Protective effect of TSLP delivered at the gut mucosa level by recombinant lactic acid bacteria in DSS induced colitis mouse model

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1 Aury et l. Micro Cell Fct (215) 14:176 DOI /s RESEARCH Open Access Protective effect of TSLP delivered t the gut mucos level y recominnt lctic cid cteri in DSS induced colitis mouse model Cmille Aury 1,2, Christophe Michon 1,2, Florin Chin 1,2, Yolnde Chvtchenko 3, Lurence Goffin 3, Simone C. Zimmerli 4, Sylvi Leguin 1,2, Philippe Lngell 1,2, Luis Bermudez Humrn 1,2 nd Jen Mrc Chtel 1,2 Astrct Bckground: Thymic stroml lymphopoietin (TSLP) is cytokine known to mture dendritics cells, lower pro-inflmmtory IL-12 secretion, induce differentition of nti-inflmmtory FoxP3+ regultory T cells (Treg). Moreover, Crohn s disese ptients hve shown reduction of intestinl TSLP expression. To understnd the role of TSLP in inflmmtion, we constructed Lctococcus lctis strin producing TSLP () nd investigted the effect of its dministrtion on dextrn sulfte sodium (DSS)-induced colitis model in mice. Results: secrete n ctive molecule which lowers secretion of IL-12 y dendritic cells. Tretment with, increses the mount of TGF-β secreted y T cells in Mesenteric Lymph Node in helthy mice. In cute DSS-induced colitis, delyed the Disese Activity Index nd lowered histologicl score nd colonic INF-γ production. In DSS-recovery model, induced etter protective effect if the strin ws dministered t the eginning of the colitis. At Dy 4 of colitis we oserved n induction of Treg y. Conclusions: TSLP showed n nti-inflmmtory protective role in DSS-induced colitis. We hve demonstrted tht short nd erly dministrtion of is more efficient thn long lsting tretment. Keywords: TSLP, Mucosl delivery, Inflmmtory owel disese, Lctococcus lctis Bckground TSLP ws first discovered in thymic stroml cell line [1]. It is minly produced y non-hemtopoietic cells s kertinocytes nd epithelil cells [2 4] in response to stress stimuli [5, 6]. TSLP is cytokine well known in the homeostsis of TH2 immune response [5]. It hs een implied in severl diseses like sthm, llergic rhinitis, food llergy [7 9] ut lso in cncer [1, 11]. However despite its key role in llergic response, TSLP hs een shown to hve protecting effects in mouse model of colitis [4, 12, 13]. Indeed, TSLP is implied in the differentition of CD4+CD25-thymocyte into FoxP3+ regultory T cells (Treg) y the intermedite of humn myeloid Dendritic Cells (DCs) or humn plsmocyte DCs [14, 15]. Correspondence: jen mrc.chtel@jouy.inr.fr Cmille Aury nd Christophe Michon uthors shre the first uthorship 2 AgroPrisTech, UMR Miclis, 7835 Jouy en Joss, Frnce Full list of uthor informtion is ville t the end of the rticle However, in mice, TSLP doesn t ffect DCs directly ut is secreted y those cells nd llows the promotion of Treg [13]. TSLP lso cts on humn nd murine DCs y inhiiting IL-12 secretion which is pro-inflmmtory protein involved in Inflmmtory Bowel Disese (IBD) [3, 4]. IBD which gthers Crohn s disese (CD) nd Ulcertive Colitis (UC) ffects 1.4 million Americns nd the prevlence rte is 396 per 1, individuls worldwide [16]. Incidence nd prevlence re incresing in vrious regions of the world including the ones which were less impcted [17, 18]. Due to its symptoms (dirrhe, dominl pin, loss of weigh) IBD is considered s n incpcitting disese. Ptients hve higher risk fctor to develop other inflmmtory or non-inflmmtory disorders like psorisis, cncer or rthritis [19 22]. So fr no curtive tretments exist for the disese. The most powerful tretment is the injection of the recominnt ntiodies trgeting TNF-α, however even if 6 % re primry responders 215 Aury et l. This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pulicdomin/zero/1./) pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Aury et l. Micro Cell Fct (215) 14:176 Pge 2 of 1 this drops to 25 4 % still in remission fter 1 yer of tretment [23]. The lst solution in IBD is surgery where inflmed prts of the intestine re withdrwn. However surgery cn led to severe complictions s Short Bowel syndrome nd relpses re frequent. All together, this mkes IBD one of the mjor helth prolems in developed country nd the development of innovtive therpeutics or curtive strtegies is crucil. One of the wys explored to help in lleviting symptoms of the disese is the delivery of nti-inflmmtory molecules y recominnt lctic cid cteri (LAB). Recently, it hs een shown tht mice treted with LAB expressing the protese inhiitor Elfin were protected ginst gut inflmmtion [24]. LAB hve een used for 1 yers for food conservtion nd pper to e promising vehicle delivering ctive molecules [25]. They re recognized s sfe y World Helth Orgniztion, nd some strins cn hve nti-inflmmtory properties [26]. In this study we decided to construct Lctococcus lctis expressing TSLP () in order to study the effect of locl gut mucosl dministrtion of TSLP on DSS-induced inflmmtion. constructs demonstrted nti-inflmmtory properties in vitro nd protected mice from DSS-induced colitis fter orl dministrtion s shown y reduced weight loss, lower disese ctivity nd microscopic score. Moreover mice were protected even if they were fed with only during the four first dys of the colitis. Additionlly we showed tht prt of this protective effect ws due to higher recruitment of Treg. Results Secretion of iologiclly ctive TSLP y recominnt Lctococcus lctis strin In order to study the role of TSLP in mucosl inflmmtion we constructed strin of L. lctis secreting TSLP (). To this end, we cloned the tslp gene in the plsmid plb333 crrying the SICE system, composed of the promoter of the stress-induced GroESL operon, the signl peptide of the well secreted L. lctis Exp4 protein nd termintor (Fig. 1), resulting in the plsmid pgroel-tslp. The strin hd the sme growth curve in rich culture medium s the wild type strin MG1363, (dt not shown). The ility of LL- TSLP to produce the cytokine ws tested in different stress conditions such s het shock nd slt stress. We oserved significnt (P <.1) 35 nd 84 % increse of TSLP secretion in presence of 1.5 % NCl nd 3 % NCl, respectively (Fig. 1). TSLP secretion is wekly ut significntly (P <.5) enhnced y het shock t 37 nd 4 C (Fig. 1c). Finlly, to vlidte the recominnt strin, we tested the iologicl ctivity of the secreted TSLP in LPS-stimulted-BMDCs model. After 24 h of LPS stimultion, we detected IL-12 secretion in BMDCs superntnts, which significntly decresed when cells received TSLP (either commercil recominnt or concentrted from superntnt), demonstrting iologicl ctivity of the recominnt cytokine (Fig. 1d). Equivlent mount of irrelevnt protein lso lowers IL-12 secretion, even though significntly different from concentrted ddition. We thus vlidted the secretion of iologiclly ctive TSLP cytokine y recominnt L. lctis strin. Orl dministrtion of LL TSLP induced TGF β secretion y ctivted cells from mesenteric lymph node of helthy mice To ssess the sl effects of gut mucosl dministrtion of TSLP on mice, two groups (n = 8) of helthy nimls received, or y orl route. Weight nd DAI were dily monitored nd scored. We did not oserve differences in these scores, showing no chnges in the physiology of mice (dt not shown). After 14 dys of tretment, mesenteric lymph nodes (MLN) were removed nd cells were ctivted with nti-cd3 nd nti- CD-28 ntiodies. We detected significntly (P <.5) higher secretion of TGF-β when mice received compre to mice orlly dosed with (Fig. 2). We did not oserve ny significnt chnges in IL-5, IFN-γ or IL-17 concentrtions in cell superntnts (Fig. 2 d). No differences hve een seen on IL-1 either (dt not shown). TSLP delivery through recominnt L. lctis in the intestinl lumen is le to trigger TGF-β secretion. LL TSLP reduce cute inflmmtion To determine the impct of locl dministrtion on intestinl inflmmtion, we first performed n cute DSS-induced colitis model on mice tht we orlly dministered with or 7 dys efore nd during colitis induction. We did not oserve difference in the weight loss of the two groups of mice (Fig. 3). Orl dministrtion of significntly decresed the DAI t D4, showing tht TSLP-secreted L. lctis delyed clinicl signs of colitis (Fig. 3), especilly feces softening nd leeding. After 7 dys of inflmmtion, colon tissues were removed nd severl inflmmtion mrkers were nlyzed. Histologicl score ws reduced in presence of TSLP (Fig. 3c, d) demonstrting n intestinl epithelil protection y orl dministrtion of. The concentrtion of the pro-inflmmtory cytokine IFN-γ in colon wshes ws lso decresed fter orl tretment with (Fig. 3e). We did not detect ny differences in the concentrtion of the pro-inflmmtory IL-12 nd the nti-inflmmtory IL-1 in these colon wshes (dt not shown). We lso oserved n increse ut not significnt (p =.53) of TGF-β in the superntnt of ctivted

3 Aury et l. Micro Cell Fct (215) 14:176 Pge 3 of 1 pgroesl SPExp4 tslp Ter pg/ml/od ct c pg/ml/od ct % 1.5% 3% 3 C 37 C 4 C d IL-12 (µg/ml) 5 Unstimulted rtslp 4 concentrted TSLP equivlent protein mount No LPS 1 1 ±1 TSLP (ng/ml) LPS (5 ng/ml) Fig. 1 Secretion of iologiclly ctive TSLP y recominnt Lctococcus lctis. Schemtic representtion of the expression cssette: GroESL promoter (pgroesl) followed y tslp gene, flnked y the signl peptide (SP) of Exp4 nd termintor (Ter). Detection of TSLP y ELISA in superntnt frctions from NCl-induced cultures or c het-shock-induced cultures. d Detection of IL-12 y ELISA in LPS-induced-BMDCs superntnts co-incuted with commercil recominnt TSLP (rtslp) ( or 1 ng/ml), recominnt TSLP produced y cultures (concentrted TSLP) (1 ng/ml) or with equivlent mount of protein from concentrted superntnt LL-NUC cultures (negtive control). Sttisticlly significnt differences (P <.5, P <.1) cells from MLNs. No differences were detected in IL-5, IL-17 or IL-22 concentrtions in the superntnt of ctivted cells from MLNs etween the two conditions (dt not shown). LL TSLP decresed Disese Activity Index ut not weight loss in DSS recovery phse model In order to test the involvement of TSLP in the heling process, we performed n cute inflmmtion experiment followed y recovery phse consisting of 5 dys of wter. Two groups of mice were treted 7 dys efore colitis, long the inflmmtion s well s the recovery period with or. Orl TSLP dministrtion did not modify the weight loss, which ws round mximum of 2 %, etween the two group of mice (Fig. 4) ut significntly decresed the Disese Activity Index (DAI) t the erly phse of the inflmmtion (Fig. 4), s seen previously, suggesting tht TSLP hd no effect in lte inflmmtion nd recovery phse. LL TSLP delivery in the erly phse of inflmmtion diminished the loss weight nd the DAI To vlidte the effect of TSLP on the erly phse of colitis, we performed n cute inflmmtion followed y recovery phse on groups of mice treted with, nd group nmed erly phse, corresponding to

4 Aury et l. Micro Cell Fct (215) 14:176 Pge 4 of NS TGF- (pg/ml) 2 1 IL-5 (pg/ml) c 4 NS d 2 NS IFN (pg/ml) IL-17 (pg/ml) Fig. 2 Orl dministrtion of induced TGF β secretion. Mice were orlly dministered during 5 dys consecutively with or LL-wt during 5 dys consecutively nd then scrificed. Concentrtions of TGF-β (), IL-5 (), IFN-γ (c) nd IL-17 (d) were mesured in superntnts of nti-cd3 nd nti-cd-28 ctivted cells from MLN n orl dministrtion of from D7 to D4 followed y orl dministrtion of from D5 to D12 (Fig. 5). As previously shown, the difference in weight loss etween nd conditions ws not significnt (dt not shown). We oserved reduction of the weight loss when mice received erly TSLP delivery, which ws significntly different t D8, D9, D11 nd D12 compred to the condition (Fig. 5). Furthermore we oserved reduced increse of DAI in the erly phse group, with significnt differences t D5 nd D6 compred to the DAI (Fig. 5c). Histologicl scores were significntly reduced in the erly phse group compred to the group ut not in the group (Fig. 5d). At D12, cells from MLN were ctivted ut we did not detect ny differences in TGF-β secretion in these cell superntnts etween the three cteril tretments (Fig. 5e). However, we did notice significnt (P <.1) decrese of IL-17 secretion with dministrtion compre to or erly phse (Fig. 5f). These results demonstrted decrese/meliortion of some colitis symptoms even when TSLP ws delivered t the erly phse of the inflmmtion. TSLP induce Treg prolifertion in the erly phse of the colitis In order to understnd the effect of TSLP on the erly phse of colitis we nlyzed the Treg proportion in MLN t dy 4 nd dy 12 of colitis. The percentge of CD25+ FoxP3 Treg mong the CD4+ popultion ws significntly higher when mice were fed with compred to control t dy 4 (Fig. 6). This difference in line with the differences in DAI scores oserved etween treted mice nd t D4 (dt not shown). No difference in percentge of CD25+ FoxP3 Treg mong CD4+ popultion ws oserved t dy 12 mong the three groups (Fig. 6). Discussion In this study, effects of gut mucosl dministrtion of TSLP in tretment for colitis hve een investigted using the recominnt L. lctis strin LL TSLP Three min studies hve shown n importnt role of TSLP in DSS-induced colitis using two knock-out mouse models, where TSLP or the TSLP receptor (TSLPR) gene ws deleted. Tylor et l. demonstrted tht TSLPR / mice were more sensitive to DSS-induced colitis compred to the WT nimls [2]. TSLPR / mice displyed higher pthologicl score, from the first dy of DSS tretment, nd colonic shortening. These mcroscopic criteri were correlted with n increse of IFN-γ producing cells within MLNs nd IL-12/23p4 secretion in colonic tissue. In contrst, Rerdon et l. hve shown tht the lck of TSLP does not led to n enhncement of the colitis severity in DSS model ut to n sence of recovery

5 Aury et l. Micro Cell Fct (215) 14:176 Pge 5 of 1 Percentge ini l weight (%) c Histologicl score dys DAI (riitrty units) d dys Without coli s e 2 f 2 NS (p=.53) IFN- (pg/ml) TGF- (pg/ml) Fig. 3 Effects of orl dministrtion of on cute DSS-induced inflmmtion in mice. Mice were orlly dosed with or 5 dys prior DSS tretment until the end of study. Percentge initil weight () nd Disese Activity Index () were monitored during the whole DSS tretment period. c Histologicl score of colon segment, d colon histology, e IFN-γ in colon wshes nd f TGF-β concentrtions in superntnts of ctivted cells from MLN fter the inflmmtion, leding to the deth of the mice [27]. During colitis, immune cells re recruited including neutrophils tht relese high mount of neutrophil elstse (NE). A lnce etween proteolytic enzymes nd their inhiitors is very importnt in the recovery phse. They oserved tht TSLP / mice hve incresed NE ctivity nd in prllel, disply diminution of the secretory leukocyte peptidse inhiitor (SLPI), n endogenous inhiitor of NE. Moreover, mortlity rte in DSS TSLP / mice ws reduced y n intrperitonel rslpi tretment. The third study hs lso demonstrted protective role of TSLP during DSS-induced colitis followed y recovery phse [13]. In sme mnner, the lck of TSLP receptor incresed the severity of the colitis monitored y rise of weight loss, DAI nd histologicl scores. Moreover, TSLPR / hd higher percentges of Th17 cells nd Th1 cells in colonic tissue. Although there re differences in the hypotheses nd explntions, ll these dt show n importnt role of TSLP in the protection of epithelil integrity nd colitis reduction. In order to further understnd the potentil protective effect of TSLP on inflmmtion, we hve developed strtegy for TSLP delivery to the gut mucosl level y orl dministrtion of LAB producing solule functionl TSLP. LAB hve een used since mny yers now s vector for protein or DNA delivery t the mucosl level [25]. We constructed nd chrcterized L. lctis strin producing TSLP,. After 2 weeks of orl

6 Aury et l. Micro Cell Fct (215) 14:176 Pge 6 of 1 Percentge initil weight (%) DAI (Aritrry Unit) DSS 2.5% dys dys Fig. 4 Effects of orl dministrtion of on DSS-recovery colitis model in mice. Mice were orlly dosed with or 5 dys prior DSS tretment until the end of study. After DSS tretment mice were llowed to recover for 5 dys. Percentge initil weight () nd DAI () were monitored during the DSS-induced colitis phse s well s during the following 5 dys of recovery dministrtion in helthy mice we oserved n increse of TGF-β production y nti-cd3/nti-cd28 stimulted cells from mesenteric lymph nodes. In n cute DSSinduced inflmmtion model, we showed tht fter 7 dys of DSS, the DAI of mice treted with tends to e lower during the 7 dys of inflmmtion, despite sence of chnges in weight loss. We oserved significnt reduction of this score t D4, demonstrting the cpcity of to dely clinicl signs t the eginning of colitis, especilly feces softening nd leeding. Furthermore we showed tht colonic tissue integrity mesured y histologicl scores is less compromised within TSLP treted mice. Orl dministrtion of reduced the secretion of the pro-inflmmtory cytokine, IFN-γ, showing tht LAB-secreted TSLP protects the intestinl epithelium from dmges induced y chemicl tretment nd modultes inflmmtion. To ssess the effect of during the recovery phse, we performed n cute colitis model followed y 5 dys of remission. TSLP ws delivered y ll long the experiment (inflmmtion + recovery phse). We did not show ny differences in weight loss nd histologicl scores fter 5 dys of wter ut we confirmed the decrese of DAI in the erly phse of inflmmtion. The recovery phse is complex process nd ddition of TSLP seems not to e sufficient to ccelerte the decrese of mrkers of inflmmtion or intestinl epithelium repir. Next, we hypothesized tht erly tretment with LL- TSLP could e sufficient to decrese inflmmtion mrkers. A group of mice received during 7 dys efore nd 4 dys fter the induction of colitis followed y until the end of the experiment. TSLP delivery in the lumen t erly phse, until D4, diminished the weight loss nd significntly incresed the weight gin t D8, D9, D11 nd D12 compre to the. Moreover it delyed nd decresed the DAI (significntly t D5 nd D6) nd reduced the histologicl score. Therefore we conclude tht, short nd erly TSLP tretment llowed etter protection ginst colitis thn longer tretment s demonstrted y lower severity s well s dely in the disese. To decipher y which mechnisms ddition of TSLP leds to the colon protection in the erly phse of the inflmmtion, we scrificed the mice t D4. At this time we oserved higher percentge of CD4+CD25+Foxp3+ cells in mice treted with LL- TSLP, suggesting role of Treg cells in the dely of the outrek of the disese. In humn, TSLP-mtured DC re le to induce the expnsion nd the differentition of CD4+CD25+Foxp3+ cells [14, 15]. However, Iliev et l. hve demonstrted tht the retinoic cid nd TGF-β ut not TSLP re essentil to convert DC in tolerogenic DC, which induce the Treg differentition [28] nd led to the protection of mice from colitis. In prllel, TSLP hs een descried to e secreted y DC [13, 29]. DC-derived TSLP cn directly ct on Tcell y inhiiting TH17 cell development nd promoting Treg cells [13]. TSLP ws lso descried to e essentil for gut homeostsis nd TSLP-TSLPR signling elicits the expnsion of Foxp3+ helios-treg cells in response to intestinl cteri [3]. We hypothesized tht ddition of TSLP to the lumen llows n enhncement of gut homeostsis y rise of the numer of Treg cells which leds to dely of the disese. Relese of TSLP could ct directly s descried y Spdoni et l. on Treg differentition or indirectly. Indeed, TSLP is le to reinforce tight-junctions of lung epithelil cells y incresing severl cludins nd the occludin [31]. In this mnner, TSLP could protect gut epithelil integrity nd increse the relese of Retinoic cid nd TGF-β y epithelil cells s well s the Treg expnsion. Finlly, TSLP expression is reduced in colonic tissue of Crohn s disese ptients [32] nd cn e correlted to

7 Aury et l. Micro Cell Fct (215) 14:176 Pge 7 of 1 Percentge ini l weight (%) DAI (Aritrry Unit) D-7 c 1 erly phse D D4 D7 D12 (D-7 to D12) (D-7 tod4) (D5 to D12) DSS 2.5% dys dys : : erly phse erly phse d Histologicl scores e IL-17 (pg/ml) TGF- (pg/ml) f erly phse erly phse erly phse Fig. 5 Short nd erly dministrtion reduced inflmmtion during DSS-induced colitis. Schemtic representtion of cteril dministrtion protocol. Percentge initil weight. c DAI of mice treted with or LL TSLP erly phse. d Histologicl score. e TGF-β nd f IL-17 concentrtions in superntnts of nti-cd3 nd nti-cd-28 ctivted cells from MLN % of CD25+ FoxP3 mong CD Dy 4 % of CD25+ FoxP3 mong CD Dy 12 Phse 1 Fig. 6 Action of fter 4 dys of colitis. Percentge of CD25+FoxP3+ mong CD4+ popultion in MLN t D4 () nd D12 ()

8 Aury et l. Micro Cell Fct (215) 14:176 Pge 8 of 1 the filure of these ptients to promote tolerogenic DCs in the gut [4, 28]. TSLP secretion y intestinl epithelil cells is dependnt nd regulted y commensl nd proiotic cteri [3, 33, 34]. A novel tretment ginst Crohn disese is fecl trnsplnttion. In the future, it could e very interesting to trget fecl trnsplnt tht restore TSLP expression or complete ctul tretment with proiotics tht re le to increse TSLP secretion y epithelil cells to promote gut homeostsis nd longer remission periods. Conclusion Thymic stroml lymphopoietin showed n nti-inflmmtory protective role in DSS-induced colitis. We hve demonstrted tht short nd erly dministrtion of is more efficient thn long lsting tretment. We hypothesized tht TSLP induced TGF-β secretion which thus will increse the Treg popultion. Methods Bcteril strins nd growth conditions Lctococcus lctis MG1363 contining pnis-empty plsmid () nd L. lctis MG1363 contining pgroesl-tslp plsmid () were grown in M17 medium (Difco) supplemented with 1 % glucose nd chlormphenicol (1 µg/ml) t 3 C without gittion. psec plsmid nd pgroesl re oth derivtive of the rod-host rnge plsmid pwv1 [35]. Plsmid construction Plsmid contining murine tslp gene ws synthesized y Genert (Invitrogen). After digestion y BmHI/SpeI, the frgment contining the gene of interest ws integrted into BmHI/SpeI digested pgroesl plsmid (chlormphenicol resistnt, gene expressed under P groesl, flnking y signl peptide from USP45 nd termintor) from SICE system [36]. Construction ws estlished y electroportion into L. lctis MG1363 t 2.4 kv, 2 Ω, 25 µf. Trnsformnts were selected t 3 C on M17 gr contining 1 % glucose nd chlormphenicol (1 µg/ml). pgroesl-tslp plsmids were extrcted from recominnt trnsformnts nd verified y sequencing. Stress inducing cytokine secretion y L. lctis Overnight cultures of TSLP-secreting L. lctis strin ws diluted in growth medium to.1 OD 6nm nd incute t 3 C without gittion until.4.6 OD 6nm. Then different stresses were dded s following: To induce slt stress, different volumes of NCl 5M solution were dded into culture to otin 1, 5 nd 3 % NCl finl concentrtion nd incute t 3 C without gittion. To provoke het-shock, cteril cultures were centrifuged t room temperture t 47 rpm during 15 min. Pellets were resuspended with pre-wrmed culture medium t 3, 37 nd 4 C nd incute t these different tempertures without gittion. Four hours fter stress, 1 ml of cteril cultures ws hrvested nd centrifuged t 4 C t 1, rpm during 1 min. The 2 µm filtered superntnts were conserved t 2 C for cytokine quntifiction y ELISA (R&D systems). Isoltion nd culture of one mrrow derived dendritic cells (BMDC) Bone mrrow cells from BALB/c mice were hrvested septiclly nd plted into petri dish in RPMI 164 (Life Technologies) supplemented with 1 % decomplemented fetl ovine serum (FBS), penicillin/streptomycin, β-mercptoethnol 5 mm nd 2 ng/ml GMCSF (peprotech) nd cultured t 37 C in 1 % CO 2 -humidified incutor. Fifteen ml of medium were dded t dy 3 nd completely chnged t dy 5; cells were hrvest t dy 7. BMDCs were then plted t cells/well (96 wells/ plte) nd cultured in RPMI 164 supplemented with 1 % decomplemented FBS nd penicillin/streptomycin t 37 C in 1 % CO 2 -humidified incutor. LPS stimulted BMDC ssy BMDCs were stimulted in presence (5 ng/ml) or sence of LPS nd with recominnt TSLP (Biolegend or from concentrted superntnts) t 1 ng/ ml. A negtive control of the culture medium (filtered superntnt of L. lctis hroring plsmid encoding for non-relevnt protein, the nuclese Nuc) ws used nd equivlent protein mount corresponding to concentrtions used with concentrted TSLP ws dded. Twenty-four hours fter stimultion, cells superntnts were hrvested for IL-12 quntifiction y ELISA (mtech). Mice experiments After cclimtiztion during t lest 7 dys, 6 weeks old C57BL/6 mice were fed dily during the whole experiment with PBS or with 1 9 ~ Colony Forming Units of or. At D colitis ws induced y dding 2.5 % (w/v) of Dextrn Sulfte Sodium Slt (DSS) t moleculr weight of 36, 5, (MPBio) to the drinking wter for 4 dys (DSS short) or 7 dys (DSS cute nd DSS recovery). The mice were scrificed either t D4 (DSS short), D7 (DSS cute) or D12 (DSS recovery) fter the DSS induction. For DSS recovery, DSS colitis induction ws followed y 5 dys of recovery with norml drinking wter. As control DSS mice hve een fed during 12 dys without DSS induction. Mice were monitored dily for weight loss, stool consistency, nd fecl occult lood (Hemoccult, Beckmn Coulter). Disese Activity Index (DAI) hs een

9 Aury et l. Micro Cell Fct (215) 14:176 Pge 9 of 1 clculted ccording to the protocol estlished y Cooper et l. [37]. Mice hve een scrificed y cervicl disloction nd mesenteric lymphtic node (MLN) s well s colon hve een hrvested for colon wshes, protein extrction nd histologicl ssessment. Interleukin detection in colon wshes, solule protein extrcts from colonic tissues nd superntnt from induced lymphocyte The colon ws removed nd wshed with 1 ml of PBS supplemented with nti-protese to otin the colon wsh. Then the colon ws opened nd 1 cm ws removed. The slice ws grinded in 1 ml of PBS supplemented with nti-protese using GentleMcs (Miltenyi) then centrifuged 2 min t 5g. Superntnt ws stored t 2 C efore further nlysis. MLN isolted from mice were mshed nd filtered (7 µm, BD iosciences). Lymphocytes in filtrte were count y flow cytometry (Accuri C6) nd resuspended in culture medium (RPMI, Lonz) with 1 Unit of Streptomicin Penicilin, PAA Lortories nd 1 % Fetl Clf Serum (FCS) (Lonz) t cells/ml. Cell solutions were dded to 24 well pltes (Costr) pre-incuted 4 h with nti-cd3 nd nti-cd28 ntiodies, 4 µg/ml of ech ntiody (ebioscience) in PBS with.5 % FCS. Pltes were incuted 48 h t 37 C 5 % of CO 2 nd cytokine levels were ssessed in superntnt. Totl mount of proteins from the different smples ws determined using Brdford ssy (Sigm-Aldrich) nd the cytokine concentrtion (IL-1, IL-12, IFN-γ, TGF-β, IL-17, IL-22 nd IL-5) y ELISA (Mtech). The concentrtion of ech cytokine is normlized on the totl proteins mount of ech smples. Histologicl ssessment For histologicl ssessment, colon smple ws fixed in 4 % prformldehyde cid (sigm) nd emedded in prffin. Four micrometer sections were stined with hemtoxylin/eosin nd exmined lindly [12]. Regultory T cells (Treg) numertion 1 6 cells hve een tken from mshed MLN filtrtes. Treg cells hve een stined for CD4, CD32 nd FoxP3 using mouse regultory T cell Stining Kit 1 (ebioscience). Cell smples hve een run through flow cytometry (BD Accuri) nd doule positive cells for CD32 nd FoxP3 mong CD4 positive cells hve een counted. Sttistic All sttistics nd grphics hve een performed on Prism-GrphPd. Results represent men ± SEM. Sttisticl significnce ws determined y the Mnn Withney test for chrts nd y 2-wy Anov with Bonferroni post-test for curves P <.5, P <.1, P <.1. Ethicl sttement All niml ssys were performed following the Europen Guidelines for the Cre nd Use of Lortory Animls. Authoriztion numer: Authors contriutions CA nd CM performed ll the lortory experiments nd drfted the pper. FC nd SL prticipted ctively to ll the niml experiments. YC, LG, SCZ PL, LB, JMC defined the reserch theme, helped to orient the work nd revised the mnuscript. JMC designed the project, coordinted it, wrote nd revised the mnuscript. All uthors red nd pproved the finl mnuscript. 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