The Intestinal Mucosal Immune System: Modulating Intestinal T Cell Subsets During Postnatal Life

Transcription

1 The Intestinal Mucosal Immune System: Modulating Intestinal T Cell Subsets During Postnatal Life Victoria Camerini, M.D. USC Division of Neonatal Medicine Childrens Hospital Los Angeles Keck School of Medicine University of Southern California Los Angeles, CA

2 Principle: humans are mostly water fetus ~95% preterm 85% term 75% adult 60% volume of water (1 g/ml) = weight kg x % water content elderly 50%

3 All living things employ a system to survey and destroy pathogens Mechanisms for defense against foreign invaders present in all organisms Host defense in invertebrates is mediated by molecules that resemble effector mechanisms of natural (innate) immunity in higher organisms Components of the mammalian immune system appear to have arisen sequentially in phylogeny and have become specialized with evolution

4 The innate or natural immune system: capabilities we are born with Present prior to the exposure to infectious microbes or other foreign macromolecules Mechanisms of defense are not enhanced by repeated exposure All foreign substances are treated similarly Self-non-self discrimination through pattern recognition receptors (Toll-like) Scanning em of a normal PMN

5 The adaptive immune system: protection acquired throughout life Induced or stimulated in response to exposure to infectious microbes or other foreign macromolecules Mechanisms of defense are enhanced with repeated exposure (memory) Responses are exquisitely specific for distinct macromolecules Self-non-self recognition through MHC antigen complexes Scanning EM of a normal T lymphocyte

6 The immune system of the human body Primary Lymphoid Organs thymus Secondary Lymphoid Organs lymph nodes spleen bone marrow mucosal lymphoid aggregates

7 The gut associated lymphoid tissue (GALT) is the oldest component of the immune system jawless fish GALT jawed fish GALT thymus spleen amphibia GALT thymus spleen bone marrow birds GALT thymus spleen bone marrow germinal centers?lymph nodes mammals GALT thymus spleen bone marrow lymph nodes germinal centers

8 The gastrointestinal tract is the inner tube of life liver gall bladder large intestine esophagus stomach small intestine Processes food through digestion and absorbs and transport of nutrients across the epithelium Promotes the excretion of waste products Supports host-bacterial mutualism which is important in human health Functions as a barrier between the external environment and the inside of the body

9 The length and surface area of the intestine contribute to the complexity of the GALT surface area length 250 cm infant 800 cm adult 950 cm2 infant 7500 cm2 adult

10 The largest collection of lymphocytes reside in the GALT { mesenteric lymph nodes peyer s patches appendix/cecum } large intestine small intestine GALT

11 Unique challenges in immune surveillance and regulation in the GALT Physiologic and barrier functions of GALTassociated mucosal surfaces are essential for life therefore only limited damage can be tolerated Continuously exposed to a vast array of antigens (harmless, infectious and self) thereby requiring distinction of self/commensal from non-self/infection Useful for therapeutic interventions in the prevention and management of disease Infectious, malignant, immune and inflammatory diseases of the gut contribute to morbidity and mortality throughout life

12 Lymphocytes reside in anatomically defined regions within the GALT epithelium IEL LPL lamina propria Peyerʼs patch mucosal layer

13 Initiation of the immune response: antigens sampled from the lumen select T cells in the GALT antigens activated lymphoid follicle thoracic duct

14 Clonally selected T cells circulate: lamina propria, epithelium and common mucosal immune system antigens activated lymphoid follicle circulation thoracic duct

15 Lymphocytes from GALT circulate in the common mucosal immune system gut * * * * * *

16 Mucosal lymphocytes play important roles in immune homeostasis Mucosal defense from pathogens Ignore harmless bacteria and food (oral tolerance) Barrier function in the support epithelial cell growth and repair Pathogenesis of inflammatory intestinal diseases

17 Intestinal mucosal T cells Conventional T cells Thymus dependent, conventional phenotype and function in host defense Unconventional T cells Less thymus dependent, (perhaps independent) unconventional phenotype function in immune tolerance and suppression

18 Conventional T cells play an important role in host defense Earliest T cells to colonize the gut during postnatal life and following colonization from the germ free state Typical of T cells in lymphoid aggregates and within the lamina propria of the intestine More common in the large intestinal epithelium when compared to the small intestinal epithelium Exhibit effector cell responses to pathogens and antigens following immunization protocols Re-circulate as part of the common mucosal immune system

19 Unconventional mucosal T cells are distinct from conventional T cells epithelium IEL IEL Virtually restricted to the epithelial layer of the gut or have limited recirculation Comprise the majority of IEL in the small intestine (70%+) (20-30% of LI-IEL) Express CD8 composed of a-a (CD8aa) homodimer (CD8ab) Unresponsive to commensal microbial flora and self-antigens Produce anti-inflammatory cytokines such as IL-10 and TGF-b

20 Unconventional T cells may function as regulatory cells in the intestine Protective in models of colitis (Chen 2002, Inagaki-Ohara 2004) Required for the induction of oral tolerance (Mowat 2003) Participate in the elimination of damaged, infected or transformed epithelial cells (Matsuda 2001) Secrete anti-inflammatory cytokines and growth factors (Taguchi 1990)

21 Regulatory T cells balance proinflammatory responses in the intestine IEL unconventional CD8ab+ CD8aa+ Pro-inflammatory Anti-inflammatory Host Defense Tolerance (food, commensal bact) *Homeostasis*

22 Few T cells are present within the lining of the newborn intestine IEL IEL newborn adult Kuo, S et.al. (2001) Ped Res 49:

23 CD8aa + IEL accumulate after CD8 - and CD8ab+ IEL in suckling mice Podd et.al. 2007

24 Colonization by regulatory T cells is delayed during postnatal maturation increasing CD8aa+ IEL mostly CD8ab+ IEL equal proportions Postnatal Age (wks) Time of Weaning

25 Pro-inflammatory responses are dominant in early postnatal intestine IEL CD8ab+ Pro-inflammatory X Inflammation X Anti-inflammatory *Inflammation*

26 Relative immune deficiency is characteristic of the human newborn infant Epithelial Cells Lymphoid Aggregates Lamina Propria a Intraepithelial zone of risk susceptibility Gestational Age (wks)

27 Why is T cell colonization in the intestine ordered? Preserves integrity of the epithelial layer during barrier maturation and intestinal growth May allow colonization by commensal microbial flora and promote innate epithelial maturation via toll receptor signaling Kinetics may link early immune activation of pro-inflammatory T cells after weaning with an abundance of regulatory T cells

28 Does a paucity of unconventional T cells contribute to susceptibility in NEC? Few Abundant HN postnatal age (weeks) weaning Kuo et.al Ped Res.

29 Necrotizing enterocolitis (NEC) is a significant health problem First described in European literature (Schimd 1953); first US series 1960 s (Berdon/Santulli,1973) The most common and serious acquired intestinal disorder in premature infants Transmural inflammation and necrosis of the small and large bowel (ileo-colonic) Survivors often have chronic nutritional deficiencies requiring hospitalization

30 Birth Vaginal delivery Breast feeding Probiotic bacteria C- section Formula feeding Antibiotic use Environment (hospital & sterility) Promote Promote Hinder Establishment of healthy intestinal flora Predominance of bifidobacteria Development of adequate gut barrier function Appropriate balance of immune responses Reduction in NEC

31 Promoting disease: opportunistic infection and bacterial overgrowth toxic ischemic hypoxic antibiotics formula substrate Mucosal Injury Bacterial Overgrowth Abnormal Flora Immature Intestinal Barrier Immature Immune Defenses Necrotizing Enterocolitis

32 Select bacteria proposed as beneficial Elie Metchnikoff ( ) Suggested that ingested bacteria could have positive influence on microflora in the intestinal tract Hypothesized that lactobacilli were important for human health and longevity (Bulgarian example) Promoted yogurt and fermented foods as a part of the healthy diet

33 Probiotics: Probiotics and Prebiotics Nonpathogenic, live microorganisms that are capable of conferring a health benefit to the host Bifidobacteria, Lactobacilli, Yeasts (S. boulardii) Prebiotics: Non-digestible carbohydrates that selectively stimulate the growth or activity of certain bacteria in the gut that have the potential to benefit the human host Galacto-oligosaccharides, Fructo-oligosaccharides (inulin, FOS in vegetables)

34 Probiotic bacteria have diverse roles promoting intestinal immune function Live-nonpathogenic bacteria that are beneficial to the host (Lactobacillus, Bifidobacterium, Streptococcus) Diminish colonization of pathogenic bacteria by competing for binding sites and lowering the intraluminal ph (Weng et al 2005) Decrease bacterial translocation (Weng et al 2005) Upregulate IgA-secreting cells (Weng et al 2005) Increase humoral responses and mediate maturation of GALT (Rhee et al 2004)

35 Probiotic bacteria have the potential to reduce the risk of NEC in premature infants Study PI n = # infants BW/GA Probiotic Strain NEC incidence Dani n=585 <1500g <33w Lactobacillus GG 1.4% vs 2.8%, p=ns Lin, 2005 n=367 <1500g L. acidophilus & Bifidobacterium infantis 1.1% vs 5.3% p=.04 Bin-Nun, 2005 n=145 <1500g B. infantis, B. bifidus & Streptococcus thermophilus 1% vs 14%, p=.013

36 Routine supplementation of probiotic bacteria in premature infants at risk Cochrane Update of 9 eligible trials randomizing 1425 infants, >1000 g Enteral administration of live probiotics significantly reduced the risk of NEC and mortality (RR ) No systemic infection with supplemented probiotic Variability in baseline risk for NEC, timing, dose and formulation *Cochrane update :5, 1202

37 Objective/Methods To determine whether early feeding of lactobacillus GG (LGG) to suckling mice results in an earlier increase in CD8aa+ regulatory T cells in the intestine Commercially available: Sigma LGG Cultured o/n in LB broth Suspensions of CFU were fed by oral intubation on day 3 of life once Left to suckle normally euthanized weekly intervals until weaning

38 Experimental Design Model Few IEL CD8ab+ >> CD8aa+ IEL CD8aa+ =/>> CD8ab+ IEL Experiment Lactobacillus IEL analysis q wk IEL IEL Dol weeks Postnatal Age

39 Methods: Isolation and purification of IEL Introduce probiotics after birth, and examine mice at weekly intervals isolate intestine cut intestine into segments wash 20X 40X 70X log green fluorescence enrich IEL by density gradient centrifugation IEL two-color dot plot sp dn dp sp log red fluorescence release IEL from epithelial layer IEL subsets identified by electronic gating and flow cytometry fluorescence labeled mab

40 Regulatory T cells increase earlier in the small intestine in LGG fed suckling mice P= 0.01 % total CD8+ T cells CD8ab+ CD8aa+ CD8ab CFU CFU CONTROL CD8aa+ SI-IEL

41 CD8aa+ IEL in the large intestine increase earlier and to a greater degree in LGG fed suckling mice P= % total CD8+ T cells CD8ab+ CD8aa+ CD8ab+ CD8aa+ 5 0 CD8ab+ CD8ab CFU 1000 CFU CFU CFU CONTROL CONTROL CD8aa+ CD8aa CFU CFU CONTROL CD8aa CD8ab n= 18 mice/ group. LI-IEL

42 Lactobacillus feeding does not alter CD8 T cell subsets in the spleen P= 0.3 % of CD8+ T cells CD8ab+ CD8aa+ CD8ab CFU CFU CONTROL CD8aa CFU CFU CONTROL CD8aa CD8ab Spleen

43 Results CD8aa+ IEL increase in the small and large intestine in response to LGG feeding in suckling mice. T cell subsets in the spleen are not altered in response to lactobacillus feeding LGG can be cultured from the stool of fed suckling mice (data not shown) No increase in mortality following to lactobacillus feeding (data not shown)

44 Speculations Earlier increases in CD8aa+ IEL may abrogate exaggerated pro-inflammatory responses by CD8ab+ IEL following abnormal bacterial colonization in the intestine The increase in CD8aa+ IEL may improve the balance of pro-and anti-inflammatory cytokines in the intestine Probiotic supplementation may promote the reduction of inflammatory immune responses such as those observed in NEC Studies to directly test this hypothesis are dependent on appropriate model in mice

45 We re not at the bedside yet--- Encourage human milk feeding Standardize the formulation of probiotic bacteria for feeding Formula additive Nutritional supplement Drug Standardize the protocol for administration

46 Thank You