The Dynamics of Interleukin-10-afforded protection during dextran sulfate sodium-induced colitis

Ma ch 2018 | Volume 9 | A icle 4001 O iginal esea ch published: 01 Ma ch 2018 doi: 10.3389/ immu.2018.00400 F on ie s in Immunology | www. on ie sin.o g Edi ed by: Massimo Gadina, Na ional Ins i u e o A h i is and Musculoskele al and Skin Diseases, Uni ed S a es Re iewed by: Robson Cou inho-Sil a, Uni e sidade Fede al do Rio de Janei o, B azil Cinzia Fionda, Sapienza Uni e si à di Roma, I aly *Co espondence: Ma ga ida Sa ai a [email p o ec ed] †P esen add ess: Guilhe mina M. Ca iche, Ins i u e o In ec ion Immunology, TWINCORE, Cen e o Expe imen al and Clinical In ec ion Resea ch, a Join Ven u e be ween he Medical School Hanno e (MHH) and he Helmhol z Cen e o In ec ion Resea ch (HZI), Hanno e , Ge many; Valen ine Mu igneux, UQ Diaman ina Ins i u e, T ansla ional Resea ch Ins i u e, Uni e si y o Queensland, B isbane, QLD, Aus alia ‡These au ho s ha e con ibu ed equally o his wo k. §Co-senio au ho s. Special y sec ion: This a icle was submi ed o In lamma ion, a sec ion o he jou nal F on ie s in Immunology Recei ed: 11Augus 2017 Accep ed: 13Feb ua y2018 Published: 01Ma ch2018 Ci a ion: Ca dosoA, Gil Cas oA, Ma insAC, Ca icheGM, Mu igneuxV, Cas oI, CumanoA, Viei aP and Sa ai aM (2018) The Dynamics o In e leukin- 10-A o ded P o ec ion du ing Dex an Sul a e Sodium-Induced Coli is. F on . Immunol. 9:400. doi: 10.3389/ immu.2018.00400 The Dynamics o in e leukin-10- a o ded P o ec ion du ing Dex an sul a e sodium-induced coli is Ana Ca doso1,2,3,4,5,6,7, An onio Gil Cas o6,7‡, Ana Ca a ina Ma ins1,2‡, Guilhe mina M. Ca iche1,2†, Valen ine Mu igneux8,9†, Isabel Cas o6,7, Ana Cumano3,4,5, Paulo Viei a3,4,5§ and Ma ga ida Sa ai a1,2*§ 1 i3S – Ins i u o de In es igação e Ino ação em Saúde, Po o, Po ugal, 2 IBMC – Ins i u o de Biologia Molecula e Celula , Uni e sidade do Po o, Po o, Po ugal, 3 Depa men o Immunology, Uni é Lymphopoièse, Ins i u Pas eu , Pa is, F ance, 4 Uni e si y Pa is Dide o , So bonne Pa is Ci é, Cellule Pas eu , Pa is, F ance, 5 INSERM U1223, Pa is, F ance, 6 ICVS, Uni e si y o Minho, B aga, Po ugal, 7 ICVS/3B’s – PT Go e nmen Associa e Labo a o y, B aga, Po ugal, 8 Depa men o Immunology, Uni é In ég i é du génome, immuni é e cance , Ins i u Pas eu , Pa is, F ance, 9 Depa men o Genomes and Gene ics, Uni é In ég i é du génome, immuni é e cance , Ins i u Pas eu , Pa is, F ance In lamma o y bowel disease encompasses a g oup o ch onic-in lamma o y condi ions o he colon and small in es ine. These condi ions a e cha ac e ized by exace ba ed in lamma ion o he o gan ha g ea ly a ec s he quali y o li e o pa ien s. Molecula mechanisms coun e ac ing his hype in lamma o y s a us o he gu o e s a egies o he apeu ic in e en ion. Among hese egula o y molecules is he an i-in lamma o y cy okine in e leukin (IL)-10, as shown in mice and humans. Indeed, IL-10 signaling, pa icula ly in mac ophages, is essen ial o in es inal homeos asis. We sough o in es iga e he empo al p o ile o IL-10-media ed p o ec ion du ing chemical coli is and which we e he unde lying mechanisms. Using a no el mouse model o inducible IL-10 o e exp ession (pMT-10), desc ibed he e, we show ha mice p econdi ioned wi h IL-10 o 8days be o e dex an sul a e sodium (DSS) adminis a ion de eloped a milde coli ic pheno ype. In IL-10-induced coli ic mice, Ly6C cells isola ed om he lamina p op ia showed a dec eased in lamma o y p o ile. Because ou mouse model leads o ansc ip- ion o he IL-10 ansgene in he bone ma ow and ele a ed se ic IL-10 concen a ion, we in es iga ed whe he IL-10 could imp in immune cells in a long-las ing way, hus con e ing sus ained p o ec ion o coli is. We show ha his was no he case, as IL-10- a o ded p o ec ion was only obse ed i IL-10 induc ion immedia ely p eceded DSS- media ed coli is. Thus, despi e he p o ec ion a o ded by IL-10 in coli is, no el s a egies a e equi ed, speci ically o achie e long-las ing p o ec ion. Keywo ds: in e leukin-10, mac ophages, in lamma ion, coli is, he apy inT ODUcTiOn In lamma o y bowel disease (IBD) comp ises a complex g oup o in lamma o y condi ions o he gas oin es inal ac (1) a ec ing an inc easing numbe o pa ien s wo ldwide (2–4). Bo h o ms o IBD, C ohn’s disease (CD) and ulce a i e coli is (UC), esul om al e a ions in he immune homeos asis o he in es inal issue leading o local uncon olled in lamma ion (5, 6). The gu is a e y pa icula si e in e ms o immune epe oi e and egula ion, as e en in homeos a ic condi ions cons an exposu e o an igens occu s (7). Thus, he main enance o in es inal homeos asis, p ima ily 2 Ca doso e al. IL-10 Amelio a es DSS-Induced Coli is F on ie s in Immunology | www. on ie sin.o g Ma ch 2018 | Volume 9 | A icle 400 ca ied ou by in es inal mac ophages, equi es a cons an and ine- uned balance be ween he s a e o ole ance and in lam- ma ion (8). In he gu en i onmen , mac ophages encoun e a ple ho a o s imuli, om die a y an igens o commensal bac e ia, ye , due o hei unique issue-speci ic cha ac e is ics, emain ole an (9). In he p edisease s age, he epi helial o mucosal ba ie s become comp omised allowing bac e ia om he lumi- nal side o in ade he lamina p op ia o he gu (10). This e en igge s an acu e in lamma o y esponse due o he ac i a ion o immune cells by di ec con ac wi h bac e ial p oduc s (10). The induced in lamma ion esul s ei he in elimina ion o he o eign bac e ial incu sion o in an exace ba ed immune esponse ha can esul in issue damage. The damage caused by de egula ed in lamma ion will pe pe ua e he ac i a ion o e ec o cells and ul ima ely lead o he clinical onse o IBD (10, 11). Epidemiological s udies ha e shown ha he e iology o IBD is mul i ac o ial, wi h gene ic p edisposi ion, dys unc ional in es- inal ba ie and imbalances o he mic obiome all con ibu ing o his condi ion (12–15). Genome-wide associa ion s udies e ealed ha he main gene ic al e a ions associa ed wi h IBD a e ound in genes encoding p o eins linked o inna e o adap- i e immuni y, such as he nucleo ide-binding oligome iza ion domain-con aining p o ein 2, Janus kinase (JAK) 2, and umo nec osis ac o supe amily 15 (16–18). O he al e a ions a e associa ed wi h molecules in ol ed in leukocy e a icking, egula ion o ba ie unc ion and sec e ion o de ensins (17). Two epo s associa e loss-o - unc ion mu a ions in in e leukin (IL)-10 o IL-10R subuni s wi h se e e IBD (19, 20). These mu a- ions esul in se e e en e ocoli is, wi h onse be o e one yea o age, and un esponsi eness o immunosupp essi e he apies. The only a ailable he apy o hese pa ien s is immune econs i u- ion wi h hema opoie ic s em cells (21–23). Al hough comple e loss-o - unc ion mu a ions in IL-10 and IL-10R s ongly co ela e wi h IBD, hey ha e an ex emely low occu ence a e (19, 24). The mos equen mu a ions a ec ing he IL-10 genes associa ed wi h IBD a e in ac single-nucleo ide polymo phisms associa ed wi h low exp ession o his molecule (25). Howe e , ha bo ing such mu a ions does no always ansla e in low se um le els o IL-10 (23) du ing he disease s age. This is likely due o he signi ican inc ease on he numbe o IL-10-p oducing myeloid cells in CD pa ien s (26–29), o he ex en ha ele a ed se um le els o IL-10 co ela e wi h disease ac i i y in CD (30–32). The ole o IL-10 in in es inal in lamma ion is also seen in he mouse model, as IL-10-de icien mice de elop mic obiome- dependen spon aneous en e ocoli is (33). Fu he mo e, mice wi h mac ophage es ic ed IL-10R de iciency also de elop a spon aneous coli ic p o ile (34), s essing he c i ical ole o he monocy e/mac ophage axis in he immunologic e en s leading o IBD. In e es ingly, i has been shown, in a model o in ec ion ha IL-10 can exe a di ec e ec on monocy es/mac ophages subse s, leading o changes in hei in lamma o y p o ile and su i al (35). Mo eo e , IL-10 has been shown o con e p o ec ion om hype - in lamma o y s a es by he induc ion o he JAK1/STAT3 signaling pa hway ha supp esses exp ession o p oin lamma o y media o s and ac i a es exp ession o an i-in lamma o y genes (36). Taking in o accoun he esul s ob ained in mu ine models o IL-10 pe u ba ion, he gene ic co ela ion es ablished in humans, and he an i-in lamma o y p ope ies o IL-10, his cy okine eme ged as a e y p omising candida e o IBD he apy. Howe e , in IBD pa ien s IL-10-based he apy has no esul ed in subs an ial clinical imp o emen s (37). The main ca ea s in hese clinical ials we e he subcu aneous ou e o adminis a ion and he concen a ion o he ecombinan molecule ha did no ensu e ha IL-10 le els eached he mucosal si es, poin ing ou he impo ance o no el-locally a ge ed he apeu ic s a egies. Fu he mo e, IL-10 adminis a ion o IL-10-de icien mu ine models only p o ec ed om coli is i adminis e ed be o e disease es ablishmen (38). In his s udy, we epo a no el mouse model o IL-10 o e - exp ession ( he pMT-10 mouse) and use i o be e explo e he mechanisms o immune egula ion elici ed by IL-10 in he con ex o in es inal in lamma ion. We show ha a sho pe iod o IL-10 o e exp ession p io o he induc ion o coli is amelio a es he disease ou come, despi e he p esence o CD11b+ Ly6C+ cells in he gu , p e iously associa ed wi h he de elopmen o de imen al in lamma ion. As compa ed o con ol animals ha do no o e exp ess IL-10, Ly6C cells isola ed om he gu lamina p op ia o coli ic pMT-10 mice showed a dec eased in lamma o y p o ile. Thus, we p opose ha IL-10 o e exp ession impai ed he esponse o hese cells o he s imulus. In addi ion o he local e ec o IL-10 in con olling exace ba ed immune esponses, ou model allows o he s udy o IL-10 in imp in ing de no o gene - a ed and ci cula ing monocy es. This is because, cons an IL-10 exp ession is ound in speci ic issues, in pMT-10 mice, culmina - ing in a sys emic e ec . The e o e, IL-10 is likely o a ec o he impo an compa men s, such as he bone ma ow (BM) and spleen. IL-10-a o ded p o ec ion was only seen i IL-10 igge - ing immedia ely p eceded dex an sul a e sodium (DSS)-induced coli is, hus calling o no el s a egies ha sus ain he e ec o IL-10 o o e long-las ing p o ec ion. MaTe ials anD MeThODs e hics s a emen In Po ugal, all animal expe imen s we e pe o med in s ic acco dance wi h ecommenda ions o he Eu opean Union Di ec i e 2010/63/EU and p e iously app o ed by Po uguese Na ional Au ho i y o Animal Heal h–Di eção Ge al de Alimen ação e Ve e iná ia (DGAV). Mice we e eu hanized by CO2 inhala ion wi h e o s o minimize su e ing. In F ance, all animal p ocedu es we e app o ed by he Pas eu Ins i u e Sa e y Commi ee and conduc ed acco ding o F ench and Eu opean Communi y Ins i u ional guidelines. animals The s udy in ol ed he use o he ollowing 7–14-week-old emale mice: wild- ype C57BL/6j, pMT-10-IL-10 inducible mice, and pMT-10 c ossed wi h IL-10Rα-de icien mice (39) (pMT-10. IL-10Rα−/−). Food was adlibi um o all animals. gene a ion o pMT-10 Mice pMT10 mice we e gene a ed by A. Gil Cas o and Paulo Viei a. Mouse IL-10 cDNA was cloned in o he p169ZT ec o , which FigU e 1 | A no el mouse model o inducible in e leukin (IL)-10 exp ession: pMT-10 mice. (a) Schema ic ep esen a ion showing he a ge ing ec o and inse ion si e. (B) Kine ics o IL-10 o e exp ession in he se um a di e en ime poin s pos Zn adminis a ion and Zn wi hd awal. pMT-10 mice we e ed wi h no mal (pMT-10-Zn) o Zn-en iched (pMT-10+Zn) wa e and a he indica ed ime poin s blood was ha es ed and he amoun o IL-10 in se um measu ed by immunoassay. (c) qRT-PCR iden i ied CD45− TER119− cell subse s om skin, bone ma ow, and small in es ine (SI) as he main p oduce s o IL-10 in pMT-10 mice ed o 8days wi h Zn-en iched wa e . In bo h (B,c), each poin o ba ep esen s he mean±SEM o h ee independen mice. Da a we e analyzed wi h (B) wo-way analysis o a iance (Sidak’s mul iple compa isons es ) o (c) S uden ’s - es , *p<0.05; **p<0.01; ***p<0.001. 3 Ca doso e al. IL-10 Amelio a es DSS-Induced Coli is F on ie s in Immunology | www. on ie sin.o g Ma ch 2018 | Volume 9 | A icle 400 ca ies a sheep me allop o ein (MT) 1a p omo e , a β-globin splice si e and a SV40 polyadenyla ion (polyA) signal. The esul - ing ec o —pMT-10 (see Figu e 1A)—was hen injec ed in o C57BL/6j eggs and ansgenic ounde s we e iden i ied by PCR using MT and IL-10-speci ic p ime s. IL-10 o e exp ession was induced by eeding he mice a 2% suc ose solu ion wi h 50mM o zinc (Zn) sul a e. Dss-induced coli is Mice we e ed o 8days wi h 3% DSS (TdB consul ancy) in he d inking wa e , and we e moni o ed, daily, o weigh loss and disease p og ession. Coli is p og ession was measu ed by he Disease Ac i i y Index (DAI), as p e iously desc ibed [Table 1 (40)]. assessmen o in es inal in lamma ion Mice we e eu hanized on day 8 pos -DSS adminis a ion o ea lie i he symp oms o clinical disease (signi ican weigh loss o dia hea) became appa en . Samples om colons we e immedia ely ixed in 4% pa a o maldehyde. Then, 5µm pa a in- embedded sec ions we e s ained wi h hema oxylin and eosin, and in lamma ion was assessed in a blinded ashion using a p e iously desc ibed sys em [Table2 (41)]. Samples we e g aded semiquan- i a i ely om 0 o 3 o he ou ollowing c i e ia: (i) deg ee o epi helial hype plasia and goble deple ion; (ii) leukocy e in il a ion in he lamina p op ia; (iii) a ea o issue a ec ed; and (i ) he p esence o ma ke s o se e e in lamma ion such as c yp abscesses submucosal in lamma ion and ulce s. Fo each sample, c i e ia sco es we e added o gi e an o e all in lamma ion sco e o 0–12. cy okine Quan i ica ion In e leukin-10 concen a ion in he se um was quan i ied using a comme cially a ailable ELISA ki (R&D sys ems). P epa a ion o cell suspensions Isola ion o non-hema opoie ic cells (CD45− TER119−) o hema - opoie ic (CD45+) BMcells ollowed s anda d p o ocols. B ie ly, hema opoie ic BMcells we e ex ac ed by lushing he emu s and ibias wi h 2 mL o HBBS epea edly. To ob ain he BM non-hema opoie ic cells, he bone agmen s we e incuba ed in RPMI medium wi h Libe ase TL (0.5mg/ml; Roche) o 30min a 37°C. To help dissocia ion o non-hema opoie ic cells om he bone, a e each incuba ion pe iod, he emu s and ibias we e lushed wi h RPMI. We epea ed his s ep h ee imes a e which we lushed he bones one las ime, ha es ed he cell suspen- sions and added 1 olume o RPMI con aining 10% FCS. Small in es ine (SI), non-hema opoie ic cells we e isola ed as p e iously desc ibed (42). Skin non-hema opoie ic cells we e isola ed om ea samples. Samples we e ha es ed and he epide mis exposed by sepa a ing he ex e nal laye s. Epide mis was incuba ed o 45min a 37°C wi h Libe ase HL (0.5mg/ml; Roche) and DNase I (1U/mL; In i ogen). A e 45min, non-hema opoie ic cells we e dissocia ed om he issue by mechanical dis up ion, collec ed, washed in HBSS con aining 10% FCS and eco e ed. A he end, non-hema opoie ic cells we e so -pu i ied by excluding all TaBle 2 | Pa ame e s o his ological analysis o coli is se e i y. sco e epi helial hype plasia and goble deple ion leukocy e in il a ion in he Lamina P op ia a ea a ec ed Ma ke s o se e e in lamma ion 0 None None/ a e None None 1 Minimal Inc ease 1/3 Minimal 2 Mild Con luen 2/3 Inc eased 3 Ma ked T ansmu al All Con luen The inal sco e is ob ained by he sum o indi idual sco es. Ma ke s o se e e in lamma ion included ulce a ion and c yp abscesses. TaBle 1 | Disease Ac i i y Index (DAI) pa ame e s. sco e Weigh loss s ool consis ency Bleeding 0 No loss No mal No blood 1 1–5% Mild so B own colo 2 5–10% Ve y so Reddish colo 3 10–20% Dia hea Bloody s ool 4>20% G oss bleeding DAI is ob ained by he sum o each indi idual sco e. 4 Ca doso e al. IL-10 Amelio a es DSS-Induced Coli is F on ie s in Immunology | www. on ie sin.o g Ma ch 2018 | Volume 9 | A icle 400 CD45+ TER119+ cells (Figu e S1A in Supplemen a y Ma e ial). Cell suspensions o all o he o gans we e ob ained by mechanical dis up ion. Lamina p op ia leukocy es (LPLs) we e p epa ed as p e iously desc ibed (43). B ie ly, LPLs we e ha es ed, dissocia ed and esuspended in Hank’s Balanced Solu ion (HBSS) supplemen ed wi h 1% e al cal se um (FCS; Gibco). To isola e LPLs, he colon was lushed wi h phospha e-bu e ed saline (PBS; Gibco), opened and cu in o 1cm pieces. To elimina e epi helial cells hese ag- men s we e incuba ed a 37°C in Ca- and Mg- ee PBS con aining 10% FCS and 5.0mM EDTA unde s ong agi a ion o 30min. Fo LPL isola ion, he emaining agmen s we e incuba ed in RPMI medium wi h Libe ase TL (0.5mg/ml; Roche) o 30min a 37°C. To comple e he diges ion, he suspension was epea edly passed h ough a 10ml sy inge o 5min, il e ed h ough a 40μm cell s aine (BD Bioscience) and collec ed by cen i uga ion. The cell pelle was esuspended in 44% Pe coll (GE Heal hca e), laid o e 67% Pe coll, and cen i uged a 600g o 20min a 20°C. Cells a he in e ace we e collec ed, washed in HBSS con aining 1% FCS and eco e ed. an ibodies An ibodies we e conjuga ed o luo och omes (FITC, PE, PECy7, APC, APCCy7, Paci ic Blue, and BV711) and we e speci ic o he ollowing mouse an igens: CD3 (145-2C11; Biolegend), CD11b (M1/70; Sony), CD11c (HL3; Biolegend), CD19 (6D5; Sony), CD45.2 (104; Biolegend), Ly6C (Hk1.4; eBioscience), Ly6G (RB6-8C5; BD Pha mingen), CD45 (30F11; Sony), and TER119 (Te 119; BD Pha mingen). cell so ing and Mul iplex eal Time-Pc analysis CD45− TER119− o CD45+ cells we e so -pu i ied based on he exp ession o CD45 and TER119 using an A ia so e (BD). Dead cells we e elimina ed by exclusion wi h p opidium iodide (PI). mRNA om so ed cells was ex ac ed using RNeasy Mic o ki (Qiagen) and con e ed in o cDNA by e e se ansc ip ion wi h P imeSc ip RT Reagen ki (Taka a, Clone ech). qRT-PCR was pe o med using Taqman p ime s (see Table S1 in Supplemen a y Ma e ial o e e ences) and Taqman Uni e sal Mas e Mix (Applied Biosys ems). qRT-PCR eac ions we e pe o med on a ABI 7300 he mocycle (Applied Biosys ems). Lamina p op ia leukocy es we e FACS-so pu i ied based on he exp ession o CD45.2, CD11b and Ly6C, using an A ia III so e (BD). Cells exp essing CD3, CD19, CD11c, and Ly6G we e excluded. Dead cells we e elimina ed by exclusion wi h PI. CD45.2+ CD11b+ Ly6C+ cells we e so ed di ec ly in o a mix o 9µl o CellsDi ec One-S ep qRT-PCR ki (Li e Technologies), con ain- ing a mix u e o dilu ed p ime s (0.05× inal concen a ion, see Table S1 in Supplemen a y Ma e ial o e e ences). P eampli ied cDNA (18 cycles), was ob ained acco ding o he manu ac u e ’s ins uc ions and was dilu ed 1:5 in TE bu e (pH=8; Ambion). The sample mix u e was as ollows: dilu ed cDNA (2.9µl), Sample Loading Reagen (0.32µl; Fluidigm), and Taqman Uni e sal PCR Mas e Mix (3.5µl; Applied Biosys ems). The assay mix u e was as ollow: Assay Loa ing Reagen (Fluidigm) and Taqman Mix. A 48×48 Dynamic A ay in eg a ed luidic ci cui (IFC; Fluidigm) was p imed wi h con ol line luid, and he chip was loaded wi h assays and samples wi h and X IFC Con olle (Fluidigm). The expe imen s we e un on a BioMa k HD (Fluidigm) o 40 cycles. Gene exp ession was no malized o Hp and assessed by he 2ΔC me hod. s a is ical analysis S a is ical analysis was pe o med wi h he S uden ’s - es o wo- way analysis o a iance as indica ed in he igu e legends. The analysis was pe o med wi h P ism So wa e (G aphPad). G aphs con aining e o s ba s show means±SEM. S a is ical signi icance is ep esen ed as ollows: *p<0.05, **p<0.01, and ***p<0.001. esUlTs gene a ion o a no el Mouse Model o il-10 O e exp ession To s udy he biological impac o IL-10 o e exp ession in di - e en se ings, we enginee ed a no el mouse model o allow o inducible IL-10 exp ession, he pMT-10 mouse (44). Fo his, a cons uc con aining he IL-10 cDNA unde he con ol o he inducible sheep MT p omo e was in oduced in he genome o wild- ype BL/6 mice (Figu e1A). Whole genome sequencing e ealed a single inse ion o he ansgene in ch o- mosome 10, be ween posi ions 78.813.656 and 78.992844bp (Figu e1A). We es ima ed, by qRT-PCR, he numbe o cop- ies o he ansgene o be 50–100 (da a no shown). The MT p omo e is ac i a ed in he p esence o 50mM o Zn in he o ganism, adminis e ed in he d inking wa e . Kine ic analysis o IL-10 in he se um o pMT-10 mice ed wi h Zn-en iched wa e showed a apid inc ease o ci cula ing IL-10 (Figu e1B). Indeed, as soon as day 3 a e IL-10 induc ion, he le els o his cy okine in he se um we e e y high (7–12 ng/ml) FigU e 2 | Dex an sul a e sodium (DSS)-induced pa hology is amelio a ed by p eexposu e o in e leukin (IL)-10. (a) BL/6, pMT-10, and pMT-10. IL-10Rα−/− mice we e ed o 8days wi h no mal (BL/6-Zn, pMT-10-Zn, and pMT-10.IL-10Rα−/− Zn, espec i ely) o Zn-en iched (BL/6+Zn, pMT- 10+Zn, and pMT-10.IL-10Rα−/−+Zn) wa e , ollowed by 8days o 3% DSS adminis a ion also in he d inking wa e . (B,F) Disease p og ession based on DAI pa ame e s was egis e ed e e y day o 8days. Each poin ep esen s he mean±SEM o h ee o i e independen mice, in wo independen expe imen s. (c) Colon leng h measu emen a day 8 o DSS adminis a ion. (D) Rep esen a i e H&E-s ained sec ions o la ge bowel a 40× magni ica ion (scale ba =200µm). (e) Coli is sco es de i ed om e alua ion o colon and cecum om ei he g oup. Each do ep esen s one independen animal; ep esen ed is also mean±SEM. Da a we e analyzed wi h (B,F) wo-way analysis o a iance (Sidak’s mul iple compa isons es ) o (c,e) S uden ’s - es . (B) # compa e BL/6-Zn agains BL/6+Zn; ^ compa e BL/6-Zn agains pMT-10-Zn; • compa e BL/6-Zn agains pMT-10+Zn; ∅ compa e BL/6+Zn agains pMT-10-Zn; x compa e BL/6+Zn agains pMT-10+Zn, * compa e pMT-10-Zn agains pMT-10+Zn. One symbol, p<0.05; wo symbols, p<0.01; h ee symbols, p<0.001. 5 Ca doso e al. IL-10 Amelio a es DSS-Induced Coli is F on ie s in Immunology | www. on ie sin.o g Ma ch 2018 | Volume 9 | A icle 400 (Figu e1B). Mo eo e , suspending Zn adminis a ion led o a sha p d op in IL-10 in se a in only 24h, o below de ec- ion le els in only 48h (Figu e1B). As expec ed, ci cula ing IL-10 was unde ec able in pMT-10 mice ed wi h no mal wa e (Figu e1B). T ansc ip ional analysis o di e en o gans and cellula compa men s o induced pMT-10 mice e ealed ha he exp ession o he exogenous IL-10 cDNA was es ic ed o CD45−TER119− cells om he SI, skin and, o a less ex en , BM (Figu e1C). IL-10 induc ion was no de ec ed in he o he o gans analyzed (li e , spleen, kidney, cho oid plexus, lung, and colon) no in hema opoie ic cells isola ed om he BM (Figu e S1B in Supplemen a y Ma e ial). Thus, he pMT-10 mouse model allows o imely con olled IL-10 o e exp es- sion in speci ic ana omic loca ions, accompanied by a s ong inc ease o he le els o his cy okine in he se um. Dss-induced coli is is amelio a ed in il-10 P eexposed Mice Despi e he clea link be ween low le els o IL-10 and suscep- ibili y o coli is in human (45) as well as in mouse models (33, 34), adminis a ion o IL-10 o ea his condi ion showed only limi ed e ec s (37). A possible eason may be he poo acces- sibili y o IL-10 o he si e o in lamma ion. In his con ex , and in iew o he high exp ession seen in he SI o induced mice, he pMT-10 mouse model o e s an oppo uni y o u he add ess he e ec s o IL-10 exp ession in he gu in he con ex o coli is. Fo his, we used he DSS expe imen al model, a highly eliable and ep oducible way o causing UC-like symp oms in he mouse model by inducing acu e in lamma ion wi h he ec ui men o in lamma o y cells (46). We in es iga ed he impac o IL-10 o e exp ession p io o DSS-induced coli is. Fo his, pMT-10 mice we e induced o o e exp ess IL-10 o 8days, be o e ini ia ion o DSS adminis- a ion (Figu e2A). As con ols, non-induced pMT-10 o BL6 mice ed wi h con ol o Zn-en iched wa e we e used. In ou expe imen al se ing, wild- ype BL/6 mice s a ed o show signs o disease om days 4 o 5 a e adminis a ion o 3% DSS in he d inking wa e (Figu e2B). Con ol pMT-10 mice showed a p og ession o he DAI e y simila o BL/6 mice (Figu e2B). As compa ed o pMT-10 o BL6 ed wi h con ol wa e , mice p econdi ioned wi h IL-10 showed signi ican ly lowe DAI a e day 5 (Figu e2B) indica ing ha IL-10 con e ed pa ial p o ec ion. Zn adminis a ion o BL6 mice p io o DSS, esul ed in pa ial p o ec ion. Indeed, by day 7 o DSS adminis a ion, a signi ican ly lowe DAI was obse ed in BL/6 mice ed wi h Zn-en iched wa e as compa ed o BL6 con ol (Figu e 2B). Ne e heless, he maximal p o ec ion was obse ed o pMT-10 mice o e exp essing IL-10, which sugges s a syne gis ic e ec o IL-10 and Zn in he amelio a ion o he disease (Figu e2B). The DAI encompasses h ee sco es, one o which is he weigh loss. Rela i ely o con ol animals, bo h pMT-10 and BL/6 mice ed wi h Zn p io o DSS adminis a ion showed less weigh loss (Figu e S2 in Supplemen a y Ma e ial). pMT-10 mice p eexposed o IL-10 showed he leas educ ion in colon leng h as compa ed o all con ol g oups (pMT-10 o BL/6 ed wi h con ol wa e , and BL/6 ed wi h Zn-en iched wa e – Figu e2C), in line wi h maximal p o ec ion being con e ed by IL-10. His ologic analysis o he o gan, comp ising he analysis o in lamma o y in il a es, a chi ec u al dis o ion (c yp sho ening and b anching) and FigU e 3 | Ly6C+ cells p eexposed o in e leukin (IL)-10 e eal a less in lamma o y p o ile upon DSS-induced coli is han hose p eexposed o Zn. (a) pMT-10 o BL/6 mice we e ed wi h Zn-en iched wa e o 8days, ollowed by 4days o 3% DSS adminis a ion. (B) A he end o he DSS ea men , Lamina p op ia leukocy es (LPLs) we e isola ed and Ly6C+ cells so -pu i ied. Shown is he ga ing s a egy o Ly6C+ cells pu i ica ion. (c) So -pu i ied Ly6C+ cells (n=25 cells) we e analyzed by qRT-PCR o a o al o 22 genes using he BioMa k HD sys em. Samples we e no malized o Hp exp ession. Rep esen ed is he exp ession hea map compiling he genes which exp ession was de ec ed in ei he mouse g oup. Each hea map ec angle ep esen s he mean o gene exp ession ob ained o cells isola ed om i e independen mice. (D) The equency o he di e en leukocy e subse s was de e mined upon s aining o LPLs o Ly6C+ cell so ing. Each do ep esen s one independen animal; ep esen ed is also mean±SEM. Da a we e analyzed wi h S uden ’s - es , *p<0.05. 6 Ca doso e al. IL-10 Amelio a es DSS-Induced Coli is F on ie s in Immunology | www. on ie sin.o g Ma ch 2018 | Volume 9 | A icle 400 ulce a ion, showed an imp o emen in pMT-10 mice p eexposed o IL-10 as compa ed o BL/6 p eexposed o Zn (Figu es2D,E). Howe e , despi e a educ ion in he his ological sco e o some pMT-10 mice p eexposed o IL-10, he o e all g oup did no each s a is ical signi icance when compa ed o he o he con ol g oups (Figu es2D,E). Thus, ou da a showed ha IL-10 o e exp ession p io o in es inal insul a o ded a signi ican deg ee o p o ec ion om DSS-induced coli is. Addi ionally, ou da a also sugges a syne gis ic e ec o IL-10 and Zn in he amelio a ion o he disease. To in es iga e his issue, we epea ed he expe imen using pMT-10 mice c ossed wi h IL-10Rα−/− mice. Since pMT- 10.IL-10Rα−/− double mu an mice a e un esponsi e o IL-10, he e ec s obse ed would only be due o Zn adminis a ion. In hese mice, we obse ed an accele a ed disease p og ession upon DSS adminis a ion, wi h an ele a ed DAI sco e as ea ly as day 3, in line wi h he known ole o IL-10 in con olling he disease (Figu e2F). In hese mice, Zn adminis a ion did no con e p o- ec ion agains DSS-induced coli is (Figu e2F). Taken oge he , ou indings suppo he no ion ha he p o ec ion con e ed by Zn equi es IL-10 signaling. P eexposu e o il-10 P omo es a Mo e con olled in lamma o y esponse P e ious s udies ha e shown ha monocy es and mac ophages a e he majo e ec o subse s o colonic in lamma ion (34, 47). Mice wi h mac ophage-speci ic IL-10R de iciency de elop a spon aneous coli ic p o ile, emphasizing he impo ance o IL-10 in egula ing he mac ophage esponse o p e en uncon olled in lamma ion (34). Thus, we nex in es iga ed whe he IL-10 amelio a ed DSS-induced coli is by es ic ing he monocy e/ mac ophage esponse. Conside ing ha Zn adminis a ion also imp o ed he ou come o DSS-induced coli is in BL/6 mice, we compa ed he ansc ip ional p o ile o monocy es/mac ophages om BL/6 o pMT-10 mice p eexposed o Zn and subjec ed o DSS adminis a ion o 4days. We chose his ime poin , since signs o coli is induced by DSS in bo h BL/6 and pMT-10 mice only become ob ious a e day 4 o DSS adminis a ion. Thus, BL/6 and pMT-10 mice we e ed wi h Zn-en iched wa e o 8days and hen ecei ed DSS o 4days (Figu e3A). A his ime poin , Ly6C+ cells om each mouse om he di e en g oups we e FACS pu i ied (Figu e3B). Exp ession o 22 genes (Table S1 in Supplemen a y Ma e ial) associa ed wi h he uncon olled FigU e 4 | The in e leukin-10 p o ec ion con e ed agains DSS-induced coli is is no long las ing. (a) pMT-10 mice we e ed wi h con ol (pMT-10-Zn) o Zn-en iched (pMT-10+Zn) wa e o 8days, ollowed by a 7- o 21-day es ing pe iod whe e only no mal wa e was a ailable, and by 8days o 3% DSS. (B) Disease p og ession based on Disease Ac i i y Index (DAI) pa ame e s was egis e ed e e y day o 8days. (c) Colon leng h measu emen a day 8 o DSS adminis a ion. (D) Rep esen a i e H&E-s ained sec ions o la ge bowel a 40× magni ica ion (scale ba =200µm). (e) Coli is sco es de i ed om e alua ion o colon and cecum om bo h g oups. Each do ep esen s one independen animal, in wo independen expe imen s; ep esen ed is also mean±SEM. Da a we e analyzed wi h (B) wo-way analysis o a iance (Sidak’s mul iple compa isons es ) o (c,e) S uden ’s - es . 7 Ca doso e al. IL-10 Amelio a es DSS-Induced Coli is F on ie s in Immunology | www. on ie sin.o g Ma ch 2018 | Volume 9 | A icle 400 immune esponse de eloped in IBD we e analyzed by mul i- plex RT-PCR. All samples, om bo h g oups, exp essed h ee house-keeping genes (Hp , Ac b, and Gapdh). O he 22 genes analyzed, we ailed o de ec exp ession o 9 (Il4, Il9, Il12α, Il12β, Il13, Il17, Il23, I nγ, and Cx3cl1) in Ly6C+ cells isola ed om he lamina p op ia in bo h g oups. We de ec ed exp ession o he 10 emaining genes in Ly6C+ cells, in Zn- ed BL/6 and pMT-10 mice a e DSS adminis a ion (Figu e3C), bu no exp ession in he absence o insul (da a no shown). Thus, Ly6C+ cells al e hei exp ession p o ile in esponse o DSS insul . Mos in e es ingly, on day 4 pos -DSS adminis a ion, Ly6C+ cells isola ed om induced pMT-10 mice p esen ed an o e all less in lamma o y p o ile han hose isola ed om BL/6 mice (Figu e3C). In he case o Tn α and Cd86, he di e ences obse ed be ween he wo mouse g oups we e s a is ically signi ican (Figu e3C). In all, hese indings sugges ha exposu e o IL-10 be o e DSS induc- ion ac s by p e en ing an in lamma o y p o ile in Ly6C+ cells. O no e, he equency o in lamma o y mac ophages ec ui ed o he in lamed gu was simila be ween he wo g oups, and he same was ue o CD3 Tcells and CD19 Bcells, showing ha IL-10 o e exp ession does no impac he ec ui men o immune cells o he gu (Figu e3D). il-10 P o ec ion agains Dss-induced coli is is no long las ing In ou mouse model, IL-10 is also o e exp essed in he BM and is ound a high le els in he se um, possibly c ea ing an an i- in lamma o y en i onmen ha could p econdi ion de no o gene a ed o ci cula ing monocy es. In his se ing, he ci cula - ing monocy es could hus be educa ed o be less esponsi e once ec ui ed o he colon du ing DSS-induced coli is. To s udy his possibili y, we combined a pe iod o IL-10 o e exp ession wi h a es ing pe iod o 7 o 21 days p io o DSS adminis a ion (Figu e4A). As shown in Figu e1B, he le els o IL-10 e u n 8 Ca doso e al. IL-10 Amelio a es DSS-Induced Coli is F on ie s in Immunology | www. on ie sin.o g Ma ch 2018 | Volume 9 | A icle 400 o basal ones as soon as 24 h a e he zinc adminis a ion is suspended. Thus, a e a 7-day es , he ci cula ing le els o IL-10 a e no malized. Fu he mo e, we chose he 21days ime-poin , because p e ious epo s showing ha esiden in es inal mac- ophages ha e a li e span o app oxima ely 3weeks (48). A e 7-day es pe iod, he p o ec ion in DAI a o ded by p e ious IL-10 exposu e was pa ly los (Figu e4B), being less p onounced han when no es ing was pe o med and only obse ed a la e ime-poin s. A e he 21-day es ing pe iod, he p o ec ion in DAI o pMT-10 p e iously o e exp essing IL-10 was comple ely abla ed (Figu e4B). Fu he mo e, independen ly o he es ing pe iod, no di e ences we e obse ed be ween he expe imen al g oups in wha conce ned colon leng h (Figu e4C) o his ology (Figu es 4D,E). Thus, we conclude ha IL-10 o e exp ession, o e a pe iod o 8days, does no con e long-las ing p o ec ion agains in es inal in lamma ion. DiscUssiOn Despi e he ac ha IBD is a ea able condi ion, he e a e many limi a ions o he he apeu ic app oaches cu en ly a ailable (5). Majo obs acles in his con ex a e he he e ogenei y o he disease, which implies ha dosage and schedule may di e ac oss disease condi ions, and he equi emen o a localized ac ion o he he apeu ic agen . In iew o he s ong immune componen associa ed wi h disease de elopmen , i is no su p ising ha he apeu ic manipula ions o he immune esponse ha e been widely sough app oaches o ackle IBD. Indeed, a commonly used he apy o IBD is he adminis a ion o an i- umo nec osis ac o alpha (TNF-α) an ibodies (49). Howe e , in line wi h he abo e men ioned limi a ions, up o one- hi d o IBD pa ien s do no espond o his he apy, and hose who espond e en ually de elop some deg ee o in ole ance o he medica ion (50). In his con ex , se e al animal models o IBD, bo h spon aneous and expe imen ally induced (such as DSS), we e de eloped o in es iga e he ole o a ious ac o s on he pa hogenesis o he disease and o e alua e he di e en he apeu ic op ions. A mol- ecule ha has been widely s udied in he con ex o IBD is IL-10. This cy okine keeps in es inal in lamma ion in check by exe ing a di ec e ec on monocy e/mac ophage popula ions (34). Thus, i is no su p ising ha IL-10-based he apies ha e been es ed in IBD. Howe e , bo h in human (51–54) and mouse models (38), adminis a ion o IL-10 did no signi ican ly imp o e in es inal in lamma ion, pe haps in pa due o he ac ha adminis e ed IL-10 did no each he in lamed issue. In his s udy, we epo a no el ansgenic mouse model o inducible IL-10 o e exp ession, he pMT-10 mice, in which high IL-10 ansc ip ion is obse ed in he in es ine, skin and BM. Upon induc ion o he ansgene, high le els o IL-10 a e de ec ed also in he se um. Taking ad an age o his no el mouse model, we in es iga ed he dynamics o IL-10 a o ded p o ec ion du ing DSS-induced coli is. We ound ha induc ion o IL-10 p io o DSS adminis a ion impac ed he p og ession o coli is. We show ha a sho pe iod o IL-10 o e exp ession be o e he induc ion o coli is amelio a ed he disease ou come, despi e he p esence o CD11b+ Ly6C+ cells in he gu , p e iously associa ed wi h he de elopmen o de imen al in lamma ion. Howe e , in compa ison o con ol animals ha do no o e exp ess IL-10, Ly6C cells isola ed om he lamina p op ia o coli ic pMT-10 mice showed a dec eased in lamma o y p o ile. Thus, we hypo h- esize ha IL-10 o e exp ession impai s he esponse o hese cells o he insul , ea i ming bo h he c i ical ole o hese cells on in es inal in lamma ion (47) and ha o IL-10 in egula ing hei in lamma o y esponses (34). In line wi h a p e iously desc ibed p o ec i e ole o Zn in he con ex o in es inal in lamma ion (55, 56), we show some e ec o Zn in educing he se e i y o coli is, which occu ed bo h la e and o a lowe ex en han ha obse ed o he combined condi ion Zn+IL-10. In addi ion, he p o ec i e e ec o Zn ailed o o e come he exace ba ed coli is obse ed in mice ha did no espond o endogenous IL-10. The mechanis ic bases unde lying he p o ec ion con e ed by Zn alone emain unknown. One possibili y is ha Zn may con ibu e o diminish he amoun o ee adical species gene a ed du ing acu e coli is which con ibu e o p o ein, DNA chain and lipid damage (57). In any case, as IBD pa ien s o en p esen a Zn de i- ciency and espond well o Zn supplemen a ion he apy (58), he exploi a ion o combined IL-10 and Zn he apies may be wo h conside ing. In line wi h his, he bene i s o combined Zn and an i-TNF he apy we e p e iously desc ibed (55). The pMT-10 mouse model allows o local, as well as sys emic, IL-10 o e exp ession. The ac ha we de ec ed inc eased an- sc ip ion o he IL-10 ansgene in he BM and ele a ed le els o se ic IL-10 led us o hypo hesize ha p eexposu e o IL-10 migh induce long-las ing ansc ip ional changes in ci cula ing mono- cy es, o example h ough epigene ic imp in ing. I his we e he case, we migh be able o educa e hese cells o gain long-las ing ole ance o DSS-induced coli is. Ou da a ob ained a e a 1- o 3-week es pos -IL-10 exposu e show ha ecen IL-10 exposu e is equi ed o maximal p o ec ion. Thus, he p o ec i e e ec s o IL-10 we e no sus ained o e ime, implying ha IL-10 p esence a he ime o insul is necessa y o p e en coli is. The e o e, inducing IL-10 exp ession in ou mouse model a he beginning o disease would be o in e es . Un o una ely, we we e unable o explo e his possibili y because he Zn necessa y o ac i a e he ansgene p ecipi a es in he p esence o DSS, when bo h a e p o ided in he d inking wa e . In addi ion, we we e unable o induce high le els o IL-10 exp ession in he se um o mice ed wi h a Zn-en iched die and e hical issues p e en ed us om a emp ing o induce sus ained high le els o IL-10 by equen ga age o i.p. injec ions o Zn-con aining p epa a ions. In conclusion, we he ein p esen a no el mouse model o inducible IL-10 o e exp ession. We also show he po en ial o his model o he s udy o he IL-10 biology in he speci ic se ing o DSS-induced coli is. Ou da a u he suppo he p o ec i e ole o IL-10 in in es inal in lamma ion, showing ha his cy okine delays disease p og ession e en when deli e ed be o e DSS adminis a ion. Howe e , he e ec is no long-las ing, which calls o al e na i e app oaches o p e en IBD. eThics sTaTeMenT In Po ugal, all animal expe imen s we e pe o med in s ic acco dance wi h ecommenda ions o he Eu opean Union Di ec i e 2010/63/EU and p e iously app o ed by Po uguese 9 Ca doso e al. IL-10 Amelio a es DSS-Induced Coli is F on ie s in Immunology | www. on ie sin.o g Ma ch 2018 | Volume 9 | A icle 400 Na ional Au ho i y o Animal Heal h–Di eção Ge al de Alimen ação e Ve e iná ia (DGAV). Mice we e eu hanized by CO2 inhala ion wi h e o s o minimize su e ing. In F ance, all animal p ocedu es we e app o ed by he Pas eu Ins i u e Sa e y Commi ee and conduc ed acco ding o F ench and Eu opean Communi y Ins i u ional guidelines. Mice we e eu hanized by CO2 inhala ion wi h e o s o minimize su e ing. aUThO cOnT iBUTiOns AC, AM, and GC pe o med he expe imen s. AGC and PV made he pMT-10 mice. VM sequenced he genome o he pMT-10 mouse. AC, AM, AGC, IC, AC, PV, and MS planned he expe i- men s and analyzed da a. AC, AGC, PV, and MS w o e he a icle. acKnOWleDgMenTs We a e g a e ul o D . We ne Mülle (Facul y o Biology, Medicine and Heal h, Uni e si y o Manches e , Uni ed Kingdom) o p o iding he IL-10R-de icien mice. We also hank he excel- len suppo gi en by he animal house wo ke s a IBMC-i3S. FUnDing We acknowledge he Po uguese Founda ion o Science and Technology (FCT) o p o iding a PhD g an o AC (SFRH/ BD/84704/2012). This a icle is a esul o he p ojec No e-01- 0145-FEDER-000012—S uc u ed p og am on bioenginee ed he apies o in ec ious diseases and issue egene a ion, suppo ed by No e Po ugal Regional Ope a ional P og amme (NORTE 2020), unde he PORTUGAL 2020 Pa ne ship Ag eemen , h ough he Eu opean Regional De elopmen Fund (FEDER). The MS lab is also inanced by a FCT-ANR g an (FCTANR/ BIM-MEC/0007/2013). This wo k was also backed by he COST Ac ion BM1404 Eu opean Ne wo k o In es iga o s T igge ing Explo a o y Resea ch on Myeloid Regula o y Cells (h p://www. mye-euni e .eu), which is suppo ed by he Ho izon 2020—EU F amewo k P og am Resea ch and Inno a ion P og amme. MS is a FCT Associa e In es iga o . AGC lab: This wo k was de eloped unde he scope o he p ojec NORTE-01-0145- FEDER-000013, suppo ed by he No he n Po ugal Regional Ope a ional P og amme (NORTE 2020), unde he Po ugal 2020 Pa ne ship Ag eemen , h ough he Eu opean Regional De elopmen Fund (FEDER); by he p ojec NORTE-01-0145- FEDER-000023, suppo ed by he No he n Po ugal Regional Ope a ional P og amme (NORTE 2020), unde he Po ugal 2020 Pa ne ship Ag eemen , h ough FEDER; and by FEDER, h ough he Compe i i eness Fac o s Ope a ional P og amme (COMPETE), and by Na ional unds, h ough he Founda ion o Science and Technology (FCT), unde he scope o he p ojec POCI-01-0145-FEDER-007038. PV is unded by ANR, h ough he p ojec MYELOTEN (ANR-13-ISV1-0003-01). sUPPleMenTa Y MaTe ial The Supplemen a y Ma e ial o his a icle can be ound online a h p://www. on ie sin.o g/a icles/10.3389/ immu.2018.00400/ ull#supplemen a y-ma e ial. eFe ences 1. S obe W, Fuss I, Mannon P. The undamen al basis o in lamma o y bowel disease. J Clin In es (2007) 117(3):514–21. doi:10.1172/JCI30587 2. Cosnes J, Gowe -Rousseau C, Seksik P, Co o A. Epidemiology and na u al his o y o in lamma o y bowel diseases. Gas oen e ology (2011) 140(6):1785– 94. doi:10.1053/j.gas o.2011.01.055 3. Lo us EV J . Clinical epidemiology o in lamma o y bowel disease: inci- dence, p e alence, and en i onmen al in luences. Gas oen e ology (2004) 126(6):1504–17. doi:10.1053/j.gas o.2004.01.063 4. Shi ananda S, Lenna d-Jones J, Logan R, Fea N, P ice A, Ca pen e L, e al. Incidence o in lamma o y bowel disease ac oss Eu ope: is he e a di e ence be ween no h and sou h? Resul s o he Eu opean Collabo a i e S udy on In lamma o y Bowel Disease (EC-IBD). Gu (1996) 39(5):690–7. doi:10.1136/ gu .39.5.690 5. Schi bel A, Fiocchi C. In lamma o y bowel disease: es ablished and e ol - ing conside a ions on i s e iopa hogenesis and he apy. J Dig Dis (2010) 11(5):266–76. doi:10.1111/j.1751-2980.2010.00449.x 6. Xa ie RJ, Podolsky DK. Un a elling he pa hogenesis o in lamma o y bowel disease. Na u e (2007) 448(7152):427–34. doi:10.1038/na u e06005 7. Mowa AM, Agace WW. Regional specializa ion wi hin he in es inal immune sys em. Na Re Immunol (2014) 14(10):667–85. doi:10.1038/n i3738 8. Zigmond E, Jung S. In es inal mac ophages: well educa ed excep ions om he ule. T ends Immunol (2013) 34(4):162–8. doi:10.1016/j.i .2013.02.001 9. Bain CC, Sco CL, U onen-Hansson H, Gudjonsson S, Jansson O, G ip O, e  al. Residen and p o-in lamma o y mac ophages in he colon ep esen al e na i e con ex -dependen a es o he same Ly6Chi monocy e p ecu so s. Mucosal Immunol (2013) 6(3):498–510. doi:10.1038/mi.2012.89 10. Xu XR, Liu CQ, Feng BS, Liu ZJ. Dys egula ion o mucosal immune esponse in pa hogenesis o in lamma o y bowel disease. Wo ld J Gas oen e ol (2014) 20(12):3255–64. doi:10.3748/wjg. 20.i12.3255 11. Neu a h MF. Cy okines in in lamma o y bowel disease. Na Re Immunol (2014) 14(5):329–42. doi:10.1038/n i3661 12. Mon gome y SM, Mo is DL, Pounde RE, Wake ield AJ. Asian e hnic o igin and he isk o in lamma o y bowel disease. Eu J Gas oen e ol Hepa ol (1999) 11(5):543–6. doi:10.1097/00042737-199905000-00013 13. Ca I, Maybe y JF. The e ec s o mig a ion on ulce a i e coli is: a h ee-yea p ospec i e s udy among Eu opeans and i s - and second-gene a ion Sou h Asians in Leices e (1991–1994). Am J Gas oen e ol (1999) 94(10):2918–22. doi:10.1016/S0002-9270(99)00494-3 14. Helman J. Congeni al amilial achei ia associa ed wi h an in e en icula sep al de ec and di a ica ion o he uppe abdominal ec us muscles. S A Med J (1979) 56(23):982. 15. O holm M, Munkholm P, Langholz E, Nielsen OH, So ensen TI, Binde V. Familial occu ence o in lamma o y bowel disease. N Engl J Med (1991) 324(2):84–8. doi:10.1056/NEJM199101103240203 16. Kho B, Ga de A, Xa ie RJ. Gene ics and pa hogenesis o in lamma o y bowel disease. Na u e (2011) 474(7351):307–17. doi:10.1038/na u e10209 17. Van Limbe gen J, Rad o d-Smi h G, Sa sangi J. Ad ances in IBD gene ics. Na Re Gas oen e ol Hepa ol (2014) 11(6):372–85. doi:10.1038/n gas o.2014.27 18. Shih DQ, Ta gan SR, McGo e n D. Recen ad ances in IBD pa hogenesis: gene ics and immunobiology. Cu Gas oen e ol Rep (2008) 10(6):568–75. doi:10.1007/s11894-008-0104-x 19. Glocke EO, Ko la z D, Boz ug K, Ge z EM, Scha e AA, Noyan F, e al. In lamma o y bowel disease and mu a ions a ec ing he in e leukin-10 ecep o . N Engl J Med (2009) 361(21):2033–45. doi:10.1056/NEJMoa0907206 20. Shou al DS, Biswas A, Goe el JA, McCann K, Conaway E, Redhu NS, e al. In e leukin-10 ecep o signaling in inna e immune cells egula es mucosal immune ole ance and an i-in lamma o y mac ophage unc ion. Immuni y (2014) 40(5):706–19. doi:10.1016/j.immuni.2014.03.011 21. Ai hal GP, C aggs A, Day CP, Wel a e M, Daly AK, Mans ield JC, e  al. Role o polymo phisms in he in e leukin-10 gene in de e mining disease