binnen de flora en fauna. Een promotietraject is hiervan doorspekt. Bij deze dank aan alle facultatief symbionten en commensalen en een aantal in het bijzonder. Professor dr. J.L.L. Kimpen, beste Jan. Hartelijk dank dat je mijn opleider en promotor bent (geweest). Jouw arbeidsmoraal die ogenschijnlijk nooit verslapt is voor mij aanstekelijk. Je bent een sterke motivator. De manier waarop we, zeker het laatste anderhalf jaar (dat hadden we eerder moeten ontdekken) gewerkt hebben werkte katalyserend en was bovenal gezellig. Dankwoord Dr. M.O. Hoekstra, beste Maarten. Dat ik me in de afgelopen jaren niet verloren heb in de enorme 197 hoeveelheid aan data die het onderzoek opleverde is jouw verdienste. Jij bracht het onderzoek indien nodig terug naar de kern. Dank voor de ruimte om mijn (indien nodig kritische) mening te ventileren. Dr. G.T. Rijkers, beste Ger. Er is niemand die ik ken met zo n groot oplossend vermogen, op welke vraag dan wel probleem mijnerzijds had jij altijd een toepasbaar antwoord dan wel creatieve oplossing voorhanden. Ik waardeer je snelheid van begrip en de snelheid waarmee wij vrijwel altijd op één lijn zaten.

Harro, jouw betrokkenheid bij mijn promotie onderzoek was zeer waardevol. Jouw inzet leidde tot de vruchtbare samenwerking met Wageningen en ik waardeer je positief kritische houding. Nathalie, honderden bloedmonsters zijn door jouw verwerkt en de data die dat opleverde maken jou onmisbaar in mijn promotie traject. Voor de werkzaamheid in het lab is het prettig als je tot een groep behoort en daarom is het mooi dat jij je plek binnen de RSV-groep gevonden hebt. Grada van Bleek, dank voor je scherpe analyses en kritische mening. Patricia de Graaff, alles wat jij wist over (het kweken van) dendritische cellen heb jij mij bijgebracht. Je hulp was uitermate waardevol en ik waardeer het zeer dat je naast je eigen werkzaamheden tijd vrij maakte om mij, die nog nooit een pipet had vast gehouden, op weg te helpen bij mijn eigen experimenten. Dankwoord Martien Kapsenberg en Hermelijn Smits, dank voor jullie interesse, leuke discussies en waardevolle suggesties. 198 Rocio and Hauke, your help with the intestinal microbiota was indispensable. I am grateful for our collaboration although it was regularly stalled due to maternity leaves but nevertheless very fruitful. Joost Frenkel, dank voor jouw bereidheid creatieve oplossingen te zoeken binnen het opleidingsschema. Het kost je regelmatig hoofdbrekers maar je bood mij de mogelijkheid tot een voorspoedige en prettige versnelling van mijn onderzoek. Laboratorium pediatrische immunologie, ik was één van de hotelgasten van het lab. Zeker anderhalf jaar vrijwel dagelijks aanwezig en daarna nog regelmatig de deur plat lopend. Het ritme van werken op een lab is in mijn ervaring vele malen anders dan in de kliniek. Ik heb echter nog nooit zo vaak mee gedaan aan voetbalpooltjes, tour de france pooltjes etcetera. Ik ben nog steeds dankbaar voor de originaliteitprijs van de koekjes-bak-wedstrijd voor mijn

moederkoekjes ten tijde van de experimenten met navelstrengbloed. In het bijzonder Koos hartelijk dank voor je interesse en hulp indien nodig. En Pirkko voor het lekkere Finse gebak en je hartelijkheid. Studenten, Jeanine Splinter, Annemijn Aarts, Saskia Bakker, Sanne Nijhof, Anne van der Gugten, en Florien Sengers, hartelijk dank voor jullie bijdrage aan de Panda studie. Zonder jullie was het combineren van het onderzoek met klinische stages niet mogelijk geweest. Het is mooi om te zien dat jullie allemaal in de loop van de tijd jullie eigen traject gevonden hebben. Pieter Mees en Evelien Burks, met jullie experimenten bewandelden jullie een aantal zijpaden die her en der tot een aantal interessante bevindingen leidden. Panda studie prikclub : hartelijk dank aan de vele collega s die indien nodig tijdens de followup bezoeken beschikbaar waren voor het verrichten van vena puncties bij de deelnemende kinderen. Judith, José en Margreet, jullie hebben het verrichten van huidpriktesten in de vingers en brachten het mij onder de knie. Dankwoord Geen promotietraject zonder kamergenoten en mede onderzoekers: 199 Joost, samen onderzoek doen schept een band die we als vriendschap zeker voort moeten zetten. Dank voor je interesse, humor, frustratie management, en boterhammen met appelstroop en nutella hazelnootpasta. Evelien, met 3 onderzoekers op nog geen 6 m2 maakt meteen duidelijk waarom voor de naam flex(ibiliteit) kamers gekozen is, maar het was zeker gezellig. Fijn dat voor jouw onderzoek en boekje nu ook de afronding nadert. Lianne, we hebben regelmatig de stoel achter ons bureau op de onderzoekskamer voor elkaar warm gehouden. Ik ben blij dat ik je regelmatig heb kunnen voorzien van adeno-specifieke-t-cellen voor het fraaie proefschrift dat ook jij binnenkort zeker af zult hebben! Berber, afkomstig uit dezelfde onderzoeksstal bood jij bruikbaar extern relativeringsvermogen. Marloes, in de slechts 3 maanden bij jou op de kamer kreeg delegeren een andere dimensie. Succes met VAART en interne geneeskunde. Marije, succes met jou Leidsche Rijn onderzoek.

Paranimfen Marieke en Bram, het voelt erg goed jullie naast me te hebben. Marieke, sinds eind 2001 volgen we elkaar en ik had en zou het niet willen missen. Er wacht zeker een mooie toekomst voor jou! Bram, wat zo n 15 jaar geleden begon met een kermishorloge is inmiddels uitgegroeid tot een mooie vriendschap die we hoop ik nog lange tijd voortzetten. Kees en Joke, hartelijk dank voor jullie zorg en interesse. Jullie enthousiasme voor jullie kleinkinderen is aandoenlijk en jullie bereidheid bij te springen indien nodig én vaker waardeer ik zeer. Pap en mam, jullie zorgen tot op de dag van vandaag voor mijn stevige fundament en een altijd veilige thuishaven. Dat geeft rust waar ik zeer dankbaar voor ben. Mam, deze is voor jou! In andere tijden heb jij je keuze moeten maken. Pap, jouw zorg en verantwoordelijkheidsgevoel voor je patiënten waren grenzeloos en zeer bewonderenswaardig en hebben me onder andere Dankwoord gemaakt tot de arts die ik nu ben. Monique, als grote zus ben je toch een beetje mijn voorland en dichtbij voor kleine vragen, grote vragen en extra relativeringsvermogen. Ik waardeer je sterke eigen wil en hoop dat die je nog 200 ver gaat brengen. Dirk, sinds een aantal jaren letterlijk op grote afstand. Maar desondanks blijf je altijd betrokken en geïnteresseerd. Ik ben trots op je dat jij nu ook papa bent en dat je ogenschijnlijk onverstoorbaar datgene bereikt wat je nastreeft. Jeroen, Stijn en Jasper, jullie zijn het allermooiste wat er is. Stijn, het is geweldig jouw ontdekkingsreis van de wereld en de vele hilarische momenten die dat oplevert met je mee te maken. Jasper, jouw ontdekkingsreis is pas net begonnen maar gaat nu al gepaard met een heldere blik en een stralende glimlach. Jeroen, het leven met jou is mooi, ik ben uitermate blij dat jij er voor mij en ons bent en samen kunnen we elke toekomstige uitdaging aan.

CURRICULUM VITAE Titia Niers werd geboren op 17 maart 1976 te Eindhoven. Zij behaalde haar VWO diploma in 1994 aan het Hertog Jan College te Valkenswaard. In datzelfde jaar begon zij met de studie Geneeskunde aan de Katholieke Universiteit Nijmegen (thans Radboud Universiteit Nijmegen), waar zij in 1998 het doctoraal examen behaalde. In 1998 verbleef zij, voor de start van haar coschappen, enkele maanden in Brazilië in het kader van de klinische stage Rural and Communitary Training Centre of Ana Bezerra Hospital, Federal University of Rio Grande do Norte (UFRN) te Santa Cruz, onder leiding van Dr. Petrônio Souza Spinelli. In 2000 verrichtte zij gedurende een half jaar wetenschappelijk onderzoek naar de mogelijkheden van prenatale diagnostiek van ademhalingsketendefecten aan het Nijmegen Center for Mitochondrial Disorders (NCMD), UMC St. Radboud onder leiding van Prof. Dr. Jan Smeitink en Dr. Bert van den Heuvel. In april 2001 behaalde zij haar artsexamen, waarna zij enkele maanden werkzaam was als arts-assistent Kindergeneeskunde in het Radboud Ziekenhuis te Nijmegen. In oktober 2001 startte zij als assistent geneeskundige in opleiding met de opleiding Kindergeneeskunde in het Wilhelmina Kinderziekenhuis, UMC Utrecht (Opleider Prof. Dr. Jan Kimpen, thans Dr. Joost Frenkel). Het onderzoek dat resulteerde in dit proefschrift startte in Curriculum vitae januari 2003 in combinatie met haar opleiding tot kinderarts onder supervisie van Prof. Dr. Jan Kimpen, Dr. Maarten Hoekstra en Dr. Ger Rijkers. In het kader van haar perifere stage was zijn van 201 januari 2007 tot juli 2008 werkzaam in het Sint Antonius Ziekenhuis te Nieuwegein (Opleider Dr. Hans Schipper). Titia woont samen met Jeroen van der Hilst en samen hebben zij twee zoons, Stijn (2007) en Jasper (2009).

Curriculum vitae 202

LIST OF PUBLICATIONS Laetitia E. M. Niers, Nathalie O. P. van Uden, Jan L. L. Kimpen, Maarten O. Hoekstra, Ger T. Rijkers. Probiotic bacteria and modulation of the developing immune system Manuscript in preparation Laetitia E. M. Niers, Rocio Martin, Ger T. Rijkers, Susanna Fuentes, Henk Panneman, Harro M. Timmerman, Maarten O. Hoekstra, Hauke Smidt. Dynamic changes in intestinal microbiota in infants at risk of eczema during supplementation of probiotics Submitted Richarde M. de Voer, F. R. van der Klis, Laetitia E.M. Niers, Ger T. Rijkers, G. A. Berbers. AbAbsence of Neisseria meningitidis Serogroup C-Specific Antibodies during the first year of life in the Netherlands: An Age-Group at Risk? Clin Vaccine Immunol. 2009, in press Laetitia E. M. Niers, Rocio Martin, Ger T. Rijkers, Florien B. Sengers, Harro T. Timmerman, Nathalie List of publications O.P. van Uden, Hauke Smidt, Jan L.L. Kimpen, Maarten O. Hoekstra. The effects of selected probiotic strains on the development of eczema (The PandA study) 203 Allergy 2009;64:1349-58 Florien B. Sengers, Laetitia E. M. Niers, Ger T. Rijkers, Maarten O. Hoekstra. Pre- en probiotica bij allergie: huidige klinische inzichten. Nederlands Tijdschrift voor Allergie 2008;8(6):201-11 Laetitia E. M. Niers, Maarten O. Hoekstra, Harro M. Timmerman, Nathalie O. P. van Uden, Patricia M. A. de Graaff, Hermelijn H. Smits, Jan L. L. Kimpen, Ger T. Rijkers. Selection of probiotic bacteria for prevention of allergic diseases: immunomodulation of neonatal dendritic cells. Clin Exp Immunol. 2007 Aug;149(2):344-52

Harro M. Timmerman, Laetitia E. M. Niers, B.U. Ridwan, Carin J. M. Koning, Linda Mulder, Louis M. A. Akkermans, Frans M. Rombouts, Ger T. Rijkers. Design of a multispecies probiotic mixture (Ecologic 641) to prevent infectious complications in critically ill patients. Clin Nutr. 2007 Aug;26(4):450-459 Laetitia E. M. Niers, Marianne Stasse-Wolthuis, Frans M. Rombouts, Ger T. Rijkers Nutritional support for the infant s immune system. Nutr Rev. 2007 Aug;65(8 Pt 1):347-60 Laetitia E. M. Niers, Harro M. Timmerman, Ger T. Rijkers, Grada M. van Bleek, Nathalie O. P. van Uden, Edward F. Knol, Martien L. Kapsenberg, Jan L. L. Kimpen, Maarten O. Hoekstra. Identification of strong interleukin-10 inducing lactic acid bacteria which downregulate T helper type 2 cytokines. Clin exp Allergy 2005;35 (11): 1481-9 List of publications Laetitia E.M. Niers, Trinette J. Steenhuis, Maarten O. Hoekstra. The hygiene hypothesis: the role of microbes in the prevention of atopy and atopic disease. In: Respiratory Tract Infections: Pathogenesis and emerging strategies for control. J.L.L. Kimpen and Ramilio T, Horizon Scientific Press Ltd., Norfolk, UK, 2004 204 Maarten O. Hoekstra, Laetitia E. M. Niers Effects of probiotics on atopic dermatitis? Additional studies are still needed! Arch Dis Child. 2006 Mar;91(3):276 Maarten O. Hoekstra, Laetitia E. M. Niers, Trinette J. Steenhuis, Maroeska M. Rovers, Edward F. Knol, Cuno S. Uiterwaal Is randomization of breast-feeding feasible? J Allergy Clin Immunol. 2005 Jun;115(6):1324 Laetitia E. M. Niers, Ger T. Rijkers, Harro M Timmerman, Edward F. Knol, Maarten O. Hoekstra Probiotics for cow s milk allergy: classification after intervention. J Allergy Clin Immunol. 2005 Feb;115(2):423

Laetitia E.M. Niers, Ger T. Rijkers, Edward F. Knol, Yolanda Meijer, Maarten O. Hoekstra. Probiotics and prevention of atopic disease? Lancet 2003;362:496 Laetitia E.M. Niers, Maarten O. Hoekstra. Probiotica: feiten, vragen en toekomstperspectieven. Nederlands Tijdschrift voor Allergie 2003;4:155-159 Antoon J.M. Janssen, Laetitia E.M. Niers, Jan A.M. Smeitink, Rob C.A. Sengers, J.M. Frans Trijbels. Prenatal Diagnosis in oxidative phosphorylation disorders. In: Oxidative phosphorylation in health and disease. J.A.M Smeitink, J.M.F. Trijbels en R.C.A. Sengers, Landes Bioscience and Kluwer Academic/Plenum publishers, 2004 Laetitia E.M. Niers, Lambert P. van den Heuvel, J.M. Frans Trijbels, Rob C.A. Sengers, Jan A.M. Smeitink. Prerequisites and strategies for prenatal diagnosis of respiratory chain deficiency in chorionic villi. Journal of Inherited Metabolic Disease 2003;26:647-658 List of publications Laetitia E.M. Niers, Jan A.M. Smeitink, J.M. Frans Trijbels, Rob C.A. Sengers, Lambert P. van den Heuvel. 205 Prenatal Diagnosis of NADH: ubiquinone oxidoreductase deficiency. Prenatal Diagnosis 2001;21:871-80.

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LIST OF ABBREVIATIONS APC APCs B BCSS BSA CCA CFU CHO CRTH2 DC Allophycocyanin Antigen presenting cells Bifidobacterium basic clinical scoring system bovine serum albumine Canonical correspondence analysis Colony forming units Chinese hamster ovary chemoattractant-homologous receptor expressed on Th2 cells Dendritic cells DGGE EDTA FACS FCS FISH Denaturing gradient gel electrophoresis Ethylenediamine tetraacetic acid Fluorescence activated cell sorter Fetal calf serum Fluorescence in situ hybridization List of abbreviations FITC GI HLA-DR IDC IECs IFN IL L LAB Lb Lc LPS mab Fluorescein isothiocyanate Gastrointestinal Human leucocyte antigen D-related Immature dendritic cells intestinal epithelial cells Interferon Interleukin Ligand Lactic acid bacteria Lactobacillus Lactococcus lipopolysaccharide Monoclonal Antibody 207

MCPC MCPP MF NFkB PBMC PBS PCR PE PERCP PFA PHA PMA Microbial community profiling and characterization Monte Carlo Permutation Procedure Maturation factors Nuclear factor kappa B Peripheral blood mononuclear cell Phosphate-buffered saline Polymerase Chain Reaction Phycoerythrin Peridinin chlorophyll protein paraformaldehyde Phytohemagglutinin Phorbol myristate acetate List of abbreviations PRC qpcr R RDA RM Principle Response Curve quantitative (real time) PCR Recombinant Redundancy Analysis Repeated Measurements analysis 208 rrna SCORAD SEB SEM SPT ribosomal RNA Scoring atopic dermatitis Staphylococcus aureus enterotoxin B Standard error of the mean Skin prick test Th1 T helper cell type 1 Th17 IL-17 producing T helper cell Th2 T helper cell type 2 TNF TLR Treg TRF T-RFLP Tumour necrosis factor Toll like receptor Regulatory T cell Terminal Restriction Fragment Terminal Restriction Fragment Length Polymorphism