Interferon- and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection

Transcription

1 Intrfron- n intrlukin t synrgistilly for th inution of intrfron-stimult gns n ontrol of rotvirus inftion Ntur Amri, In. All rights rsrv. Pro P Hrnánz,, Tnl Mhlkõiv,,, Ins Yng,7, Vr Shwirzk,, Nm Nguyn, Fin Gunl,,8, Konr Gronk,8, Brnhr Ryffl 9, Christoph Hölshr,, Lur Dumoutir, Jn-Christoph Rnul, Sstin Surum,7, Ptr Sthli & Anrs Difnh,,8 Th pithlium is th min ntry point for mny viruss, ut th prosss tht prott rrir surfs ginst virl inftions r inompltly unrstoo. Hr w intifi intrlukin (IL-) prou y innt lymphoi ll group (ILC) s n mplifir of signling vi intrfron-l (IFN-l), synrgism n to urtil th rplition of rotvirus, th ling us of hilhoo gstrontritis. Cooprtion twn th rptor for IL- n th rptor for IFN-l, oth of whih wr prfrntilly xprss y intstinl pithlil lls (IECs), ws rquir for optiml tivtion of th trnsription ftor STAT n xprssion of intrfron-stimult gns (ISGs). Ths t suggst tht pithlil lls r prott ginst virl rplition y o-option of two volutionrily rlt ytokin ntworks. Ths t my inform th sign of novl immunothrpy for virl inftions tht r snsitiv to intrfrons. Mny viruss, suh s hptitis B virus, hptitis C virus, humn immunofiiny virus, influnz virus, rotvirus n poliovirus, us rplition in pithlil lls to stlish inftion of multillulr hosts, ut th molulr ntworks tht prott rrir surfs ginst virl inftions r inompltly unrstoo,. It is gnrlly liv tht intrfrons, suh s typ I intrfrons (i.., intrfron-α, (IFN-α) n IFN-β) n typ III intrfrons (i.., IFN-λ n IFN-λ in mi; olltivly ll IFN-λ hr), r ssntil in ntivirl fns. Intrfrons r prou y virus-inft pithlil lls n inu n nti-virl stt in lls riving n intrfron signl n thry onstitut n ffiint innt fns mhnism,. Whil typ I n typ III intrfrons n thir rptors r only istntly rlt,, thy show lmost ompltly intil signling in tht thy tivt th STAT-STAT trnsription ftor signling pthwy, whih ls to formtion of th trnry ISGF ( intrfron-stimult gn (ISG) ftor ) omplx, onsisting of STAT, STAT n th trnsription ftor IRF9, tht ontrols trnsription of mor thn ISGs noing prouts tht t in onrt to rstrit th rplition of viruss,7. IFN-λ n its rptor (IFN-λR) shr homology with ytokins of th IL- fmily n thir rptors. In prtiulr, th gn noing th α-hin of th IL- rptor (Ilr) is th losst rltiv of th gn noing th IFN-λR hin (Ifnlr),. Ilr n Ifnlr r jnt on hromosom in mi or hromosom in humns,, n oth IL-Rα n IFN-λR ssoit with th IL-Rβ hin (IL-R) to form funtionl, htroimri rptor omplxs. Th los rltionship twn IFN-λR n IL-R is notl us oth rptors r prfrntilly xprss y pithlil lls,, whih inits signt rol in ontrolling pithlil funtion in rspons to inftion. Although IFN-λR is onnt to signling vi STAT n STAT, IL-R nggs th STAT signling pthwy, n STAT signling hs n link to lmost ll of th known iologil funtions of IL-, in prtiulr to uprgultion of th xprssion of gns noing ntitril ftors n of gns noing prouts tht promot tissu rpir. Inftion with rotvirus, oul-strn RNA virus of th Roviri fmily, is suitl mol systm with whih to nlyz th ytokin ntworks rquir for prottion of th intstinl rrir ginst virl inftion us rotvirus shows prfrntil tropism for th villous pithlium of th smll intstin of infnt humns n mi. virus inftion is th ling us of virl gstrontritis in infnts n uss mor thn, ftlitis worlwi, nnully 7. Rsrh Cntr Immunology n Institut of Mil Miroiology n Hygin, Univrsity of Minz Mil Cntr, Minz, Grmny. Dprtmnt of Mil Miroiology n Hygin, Institut for Mil Miroiology n Hygin, Friurg Univrsity Mil Cntr, Friurg, Grmny. Mx-Plnk-Institut for Immunoiology n Epigntis, Friurg, Grmny. Dprtmnt of Mil Miroiology n Hygin, Institut for Virology, Friurg Univrsity Mil Cntr, Friurg, Grmny. Spmnn Grut Shool of Biology n Miin, Univrsity of Friurg, Friurg, Grmny. Institut of Mil Miroiology n Hospitl Epimiology, Hnnovr Mil Shool, Hnnovr, Grmny. 7 Grmn Cntr for Inftion Rsrh, Hnnovr, Grmny. 8 Rsrh Trining Group (GRK) of Orgnognsis, Friurg, Grmny. 9 INEM UMR7, Molulr Immunology, Univrsity of Orlns, Orlns, Frn. CNRS, Orlns, Frn. Institut of Inftious Diss, Univrsity of Cp Town, Cp Town, South Afri. Inftion Immunology Rsrh, Rsrh Cntr Borstl, Borstl, Grmny. Clustr of Exlln Inflmmtion t Intrfs, Borstl-Kil-Lük-Plön, Grmny. Luwig Institut for Cnr Rsrh, Univrsité Ctholiqu Louvin, Brussls, Blgium. Ths uthors ontriut qully to this work. Corrsponn shoul rss to A.D. (ifnh@uni-minz.). Riv Jnury; pt 7 April; pulish onlin My ; oi:.8/ni.8 ntur immunology DVANCE ONLINE PUBLICATION

2 Ntur Amri, In. All rights rsrv. virus is wll pt to its host, n homologous niml mols of rotvirus inftion hv shown tht lss thn inftious units of virus rplit vigorously n initit irrhl iss, inluing virl shing n ffiint spr to uninft mi. Suh ffiint virl rplition is mit in prt y th ffiint mhinry of th virus tht llows it to v rognition y th immun systm of th host n/or to supprss intrfron-mit innt immun rsponss 8. Th muosl typ III intrfron systm, ut not typ I intrfrons, hs n importnt rol in urtiling rly rotvirus rplition in its homologous host to minimiz tissu mg,. Givn th los rltionship twn IL- n IFN-λ n thir onrt tion on pithlil lls, w invstigt th rol of IL- in prottion ginst murin virus with prfrntil tropism to pithlil lls (IECs) of th smll intstin. W foun tht group innt lymphoi ll (ILC) riv IL- ws rquir for ontrol of inftion with murin rotvirus. IL-α, n pithlil lrmin rls y virus-inft lls, ut not IL- or IL-β, mit high xprssion of IL- y ILC lls following inftion with rotvirus. IL--mit prottion ginst rotvirus-mit tissu mg i not rquir STAT, trnsription ftor involv in lmost ll prviously rport iologil tivitis of IL-. Inst, IL- t in synrgy with IFN-λ for optiml tivtion of STAT n xprssion of ISGs. Our t intify prviously unknown synrgy twn two ytokins ting on IECs for prottion of th intstinl rrir ginst virl inftion. Suh synrgy twn ths volutionrily rlt ytokins might hrnss for th linil thrpy of hroni virl inftion. RESULTS IL- urtils rotvirus rplition in its homologous host Thr is puity of informtion out whthr mplifirs of intrfron signling xist tht oul hrnss for ttr ontrol of virl inftion. Th rlt ytokins IL- n IFN-λ t on th sm llulr trgt (i.., IECs), ut ooprtion twn ths two muosl ytokin ntworks hs not n xplor. W fostr Il / mi, Ifnlr / mi n wil-typ mi to ithr wil-typ mothrs (Fig.,) or Ifnlr / mothrs (Fig.,) n inft thm t y 7 of g ( sukling mi ) with 7 ID (th min (%) inftious os) of homologous murin rotvirus strin EDIM ( pizooti irrh of infnt mi ) n ssss virl lo y quntittiv RT-PCR nlysis of RNA from tissus of th smll intstin (Fig.,), y msuring virus sh into th olon lumn y nzymlink immunosornt ssy (ELISA) (Fig.,) n y visulizing virl ntign in th smll intstin y immunofluorsn stining (Fig. ). In n xtnsion of pulish work, Ifnlr / mi h signifintly highr virl urn t y (th pk of virl rplition; Supplmntry Fig. ) n t y of rotvirus inftion (pk of virl shing; Fig.,) thn i wil-typ mi. Th grtr virl rplition in Ifnlr / mi ws proly inpnnt of th xprssion of wil-typ Mx lll, n ISG whos prout is involv in rstriting virl rplition, us Mx sttus i not fft th ontrol of rotvirus rplition (Supplmntry Fig. ). Strikingly, Il / mi lso h signifint grtr virl lo, lit to lssr xtnt thn i Ifnlr / mi (Fig. ). Thorough nlysis of intstinl pthology in inft mi ovr th ntir xtnsion of th smll intstin rvl itionl insights. Th pthologil lsions tht follow rotvirus inftion r isontinuous, n rs of histopthologil mg r intrsprs with hlthy tissu. Thrfor, w nlyz th ntir smll intstin to ssss th xtnt of tissu mg. In wil-typ mi, rpliting rotvirus ws rstrit to th istl thir of th smll intstin, whrs in oth Il / n Ifnlr / mi, virl rplition ws prsnt throughout th full xtnsion of th smll intstin (Fig.,f). Ths t init tht signling vi IL- n IFN-λ prott in prtiulr th proximl prts of th smll intstin ginst rotvirus inftion. Dspit fftiv virl rplition n irrh in ll mi ssss, histologil nlysis of tissus of th smll intstin from inft wil-typ mi show only mort mg of th pithlil rrir in th istl smll intstin (Fig. g,h). Inft Il / mi n Ifnlr / mi xhiit mor svr pthology, hrtriz y vuoliztion, pithlil rosions n villus isruption ross th ntir smll intstin (Fig. g,h n Supplmntry Fig. ). Ths t init tht IL- n IFN-λ supprss virl rplition to n xtnt tht prvnt th vlopmnt of svr pithlil mg. Uninft mi of ll gnotyps i not show ny signs of pthology (t not shown). In ition, rotvirus-inft Il / n Ifnlr / mi show rtrtion of wight gin ompr with tht of thir inft wil-typ ountrprts, n thir wight ws not normliz t lmost wks ftr inftion (Fig. i), whn th virus h om unttl in ll mous strins invstigt. Notly, th wight gin of uninft Il / n Ifnlr / mi i not iffr from tht of wil-typ mi (Fig. i), whih init tht th Il / mi n Ifnlr / mi h no gntilly trmin growth rtrtion. In ontrst to sukling mi, ult wil-typ mi inft with. ID rotvirus i not vlop linilly ovrt symptoms of rotvirus inftion, lthough thy h low (ut ttl) virl titrs oth in tissus n in fs (Supplmntry Fig.,), whih init tht ll mi wr inft. Ault mi gntilly fiint in Il or Ifnlr lso show impir ontrol of rotvirus rplition (Supplmntry Fig.,). Thus, IL- ws n to urtil rotvirus rplition in oth sukling mi n ult mi. Although IL- hs n link to tissu rpir ftr virl inftion, w i not fin ny signifint iffrns (P >.) mong inft wil-typ, Il / n Ifnlr / mi in th prolifrtion of rypt-rsint lls (i.., stm lls) (Supplmntry Fig.,f) or in th upwr migrtion of iffrntiting IECs (Supplmntry Fig. g,h), whih r prosss tht fftivly rpl mg lls following virl inftion. It hs n not tht muttions in ytokin-noing gns n l to iffrns in miroil ommunitis, whih oul fft th ours of rotvirus inftion. Bus istint miroiot n quir through mtrnl trnsmission, w fostr nworn Il /, Ifnlr / n wil-typ pups with ithr wil-typ mothrs or Ifnlr / mothrs. W osrv no iffrn in th ours of th inftion or in th svrity of pthology (Fig. n t not shown). W lso irtly xmin th miroiot of th smll intstin in nworn n ult mi y high-throughput squning of S rdna mplions. Diffrns in olon-rsint miroil ommunitis in ult Il / mi hv n rport. Howvr, w foun no signifint iffrn twn ult wil-typ mi n ult Il / mi, twn littrs of ult mi, or twn gnotyps within littrs of ult mi in th omposition of th miroiot of th smll intstin (Supplmntry Fig. n Supplmntry Tl ). By prinipl-oorint nlysis n nlysis of molulr vrin, w foun th miroiot of sukling mi to highly littr spifi (Supplmntry Fig. f n Supplmntry Tl ). Thus, th ours of rotvirus inftion in th mous strins invstigt ws not trmin y iffrns in miroil ommunitis. Proution of IFN- y rotvirus-inft IECs IFN-λ proution ws inu sustntilly following rotvirus inftion (Fig. ) rgrlss of th Mx sttus of th mi (Supplmntry Fig. ), ut th llulr sours of IFN-λ proution DVANCE ONLINE PUBLICATION ntur immunology

3 Ntur Amri, In. All rights rsrv. Figur Control of rotvirus inftion rquirs IL-. ( ) Virl rplition in wil-typ (), Il / or Ifnlr / pups fostr with wiltyp mothrs (,) or Ifnlr / mothrs (,) s nworns, thn orlly inft with 7 ID rotvirus t y 7 of g n ssss or ftr inftion y quntittiv RT-PCR nlysis of mrna noing rotvirus protin VP ( VP) mong RNA in tissus of th smll intstin (,) n y ELISA of rotvirus protin () in olon homognts (,); mrna rsults (,) wr lult y th hng-in-yling-thrshol mtho ( Ct ) n r prsnt rltiv to thos of th ontrol gn Hprt. (,f) Immunofluorsn mirosopy of tissu from th smll intstin of wil-typ, Il / or Ifnlr / mi s in, ftr inftion with rotvirus, stin with th DNA-ining y DAPI, ntioy to E-hrin (E-) n ntioy to rotvirus (), n istriution ( sli siz) n quntifition (plot siz) of inft lls in th smll intstin (f). Clls pr visul fil in f (mn ± s..m.): wil-typ, 9 ± 8; Il /, 98. ± ; Ifnlr /,,87. ± 8. (g) Hmtoxylinn-osin stining of tissu stions of th smll intstin of mi s in,. (h) Clinil sor, oring to thr prmtrs (ky), for mi s in,. (i) Boy wight of mi s in, lft uninft (UI) or t y 9 ftr inftion, prsnt rltiv to oy wight t y, st s %. Sl rs (,g), µm. Eh symol (,,i) rprsnts n iniviul mous; smll horizontl lins init th mn (±s..m.)., not signifint (P >.); P <., P <. n P <. DAPI E- virus (on-wy nlysis of vrin (ANOVA)). Dt r rprsnttiv of four inpnnt xprimnts (,; mn n s..m. of n mi pr group), two inpnnt xprimnts ( g; mn n s..m. of n mi pr group in,,f) or two xprimnts (h; mn n s..m.) or r pool from two inpnnt xprimnts (i; n mi pr group). rmin osur. IFN-λ proution hs n oumnt for virusinft pithlil lls n for hmtopoiti lls (for xmpl, mononulr phgoyts n plsmytoi nriti lls) 7,8. Using th EDTA issoition mtho, w sprt pithlil lls n hmtopoiti lls from uninft n rotvirus-inft mi. W foun lrgr mounts of Ifnl n Ifnl trnsripts (whih no IFN-λ n IFN-λ, rsptivly; th primr pir tt oth Ifnl n Ifnl) in th pithlil frtion of oth rotvirusinft sukling mi (Fig. ) n rotvirus-inft ult mi (Supplmntry Fig. ). W onfirm th purity of th isolt lls y iffrns in th unn of rotvirus trnsripts ( prfrntilly prsnt in IECs; Supplmntry Fig. ) n of th xprssion of tissu-spifi gns, whih llow isrimintion of hmtopoiti lls of th lmin propri (with highr xprssion of Ptpr, whih nos CD, mrkr of ll hmtopoiti lls) vrsus IECs (tht xprss Ch, whih nos E-hrin) (Supplmntry Fig.,). As th pithlil frtion ontin IECs n intrpithlil lukoyts, w purifi IECs n hmtopoiti lls to high purity on th sis of iffrntil xprssion of th hsion molul EpCAM n lukoyt mrkr CD (Supplmntry Fig. ) n foun Ifnl n Ifnl trnsripts prominntly in EpCAM + CD IECs (Fig. ). Thus, IECs, not hmtopoiti lls, wr th min sour of IFN-λ following rotvirus inftion. To trmin th ll typ tht ws rsponing to IFN-λ, w monitor th xprssion of thr ISGs in IECs or hmtopoiti lls following rotvirus inftion in wil-typ n Ifnlr / mi. In IECs (EpCAM + lls) of VP (fol ) g i..... Dy Dy Wight (%) Dy 8 VP (fol) Il / Ifnlr / Il / Ifnlr / UI Dy (ID ) mothr virus Dy Colon Il / Ifnlr / Il / Ifnlr / Dy VP (fol) f Il / Ifnlr / h Clinil sor Proximl Mil Distl Epithlil rosion Villus isruption Vuoliztion th smll intstin, ISG xprssion ws ntirly IFN-λR pnnt (Supplmntry Fig. f h). Thus, IECs not only wr th min prours of IFN-λ following rotvirus inftion ut lso rspon to IFN-λ, whih init tht th IFN-λ ntwork ws n IECutonomous nti-virl fns pthwy. virus inftion nhns IL- proution y ILC lls Although IECs wr th min sour of IFN-λ in rotvirus-inft mi, Il ws xprss xlusivly y hmtopoiti lls of th lmin propri of th smll intstin in oth sukling mi (Fig. ) n ult mi (Supplmntry Fig. ), n rotvirus inftion l to sustntilly nhn Il xprssion (Fig. n Supplmntry Fig. ). IL- n prou y ILC lls 9, th T H 7 sust of hlpr T lls, susts of γδ T lls n possily nutrophils. Th IL--prouing lls in rotvirusinft mi xprss th trnsription ftor RORγt ut not th intgrin CD (α M ) or th signl-trnsution rptor CD9 (Supplmntry Fig.,) or th invrint signling protin CD (Fig. ), whih xlu th possiility tht nutrophils or T lls wr sours of sustntil IL- ftr rotvirus inftion. IL- proution y RORγt + non-b, non-t lls ws initiv of ILC susts. On th sis of iffrntil xprssion of th hmokin rptor CCR, two mjor ILC popultions hv n intifi. As th nworn intstin ontins only smll numrs of CCR ng lo RORγt + ILC lls (inluing th NKp + sust of ILC lls), th min sour of IL- in rspons to rotvirus inftion ws prntlly Il / Ifnlr / Dy Il / Ifnlr / mothr Ifnlr / (ID ) Il / Ifnlr / Dy Colon ntur immunology DVANCE ONLINE PUBLICATION

4 Ntur Amri, In. All rights rsrv. IFN-λ (ng/ml) 8 Ifnl (fol).... IEC LPL mrging CCR + ILC lls (Supplmntry Fig. ). Thus, two volutionrily rlt ytokins simultnously ting on pithlil lls, IFN-λ prou y inft IECs n IL- prou y CCR + ILC lls, urtil rotvirus inftion n prott ginst svr mg to th pithlil rrir. Following rotvirus inftion, oth th frtion of ILC lls prouing IL- (Fig.,f) n th solut numr of IL--prouing ILC lls (Fig. g) inrs signifintly. W ssss whthr ILC fiiny woul l to inrs virus rplition. In, mi lking IL--prouing ILC lls (Ahr ILC,T mi n Ror / mi), wr unl to fftivly ontrol rotvirus inftion s sukling mi (Fig. h) n s ult mi (Supplmntry Fig. ). This monstrt tht RORγt + ILC lls wr ntrl omponnt of th mpning of rplition of virus with prfrntil tropism for th pithlium of th smll intstin. In n xtnsion of pulish t, w foun tht shing of rotvirus in mi lking ptiv immun systm omponnts (i.., Rg / mi, whih lk ll T lls n B lls, n Tr / Tr / mi, whih lk αβ n γδ T lls) ws omprl to tht osrv in wil-typ mi (Supplmntry Fig. ). In ontrst, lymphoi Rg / Ilrg / mi, whih hv lrgly norml mononulr phgoyt omprtmnt ut lk ll ILC susts s wll s onvntionl lymphoyts, h highr rotvirus urn, similr to Ifnlr / mi n Il / mi (Fig. h n Supplmntry Fig. ); ths rsults sri n importnt rol to ILCs in urtiling rotvirus inftion. IL- nhns IL- proution y ILC lls W sought to trmin whih virus-inu ftors wr rquir for th nhn xprssion of IL- y ILC lls. IFN-λ signls wr not rquir for th IL- proution, us Ifnlr / mi show no rution in xprssion of IL- (Supplmntry Fig. f) or its rptor (Supplmntry Fig. g) rltiv to tht in wil-typ mi, t sty stt or following rotvirus inftion. In ft, IL- proution ws nhn in Ifnlr / mi t y following rotvirus inftion (Supplmntry Fig. f); this orrlt with inrs xprssion of th ntitril ltin RgIIIγ (Supplmntry Fig. h,i), whih is unr th ontrol of IL- (rfs.,9). Pulish work hs Ifnl (fol).... EpCAM + CD EpCAM CD +... ND Figur virus inftion inus proution of IFN-λ y IECs n of IL- y ILC lls. () ELISA of IFN-λ in tissu of th whol smll intstin of wil-typ sukling mi t y ftr inftion with rotvirus () or mok inftion (). Eh symol rprsnts n iniviul mous; smll horizontl lins init th mn (±s..m.). (,) Quntittiv RT-PCR nlysis of Ifnl n Ifnl (with primr tht tts oth; olltivly ll Ifnl hr) in smll IECs n LPLs isolt y th EDTA-issoition mtho () or in pithlil lls (EpCAM + CD ) or hmtopoiti lls (EpCAM CD + ) highly purifi y sorting () t y ftr inftion s in (prsnt s in Fig.,). () Quntittiv RT-PCR nlysis of Il in IECs n LPLs isolt y th EDTA-issoition Il (fol) IEC LP ND CD g IL- + lls ( ).... IL- Ctrl UI CD + CD ssign n importnt rol to th mononulr phgoyt riv ytokins IL- (rf. 7) n IL-β 8 in ontrolling IL- proution y ILC lls. Howvr, th proution of IL- (Fig. ) n IL-β (Fig. ) ws not signifintly nhn following rotvirus inftion, n trnsripts noing ths ytokins wr lvt only slightly ftr inftion (Supplmntry Figs.,f n,). Ths ytokin-noing mrnas wr prsnt in lmin propri lukoyts (LPLs), ut thir unn ws not lvt in IECs (Supplmntry Fig.,). In ontrst, rotvirus inftion signifintly nhn xprssion of th lrmin IL-α y IECs, t th lvl of oth protin (Fig. ) n mrna (Fig. n Supplmntry Fig. ). LPLs ontin fw Il trnsripts (Fig. ). Th mount of IL-α protin vill ftr rotvirus inftion ws mor thn tnfol lrgr thn tht of IL- or IL-β (Fig. ). Mi fiint in Il (whih nos th p9 suunit of IL-) h norml xprssion of IL- (Fig. ) n IFN-λ (Supplmntry Fig. ) n h rotvirus titrs (t y ) similr to thos of thir wil-typ ountrprts (Fig. f), whrs Ilr / mi, whih lk signling vi oth IL-α n IL-β, show signifintly nhn virl rplition rltiv to tht of thir wil-typ ountrprts (Fig. f). Th iminish virl ontrol in Ilr / mi ws proly u to lowr xprssion of Il in thir LPLs (Fig. ), us thir xprssion of IFN-λ ws not ru (Supplmntry Fig. ). To intify th rlvnt typ of IL- rquir for IL- proution n for urtiling rotvirus rplition, w injt nutrlizing ntiois spifi for ithr IL-α or IL-β into mi for infting th mi with rotvirus. Nutrliztion of IL-α rogt th rotvirusinu IL- xprssion (Fig. g n Supplmntry Fig. f) n l to nhn rplition of rotvirus (Supplmntry Fig. g) n shing of rotvirus (Fig. h) n sustntilly ggrvt pithlil pthology (Fig. i,j). Nutrliztion of IL-β h no suh fft (Fig. g j n Supplmntry Fig. g). W monitor th fftivnss of th ntioy trtmnt y vluting th xprssion of IL--inu gns n foun this xprssion ws ru ftr injtion of ntiois to IL-α or IL-β (Supplmntry Fig. h,i), whih onfirm fftiv nutrliztion of ths ytokins. Ex vivo,.9. h VP (fol).97. frorγ t+ IL-+ lls (%) CD + CD UI Ror Ahr ILC,T Ifnlr mtho t y ftr inftion s in (prsnt s in Fig.,). () Flow ytomtry nlyzing th xprssion of CD n IL- y gt RORγt + LPLs t y (uninft (UI)) or y () ftr inftion s in. Numrs in qurnts init prnt lls in h throughout. (f,g) Frquny (f) n solut numr(g) of RORγt + IL- + CD + n IL- + CD LPLs from mi lft uninft or t y ftr inftion s in. (h) Virl lo in wil-typ, Ror /, Ahr ILC,T n Ifnlr / sukling mi t y ftr inftion with rotvirus (ssss (in tissus from th whol smll intstin) n prsnt s in Fig.,). ND, not ttl (or not l to isply on th sl hr). P <., P <. n P <. (Stunt s t-tst (,), two-wy ANOVA (,,f,g) or on-wy ANOVA (h)). Dt r rprsnttiv of two inpnnt xprimnts (,,h; mn n s..m. of n () n () or n (h) mi pr group) or thr inpnnt xprimnts (, g; mn n s..m. of n (,) or n = (f,g) mi pr group). DVANCE ONLINE PUBLICATION ntur immunology

5 Ntur Amri, In. All rights rsrv. IL- (pg/ml) g CD 8..9 IL- 8,. 8.,... UI UI UI h UI Anti-IL-α Anti-IL-β IL-β (pg/ml) 8..9 IL-α ws s ffiint s IL-β in inuing IL- proution from ILC lls (Fig. k,l). Thus, th pithlil lrmin IL-α prou n rls in rspons to rotvirus inftion ws rquir for th nhn proution of IL- y RORγt + ILC lls n for th urtilmnt of rotvirus rplition. IL-α (pg/ml) Il (fol) (ID ) UI Anti-IL-β IEC LPL Anti-IL-α Figur IL-α prou y IECs ontrols rotvirus-inu proution of IL- y ILC lls. ( ) ELISA of IL- (), IL-β () n IL-α () in tissus of th smll intstin from wil-typ sukling mi lft uninft n or t ftr inftion with rotvirus. (,) Quntittiv RT-PCR nlysis of Il () n Il () in LPLs n IECs () or tissus () of th smll intstin of mi s in (prsnt s in Fig.,). (f) Virl lo in th smll intstin of wil-typ, Ilr / n Il / sukling mi t y ftr inftion with rotvirus (ssss n prsnt s in Fig.,). (g) Flow ytomtry nlyzing CD n IL- in RORγt + LPLs from wil-typ mi lft uninft (UI) or trt with or nutrlizing ntioy to IL-α or IL-β (ov plots) for inftion with rotvirus () n ssss ftr inftion. Il (fol)... i jclinil sor UI Ilr / Il / Synrgism of IL- n IFN- for optiml xprssion of ISGs W nxt sought to trmin how IL- limit virl rplition. IL- ws not rquir for th xprssion of IFN-λ (Fig. ) or its rptor (Fig. ). Although xprssion of Ifnl n Ifnl ws similr in wil-typ mi n Il / mi t y following inftion, Il / f VP (fol) Anti-IL-α Anti-IL-β Anti-IL-α Anti-IL-β Epithlil rosion Villus isruption Vuoliztion (h) ELISA of virl lo in oloni tissu of wil-typ mi trt with or nutrlizing ntioy to IL-α or IL-β for n ftr inftion with rotvirus () n ssss ftr inftion. (i) Hmtoxylin-n-osin stining of tissu stions of th smll intstin of mi trt s in h, ssss ftr inftion. (j) Clinil sor of mi trt s in h, ssss ftr inftion. (k) Quntittiv RT-PCR nlysis of Il in ultur ILC lls mong lls otin from th lmin propri of th smll intstin of wil-typ mi n highly purifi y sorting s CD CD9 CD KLRG NK. IL-7R + -Kit + lls, thn ultur for in th prsn of IL-7 n SCF, n lft unstimult (Control) or stimult with IL-α or IL-β (ky) (prsnt s in Fig.,). (l) ELISA of IL- in suprntnts of ultur ILC lls s in k. Eh symol (,h) rprsnts n iniviul mous; smll horizontl lins init th mn (±s..m.). P <., P <. n P <. (Stunt s t-tst (,), two-wy ANOVA (,) or on-wy ANOVA (f,j l)). Dt r rprsnttiv of two inpnnt xprimnts (,h l; mn n s..m. in j l) or thr inpnnt xprimnts (,,g; mn n s..m. in,). k Il (fol) Control IL-α IL-β ND l IL- (ng/ml).... Ilr / Il / Control IL-α IL-β Ifnl (fol).. ND ND Dy Il / Dy Dy Ifnlr (fol)... Il / VP (fol) Figur Control of rotvirus rplition y IL- pns on signling vi IFN-λR. (,) Quntittiv RT-PCR nlysis of Ifnl () n Ifnlr () in th smll intstin of wil-typ or Il / sukling mi t y ftr mok inftion () or y () or y (,) ftr inftion with rotvirus () (prsnt s in Fig.,). (,) Virl lo in wil-typ, Il /, Ifnlr / or Il / Ifnlr / sukling mi t y ftr inftion with rotvirus, ssss y quntittiv RT-PCR nlysis of VP mrna () or y ELISA () (prsnt s in Fig., () or prsnt rltiv to rsults for wil-typ mi ()). ( h) Virl lo in -wk-ol wil-typ mi (,f), Il / mi (g) Il / Ifnlr / Il / Ifnlr / f (ID ) 8 (ID fol) Colon Colon IL- IFN-λ g VP (fol) Il / Ifnlr / Il / Ifnlr / Il / IFN-λ h VP (fol) or Ifnlr / mi (h) givn suutnous injtion of, mous IL- or mous IFN-λ (kys) 8 h for inftion with rotvirus, follow y rpt injtion on ys n ftr inftion n nlysis on y ftr inftion (ssss n prsnt s in Fig.,) (,g,h) or y ELISA of olon homognts (f). Eh symol (,f) rprsnts n iniviul mous; smll horizontl lins init th mn (±s..m.). P <., P <. n P <. (on-wy ANOVA ( f) or Stunt s t-tst (g,h)). Dt r rprsnttiv of thr inpnnt xprimnts (,; mn n s..m. of n mi pr group) or two inpnnt xprimnts (, h; mn n s..m. of n (), n (,f) or n (g,h) mi pr group) or r pool from two inpnnt xprimnts (; n mi pr group)). VP (fol) Ifnlr / IL- IFN-λ IL- ntur immunology DVANCE ONLINE PUBLICATION

6 Ifn Il / Ifnlr / UI Mx Cxl Os Iigp Fosl Os Ifil Osl Hr Stt Sos Trx Tp Io Psm8 Os Ifih Mmp Gp Irf7 Dx8 Nmi Ul Cl Egr Il s9 Apol8 s8 Tp Ul Ar Mu Ifitm Srpin Klf.. Isg (fol) f Isg (fol) IFN-λ (ng/ml) IFN-λ (µg) IL- +IL- IEC. IL- +IL- g Osl (fol) IFN-λ (µg) Osl (fol) IFN-λ (ng/ml) IL- +IL-.... IEC h Isg (fol) IL- +IL- Ifnlr ISG (fol) IFN-λ (ng/ml) i Osl (fol) Co IL- +IL- OASL (fol)... IFN-λ (ng/ml) j k 8 7 IL- IFN-λ Ifnlr IFN-λ + IL- Co Poliovirus (pfu/ml) VSV (pfu/ml) Co IEC IL- +IL- IL- IFN-λ IFN-λ + IL- Ntur Amri, In. All rights rsrv. Figur Cooprtion twn IL- n IFN-λ is rquir for inution of n ffiint ntivirl stt in IECs. () Ht mp of gn xprssion in IECs isolt from sukling mi t y ftr inftion with rotvirus, prsnt s xprssion in inft wil-typ mi rltiv to tht in uninft wiltyp mi ( Inf/ UI), or xprssion in inft Il / mi rltiv to tht in inft wil-typ mi (Il / /) or in inft Ifnlr / mi rltiv to tht in inft wil-typ mi (Ifnlr / /). ( ) Quntittiv RT-PCR nlysis of th ISGs Isg (), Osl (), ISG () n OASL () in th rt IEC lin IEC (,) n humn IEC lin Co (,) stimult for h with vrious oss (horizontl xs) of mous IFN-λ (,) or humn IFN-λ (,) lon ( IL-) or in omintion with mous IL- (,) or humn IL- (,) t os of ng/ml (+IL-) (prsnt s in Fig.,). (f,g) Quntittiv RT-PCR nlysis of Isg (f) n Osl (g) in IECs from wil-typ sukling mi h ftr suutnous injtion of vrious oss (horizontl xs) IFN-λ lon ( IL-) or togthr with µg of IL- (+IL-) (prsnt s in Fig.,). (h,i) Quntittiv RT-PCR nlysis of Isg (h) n Osl (i) in IECs isolt from th smll intstin of wil-typ or Ifnlr / ult mi h ftr suutnous injtion of µg of IL- (prsnt s in Fig.,). (j,k) Quntifition of poliovirus (j) or vsiulr stomtitis virus (VSV) (k) in Co lls (j) or IEC lls (k) trt with IFN-λ ( ng/ml) n/or IL- ( ng/ml) for inftion with poliovirus (j) or vsiulr stomtitis virus (k). P <. n P <. (Stunt s t-tst). Dt r rprsnttiv of thr inpnnt xprimnts ( g,j,k; mn n s..m. of n mi pr group (), n = iologil rplits ( ), n mi pr group (f,g) or n (j,k)) or two inpnnt xprimnts (h,i; mn n s..m. of n = mi pr group (h,i)). mi prou roughly twofol mor IFN-λ t y following rotvirus inftion thn i wil-typ mi (Fig. ), proly us of th highr virl titrs in Il / mi (Fig. ). Howvr, th unn of Ifnl n Ifnl trnsripts ws fiv- to ightfol lowr t y thn t y in oth gnotyps (Fig. n Supplmntry Fig. ). virus inftion l to signifint ownrgultion of xprssion of mrna noing IFN-λR, rgrlss of th prsn or sn of IL- (Fig. n Supplmntry Fig. i), whrs th xprssion of mrna noing IL-R ws unhng (Supplmntry Fig. g). Thus, IFN-λ signling might om limiting uring rotvirus inftion, prhps us of th ownrgultion of IFN-λR xprssion y IECs or of othr prturtions of th IFN-λ signling pthwy 9,,. W ompr virl ontrol in Il / mi, Ifnlr / mi n mi ouly fiint in oth IL- n IFN-λR (Il / Ifnlr / mi). Il / Ifnlr / mi i not hv signifintly highr virl titrs thn thos of Ifnlr / mi or Il / mi (Fig.,). Th sn of ny synrgisti or itiv ffts of omin fiiny in Ifnlr n Il rltiv to th fft of singl fiiny in Ifnlr might init tht IL--mit virus ontrol rquirs funtionl IFN-λ signling pthwy. To xprimntlly rss this, w suutnously injt IL- or IFN-λ into -y-ol mi 8 h for rotvirus inftion n t ys n ftr inftion n nlyz virl rplition t y ftr inftion. Although injtion of IL- or IFN-λ into wiltyp mi (Fig.,f) or of IFN-λ into Il / mi (Fig. g) l to signifint rution in virl titrs, injtion of IL- into Ifnlr / mi i not fft rotvirus rplition (Fig. h). This monstrt tht th fft of IL- on rotvirus rplition rquir funtionl IFN-λ signling pthwy. As IL- n IFN-λ t on IECs, w isolt pithlil lls from wil-typ, Il / n Ifnlr / mi t y (Fig. n Supplmntry Fig. 7) n y (Supplmntry Fig. 7) ftr rotvirus inftion n profil th llulr trnsription. At y of inftion, gns tht r trgts of IL- (for xmpl, Rgg n Rg) wr not uprgult in Il / mi ut wr ovrxprss in Ifnlr / mi (Supplmntry Fig. 7), whih rflt th inrs IL- xprssion in Ifnlr / mi (Supplmntry Fig. f). ISGs wr not inu or wr only vry wkly inu in Ifnlr / mi (Fig. n Supplmntry Fig. 7 ). Th uprgultion of ISGs ws lso iminish in Il / mi rltiv to tht in wil-typ mi (Fig. n Supplmntry Fig. 7 ). In gnrl, th uprgultion of ISGs ws mor profounly impir in Ifnlr / mi thn in Il / mi (Fig. n Supplmntry Fig. 7 ). Howvr, th vrious ISG fmilis iffr in th xtnt to whih thy pn on IFN-λR, IL- or oth. This proly rsult from th omplx mhnisms for th rgultion of ISG inution n from th ft tht th vrious ISGs ontin iffrnt numrs of intrfron-stimult rspons lmnts n/or IFN-γ-tivt sit squns in thir promotrs 9. Furthrmor, w tt -fol inution of Stt mrna xprssion in wil-typ mi upon rotvirus inftion, whih ws impir in Il / mi n ws vn mor impir in Ifnlr / mi (Fig. n Supplmntry Fig. 7). Th positiv fk loop of tivtion y STAT n nwly synthsiz STAT protin is rquir for th full inution of ISGs n is impir in Il / mi 9. W xprimntlly ssss whthr IL- ws l to t in synrgy with IFN-λ for optiml inution of ISGs y ssssing th xprssion of six rprsnttiv ISGs (i.., Isg, Osl, Gp, Mx, Eifk n Rs) in rt IEC lin (IEC) n humn IEC lin (Co; humn pithlil olortl norinom lls) tht r h rsponsiv to IL- n IFN-λ. Although trtmnt with DVANCE ONLINE PUBLICATION ntur immunology

7 Ntur Amri, In. All rights rsrv. IL- lon i not inu ISG xprssion, it potntly nhn IFN-λ-inu ISG xprssion, n this fft ws most signifint whn IFN-λ onntrtions wr limiting (Fig. n Supplmntry Fig. 7 l). W otin similr rsults for wil-typ mi trt in vivo with IL- n IFN-λ for nlysis of th xprssion of Isg n Osl in IECs (Fig. f,g). In vivo, IL- lon ws l to inu roust ISG xprssion in ult mi (Fig. f,g). This rquir onstitutiv, low xprssion of IFN-λ y IECs n signling vi IFNλ within IECs (Supplmntry Fig. ), us injtion of IL- lon i not uprgult ISG xprssion in Ifnlr / mi (Fig. h,i). Notly, IL- n IFN-λ t in synrgy to urtil th rplition of two othr viruss tht n inft pithlil lls: poliovirus (Fig. j) n vsiulr stomtitis virus (Fig. k). Thus, IL- limit virl rplition in pithlil lls y potntiting IFN-λ-pnnt ISG xprssion in IECs. IL--mit virus ontrol is inpnnt of STAT Almost ll of th iologil ffts of IL- intifi so fr r liv to mit y th inution of STAT signling ownstrm of th IL-R in pithlil lls,. To rss th rol of STAT signling within IECs in th ontxt of rotvirus inftion, w gnrt mi with ltion of Stt in ll IECs vi ltion of loxp-flnk Stt llls (Stt fl/fl ) y Cr romins xprss from th IEC-spifi Vil promotr (Stt fl/fl Vil-Cr; ll hr; Supplmntry Fig. 8,). In n xtnsion of our t monstrting norml prolifrtion n migrtion of stm lls in inft Il / mi (Supplmntry Fig. h), mi show no vition in rotvirus titrs rltiv to tht of wil-typ mi (Fig. ) n h norml xprssion of Isg (Fig. n Supplmntry Fig. 8), VP (fol) Isg (fol) 8 Figur IL- n IFN-λ t synrgistilly for th phosphoryltion of STAT. () Virl lo in IECs from wil-typ n mi t y ftr inftion with rotvirus, ssss y quntittiv RT-PCR nlysis of VP mrna (prsnt s in Fig.,). ( h) Quntittiv RT-PCR nlysis of th STAT-pnnt gn Isg () or th STAT-pnnt i Rgg (fol) IL- (ng/ml) IFN-λ(ng/ml) Cll ount 8 8. p-stat 7.8 p-stat Bir (fol) Lrg (fol)... lthough thir xprssion of gns tht r rogniz trgts of STAT (for xmpl, Rgg, Bir n Plg) ws signifintly lowr uring sty stt (Supplmntry Fig. 8 f) n ftr rotvirus inftion (Fig. h) thn tht in wil-typ mi. Thus, th IL--inu n STAT-ontroll gn-xprssion progrm in pithlil lls ws ispnsl for th IL--mit supprssion of virl rplition. Synrgism of IL- n IFN- for optiml tivtion of STAT Pulish t otin with hptom ll lins hv init tht IL- might lso onnt to th STAT signling pthwy 7,,, whih provis tstl hypothsis for how IL- might t togthr with IFN-λ signling. W stimult IECs with gr oss of IFN-λ in th prsn or sn of IL- n nlyz th phosphoryltion of STAT protins. In ll lins, IL- or low oss of IFN-λ inu only wk phosphoryltion of STAT, whrs high oss of IFN-λ l to roust phosphoryltion of STAT (Fig. i). Notly, omintion of low os of IFN-λ n stimultion with IL- l to phosphoryltion of STAT omprl to tht hiv y stimultion with high os of IFN-λ (Fig. i). Applition of IL- or IFN-λ in vivo l to wk phosphoryltion of STAT in IECs (Fig. j n Supplmntry Fig. 8g). Comin pplition of IL- n IFN-λ l to synrgistilly nhn formtion of phosphorylt STAT (Fig. j) n high xprssion of mrna from ISGs (t not shown). W otin similr rsults in vitro with two inpnnt IEC lins (Fig. k n Supplmntry Fig. 8h,i). Although phosphoryltion of STAT ws inu only y IL- (Fig. i k n Supplmntry Fig. 8g i), phosphoryltion of STAT rquir th prsn of IFN-λ n ws not inu y IL- (Fig. k). Injtion of IL- fil to.7. j p-stat STAT Atin p-stat STAT Atin f Plg (fol) Intstin IL- + + IFN-λ + + k g Plg (fol)... IL- IFN-λ p-stat STAT Atin p-stat p-stat STAT Atin IEC h My (fol) 8 Co gns Rgg (), Bir (), Lrg (), Plg (f), Plg () n My (h) in IECs from wil-typ n mi t y ftr mok inftion () or inftion with rotvirus (prsnt s in Fig.,). (i) Flow ytomtry nlyzing phosphorylt (p-) STAT (top row) n STAT (ottom row) in IEC lls lft unstimult (gry shing) or stimult for min with vrious onntrtions (ov plots) of IL- n/or IFN-λ (lk lins). Numrs ov rkt lins init prnt lls with phosphoryltion of STAT (top row) or STAT (ottom row). (j) Immunolot nlysis of totl n phosphorylt STAT n STAT, n tin (loing ontrol), in tissus of th smll intstin of wil-typ mi h ftr suutnous injtion of vrious omintions (ov lns) of IFN-λ ( µg) n/or IL- ( µg). (k) Immunolot nlysis of totl n phosphorylt STAT n STAT, n tin, IEC lls (lft) n Co lls (right) stimult for min with vrious omintions (ov lns) of IFN-λ ( ng/ml) n/or IL- ( ng/ml). P <., P <. n P <. (Stunt s t-tst () or two-wy ANOVA ( h)). Dt r rprsnttiv of two inpnnt xprimnts ( h; mn n s..m. of n mi pr group), thr xprimnts (i,k) or two xprimnts (j). ntur immunology DVANCE ONLINE PUBLICATION

8 Isg (fol) 8 IL- Stt / Osl (fol).8... IL- Stt / Isg (fol) IL- IFN-λ Ctrl STAT STAT VP (fol ) Dy Dy Stt / Il / Ifnlr / Isg (fol) Dy Dy Stt / Il / Ifnlr / f (ID ) Stt / Il / Ifnlr / Dy Colon Figur 7 Enhnmnt of IFN-λ-pnnt ISG xprssion y IL- rquirs STAT signling. (,) Quntittiv RT-PCR nlysis of Isg () n Osl () in RNA isolt from tissu of th smll intstin of ult wil-typ or Stt / mi h ftr trtmnt with IL- ( µg) (prsnt s in Fig.,). () Quntittiv RT-PCR nlysis of Isg in IEC lls trt with ontrol (Ctrl) or STAT- or STAT-spifi smll intrfring RNA (ky) n stimult for h with vrious omintions (low plot) of IL- ( ng/ml) n/or IFN-λ ( ng/ml) (prsnt s in Fig.,). () virus rplition in th smll intstin of wil-typ, Stt / or Il / Ifnlr / sukling mi t y or y ftr inftion with rotvirus (ssss n prsnt s in Fig.,). () Quntittiv RT-PCR nlysis of Isg in IECs of th smll intstin of mi s in (prsnt s in Fig.,). (f) ELISA of th shing of virus into olon tissu of mi s in t y ftr inftion. Eh symol rprsnts n iniviul mous; smll horizontl lins init th mn (± s..m.). P <., P <. n P <. (two-wy ANOVA). Dt r rprsnttiv of two inpnnt xprimnts (,, f; mn n s..m. of n (,) or n ( f) mi pr group) or four inpnnt xprimnts (; mn n s..m. of n mi pr group). Ntur Amri, In. All rights rsrv. nhn ISG xprssion in Stt / mi (Fig. 7,), whrs mi show norml ISG xprssion following rotvirus inftion (Fig. ). W lso ssss th xprssion of th gns noing SOCS n SOCS, whih r inhiitory rgultors of STAT signling. Th xprssion of Sos n Sos ws not highr in Il / mi thn in wil-typ mi (Supplmntry Fig. 8j), whih rul out th possiility tht th nhn rplition of rotvirus in th sn of IL- ws u to nhn xprssion of ngtiv rgultors of STAT signling. Sos xprssion ws not ltr in Ifnlr / mi rltiv to tht in wil-typ mi, whrs Sos xprssion ws lowr in Ifnlr / mi thn in wil-typ mi (Supplmntry Fig. 8j), onsistnt with th fining tht Sos xprssion n inu y STAT signling. Thus, our t monstrt tht IL- n IFN-λ t synrgistilly for th phosphoryltion of STAT, whih ws n to urtil virl rplition in IECs n to limit tissu mg. To mor finitivly rss th rol of STAT in th nhn xprssion of ISGs following stimultion with IFN-λ n IL-, w us smll intrfring RNA to mit th knokown of STAT or STAT in IECs (Supplmntry Fig. 8k) n ssss th xprssion of ISGs. Th omintion of IL- n IFN-λ l to signifintly highr xprssion of mrna from ISGs thn i stimultion with IFN-λ lon, n this synrgy ws ntirly pnnt on STAT, not STAT (Fig. 7 n Supplmntry Fig. 8k). W rson tht if STAT signling wr th ntrl signling pthwy tht mit th ooprtion twn IFN-λ n IL-, thn th phnotyp of Stt / n Il / Ifnlr / mi woul mostly similr. Th rplition of rotvirus (Fig. 7) n inution of Isg (Fig. 7) wr lrgly similr in Stt / mi n Il / Ifnlr / mi. Howvr, Stt / mi show signifintly mor shing of virus vi th fs thn i Il / Ifnlr / mi (Fig. 7f), suggstiv of itionl STAT-mit ffts on th shing of virus tht wr inpnnt of IL- n IFN-λ. Thus, th ooprtion twn th muosl ytokins IL- n IFN-λ for th optiml inution of ISGs n for th urtilmnt of rotvirus rplition oul xplin to lrg xtnt y th ooprtion of ths ytokins in th optiml tivtion of STAT. DISCUSSION Borr surfs suh s thos of th intstinl pithlium r mjor ntry ports for viruss, n thy n multi-lyr n fil-sf mhnisms of prottion. Hr w hv intifi los ooprtion of two volutionrily rlt muosl ytokin ntworks (i.., IFN-λ n IL-), tht t synrgistilly to urtil muosl virl inftion, inluing inftion with rotvirus, th ling us of hilhoo gstrontritis worlwi. Our t monstrt tht IFN-λ proution y IECs lon, lthough rquir, ws not suffiint to limit rotvirus-inflit mg to IECs. Inst, th onrt tion of IL- provi y ILC lls n IFN-λ ws rquir for fftiv ontrol of rotvirus rplition. Th rlt rptors for IL- n IFN-λ wr xprss lmost xlusivly y pithlil lls, n simultnous nggmnt of oth rptors on IECs ws rquir for fftiv inution of ISGs tht onfrr n ntivirl stt to IECs. It is notl tht typ I intrfrons m only minor ontriution to immunity to homologous rotvirus inftion, whrs thy r rquir for immunity to inftion with htrologous rotviruss (for xmpl, inftion of mi with rhsus mqu rotvirus),. Suh finings rflt th low xprssion of th rptor for typ I intrfrons y IECs n strong pttion of rotvirus strins to thir homologous hosts n th xistn of strtgis for vsion of th immun systm tht trgt th intrfron pthwys 9,,. It hs n monstrt tht IFN-λ, not typ I intrfrons, ontrols prsistnt ntri inftion with nothr importnt n hlth-rlvnt intstinl virus, norovirus,. Colltivly, suh t intify IFN-λ s n importnt ntivirl fns strtgy t th intstinl rrir. Following inftion with rotvirus, IECs prou n rls IL-α, n pithlil lrmin tht nhn IL- proution y CCR + ILC lls. IL- (rf. 7) n IL-β 8 xprss y hmtopoiti lls hv n shown to mjor rgultors of IL- xprssion t sty stt n in th ontxt of inflmmtory rsponss. Howvr, uring rotvirus inftion, nithr IL- nor IL-β ws n to sustin IL- xprssion. Inst, only nutrliztion of IL-α inhiit th proution of IL- y ILC lls ftr rotvirus inftion. Anothr ytokin of th IL- fmily, IL-8, is prou y pithlil lls, n rls of IL-8 is rquir for th trimntl unnt proution of IL- following inftion with Toxoplsm gonii. Colltivly, ths t to th mrging onpt tht ytokins of th IL- fmily tht r prou y pithlil lls (i.., IL-α n IL-8) r n importnt trminnt of IL- proution y ILC lls. IL- hs n link to rgultion of th xprssion of ntimiroil pptis in IECs rquir for prottion ginst tril inftion,7 n to th promotion of tissu prottion n rpir DVANCE ONLINE PUBLICATION ntur immunology

9 Ntur Amri, In. All rights rsrv. following vrious typs of pithlil insults, suh s xtrn soium sulft mit olitis, irrition 8, grft-vrsus-host iss 9 n inftion with influnz virus. Almost ll of th iologil funtions ssign to IL- hv n link to STAT signling in pithlil lls, lthough it is known tht IL- n l to wk tivtion of STAT in svrl ll lins 7,,. Howvr, th iologil onsquns of suh IL--rivn tivtion of STAT hv not n xplor. Pulish t hv shown tht injtion of IL- into rotvirus-inft mi ls to rution in virl lo, n fft inpnnt of typ I n typ II intrfrons. Howvr, th mhnism y whih IL- rstrins virl rplition rmin unknown, n whthr nognously prou IL- hs ny rol in rstriting rotvirus inftion ws not rss. Our t hv now monstrt tht ooprtion twn IL- n IFN-λ for synrgisti tivtion of STAT ws rquir for optiml trnsription of ISGs n for limiting th rplition of muosl viruss suh s rotvirus, poliovirus n vsiulr stomtitis virus. Colltivly, our t ssign notl n prviously unpprit rol to IL- for urtiling virl inftion. Th ooprtion twn n pithlil ll utonomous ntivirl fns systm (typ III intrfron) with n volutionrily rlt ut hmtopoiti ll riv ytokin (IL-), oth of whih t on pithlil lls, might init son lyr of virl ontrol tht m nssry, throughout th o-volution of viruss n vrtrt hosts, for thos hosts to fn off strtgis y whih viruss v th immun systm, suh s ownrgultion of IFN-λR xprssion n intrfrn with STAT signling y rotvirus 8. Notly, in humns n mi, rotvirus infts minly infnts. Thrfor, our t ontriut to th unrstning of immunity in rly lif, whn omponnts of ptiv immun systm r not yt fully oprtiv. Ths rsults rprsnt n vn in th unrstning of how muosl ytokin ntworks t togthr for ffiint ntivirl immunity to limit mg t th pithlil rrir. Mthos Mthos n ny ssoit rfrns r vill in th onlin vrsion of th ppr. Assion os. Europn Nuloti Arhiv: miroiot S rdna mplion t, PRJEB899. Not: Any Supplmntry Informtion n Sour Dt fils r vill in th onlin vrsion of th ppr. Aknowlgmnts W thnk V. Sxl (Univrsity of Vtrinry Miin Vinn) for mi with Stt fl lll (provi with prmission from V. Poli, Univrsit i Torino); M. Stmmlr (Mx-Plnk-Institut of Immunoiology & Epigntis, Friurg) for Vil-Cr mi (provi with prmission from S. Roin. Institut Curi, Pris); C. Johnr (Mx-Plnk-Institut of Immunoiology & Epigntis, Friurg) for Rg / n Rg / Ilrg / mi; D. Littmn (Skirll Institut of Biomolulr Miin) for Ror-Cr TG mi; N. Ghilri (Gnnth) for Il / mi; M. Hornf (Hnnovr Mil Shool) for th rt IEC lin IEC n th mous rotvirus strin EDIM n for isussions; S. Koik (Tokyo Mtropolitn Institut of Mil Sin) for poliovirus typ I Mhony strin; G. Häkr for support; mmrs of th Difnh lortory for isussions; A. Trintfyllopoulou for ritil ring of th mnusript; K. Orl, S. Woltmt n Z. Fiig for thnil ssistn; n M. Follo, K. Gigr n J. Boink-Wrsing for ll sorting. Support y ERC (77 to A.D.), Dutsh Forshungsgminshft (Priority Progrm Intstinl Miroiot DI7/ to A.D. n SU/9 to S.S.; GRK to A.D. n F.G.; n SFB9/Z to S.S.) n th Exlln Inititiv of th Dutsh Forshungsgminshft n th Grmn Sin Counil (Spmnn Grut Shool of Biology n Miin (GSC- to T.M.); n th Clustr of Exlln Inflmmtion t Intrfs (Borstl-Kil-Lük-Plön) EXC to C.H.). AUTHOR CONTRIBUTIO P.P.H. n T.M. prform most of th xprimnts with th hlp of V.S., N.N., F.G. n K.G.; I.Y. n S.S. prform th miroiot nlysis; B.R., C.H., L.D. n J.-C.R. provi mi, rgnts n input on th mnusript; P.S. n A.D. sign th stuy; P.P.H., T.M., P.S. n A.D. nlyz th t; n A.D. irt th stuy n wrot th mnusript (with ontriutions from P.P.H., T.M. n P.S.). COMPETING FINANCIAL INTERESTS Th uthors lr no ompting finnil intrsts. Rprints n prmissions informtion is vill onlin t rprints/inx.html.. Moon, C. & Stppnk, T.S. Virl intrtions with th host n miroiot in th intstin. Curr. Opin. Immunol., ().. Durkop, B.A. & Hoopr, L.V. Rsint viruss n thir intrtions with th immun systm. Nt. Immunol., 9 ().. Ktz, M.G., H, Y. & Gl, M. Jr. Viruss n intrfron: fight for suprmy. Nt. Rv. Immunol., 7 87 ().. Durin, R.K., Kotnko, S.V. & Durin, J.E. Intrfron inution n funtion t th muosl surf. Immunol. Rv., 9 ().. Kotnko, S.V. t l. IFN-lms mit ntivirl prottion through istint lss II ytokin rptor omplx. Nt. Immunol., 9 77 ().. Shppr, P. t l. IL-8, IL-9 n thir lss II ytokin rptor IL-8R. Nt. Immunol., 8 (). 7. Dumoutir, L. t l. Rol of th intrlukin (IL)-8 rptor tyrosin rsius for ntivirl n ntiprolifrtiv tivity of IL-9/intrfron-λ: similritis with typ I intrfron signling. J. Biol. Chm. 79, 9 7 (). 8. Ank, N. t l. Lm intrfron (IFN-λ), typ III IFN, is inu y viruss n IFNs n isplys potnt ntivirl tivity ginst slt virus inftions in vivo. J. Virol. 8, 9 (). 9. Doyl, S.E. t l. Intrlukin-9 uss typ intrfron-lik progrm to promot ntivirl rsponss in humn hptoyts. Hptology, 89 9 ().. Slr, A.J. & Willims, B.R. Intrfron-inuil ntivirl fftors. Nt. Rv. Immunol. 8, 9 8 (8).. Kotnko, S.V. IFN-lms. Curr. Opin. Immunol., 8 9 ().. Wolk, K. t l. IL- inrss th innt immunity of tissus. Immunity, ().. Sommryns, C., Pul, S., Sthli, P. & Mihils, T. IFN-lm (IFN-λ) is xprss in tissu-pnnt fshion n primrily ts on pithlil lls in vivo. PLoS Pthog., 7 (8).. Rutz, S. & Ouyng, W. Rgultion of intrlukin- n intrlukin- xprssion in T hlpr lls. Curr. Opin. Immunol., ().. Zhng, Y. t l. Intrlukin- mits rly host fns ginst tthing n ffing tril pthogns. Nt. M., 8 89 (8).. Pikrt, G. t l. STAT links IL- signling in intstinl pithlil lls to muosl woun hling. J. Exp. M., 7 (9). 7. Rmig, R.F. Pthognsis of intstinl n systmi rotvirus inftion. J. Virol. 78, (). 8. Sn, A., Rott, L., Phn, N., Mukhrj, G. & Grnrg, H.B. virus P protin inhiits intrfron-mit STAT tivtion. J. Virol. 88, (). 9. Hollowy, G. & Coulson, B.S. Innt llulr rsponss to rotvirus inftion. J. Gn. Virol. 9, ().. Arnol, M.M., Sn, A., Grnrg, H.B. & Ptton, J.T. Th ttl twn rotvirus n its host for ontrol of th intrfron signling pthwy. PLoS Pthog. 9, ().. Pott, J. t l. IFN-λ trmins th intstinl pithlil ntivirl host fns. Pro. Ntl. A. Si. USA 8, ().. Fng, N. t l. Rol of intrfron in homologous n htrologous rotvirus inftion in th intstins n xtrintstinl orgns of sukling mi. J. Virol. 8, (8).. Sn, A. t l. Innt immun rspons to homologous rotvirus inftion in th smll intstinl villous pithlium t singl-ll rsolution. Pro. Ntl. A. Si. USA 9, 7 7 ().. Kumr, P., Thkr, M.S., Ouyng, W. & Mlrknnn, S. IL- from onvntionl NK lls is pithlil rgnrtiv n inflmmtion prottiv uring influnz inftion. Muosl Immunol., 9 8 ().. U, C. t l. Fmilil trnsmission rthr thn ftiv innt immunity shps th istint intstinl miroiot of TLR-fiint mi. J. Exp. M. 9, ().. Znwiz, L.A. t l. IL- fiiny ltrs oloni miroiot to trnsmissil n olitogni. J. Immunol. 9, (). 7. Lutrh, H. t l. Mous CD8lph+ DCs n humn BDCA + DCs r mjor prours of IFN-lm in rspons to poly IC. J. Exp. M. 7, 7 77 (). 8. Yin, Z. t l. Typ III IFNs r prou y n stimult humn plsmytoi nriti lls. J. Immunol. 89, 7 7 (). 9. Snos, S.L. t l. RORγt n ommnsl miroflor r rquir for th iffrntition of muosl intrlukin -prouing NKp + lls. Nt. Immunol., 8 9 (9).. Ling, S.C. t l. Intrlukin (IL)- n IL-7 r oxprss y Th7 lls n ooprtivly nhn xprssion of ntimiroil pptis. J. Exp. M., 7 79 (). ntur immunology DVANCE ONLINE PUBLICATION

10 . Mrtin, B., Hirot, K., Cu, D.J., Stokingr, B. & Vlhon, M. Intrlukin-7- prouing γδ T lls sltivly xpn in rspons to pthogn prouts n nvironmntl signls. Immunity, (9).. Zinl, C.L. t l. IL--prouing nutrophils ontriut to ntimiroil fns n rstitution of oloni pithlil intgrity uring olitis. Pro. Ntl. A. Si. USA, ().. Klos, C.S. t l. A T-t grint ontrols th ft n funtion of CCR-RORγt + innt lymphoi lls. Ntur 9, ().. Kiss, E.A. t l. Nturl ryl hyroron rptor ligns ontrol orgnognsis of intstinl lymphoi follils. Sin, ().. Erl, G. t l. An ssntil funtion for th nulr rptor RORγ(t) in th gnrtion of ftl lymphoi tissu inur lls. Nt. Immunol., 7 ().. Frno, M.A. & Grnrg, H.B. Immunity to rotvirus inftion in mi. J. Inft. Dis. 79 (suppl. ), S S9 (999). 7. Kinnrw, M.A. t l. Intrlukin proution y intstinl CD + CD + nriti lls in rspons to tril flgllin nhns muosl innt immun fns. Immunity, 7 87 (). 8. Rynrs, A. t l. Intity, rgultion n in vivo funtion of gut NKp + RORγt + n NKp + RORγt lymphoi lls. EMBO J., 9 97 (). 9. Shoggins, J.W. & Ri, C.M. Intrfron-stimult gns n thir ntivirl fftor funtions. Curr. Opin. Virol., 9 ().. Bhmnn, M., Ulziit, S., Hrl, L., Pfilshiftr, J. & Muhl, H. IFNα onvrts IL- into ytokin ffiintly tivting STAT n its ownstrm trgts. Biohm. Phrmol. 8, 9 ().. Ljun, D. t l. Intrlukin- (IL-) tivts th JAK/STAT, ERK, JNK, n p8 MAP kins pthwys in rt hptom ll lin. Pthwys tht r shr with n istint from IL-. J. Biol. Chm. 77, 7 8 ().. Di, X. t l. SOCS-ngtiv fk of STAT tivtion is ky pthwy in th srna-inu innt immun rspons of humn krtinoyts. J. Invst. Drmtol., 7 8 ().. Angl, J., Frno, M.A., Grnrg, H.B. & Bss, D. Lk of rol for typ I n typ II intrfrons in th rsolution of rotvirus-inu irrh n inftion in mi. J. Intrfron Cytokin Rs. 9, 9 (999).. Ni, T.J. t l. Intrfron-λ urs prsistnt murin norovirus inftion in th sn of ptiv immunity. Sin 7, 9 7 ().. Blrig, M.T. t l. Commnsl miros n intrfron-λ trmin prsistn of ntri murin norovirus inftion. Sin 7, 9 ().. Muñoz, M. t l. Intrlukin- inus intrlukin-8 xprssion from pithlil lls uring intstinl inftion. Immunity, (). 7. Aujl, S.J. t l. IL- mits muosl host fns ginst Grm-ngtiv tril pnumoni. Nt. M., 7 8 (8). 8. Dukov, J.A. t l. Intrlukin- rivs nognous thymi rgnrtion in mi. Sin, 9 9 (). 9. Hnsh, A.M. t l. Intrlukin- protts intstinl stm lls from immunmit tissu mg n rgults snsitivity to grft vrsus host iss. Immunity 7, 9 ().. Zhng, B. t l. Virl inftion. Prvntion n ur of rotvirus inftion vi TLR/NLRC-mit proution of IL- n IL-8. Sin, 8 8 (). Ntur Amri, In. All rights rsrv. DVANCE ONLINE PUBLICATION ntur immunology

11 Ntur Amri, In. All rights rsrv. ONLINE METHODS Mi. Convntionl (spifi pthogn fr) C7BL/ mi wr from Jnvir Lortoris. Tr / (rf. ) n Tr / mi from Jkson Lortoris wr ross in th niml fility of th Dprtmnt of Mil Miroiology n Hygin of Friurg Univrsity Mil Cntr, to otin Tr / Tr / mi. Mi with Stt fl lll wr provi y V. Sxl (with prmission from V. Poli). Vil-Cr mi wr provi y M. Stmmlr (with prmission from S. Roin). Mi with IEC-spifi ltion of Stt ( ) wr gnrt y ring of Stt fl/fl mi with Vil-Cr mi. Rg / n Rg / Ilrg / mi wr provi y C. Johnr. Il / mi, B.AG-Mx wil-typ mi rrying intt Mx llls (wil-typ), B.AG-Mx-Ifnlr / mi lking funtionl typ III intrfron rptors (Ifnlr / ), B.AG- Mx-Ifnlr / Il / oul-knokout mi (Ifnlr / Il / ), Ahr ILC,T mi n Stt / mi 7 wr r in th niml fility not ov. Ror- Cr TG mi 8 wr gift from D. Littmn. Ilr / mi 9 n Il / mi wr provi y B.R. n C.H., rsptivly. Animls wr hous in orn with th guilins fin y th Frtion for Lortory Animl Sin Assoitions n th ntionl niml wlfr oy Gsllshft für Vrsuhstirkun, n xprimnts wr in omplin with th Grmn niml prottion lw (TirShG) n wr pprov y th lol niml wlfr ommitt of th Univrsity of Friurg. Cll lins n rgnts. Th humn pithlil olortl norinom ll lin Co, th humn olon rinom ll lin HT9, th Afrin grn monky kiny ll lin Vro n th Min-Dry nin kiny ll lin MDCK wr ultur in Dulo s moifi Egl mium (DMEM) (Invitrogn) supplmnt with % FCS (Sigm), glutmin (Invitrogn),. g/l gluos (Sigm) n pniillin-strptomyin. Th rt IEC lin IEC ws provi y M. Hornf. All ll lins wr mintin t 7 C n in humiifi tmosphr of % CO. Rominnt mous n humn IFN-λ n IL- wr from Pproth. Cytokins wr ilut in phospht-uffr slin (). Isoltion of LPLs n IECs. LPLs wr isolt s sri. Intstins wr isolt, ut opn longituinlly n wsh rifly in. Epithlil lls wr issoit y shking of smpls (t r.p.m. pr min) for min t 7 C in Hnk s uffr-slt solution ontining mm EDTA n mm HEPES. Epithlil lls wr ollt n thn wr ntrifug for min t, r.p.m./min t C, n ll pllts wr rsuspn in Trizol with FstPrp- instrumnt (MP Biomils). To otin highly purifi pithlil lls, th ll pllts wr igst for min in Disps ( U/ml; ; BD), ollgns D (. mg/ml; 78; Roh) n DNs A (. mg/ml; D9; Sigm-Alrih). Clls wr thn stin with DAPI (,-imiino- -phnylinol), nti-cd n nti-epcam (oth ntiois intifi low) n wr sort on FACSAri III ll sortr (BD Biosins). Th rmining lmin propri frtion ws ut with slpl into pis m in surf r for nzymti igstion with th sm nzym mix s tht us for th igstion of pithlil lls. Enrihmnt for lymphoyts ws hiv y Proll grint ntrifugtion (GE7-89-; Sigm-Alrih). In vitro stimultion n intrllulr stining. For ytokin stining, lls wr stimult t 7 C for h in RPMI mium ontining µg/ml rflin A. Clls wr prmiliz with Cytofix/Cytoprm (7; BD Biosins) n wr stin with nti-il- (intifi low). For intrllulr stining of RORγt, lls wr prmiliz with Foxp stining solutions oring to th mnufturr s protool (--; Biosin) n wr stin with llophyoynin-onjugt nti-rorγt (intifi low). For ttion of intrllulr phosphorylt STAT n STAT, IEC lls wr hrvst n thn wr fix with % prformlhy, wsh with n rsuspn for min t C in i-ol mthnol. Aftr ing wsh, lls wr inut for min t room tmprtur with F Blok n ntioy to phosphorylt STAT n ntioy to phosphorylt STAT (oth ntiois intifi low). Antiois n ELISA kits. Th following fluorophor-onjugt ntiois rtiv to mous ntigns wr us: nti-cd (-C), nti-cd (LT), nti-cd (M/7), nti-cd9 (D), nti-cd. (), nti-cd (F), nti-cd7 (B8), nti-cd7 (A7R), nti-klrg (F), nti- NK. (PK), nti-il- (H8PWSR), nti-rorγt (BD) n nti-bru (PRB-) (ll from Biosin); n nti-epcam (G8.8), nti-e-hrin (), ntioy to STAT phosphorylt t Tyr7 (), ntioy to STAT phosphorylt t Tyr7 (), ntioy to purifi CD/CD (.G) (ll from BD Biosins). Polylonl got ntioy to rotvirus (NCDV) ws from Mriin Lif Sin. Th sonry ntiois Alx Fluor 88 onjugt onky nti-rit (A-) n Alx Fluor or Alx Fluor 88 onjugt onky nti-got (A- n A-, rsptivly) wr from Invitrogn. Nutrlizing ntioy to mous IL-α (ALF-) n nutrlizing ntioy to mous IL-β (B) wr from BioXCll. For nlysis of IFN-λ, IL-α, IL-β, IL- n IL-, ELISA kits from Biosin wr us. Flow ytomtry n ll sorting. Aftr immunostining, singl-ll suspnsions of lmin propri lymphoyts wr quir with FACSCnto II flow ytomtr n FACSDiv softwr (BD Biosins). For t nlysis, FlowJo V9. softwr (TrStr) ws us. For sorting of IECs n lymphoyts, full smll intstins from rotvirus-inft s wll s ontrol sukling mi wr ut into pis m in surf r n sujt to nzymti igstion. Aftr lok of F rptors with nti-cd/cd (intifi ov), singlll suspnsions wr inut with fluorsn-onjugt nti-cd n nti-epcam (oth intifi ov). Aftr ing wsh, lls wr inut with DAPI for xlusion of lls n wr sort (to purity of >98%) with FACSAri III ll sortr (BD Biosins). Immunohistology. s wr fix for h on i with % prformlhy n wsh with. Ltr, th tissu ws inut ovrnight t C with % suros, thn ws m in optimum utting tmprtur ompoun (Skur; Fintk) n frozn in liqui nitrogn. For ttion of rotvirus, ftr lok of nonspifi ining with % onky srum, stions wr inut with nti-rotvirus n llophyoynin-onjugt E-hrin follow y sonry Alx Fluor 88 nti-got (ll ntiois intifi ov). For BrU puls-hs ssys, mi wr givn intrpritonl injtion of BrU ( mg pr kg oy wight) (Sigm) n thn kill t th pproprit tim ftr injtion. Frozn stions wr trt for h with N HCl n wr inut for h with fluorsin isothioynt (FITC) nti-bru (intifi ov). Bfor ing mount with mounting mium (PrmFluor; Thrmo Sintifi), stions wr inut with DAPI for nulr ountrstining. Tissu stions wr visuliz with n ApoTomquipp Axiopln mirosop onnt to n AxioCm igitl mr (Crl Ziss MiroImging). Altrntivly, smll intstins wr fix ovrnight t room tmprtur with % prformlhy n wr m in prffin. Aftr utting n prffiniztion, stining with hmtoxylin n osin ws prform oring to Myr s protool with rgnts from Roth. Epithlil vuoliztion (ssign gr of to init whthr thr ws no mg () or mg ws sn in on thir, two thirs () or ll () of th villi in visul fil), villus isruption (ssign gr of or to init th sn () or prsn () of rokn or mg pprn of th villus) n rkg of pithlil rrir (ssign gr of or to init th sn () or prsn () of loss of pithlil lls) ws ssss in istl, mil n proximl tissu stions from intstins of mi y invstigtors lin to smpl intify (xmpl of histopthologil hngs, Supplmntry Fig. ). Inft lls wr quntifi (pr visul fil) in prlll. Th linil sor ws trmin s follows: histopthologil sor (inft lls pr visul fil/). Miroiot nlysis. S rdna lirris wr onstrut n squn s sri, with moifitions. S rdna lirris wr onstrut following th Amplion Lirry Prprtion Mnul (pgs ) s of April (Roh), with h rtion ontining ng of DNA. Li-L fusion primrs ontin multiplx intifirs n th tmplt-spifi prts 8F ( -AGAGTTTGATCCTGGCTCAG- ) n R ( -T ACCGCGGCTGCTGG- with W = A/T). Positiv n no-tmplt ngtiv ontrols wr inlu for h multiplx intifir. PCR onitions inlu th following stps: 9 C for min, yls of s t 9 C, s t C n min t 7 C, follow y finl longtion for 8 min t 7 C. PCR prouts oi:.8/ni.8 ntur immunology