Immune phenotype in children with therapy-naïve remitted and relapsed Crohn s disease

Transcription

1 Online Sumissions: doi:1.3748/wjg.v16.i47.61 World J Gstroenterol 21 Decemer 21; 16(47): ISSN (print) ISSN (online) 21 Bishideng. All rights reserved. BRIEF ARTICLE Immune phenotype in children with therpy-nïve remitted nd relpsed Crohn s disese Aron Cseh, Brn Vsrhelyi, Kriszt Molnr, Blzs Szly, Peter Svec, Andrs Treszl, Antl Dezsofi, Peter Lszlo Lktos, Andrs Arto, Tivdr Tulssy, Gor Veres Aron Cseh, Brn Vsrhelyi, Kriszt Molnr, Blzs Szly, Peter Svec, Andrs Treszl, Reserch Group for Peditrics nd Nephrology, Semmelweis University nd Hungrin Acdemy of Sciences, H-183, Budpest, Hungry Brn Vsrhelyi, Deprtment of Lortory Medicine, Semmelweis University, H-183, Budpest, Hungry Antl Dezsofi, Andrs Arto, Tivdr Tulssy, Gor Veres, First Deprtment of Peditrics, Semmelweis University, H-183, Budpest, Hungry Peter Lszlo Lktos, First Deprtment of Medicine, Semmelweis University, H-183, Budpest, Hungry Author contriutions: Veres G nd Cseh A designed the reserch; Dezsofi A, Lktos PL, Arto A nd Veres G included the ptients; Cseh A, Molnr K, Szly B nd Svec P performed the nlyses; Treszl A nlyzed the dt; Cseh A, Vsrhelyi B nd Veres G wrote the pper; Tulssy T criticlly reviewed the pper. Supported y Grnts TÁMOP /1/KMR-28-4, OTKA-76316, OTKA-K81117 nd ETT-28-2 Correspondence to: Aron Cseh, MD, Reserch Group for Peditrics nd Nephrology, Semmelweis University nd Hungrin Acdemy of Sciences, H-183, Budpest, Hungry. [email protected] Telephone: Fx: Received: Mrch 1, 21 Revised: June 1, 21 Accepted: June 8, 21 Pulished online: Decemer 21, 21 Astrct AIM: To chrcterize the prevlence of supopultions of CD4 + cells long with tht of mjor inhiitor or stimultor cell types in therpy-nïve childhood Crohn s disese (CD) nd to test whether normlities of immune phenotype re normlized with the improvement of clinicl signs nd symptoms of disese. METHODS: We enrolled 26 peditric ptients with CD. 14 therpy-nïve CD children; of those, 1 children remitted on conventionl therpy nd formed the remission group. We lso tested nother group of 12 children who relpsed with conventionl therpy nd were given inflixim; nd 15 helthy children who served s controls. The prevlence of Th1 nd Th2, nïve nd memory, ctivted nd regultory T cells, long with the memers of innte immunity such s nturl killer (NK), NK-T, myeloid nd plsmocytoid dendritic cells (DCs), monocytes nd Toll-like receptor (TLR)-2 nd TLR-4 expression were determined in peripherl lood smples. RESULTS: Children with therpy-nïve CD nd those in relpse showed decrese in Th1 cell prevlence. Simultneously, n incresed prevlence of memory nd ctivted lymphocytes long with tht of DCs nd monocytes ws oserved. In ddition, the rtio of myeloid /plsmocytoid DCs nd the prevlence of TLR-2 or TLR-4 positive DCs nd monocytes were lso higher in therpy-nïve CD thn in controls. The mjority of ltertions diminished in remitted CD irrespective of whether remission ws otined y conventionl or iologicl therpy. CONCLUSION: The finding tht immune phenotype is normlized in remission suggests link etween immune phenotype nd disese ctivity in childhood CD. Our oservtions support the involvement of memers of the dptive nd innte immune systems in childhood CD. 21 Bishideng. All rights reserved. Key words: Crohn s disese; Dendritic cell; Inflixim; Lymphocyte; Monocyte; Regultory T cell; Relpse; Remission; Therpy-nïve; Toll-like receptor Peer reviewers: Dr. Stefn Wirth, Professor, Children s Hospitl, Heusnerstt. 4, Wuppertl 42349, Germny; Mshiro Iizuk, MD, PhD, Director, Akit Helth Cre Center, Akit Red Cross Hospitl, , Nkdori, Akit 1-1, Jpn Cseh A, Vsrhelyi B, Molnr K, Szly B, Svec P, Treszl A, Dezsofi A, Lktos PL, Arto A, Tulssy T, Veres G. Immune 61 Decemer 21, 21 Volume 16 Issue 47

2 Cseh A et l. Immune phenotype in childhood Crohn s disese phenotype in children with therpy-nïve remitted nd relpsed Crohn s disese. World J Gstroenterol 21; 16(47): Aville from: URL: v16/i47/61.htm DOI: i47.61 INTRODUCTION Crohn s disese (CD) is chronic gstrointestinl disese chrcterized y segmentl inflmmtion of the intestinl mucos ssocited with dysregulted ction of the mucosl immune system to the otherwise innocuous luminl ntigens in geneticlly susceptile host. Aout 1%-15% of ptients with CD re dignosed efore 18 yers of ge [1]. Certin fetures re unique to peditric CD in comprison to dult onset disese such s different disese loction, ltered response to immunosuppressive therpy nd different genetic nd immune phenotype [2,3]. Different chrcteristics my suggest differences in the pthomechnism of CD in children compred to tht in dults. Theories regrding CD pthomechnism include mlfunctioning of the immune system. Indeed, severl studies crried out in dult CD indicte the involvement of either dptive or innte immunity [4-7]. In dult CD, peripherl Treg prevlence is diminished in therpy-nïve ptients nd in the ctive stte of disese or relpses nd is incresed with therpy or in remission [8-1], while intestinl Treg prevlence is incresed in ctive CD [9-11]. Simultneously, the prevlence of effector T cells [12-14] nd ctivted T cells [15-17] is incresed in the periphery in ctive CD. The shift of T lymphocytes towrd Th1 commitment in peripherl lood nd iopsy specimens is widely oserved phenomenon in CD [18]. In ddition, the prevlence of ntigen presenting cells (APCs) including dendritic cells (DCs) decrese in remission, nd even more so in relpses t the periphery, ut is incresed in tissues, simultneously with n incresed expression of Toll-like receptor (TLR)-2 nd 4 in therpynïve nd treted CD dults [19-21]. Monocytes/mcrophges, nother group of APCs, re comprle t the periphery ut incresed in iopsies nd showed upregultion of TLR-2 nd TLR-4 in the ctive stte nd in remission of CD [22-25]. Other cell types of innte immunity such s nturl killer (NK) nd nturl killer T (NKT) cells re less prevlent in ctive CD [17,26-28]. There hve een limited numer of studies specificlly performed to investigte ltertions of the immune system in children with CD. In ddition, the mjority of ville dt re on treted CD children. While in untreted CD children some ltertions in dptive immunity hve een reported, such s the skewness of the Th1/Th2 rtio to Th2 (this finding is in contrst with tht oserved in dult CD) [29-32], recent dt suggest tht the disturnce of innte immune functions is lso mjor fctor contriuting to CD in children. Indeed, in untreted children the centrl mcrophge prevlence is incresed [33], nd TLR-2 nd 4 receptor expression is lso enhnced [34]. The role of innte immune system in the pthomechnism of CD my e inversely ssocited with ge t disese onset [2]. The immune dysregultion in CD is ffected y ongoing therpy. Aminoslicyltes nd steroids, s well s immunosuppressive drugs used s first-line therpy in CD hve strong immunomodultory effects [35]. In ddition, iologicl therpy including the tumor necrosis fctor α inhiitor inflixim (IFX) [36] lso hs sustntil effects on immune cell functions [mny of the dt re from ptients with rheumtoid rthritis (RA)] s it my increse the prevlence of peripherl nd centrl Tregs [8,37], effector nd ctivted [38,39] nd Th1 committed T cells [39,4]. IFX lso decreses the prevlence of NK cells [38], DCs [41,42] nd monocytes [43,44], s well s TLR-2 nd TLR-4 expression in peripherl cells [45]. The immunologicl impct of conventionl or IFX therpy on peripherl immune phenotype is, however, known exclusively for dult CD ptients nd hs not een explored fully in CD children. In this prospective study we imed to explore the mjor cell prevlence of the dptive nd innte immune systems in therpy-nïve CD children nd its ltertion with the improvement of CD otined y conventionl therpy or IFX tretment. MATERIALS AND METHODS We enrolled the following ptient groups into our study: (1) 14 therpy-nïve CD children. No drug ws prescried for these ptients t the time of CD dignosis. The dignosis of CD ws estlished y mens of The Porto criteri [46] ; disese ctivity ws determined ccording to the Peditric Crohn s disese ctivity index (PCDAI) [47] ; (2) During conventionl tretment [steroid, zthioprine (AZT) nd 5-minoslicylte (5-ASA)], 1 children responded forming the remission group. Clinicl remission ws defined s PCDAI < 1; (3) IFX therpy (5 mg/kg IFX t weeks, 2, nd 6) ws strted in 12 CD children who filed to respond to conventionl therpy forming the relpsed group. Non-responsiveness ws defined s modertely incresed PCDAI (PCDAI > 3) in ptients under conventionl therpy; nd (4) Fifteen ge- nd gendermtched children with functionl dominl pin served s controls. All ptients nd controls were dignosed, treted nd followed up in the Outptient Clinic of the First Deprtment of Peditrics, Semmelweis University etween Septemer 27 nd August 29. The Institutionl Ethicl Committee pproved our study; written prentl informed consent ws otined. The ptients clinicl chrcteristics re shown in Tle 1. Smll nd lrge owel ws involved in 11 of 14 tretment nïve CD ptients, ccording to the literture (L3 locliztion, Montrel criteri [48] ). Therpy-nïve CD ptients nd CD ptients with relpse hd lower ody mss index thn controls. Lower ody weight nd ody mss index is common presenting sign in peditric ptients with CD [49]. Reduced food intke, postprndil dominl crmps, systemic relese of cytokines nd mlsorptive dirrhe were listed s fctors responsile for this phenomenon [5]. 62 Decemer 21, 21 Volume 16 Issue 47

3 Cseh A et l. Immune phenotype in childhood Crohn s disese Tle 1 Clinicl dt nd ptient chrcteristics Therpy-nïve (efore conventionl (with conventionl therpy) therpy) Relpse (efore IFX therpy) IFX therpy (efore 2nd infusion) IFX therpy (efore 3rd infusion) Clinicl dt n (oys/girls) 15 (6/9) 14 (6/8) 1 (4/6) 12 (5/7) Age (yr) 12 (8-16) 1 (8.5-13) 11.5 ( ) 14 (11-16) 14.5 ( ) 14.5 (12-16) Body mss index (kg/m 2 ) 19.5 ( ) 13.9 ( ) d 18.6 ( ) c 17.4 ( ) 18.8 ( ) ( ) Body weight (percentile) 58 (35-79) 13 (4-23) d 24 (19-44) c 21 (7-28) 23 (15-63) 35 (1-74) Disese durtion (mo) - 1 (8.5-13) 1.5 ( ) 11.5 ( ) 12 (9.5-15) 12 (1-15) Activity index (PCDAI) - 45 (39-58) (-5) f 45 (25-48) j 2 (7-28) g 13 (2-22) l Locliztion (n, montrel criteri) - L1 (1), L2 (2), L3 (11) L1 (), L2 (2), L3 (8) L1 (1), L2 (2), L3 (9) Lortory dt White lood cell count (g/l) 7.9 ( ) 12.4 ( ) 11.7 ( ) 1 ( ) 7.9 ( ) 6.7 ( ) e Pltelet (g/l) 346 ( ) 657 ( ) 442 (38-624) 479 (396-75) 392 ( ) 383 (38-483) e Serum iron (mmol/l) 16 (14-21) 4 (2-12) 8 (4-1) 4 (2-7) d 5 (4-7) d 8 (6-8),e Serum lumin (g/l) 45 (44-49) 37 (34-39) d 42 (42-45) c 4 (36-41) d 41 (38-45) 42 (38-45) C-rective protein (mg/l) (-1) 21 (5-65) d 7 (2-11),c 27 (9-55) h, d 9 (3-11) d 5 (1-14) d,e P <.5, P <.1; d P <.1 vs control; c P <.5, f P <.1 vs therpy-nïve; h P <.1, j P <.1 vs first remission; e P <.5, g P <.5, l P <.1 vs relpse. IFX: Inflixim; PCDAI: Peditric Crohn's disese ctivity index; L1: Smll owel; L2: Lrge owel; L3: Smll owel nd lrge owel locliztion ccording to Montrel criteri [48]. Together with other routine lood smpling 6 ml of lithium-heprin nticogulted lood ws tken from therpy-nïve ptients t the time of dignosis, t the time of first remission in the remission group nd t the initition of IFX therpy, nd then 2 nd 6 wk lter in the relpsed groups. From peripherl lood mononucler cells (PBMCs), the identifiction of mrkers (6B11, CCR4, CD3, CD4, CD8, CD11c, CD14, CD25, CD45RA, CD45RO, CD123; CD161, CXCR3, HLA-DR nd Lin-1 BD Biosciences Phrmingen, Sn Diego, CA, USA; TLR-2 nd TLR-4 ebioscience, Sn Diego, CA, USA) nd FoxP3 ssy (ebioscience, Sn Diego, CA, USA) were performed with BD FACS Ari (BD Biosciences Phrmingen, Sn Diego, CA, USA) [51]. Briefly, from whole lood, PBMCs were seprted with grdient centrifugtion using Ficoll-Pque (GE Helthcre Life Sciences, Pittsurgh, PA, USA). The isolted PBMCs were wshed twice with Phosphte Buffered Sline ph 7.4 (PBS, Centrl Phrmcy of Semmelweis University, Budpest, Hungry) nd were stined with the pproprite fluorescent ntiodies ccording to the mnufcturers instructions. For intrcellulr stining, cells were incuted first with Fixtion/Permeiliztion Buffer (ebioscience, Sn Diego, CA, USA), then wshed twice with Permeiliztion Buffer (ebioscience, Sn Diego, CA, USA) nd stined y FoxP3 nd isotype control. At the end of stining, cells were wshed twice with PBS nd with Permeiliztion Buffer for the detection of cell surfce mrkers nd FoxP3, respectively. Smples were resuspended in PBS nd were mesured within 1 h recording t lest 5 events in the lymphocyte gte. Our dt did not follow norml distriution, therefore non-prmetric sttisticl tests nd medin nd interqurtile rnges were used. Mnn-Whitney nd Friedmn tests with Dunn s post hoc comprison nd Spermn s correltion were used for sttisticl nlysis, the level of significnce ws 5% (P <.5). All dt re expressed s medin (interqurtile rnge). Sttisticl nlysis ws performed with Sttistic 8 (Sttsoft, Tuls, OK, USA). RESULTS Mjor clinicl chrcteristics nd lortory dt re summrized in Tle 1. The investigted cell prevlence vlues nd cell rtios of the dptive nd innte immune systems re summrized in Tles 2 nd 3, respectively. We lso highlighted the most importnt ltertions in Figures 1 nd 2. First, we compred the immune phenotype in therpy-nïve CD ptients with tht of helthy controls. In CD children, the prevlence of ctivted T cells (i.e. CD4 + CD25 + cells) incresed. At the sme time, the prevlence of T cells with Th1 commitment (i.e. CD4 + CXCR3 + cells) decresed resulting in skewness of Th1/Th2 to Th2. The prevlence of memory (i.e. CD4 + CD45RO + ) cells incresed nd, therefore, shift in the nïve/memory rtio towrd memory cells ws oserved. The prevlence of regultory T (i.e. CD4 + CD25 hi FoxP3 + ) cells ws comprle etween the two groups. Striking differences in cell prevlence vlues of innte immunity were otined etween therpy-nïve CD nd helthy children. The occurrence of NK nd NKT cells (mrked s CD3 - CD161 + nd CD , respectively) ws lower in CD children. Interestingly, the prevlence of the APCs investigted differed lrgely etween CD nd helthy children. DCs (i.e. those with Lin1 - HLADR + expression) were more prevlent nd, within DC cells, the myeloid DCs (mdcs, i.e. CD11c + cells) were more prevlent, while plsmocytoid DCs (pdcs, i.e. CD123 + cells) were less frequent in CD thn in helthy children. This leds to skewness of mdcs in the mdc/pdc rtio. The prevlence of peripherl monocytes (i.e. CD14 + cells) lso incresed in therpy-nïve CD ptients. In ddition, the prevlence of DC cells nd monocytes expressing TLR-2 nd TLR-4 receptors ws lso incresed in CD. Of note, 63 Decemer 21, 21 Volume 16 Issue 47

4 Cseh A et l. Immune phenotype in childhood Crohn s disese Tle 2 Prevlence nd rtios of cellulr memers of dptive immunity Cell prevlence in prent popultion Therpy-nïve (efore (with Relpse (efore IFX IFX therpy (efore IFX therpy (efore conventionl therpy) conventionl therpy) therpy) 2nd infusion) 3rd infusion) CD4 + in PBMC ( ) ( ) ( ) ( ) ( ) ( ) Activted in CD ( ) 1.61 ( ) 5.3 ( ) g 7.84 ( ) 7.24 ( ) 9.98 ( ) Nïve in CD ( ) ( ) ( ) ( ) ( ) ( ) Effector in CD ( ) ( ) ( ) ( ) ( ) ( ),i Nïve/effector rtio 2.73 ( ) 1.57 ( ) 1.43 ( ) 2.34 ( ) 1.79 ( ) 1.52 ( ),e Th1 in CD ( ) 6.49 ( ) d ( ) c 1.9 ( ) ( ) ( ) e Th2 in CD ( ) 4.7 ( ) 5.6 ( ) 3.97 ( ) 4.61 ( ) 4.46 ( ) Th1/Th2 rtio 3.98 ( ) 1.6 ( ) d 1.95 ( ) 2.68 ( ) 2.68 ( ) 3.25 ( ) e Treg in CD ( ) 1.36 ( ) 1.41 ( ) 1.31 ( ) 1.33 ( ) 1.96 ( ),e P <.5, P <.1, d P <.1 vs control; c P <.5, g P <.1 vs therpy-nïve; e P <.5, i P <.1 vs relpse. Dt re expressed s medin (interqurtile rnge). IFX: Inflixim; PBMC: Peripherl lood mononucler cell; Activted (CD25 + ); Nïve (CD45RA + ); Effector (CD45RO + ); Th1: T helper 1 committed, CXCR3 + ; Th2: T helper 2 committed, CCR4 + ; Treg: Regultory T lymphocytes, CD25 hi FoxP3 +. Tle 3 Prevlence nd rtios of cellulr memers of innte immunity Cell prevlence in prent popultion Therpy-nïve (efore (with conventionl therpy) conventionl therpy) Relpse (efore IFX therpy) IFX therpy (efore 2nd infusion) IFX therpy (efore 3rd infusion) NKT in PBMC 1.39 ( ).7 ( ).74 ( ).72 ( ).73 ( ).83 ( ) NK in PBMC 3.17 ( ) 1.73 ( ) 1.98 ( ) 1.95 ( ) 2.14 ( ) 2.45 ( ) DC in PBMC.61 ( ) 1.5 ( ) 1.16 ( ) 1.45 ( ).86 (.3-2.8).82 ( ) mdc in DC 46.4 ( ) ( ) ( ) c 69.1 ( ),e 6.1 ( ) ( ),g pdc in DC ( ) ( ) ( ) ( ) 19.3 ( ) ( ) g mdc/pdc rtio 1.85 ( ) 3.5 ( ) d 2.35 ( ),c 3.5 ( ) d 3.4 ( ) d 2.2 ( ),g TLR-2 in DC 8.8 ( ) 55.8 ( ) d ( ) c 38.7 ( ),e ( ) 16.7 ( ) g TLR-4 in DC 2.24 ( ) ( ) 2.73 ( ) f 1.11 ( ),e 5.31 ( ) 3.32 (.1-6.5) g Monocyte in PBMC 2.1 ( ) 8.57 ( ) 5.96 ( ),c 7.3 ( ) 5.58 ( ) 5.31 ( ) TLR-2 in monocyte ( ) ( ) 2.92 ( ) c 2.63 ( ) ( ) ( ) TLR-4 in monocyte 7.17 ( ) ( ) 4.43 ( ) c 1.39 ( ) 8.26 ( ) 6.3 ( ) P <.5, P <.1, d P <.1 vs control; c P <.5, f P <.1 vs therpy-nïve; e P <.5 vs first remission; g P <.5 vs relpse. Dt re expressed s medin (interqurtile rnge). IFX: Inflixim; PBMC: Peripherl lood mononucler cell; NK: Nturl killer, CD3 - CD161 + ; NKT: Nturl killer T, CD ; DC: Dendritic cell, Lin1-HLA-DR + ; mdc: Myeloid dendritic cell, CD11c + ; pdc: Plsmocytoid dendritic cell, CD123 + ; Monocyte (CD14 + ); TLR: Toll-like receptor. 4 of the 14 therpy-nïve CD children did not respond to conventionl therpy. Their immune phenotype t the therpy-nïve phse did not differ from those children who responded to conventionl therpy (dt not shown). In the second phse of our study, we prospectively tested the ltertion in cell prevlence vlues during therpy. At the time of first remission with conventionl therpy, the Th1/Th2 rtio shifted to Th1 nd normlized long with ctivted T cell prevlence. Memory T cells remined elevted, while ll the other cell types of dptive immunity were comprle to tht mesured efore therpy. For innte immune cells, NK nd NK-T remined lower thn norml nd totl DC prevlence remined higher thn the control. However, mdc nd pdc rtios, totl monocyte prevlence nd cells expressing TLR-2/TLR-4 receptor vlues (including monocytes nd DCs) were norml. In children who relpsed with conventionl therpy, immune phenotype gin ecme comprle to tht in therpy-nïve CD. Therefore, we mesured lower Th1, incresed ctivted T, higher DC nd higher mcrophge prevlence s well s higher TLR-2 nd TLR-4 expression in comprison to controls. In ddition, the prevlence of mdcs, simultneously with TLR-2 nd TLR-4 expressing DCs ws higher in relpsed thn in remitted CD. During IFX therpy, immune cell prevlence ws mesured t two time points (i.e. 2 nd 6 wk fter the initition of therpy). Th1, ctivted T nd Treg prevlence incresed significntly y week 6 of therpy. Totl DC, mdc, pdc, totl monocyte, long with TLR-2 nd TLR-4 expressing DC nd mcrophge prevlence were norml t this time. Of note, lthough no significnt ltertion ws oserved t week 2, some tendencies were lredy present (Figures 1 nd 2). DISCUSSION In our study we investigted the mjor components of dptive nd innte immunity in simultneous mnner in CD children. While CD4 numers in therpy-nïve CD children were norml s in erly studies with dult ptients [52,53], we noticed shift to the Th2 direction in Th1/ Th2 committed T lymphocytes. This finding is in line with other reports on lood [29,3,32] or iopsy specimens of therpy-nïve CD children [31]. Similr to dults, we lso found higher thn norml prevlence of ctivted CD4 + cells nd effector memory cells nd decrese in effector cell/ nïve CD4 cell rtios [12-17]. We tested the ide tht this ws 64 Decemer 21, 21 Volume 16 Issue 47

5 Cseh A et l. Immune phenotype in childhood Crohn s disese A Activted (CD25 + ) T helper lymphocytes Therpy-nïve f Relpse IFX therpy 1 IFX therpy 2 B Effector/memory (CD45RO + ) T helpers Therpy-nïve h Relpse IFX therpy 1 IFX therpy 2 C T helper 1 committed (CXCR3 + ) lymphocytes d Therpy-nïve c e Relpse IFX therpy 1 IFX therpy 2 D Regultory (FoxP3 + ) T lymphocytes Therpy-nïve e Relpse IFX therpy 1 IFX therpy 2 Figure 1 Prevlence of cellulr memers of dptive immunity. The prevlence of ctivted (i.e. CD4 + CD25 + ) (A), effector or memory (i.e. CD4 + CXCR3 + ) (B), Th1 committed (i.e. CD4 + CXCR3 + ) (C) nd regultory T (i.e. CD4 + CD25hiFoxP3 + ) cells (D). P <.5, P <.1, d P <.1 vs control; c P <.5, f P <.1 vs therpynïve; e P <.5, h P <.1 vs relpse. due to low Treg numers, ut our dt showing norml prevlence of FoxP3 expressing CD4 cells do not support this notion. This finding is in contrst with tht suggesting diminution of Treg cells in untreted CD dults. However, in n erlier study Tregs were identified ccording to CD4 + CD25 high positivity [8] currently not regrded s sensitive mrker for this cell type. Our study provides novel informtion on the possile contriution of the innte immune system to Th2 shift in therpy-nïve CD children. While some uthors suggested tht ltered NK nd NKT function my e component in dult CD [17,26-28], our dt re the first to show lower thn norml NK nd NKT prevlence in therpynïve CD children. In ddition, we lso oserved mrked increse in monocyte nd DC prevlence with n increse in the mdc/pdc rtio. These cell popultions re mjor triggers of immune response nd my e linked with the increse in memory T cell prevlence. As recent studies emphsized, mdc nd pdc hve distinct regultory properties s mdc my shift the immune response not only towrd Th1, ut lso in the Th2 direction [54], while pdc cn induce Tregs [55]. Therefore, n increse in the mdc/ pdc rtio in our ptients my contriute to lower Th1/ Th2 rtio. Furthermore, we lso mesured n incresed prevlence of TLR-2 nd TLR-4 expressing monocytes nd DCs tht my lso ply role in the ctivtion of immune cells. This finding is in ccordnce with our previous oservtion of high TLR-2 nd TLR-4 expression in the colonic mucos of therpy-nïve CD children [34]. We tested prospectively the link etween immune phenotype nd disese ctivity index in our ptients. The mjority of immune system ltertions in therpy-nïve CD re normlized with the normliztion of PCDAI. While Th1 prevlence significntly incresed compred to the therpy-nïve stte nd lmost normlized during therpy, Th1/Th2 ws still in the norml rnge t first remission suggesting difference in CD immune phenotype etween dults nd children [4-7]. In remitted ptients, NK nd NKT prevlence incresed nd the difference etween therpy-nïve CD nd helthy controls disppered. This finding does not support previous reports on decresed NK nd NKT numers in treted CD dults [17,26-28]. We lso oserved tht the mdc/pdc rtio, prevlence of monocytes nd tht of TLR-2 nd TLR-4 expressing DCs nd monocytes were lso normlized. This my suggest tht normliztion of immune phe- 65 Decemer 21, 21 Volume 16 Issue 47

6 Cseh A et l. Immune phenotype in childhood Crohn s disese A Nturl killer T (CD ) cells B Nturl killer (CD3 - CD161 + ) cells Therpy-nïve Relpse IFX therpy 1 IFX therpy 2 Therpy-nïve Relpse IFX therpy 1 IFX therpy 2 C 6 D 25 c Dendritic (Lin1-HLADR + ) cells 4 2 Peripherl monocytes (CD14 + ) Therpy-nïve Relpse IFX therpy 1 IFX therpy 2 Therpy-nïve Relpse IFX therpy 1 IFX therpy 2 E 8 d c e g F 4 f e g TLR-2 expressing dendritic cells TLR-4 expressing dendritic cells Therpy-nïve Relpse IFX therpy 1 IFX therpy 2 Therpy-nïve Relpse IFX therpy 1 IFX therpy 2 Figure 2 Prevlence of cellulr memers of innte immunity. The prevlence of nturl killer T (i.e. CD ) (A), nturl killer (i.e. CD3 - CD161 + ) (B), dendritic cell (i.e. Lin1-HLA-DR + ) (C), peripherl monocyte (i.e. CD14 + ) (D), Toll-like receptor 2 expressing dendritic cell (E) nd Toll-like receptor 4 expressing dendritic cell (F). P <.5, P <.1, d P <.1 vs control; c P <.5, f P <.1 vs therpy-nïve; e P <.5 vs first remission; g P <.5 vs relpse. notype is linked to n improvement in PCDAI. This my e specific feture in childhood CD s numer of studies reported higher thn norml prevlence of ctivted nd effector T lymphocytes even in treted CD dults [12-17]. Tregs proly do not ply role in this normliztion s their prevlence ws not ltered in remission. We performed nother prospective study in 12 CD children who relpsed with conventionl therpy nd were treted with IFX. In these CD children, the immune phenotype ws similr to tht oserved in therpy-nïve CD children (with the only exception of norml prevlence of NK nd NKT cells). During IFX therpy, however, mrked chnges occurred in the 6th wk of IFX tretment nd the prevlence of Th1 cells nd the APCs investigted were normlized. Interestingly, the prevlence of ctivted T cells, memory cells nd Treg cells were elevted further. 66 Decemer 21, 21 Volume 16 Issue 47

7 Cseh A et l. Immune phenotype in childhood Crohn s disese Recently, incresed numers of Th1 cells nd high lood levels of Th1-type cytokines were found in dult ptients with rheumtoid rthritis (RA) fter IFX tretment [39,4]. Others lso found n increse in effector nd ctivted T cells with IFX [38,39]. While the explntion for this is still uncler, n ttrctive hypothesis my e tht IFX inhiits the homing of Th1 nd ctivted T cells to the inflmmtion site nd trnsiently increses their peripherl occurrence [38,4]. Theoreticlly, this immune phenotype my refer to n incresed risk for infections. However, our ptients did not exhiit mjor clinicl signs nd symptoms of infection during IFX therpy. Interestingly, simultneously with these chnges, IFX therpy lso incresed the prevlence of peripherl Treg cells. This phenomenon - which my e due to resistnce of Tregs to IFX-induced poptosis - ws lso demonstrted previously in the peripherl lood of dult CD ptients [8], nd in colonic smples from CD children [37]. In our ptients, the prevlence of monocytes, mdcs, mdc/pdc rtio nd TLR-2 nd TLR-4 expressing DCs normlized during IFX therpy indicting the possile impct of iologicl therpy on innte immunity. This is in line with the oservtion of others investigting RA nd CD ptients [41,43-45]. Our study hs three mjor limittions. First, lthough we did correct for multiplicity when performing pirwise comprisons, the study ws not powered for the multitude of sttisticl tests we performed, thus some significnces could occur y chnce lone. Second, peripherl cell prevlence vlues do not necessrily reflect the intestinl phenotype. Third, our results my hve een ffected y geing of the ptients during the follow-up period. The short durtion of our prospective study (i.e. out 1 mo until the first remission or 6 wk from the eginning of IFX therpy), however, mkes this is less likely. CD exhiits severl normlities in dptive immunity (such s decrese in Th1 cell nd n increse in memory nd ctivted T cell prevlence) nd innte immunity (such s n increse in DC, monocyte nd TLR-2 nd TLR-4 exhiiting APC prevlence). The mjority of the oserved ltertions of the innte immune system re normlized with the improvement of clinicl signs nd symptoms of CD, irrespective of whether this is otined y conventionl therpy or dd-on IFX therpy. This finding suggests link etween immune phenotype nd disese ctivity in childhood CD. If these results re reinforced y other groups, the oservtions my rise the possiility tht immune phenotype is potentil iomrker for clinicl response in CD children. COMMENTS Bckground Aout 1%-15% of ptients with Crohn s disese (CD) re dignosed efore 18 yers of ge. Peditric CD is unique sutype of CD due to different loction, ltered responsiveness to therpy nd different susceptiility fctors compred with those in dult CD. Reserch frontiers Severl studies indicte oth the mlfunction of dptive nd innte immunity in dulthood CD. Fewer dt re ville for CD children. The ltertion of immune phenotype with tretment is lso uncler in this popultion. Innovtions nd rekthroughs In this prospective study, the uthors demonstrted mrked ltertions in oth dptive nd innte immunity in childhood CD. These normlities were resolved in infnts responding to conventionl therpy ut not in non-responding children. Immune phenotype depends on disese ctivity. Applictions These oservtions support the involvement of memers of the dptive nd innte immune systems in childhood CD. They lso identify immune phenotype s possile iomrker for the follow-up of therpeutic success. Peer review In this study, the uthors prospectively investigted the mjor components of dptive nd innte immunity in CD children. The uthors showed severl normlities in dptive nd innte immunity in CD children. They lso showed tht the mjority of oserved ltertions were normlized in remission stge nd suggest link etween immune phenotype nd disese ctivity in childhood CD. Although the numer of CD ptients enrolled to this study ws smll, this prospective study demonstrted cliniclly interesting results in CD children. REFERENCES 1 Duinsky M. Specil issues in peditric inflmmtory owel disese. World J Gstroenterol 28; 14: Nieuwenhuis EE, Escher JC. Erly onset IBD: wht's the difference? Dig Liver Dis 28; 4: Shoul R, Krn A, Reif S, Weiss B, Shmir R, Tmir A, Dvidovich O, Hlevi J, Silver EL, Levine A. Disese ehvior in children with Crohn's disese: the effect of disese durtion, ethnicity, genotype, nd phenotype. Dig Dis Sci 29; 54: Zenewicz LA, Antov A, Flvell RA. CD4 T-cell differentition nd inflmmtory owel disese. Trends Mol Med 29; 15: Himmel ME, Hrdenerg G, Piccirillo CA, Steiner TS, Levings MK. The role of T-regultory cells nd Toll-like receptors in the pthogenesis of humn inflmmtory owel disese. 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Clin Exp Immunol 23; 132: Perminow G, Reikvm DH, Lycknder LG, Brndtzeg P, Vtn MH, Crlsen HS. Incresed numer nd ctivtion of colonic mcrophges in peditric ptients with untreted Crohn's disese. Inflmm Bowel Dis 29; 15: Szeeni B, Veres G, Dezsõfi A, Rusi K, Vnny A, Mrz M, Mjorov E, Artó A. Incresed expression of Toll-like receptor (TLR) 2 nd TLR4 in the colonic mucos of children with inflmmtory owel disese. Clin Exp Immunol 28; 151: Diefench KA, Breuer CK. Peditric inflmmtory owel disese. World J Gstroenterol 26; 12: Veres G, Bldssno RN, Mmul P. Inflixim therpy in children nd dolescents with inflmmtory owel disese. Drugs 27; 67: Riccirdelli I, Lindley KJ, Londei M, Qurtino S. Anti tumour necrosis-lph therpy increses the numer of FOXP3 regultory T cells in children ffected y Crohn's disese. Immunology 28; 125: Ferkolj I, Ihn A, Mrkovic S, Veceric Z, Pohr M. Inflixim reduces the numer of ctivted mucosl lymphocytes in ptients with Crohn's disese. 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Int J Clin Phrmcol Res 25; 25: Lügering A, Schmidt M, Lügering N, Puels HG, Domschke W, Kuchrzik T. Inflixim induces poptosis in monocytes from ptients with chronic ctive Crohn's disese y using cspse-dependent pthwy. Gstroenterology 21; 121: Bert FJ, D'Hens GR, Peeters M, Hiele MI, Schile TF, Shely D, Geoes K, Rutgeerts PJ. Tumor necrosis fctor lph ntiody (inflixim) therpy profoundly down-regultes the inflmmtion in Crohn's ileocolitis. Gstroenterology 1999; 116: De Rycke L, Vndooren B, Kruithof E, De Keyser F, Veys EM, Beten D. Tumor necrosis fctor lph lockde tretment down-modultes the incresed systemic nd locl expression of Toll-like receptor 2 nd Toll-like receptor 4 in spondylrthropthy. Arthritis Rheum 25; 52: Inflmmtory owel disese in children nd dolescents: recommendtions for dignosis--the Porto criteri. J Peditr Gstroenterol Nutr 25; 41: Hyms JS, Ferry GD, Mndel FS, Gryoski JD, Kiort PM, Kirschner BS, Griffiths AM, Ktz AJ, Grnd RJ, Boyle JT. 68 Decemer 21, 21 Volume 16 Issue 47

9 Cseh A et l. Immune phenotype in childhood Crohn s disese Development nd vlidtion of peditric Crohn's disese ctivity index. J Peditr Gstroenterol Nutr 1991; 12: Stsngi J, Silvererg MS, Vermeire S, Colomel JF. The Montrel clssifiction of inflmmtory owel disese: controversies, consensus, nd implictions. Gut 26; 55: Mmul P, Mrkowitz JE, Bldssno RN. Inflmmtory owel disese in erly childhood nd dolescence: specil considertions. Gstroenterol Clin North Am 23; 32: , viii 5 Diefench KA, Breuer CK. Peditric inflmmtory owel disese. World J Gstroenterol 26; 12: Svec P, Vásárhelyi B, Pászthy B, Körner A, Kovács L, Tulssy T, Treszl A. Do regultory T cells contriute to Th1 skewness in oesity? Exp Clin Endocrinol Dietes 27; 115: Sely WS, Jewell DP. T lymphocyte susets in inflmmtory owel disese: peripherl lood. Gut 1983; 24: Yun SZ, Hnuer SB, Kluskens LF, Krft SC. Circulting lymphocyte supopultions in Crohn's disese. Gstroenterology 1983; 85: Kool M, Lmrecht BN. Dendritic cells in sthm nd COPD: opportunities for drug development. Curr Opin Immunol 27; 19: de Heer HJ, Hmmd H, Kool M, Lmrecht BN. Dendritic cell susets nd immune regultion in the lung. Semin Immunol 25; 17: S- Editor Tin L L- Editor Wester JR E- Editor M WH 69 Decemer 21, 21 Volume 16 Issue 47