Donor-derived IL-17A and IL-17F deficiency triggers Th1 allo-responses and increases gut leakage during acute GVHD.

RESEARCH ARTICLE Dono -de i ed IL-17A and IL-17F de iciency igge s Th1 allo- esponses and inc eases gu leakage du ing acu e GVHD I an OdakID 1☯ , Alina Depka -Jakob 1☯ , Maleen Beck 2 , Michael Ja ek 3 , Yan Yu 4 , U sula Seidle 4 , Sascha Da id 5 , A nold Ganse 2 , Reinhold Fo ¨ s e 1 , Immo P inz 1☯ , Ch is ian KoeneckeID 1,2☯ * 1Ins i u e o Immunology, Hanno e Medical School, Hanno e , Ge many, 2Depa men o Hema ology, Hemos asis, Oncology and S em-Cell T ansplan a ion, Hanno e Medical School, Hanno e , Ge many, 3Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many, 4Depa men o Gas oen e ology, Hepa ology and Endoc inology, Hanno e Medical School, Hanno e , Ge many, 5Depa men o Neph ology, Hanno e Medical School, Hanno e , Ge many ☯These au ho s con ibu ed equally o his wo k. *koenecke.ch is ian@mh-hanno e .de Abs ac IL-17A and IL-17F cy okines a e impo an egula o s o acu e g a - e sus-hos -disease (GVHD). Howe e , con a y e ec s o hese cy okines in in lamma o y diseases ha e been epo ed. To in es iga e he e ec s o dono -de i ed IL-17A and IL-17F on GVHD, we made use o single (Il17a -/- o Il17 -/- ) and double de icien (Il17a -/- ) allogeneic dono CD4 + T cells. We could demons a e ha ansplan a ion o Il17a -/- CD4 + dono T cells led o agg a a ed GVHD. Howe e , his pheno ype was no obse ed a e ansplan a ion o single, Il17a -/- o Il17 -/- , de icien CD4 + T cells, sugges ing edundan e ec s o IL-17A and IL-17F. Mo eo e , Il17a -/- cell ecipien s showed an inc ease o sys emic IFNγ, indica ing a heigh ened p o- in lamma o y s a e, as well as in il a ion o IFNγ-sec e ing CD4 + T cells in he ecipien s’ in es- inal ac . These ecipien s exhibi ed signi ican gu leakage, and ma kedly mac ophage in il- a ion in he gas oin es inal epi helial laye . Mo eo e , we saw e idence o impai ed eco e y o gu epi helial cells in ecipien s o Il17a -/- CD4 + T cells. In his s udy, we show ha IL-17A/F double de iciency o dono CD4 + T cells leads o accele a ed GVHD and he e o e highligh he impo ance o hese cy okines. Toge he , IL-17 cy okines migh se e as a b ake o an in ensi ied Th1 esponse, leading o he exace ba ed gu damage in acu e GVHD. In oduc ion Acu e G a - e sus-Hos disease (GVHD) is s ill a majo cause o non- elapse- ela ed mo al- i y a e allogeneic hema opoie ic s em-cell o bone ma ow ansplan a ion (BMT) [1]. The cu en s anda d o ca e o highe g ade GVHD is he sys emic use o s e oids. Fu he he a- peu ic op ions, especially o s e oid- e ac o y GVHD a e spa se [2]. The e o e, iden i ica ion o new he apeu ic a ge s bo h o p ophylaxis and ea men o GVHD a e needed. Alloge- neic dono lymphocy es induce and o ches a e his highly in lamma o y disease in he lym- pho-hema opoie ic compa men and in GVHD a ge o gans, espec i ely. In pa icula PLOS ONE PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0231222 Ap il 6, 2020 1 / 16 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Ci a ion: Odak I, Depka -Jakob A, Beck M, Ja ek M, Yu Y, Seidle U, e al. (2020) Dono -de i ed IL-17A and IL-17F de iciency igge s Th1 allo- esponses and inc eases gu leakage du ing acu e GVHD. PLoS ONE 15(4): e0231222. h ps://doi.o g/ 10.1371/jou nal.pone.0231222 Edi o : Pie e Bobe ´, Uni e si e Pa is-Sud, FRANCE Recei ed: Decembe 10, 2019 Accep ed: Ma ch 18, 2020 Published: Ap il 6, 2020 Copy igh : ©2020 Odak e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y S a emen : All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. Funding: This wo k was suppo ed by Deu sche Fo schungsgemeinscha : SFB738/A8 o C.K. and SFB900/B8 o C.K. and I.P. The unde had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing in e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . CD4 + T cells show a high deg ee o plas ici y in he cou se o he disease [3]. Func ional oles o Th1, Th2 and egula o y T cells (T egs) a e well known in GVHD [4,5]. Howe e , he exac ole o IL-17 and Th17 cell esponses in acu e GVHD is less clea . The subse o CD4 + T cells e med Th17 cells is cha ac e ized by p oduc ion o i s signa u e cy okine IL-17A. Howe e , he IL-17 cy okine amily comp ises IL-17A, IL-17B, IL-17C, IL- 17D, IL-17E and IL-17F, all ha ing a simila p o ein s uc u e and sha ing be ween 62% o 88% o homology o mu ine o human [6]. The co esponding IL-17 ecep o amily consis s o i e membe s, IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE. IL-17RA o ms a he e odime wi h IL-17RC, which oge he binds IL-17A dime s, IL-17F dime s, as well as IL-17A:IL17F he - e odime s [7,8]. IL-17A and IL-17F sha e 55% homology on he amino acid le el, and a e syn e- nic bo h in mice and humans [9]. Bo h cy okines a e in ol ed in an i- ungal, bac e ial and alle gic immune esponses [10,11]. Howe e , despi e he appa en simila i ies, he e is e idence o dis inc oles o he wo cy okines in immuni y [12]. Depending on he expe imen al model, IL-17 cy okines IL-17A and IL-17F may exe ei he pa hogenic o p o ec i e e ec s, e.g. p o- mo ing espi a o y alle gy [11] o media ing p o ec ion in neph i is [13]. To da e, Janus-head oles aken by Th17 and associa ed cy okines such as IL-17A and IL-22 du ing acu e GVHD ha e been documen ed [14] In one s udy, IL-17A de iciency led o disease educ ion [15], whe eas ano he s udy showed ha he absence o IL-17A- sec e ing cells exace ba ed GVHD [16]. Howe e , expe imen al se ups and GVHD models di e ed in hose s udies. IL-17A is p o- posed o exe a p o ec i e ole du ing gu -in lamma ion by limi ing excessi e pe meabili y and he eby main aining ba ie in eg i y [17,18]. Ano he p o ec i e ole in coli is model has been a ibu ed o IL-17A by o cing he exp ession o Th1- associa ed esponses [19]. Since excessi e endo helial and epi helial pe meabili y is one o he p e equisi es o acu e GVHD [20], we hypo hesized ha dono -de i ed IL-17 cy okines exe a p o ec i e ole in acu e GVHD. In his s udy, we dissec he ole o dono -de i ed IL-17A and IL-17F o endo helial and epi helial pe meabili y in an expe imen al acu e GVHD model using single- (Il17a -/- ,Il17 -/- ) and double-de icien (Il17a -/- ) dono T cells. Ou esul s show a p o ec i e ole o mu ually edundan dono -de i ed IL-17 cy okines IL-17A and IL-17F. We u he demons a e ha inc eased gu leakage and mac ophage in il a ion occu s when dono -de i ed IL-17A and IL- 17F a e absen . Ou esul s sugges ha dono -de i ed IL-17 migh con ibu e o p o ec ion o he in es inal ba ie du ing acu e GVHD. Ma e ial and me hods Animals Wild ype (WT) C57BL/6 Thy1.2 (BL6, H-2K b ), BALB/c (H-2K d ), B6xDBA2 F1 (BDF1, H-2K bxd ) mice we e ob ained om Cha les Ri e Labo a o ies (Sulz eld, Ge many). B6.129P2-Il17a m1Yiw (Il17a –/– ) mice we e kindly p o ided by Y. Iwaku a (The Uni e si y o Tokyo, Bunkyō-ku, Japan) B6.129S6-Il17 m1Awai (Il17 –/– ) mice we e kindly p o ided by B. Beche (Uni e si y o Zu¨ ich, Zu¨ ich, Swi ze land). WT C57BL/6 Thy1.1, Il17a –/– ,Il17 –/– and Il7a -/- (C57BL/6J-Il17a/ Il17 m1Imp ) we e b ed a he cen al animal acili y o Hanno e Medical School unde spe- ci ic pa hogen- ee condi ions. All animal expe imen s we e ca ied ou in acco dance wi h ins i- u ional and go e nmen al di ec i es and we e app o ed by Niede sa¨chsisches Landesam u¨ Ve b auche schu z und Lebensmi elsiche hei (pe mi numbe : 33.14-42502-04-11/0619 and 33.19-42502-04-14/1660). Bone ma ow ansplan a ion and GVHD induc ion Fo BMT and GVHD-induc ion in he C57BL/6!BALB/c model, 8–10 weeks old BALB/c ecipien s ecei ed le hal i adia ion wi h 8 Gy om a Cs γ-sou ce. Dono cells we e PLOS ONE IL-17A and IL17F and acu e GVHD PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0231222 Ap il 6, 2020 2 / 16 ansplan ed wi hin 24 hou s a e i adia ion. All ecipien mice ecei ed 3.0–5.0x10 6 T cell- deple ed bone ma ow (TCD BM) C57BL/6 o BALB/c BM cells and 0.5x10 6 CD4 + T cells om C57BL/6 WT, Il17a –/– ,Il17 –/– o Il17a –/– mice. Single-cell suspensions we e p epa ed om pe iphe al lymph nodes (pLN) and spleen and en iched ia magne ic mic obeads (MACS, CD4 + T cell isola ion ki ; Mil enyi Bio ec, Be gisch-Gladbach, Ge many). BM cells we e ha es ed om he emu s and ibias o dono mice. BM cells we e s ained wi h bio iny- la ed an i-CD3 (clone 17A2, homemade) and sepa a ed ia s ep a idin-conjuga ed magne ic beads (Mil enyi Bio ec, Be gisch-Gladbach, Ge many) in o de o deple e T cells. A e ans- plan a ion, mice we e kep on an ibio ic wa e (Co imoxazol; Ra iopha m, Ulm, Ge many) un il he end o he expe imen . Su i al, weigh loss and clinical GVHD-signs o ecipien mice we e moni o ed and sco ed acco ding o Cooke e al [21]. Clinical signs o acu e G HD, such as u led u , weigh loss (mild >10% o ini ial body weigh ; se e e >25% o ini ial body weigh ), hunched back, inac i i y and dia hea, we e moni o ed wo imes pe day. Se e i y o each clinical sign was sco ed (no = 0; mild = 1; se e e = 2) and animals wi h a o al sco e o �6 we e sac i iced immedia ely by ce ical disloca ion and coun ed as GVHD le hali y. All ans- plan ed mice we e p o ided wi h mois ened ood o allow easie eeding and aid hyd a ion. Hanno e Medical School p o ided he esea ch s a wi h special aining in animal handling. Despi e equen moni o ing, occasionally, mice we e ound dead wi hou clinical signs o GVHD wi hin he i s 10 days a e ansplan a ion. This was limi ed o ewe han 5% o mice in ol ed in he s udy and was conside ed as non-GVHD mo ali y, and hose mice we e excluded om he inal analysis. Cell p oli e a ion assay P oli e a ion o Il17a –/– o WT T cells a e unspeci ic s imula ion wi h CD3/CD28 beads (The moFishe , Schwe e, Ge many) o allogeneic BM-de i ed DCs was de e mined by 3 H-Thymidine up ake as ollows: 5,000 cells we e incuba ed in a 96-well pla e wi h 150μl RPMI medium supplemen ed wi h 10% FCS, 1% L-glu amine, 1% Pen-S ep and 0.04% gen a- mycine. The cells we e incuba ed o wo days in 95% humidi ied a mosphe e by 5% CO 2 a 37˚C, be o e 0.8 mCi 3 H-Thymidine (Ha man) was added pe well. The inco po a ion o adioac i e hymidine was measu ed 16h la e in a Mic obe a wo ks a ion (Pe kin Elma ). B dU p oli e a ion assay Day 20 a e BMT, mice we e i.p. injec ed wi h 3mg 5-B omo-20-deoxyu idine (B dU) (Sigma- Ald ich) and se on 0.8mg/ml B dU con aining wa e o e nigh . A e 24h, mice we e sac i- iced 24h and SI and colon we e ixed in 4% o maline (Sigma-Ald ich) o e nigh and p o- cessed as desc ibed abo e. De ec ion o inco po a ed B dU was pe o med wi h he B dU In- Si u De ec ion Ki (Ca No. 550803, BD Biosciences) acco ding o he manu ac u e ’s manual. Pic u es we e acqui ed wi h an Olympus BX61 (Olympus, Hambu g, Ge many) con ocal mic oscope and p ocessed wi h he cellSens Dimensions 1.9 so wa e (Olympus, Hambu g, Ge many). 16S DNA Illumina sequencing o s ool bac e ia in GVHD si ua ion F esh s ool samples we e collec ed om il17a –/– and WT ecipien s a day 14 and day 21 a e BMT. Bac e ial DNA was isola ed using QIAamp DNA S ool Mini Ki (Qiagen) acco ding o manu ac u e ’s manual. In he i s PCR ound, 16S V3-V4 egions we e ampli ied by 15 PCR cycles using published p ime s [22]: S-D-Bac -0341F (5’-acac c ccc acacgacgc c ccga c CCTACGGGNGGC WGCAG-3’) and S-D-Bac -0785R (5’- PLOS ONE IL-17A and IL17F and acu e GVHD PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0231222 Ap il 6, 2020 3 / 16 g gac ggag cagacg g gc c ccga c GACTACHVGGGTATCTAATCC-3’). In he second PCR wi h 13 cycles Illumina-adap e s we e added using ollowing p ime s: adap- e _ o : (5’ aa ga acggcgaccaccgaga c acac c ccc ac 3’) and adap e _- e : (5’-caagcagaagacggca acgaga XXXXXXg gac g-3’). The XXXXXX bases ep esen he MID egion, each sample was coded wi h a dis inc DNA agmen o la e assignmen . All PCR s eps we e pe o med wi h Ad an age2 PCR ki (Taka a). PCR p oduc s we e sepa a ed wi h a 2% aga ose gel in TAE bu e . Fo gel ex ac ion, QIAquick Gel ex ac- ion ki (Quagen) was used acco ding o manu ac u e ’s manual. Lib a y concen a ion was adjus ed and 250bp pai ed-end sequencing was pe o med on he Illumina MiSeq sys em ol- lowing s anda d p o ocol. Quali y con ol and adap e clipping o he sequences was done using as q-mc ool o ea-u ils [23]. Sequencing da a we e p ocessed acco ding o he wo k- low lis ed: Pai ed- eads we e joined using o ea-u ils. Chime as we e excluded using Usea ch [24] sequence analysis ool wi h uchime [25] command based on chime aslaye gold 16s RNA da abase ( elease 4.29.2010) as e e ence. Taxonomy assignmen was pe o med by RDP-classi ie 2.8 [26] wi h con idence alue o 0.5. T ansendo helial elec ical esis ance (TEndoR) Human umbilical ein endo helial cells (HUVECs) we e g own o con luence in polyca bona e wells con aining e apo a ed gold mic oelec odes in se ies wi h a la ge gold coun e connec ed o a phase-sensi i e lock-in ampli ie as desc ibed p e iously. TEndoR was measu ed using an elec ical cell-subs a e impedance sensing sys em (ECIS) (Applied BioPhysics Inc.) as desc ibed elsewhe e [27]. Each condi ion’s endpoin esis ance was di ided by i s s a ing esis ance o gi e he no malized TEndoR. When he cells eached he esis ance mo e han 1500 ohm, cells we e ea ed wi h 10ng/ml IL-17 (Sigma-Ald ich, S -Luis, MO) and TEndoR was measu ed he ea e in eal- ime. T ansepi helial esis ance (TEpiR) and luo escein pe meabili y measu emen s Epi helial pe meabili y: Caco-2Bbe cells we e seeded on 0.4 μm snapwells (Co ning Li e Sci- ences, USA) a 1.5 ×104/cm 2 and g own o 14 days. TEpiR was measu ed daily using a EVOM2 ol ohmme e (Wo ld P ecision Ins umen s, Sa aso a, FL). On day 14, IFN-γ10ng/ ml (Sigma Ald ich, Ge many) ±IL-17 (IL-17A o IL-17F 2.5ng/ml ~ 10ng/ml) (Sigma Ald ich, Ge many) was added o he apical and basola e al ba h e e y 12 hou s. A e 24h, bo h sides o he cell monolaye we e incuba ed wi h TNF-α10ng/ml ±IL-17 (IL-17A o IL-17F 2.5ng/ml ~ 10ng/ml) in he con inuous p esence o INF-γ, and TEpiR was measu ed be o e, 2h, 4h, 8h, 12h and 24h a e TNF-αaddi ion. Subsequen ly, he cell we e ans e ed o an Ussing Cham- be sys em (Easymoun , Physiologic Ins umen s, CA), incuba ed in a bu e con aining NaCl 116 mM, KH2PO4 0.4 mM, K2HPO4 2.4 mM, MgCl2 1.2 mM, CaCl2 1.2 mM, NaHCO3 24 mM, gassed wi h 95% O2 and 5% CO2 a 37˚C. TEER was moni o ed wi h KCl aga elec odes. 100 μM luo escein o (FITC)-dex an-4000 was added o he apical side, and bo h apical and basola e al samples we e collec ed 1 hou la e and measu ed wi h a luo ome e a 520 nm (Tecan In ini e M200, Tecan, Swi ze land). Fluo escein and (FITC)-dex an-4000 pa acellula luxes we e p esen ed as he a io o he ace in he basola e al compa men and in he apical compa men . T ace luxes = basola e al FITC in ensi y / apical FITC in ensi y ×100%. Da a a e p esen ed as mean ±SEM, n = 3–4 samples in each condi ion. PLOS ONE IL-17A and IL17F and acu e GVHD PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0231222 Ap il 6, 2020 4 / 16 Da a analysis and s a is ics S a is ical analysis was pe o med wi h P ism 7 (G aph-Pad So wa e, Inc.). S a is ical di e - ences o he mean alues a e as ollows: �, P�0.05; ��, P�0.01; and ���, P�0.001. S uden ’s es , Mann Whi ney U es o ANOVA we e used o calcula ing s a is ical signi icance. The analysis o su i al da a was pe o med using Kaplan-Meie es ima ion and log- ank es . Fu he de ails on me hods a e a ailable as supplemen a y me hods and ma e ial. Resul s Dono IL-17A and IL-17F de iciency leads o agg a a ed acu e GVHD To assess he e ec s o IL-17 p oduced by dono -de i ed Th17 cells in acu e GVHD, we com- pa ed he ou come o expe imen al acu e GVHD induced by adop i e ans e o allogeneic CD4 + T cells om wild ype (WT) o Il7a -/- dono s [5] using C57BL/6 dono s and le hally i adia ed BALB/c ecipien s. Pheno ype o s eady s a e CD4 + cells om Il17a -/- mice we e analyzed by low cy ome y and showed no majo di e ences compa ed o WT CD4 + T cells (S1 Fig). Recipien s o Il17a -/- CD4 T cells showed a signi ican inc ease o GVHD se e i y and mo ali y as compa ed o WT con ols (Fig 1A and 1B). In e es ingly, ecipien s o Il17a -/- T cells su e ed om se e e dia hea ea ly a e ansplan a ion (Fig 1C). To e i y he occu ence o Th17 cells a e BMT, we analyzed IL-17 sec e ion o CD4 + T cells in hos issues by in acellula cy okine s aining. We e-isola ed dono lymphocy es om ecipien s’ colon, small in es ine (SI) and lymph nodes 21 days a e ansplan a ion and s ained o IL-17A and IL-17F. Thy1.1 was used o sepa a e dono om emaining hos CD4 + T cells ha escaped elimina ion by condi ioning. IL-17A and F sec e ion by CD4 T cells was e iden in pe iphe al lymph nodes (pLN) and GVHD a ge o gans (Fig 1D). Nex , we ana- lyzed sys emic cy okine le els a e GVHD ini ia ion. We obse ed a ma ked inc ease o IL-6, IFNγand MCP-1 le els in Il17a -/- ecipien s as compa ed o WT- ecipien s a di e en ime poin s a e ansplan a ion (Fig 1E). F om hese da a we conclude ha dono -de i ed IL-17A and IL-17F exe p o ec i e e ec s in he ea ly cou se o GVHD. Since he IL-17 iso o ms A and F bo h bind o he IL-17RA ecep o , we checked whe he ei he lack o IL-17A o F al e ed p o ec ion om acu e GVHD. To ha end, we made use o single Il17a -/- ,Il17 -/- and double IL-17-de icien (Il17a -/- ) CD4 + T cells. We obse ed ha only ecipien s ecei ing Il17a -/- , bu no Il17a -/- ,Il17 -/- CD4 + T cells, de eloped an agg a a ed GVHD as compa ed o WT con ols (Fig 2A and 2B). In o de o alida e ou expe imen al se up, we e-isola ed dono CD4 + T cells om pLNs o ecipien s and s ained o IL-17A and IL-17F o check o sec e ion o IL-17 cy okines. Indeed, we obse ed exp ession o bo h IL-17A and IL-17F om WT CD4 + cells, whe eas Il17a -/- and Il17 -/- CD4 + T cells exp essed only IL-17F and IL-17A espec i ely. Expec edly, Il17a -/- CD4 + T cells did nei he exp ess IL-17A no IL-17F (Fig 2C). IL-17 de iciency o dono T cells does no al e he mic obial lo a a e BMT The composi ion o he gu mic obiome a ec s GVHD ou come [28] and i has been shown ha Th17 cells ha e an impac on modula ion o he mic obiome in se e al expe imen al GVHD models [29]. O no e, bac e oidaceae species ha e been shown o compose he majo i y o he s ool mic obio a in pa ien s wi hou acu e GVHD [30]. The e o e, we sequenced s ool samples o Il17a -/- o WT CD4 + T cell ecipien s a day 14 and day 21 day a e ansplan a ion (Fig 3A and 3B). In e es ingly, we ound no signi ican di e ence in he o e all mic obio a di e si y be ween ecipien s ei he on day 14 o day 21 pos allo-BMT as assessed by Shannon indices (Fig 3C and 3D) o in p e alence o he bac e oidaceae amily be ween g oups (Fig 3E). PLOS ONE IL-17A and IL17F and acu e GVHD PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0231222 Ap il 6, 2020 5 / 16 Fig 1. De iciency o IL-17A and IL-17F in dono CD4 + T cells leads o agg a a ed GVHD. BALB/c mice we e le hally i adia ed and ansplan ed wi h 5x10 6 TCD BM and 0.5x10 6 CD4 + T cells om BL6 WT o Il17a -/- dono s. A) Su i al cu e o Il17a -/- and WT T cell ecipien s. Da a a e pooled om ou independen expe imen s (Il17a -/- n = 21, WT CD4 + cells n = 22). Fo s a is ical analysis he log ank es was used. B) Clinical sco e. C) Pe cen age o dia hea- ee mice. D) FACS so ed dono Thy1.1 + CD4 + T cells we e analyzed o he exp ession o IL-17A and IL-17F. Dono WT o Il17a -/- CD4 + T cells we e isola ed om BALB/c ecipien s om colon, SI and pLNs on day 21 a e BMT. Da a we e collec ed om h ee independen expe imen s o colon and SI (WT n = 10, Il17a -/- n = 11); and wo expe imen s pLNs (WT = 8, Il17a -/- n = 7). E) Concen a ions o IL-6, MCP-1 and IFNγcy okines in he se a o WT o Il17a -/- CD4 + T cell ecipien s sac i iced a day 7, 14, and 21 a e BMT. (day 7 WT n = 10, Il17a -/- n = 10; day 14 WT n = 11, Il17a -/- n = 12; day 21 WT n = 16, Il17a -/- n = 15). S a is ical signi icance was de e mined by S uden ’s es . The ba s show he mean and e o ba s show SEM. h ps://doi.o g/10.1371/jou nal.pone.0231222.g001 PLOS ONE IL-17A and IL17F and acu e GVHD PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0231222 Ap il 6, 2020 6 / 16 Lack o dono CD4 + T cell-de i ed IL-17A and IL-17F esul s in inc eased in es inal leakage Recipien s o Il17a -/- CD4 + T cells showed ea ly and se e e dia hea (Fig 1C), sugges ing al e - a ion o he in es inal ba ie . To es he unc ionali y o he in es inal ba ie a e GVHD-ini- ia ion, we applied FITC-dex an by o al ga age o BMT- ecipien s [31]. The ea e , se um FITC-Dex an concen a ions we e assessed a day 21 pos - ansplan a ion. We obse ed a sig- ni ican ly inc eased in es inal leakage in ecipien s o Il17a -/- CD4 + T cells (Fig 4A), indica ing in es inal ba ie b eakdown. We es ed whe he IL-17 cy okines exe p o ec i e e ec s in i o. Fo his pu pose, we used ECIS o analyze he e ec o IL-17 on endo helial leakage. In e es ingly, we did no see any e ec o ei he o he IL-17 cy okines in educ ion o TNFα- induced leakage in such expe imen s (Fig 4B). Fig 2. IL-17A and IL-17F a e ecip ocally compensa ed du ing GVHD. BALB/c mice we e le hally i adia ed and ansplan ed wi h 5x10 6 TCD BM and 0.5x10 6 CD4 + T cells om BL6 WT, Il17a -/- ,Il17a -/- , o Il17 -/- dono s. A) Su i al cu e o BL6 BL6 WT, Il17a -/- ,Il17a -/- , o Il17 -/- CD4 + T cell ecipien s, da a we e gene a ed in wo expe imen s (BL6 WT, Il17a -/- ,Il17a -/- n = 8, Il17 -/- n = 10) B) Clinical sco e. C) Exp ession o IL-17A and IL-17F by dono CD4 + T cells isola ed om pLNs o BL6 WT, Il17a -/- Il17a -/- , o Il17 -/- CD4 + T cell ecipien s in expe imen al GVHD a day 21 a e ansplan a ion. Fo s a is ical analysis, non-pa ame ic wo- ailed T es was used. Da a ep esen he equency and he SD o a single expe imen (n = 3 pe g oup). �p�0.05. h ps://doi.o g/10.1371/jou nal.pone.0231222.g002 PLOS ONE IL-17A and IL17F and acu e GVHD PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0231222 Ap il 6, 2020 7 / 16 The e has been eme ging e idence o a p o ec i e ole o IL-17 in os e ing gu in eg i y ia modula ion o epi helial igh junc ions, albei in a di e en expe imen al se up [32]. The e- o e, we analyzed he consequences o IL-17A and IL-17F adminis a ion in an in i o-sys em o gu -epi helial-leakage. To ha end, we used he well-cha ac e ized Caco2BBe cell line exp essing IL-17 ecep o , g own as di e en ia ed monolaye cul u es on pe meable il e s, and measu ed he TEpiR as a measu e o pa acellula pe meabili y. TEpiR was measu ed daily wi h an EVOM2 (Vol ohmme e , WPI), and a he end o he incuba ion ime in an Fig 3. IL-17 A/F de iciency does no al e he mic obial lo a a e allogeneic BMT. BALB/c mice we e le hally i adia ed and ansplan ed wi h 5x10 6 TCD BM and 0.5x10 6 CD4 + cells om BL6 WT o Il17a -/- dono s. A-B) Rep esen a i e g aph o mic obial lo a in he eces o WT and Il17a -/- T cell ecipien s a A) day 14 o B) day 21 a e BMT. Hea -maps o he ela i e abundance o bac e ial species a phylogene ic le el o amilies a e p esen ed o one ep esen a i e expe imen . Each column ep esen s one sample om indi idual mouse. (day 14 WT n = 3, Il17a -/- n = 3; day 21 WT n = 4, Il17a -/- n = 4). C) Shannon index o di e si y o all samples on he amily le el a day 14 and D) day 21 a e BMT. E) Rela i e abundance o Bac e oidaceae amily in he eces o WT and Il17a -/- T cell ecipien s a day 21 a e BMT. (day 14 WT n = 10, Il17a -/- n = 10; day 21 WT n = 13, T Il17a -/- n = 14). Sequences wi h an abundance o less han 1% we e excluded om he analysis. h ps://doi.o g/10.1371/jou nal.pone.0231222.g003 PLOS ONE IL-17A and IL17F and acu e GVHD PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0231222 Ap il 6, 2020 8 / 16 Ussing chambe se up. Addi ionally, we assessed apical o basola e al luo escein (FITC) luxes. Howe e , we saw no e idence o a p o ec i e ole o IL-17A o IL-17F agains TNFα- induced dec ease in TEpiR (Fig 4C and 4D). While TNFαincuba ion esul ed in a signi ican dec ease in TEpiR, e i ied by TEpiR measu emen s in he Ussing Chambe (Fig 4E) and by an inc ease in FITC pe meabili y, he p e-incuba ion o 24 h wi h IL-17A o IL-17F did no p e en he inc ease in epi helial pe meabili y (Fig 4F). Lack o dono -de i ed IL-17A and F cells leads o inc eased IFNγ p oduc ion o dono CD4 T cells and mac ophage in lux o he in es ine Th1 T cells p oducing he p oin lamma o y cy okine IFNγha e been shown o be de imen al in inducing GVHD [33]. Hence, we compa ed he numbe s o CD4 + T cells sec e ing IFNγin Fig 4. Recipien s o Il17a -/- CD4 + cells show gu -leakiness. BALB/c mice we e le hally i adia ed and ansplan ed wi h 5x10 6 TCD BM and 0.5x10 6 CD4 + cells om BL6 WT o Il17a -/- dono s. A) Concen a ion o ansloca ed FITC-dex an in he se a o WT and Il17a -/- CD4 + T cell ecipien s a day 21 a e BMT. (n = 6, Il17a -/- n = 4) B) Inc ease in ansendo helial elec ical esis ance (TEndoR) no malized o ehicle. C) Inc ease in ansepi helial elec ical esis ance (TepiR, measu ed daily wi h an EVOM2 ol ohmme e , o e he days in cul u e a e con luency. All ou g oups had a simila inc ease in TEpiR p io o he cy okine ea men . D) a day 14 pos con luency, he cells we e incuba ed wi h 10ng/ml in e e on-γ± IL17A o IL17F, ollowed by 10g/ml TNF-α10ng/ml ±IL-17 (IL-17A o IL-17F 10ng/ml) 24 h la e , and TEpiR was assessed a he indica ed imes. E) A e 24h, he il e s we e ans e ed o an Ussing-chambe sys em, and bo h TEpiR, and F) FITC pe meabili y was assessed. The alues in C and D a e gi en in % o he alue ea ed wi h IFN-γand TNF-αonly. All da a a e pooled om wo o h ee independen expe imen s. E o ba s ep esen SEM. Fo s a is ical analysis Log- ank es was used. �p�0.05. h ps://doi.o g/10.1371/jou nal.pone.0231222.g004 PLOS ONE IL-17A and IL17F and acu e GVHD PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0231222 Ap il 6, 2020 9 / 16 31. Hanash AM, Dudako JA, Hua G, O’Conno MH, Young LF, Singe N V., e al. In e leukin-22 P o ec s In es inal S em Cells om Immune-Media ed Tissue Damage and Regula es Sensi i i y o G a e sus Hos Disease. Immuni y [In e ne ]. 2012 Aug; 37(2):339–50. 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