The activating effect of IFN-c on monocytes/macrophages is regulated by the LIF trophoblast IL-10 axis via Stat1 inhibition and Stat3 activation

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1 Cellulr & Moleulr Immunology (214), 1 16 ß 214 CSI n USTC. All rights reserve /14 $32. RESEARCH ARTICLE The tivting effet of IFN- on monoytes/mrophges is regulte y the LIF tropholst IL-1 xis vi Stt1 inhiition n Stt3 tivtion Anghm Dllgi 1,2, Julie Girour 1,2, Jovne Hmelin-Morrissette 1,2, Rhel Dzie 1,2, Letiti Lurent 2,3, Cthy Villnourt 2,3, Julie Lfon 2,4, Christin Crrier 5 n Crlos Reyes-Moreno 1,2 Interferon gmm (IFN-) n leukemi inhiitory ftor (LIF) re key gesttionl ftors tht my ifferentilly ffet leukoyte funtion uring gesttion. Beuse IFN- inues pro-inflmmtory phenotype in mrophges n euse tropholst ells re prinipl trgets of LIF in the plent, we investigte whether n how solule ftors from tropholst ells regulte the effets of IFN- on mrophge tivtion. IFN- reues mrophge motility, ut enhnes Stt1 tivtion, pro-inflmmtory gene expression n ytotoxi funtions. Solule ftors from villous ytotropholsts (vct1lif ells) n BeWo ells (BW/ST1LIF ells) tht were ifferentite in the presene of LIF inhiit mrophge Stt1 tivtion ut inversely sustin Stt3 tivtion in response to IFN-. vct1lif ells proue solule ftors tht inue Stt3 tivtion; this effet is prtilly rogte in the presene of neutrlizing nti-interleukin 1 (IL-1) ntioies. Moreover, solule ftors from BW/ST1LIF ells reue ell prolifertion ut enhne the migrtory responses of monoytes. In ition, these ftors reverse the inhiitory effet of IFN- on monoyte/mrophge motility. BW/ST1LIF ells lso generte IFN--tivte mrophges with enhne IL-1 expression, ut reue tumor-nerosis ftor lph (TNF-), CD14 n CD4 expression s well s impire ytotoxi funtion. Aitionl ssys performe in the presene of neutrlizing nti-il-1 ntioies n exogenous IL-1 emonstrte tht reue mrophge ytotoxiity n prolifertion, ut inrese ell motility result from the ility of tropholst IL-1 to sustin Stt3 tivtion n suppress IFN--inue Stt1 tivtion. These in vitro stuies re the first to esrie the regultory role of the LIF tropholst IL-1 xis in the proess of mrophge tivtion in response to pro-inflmmtory ytokines. Cellulr & Moleulr Immunology vne online pulition, 14 July 214; oi:1.138/mi Keywors: ell migrtion/motility; emryoni ell; gesttionl ftors; inflmmtory ell; mrophge etivtion INTRODUCTION Tissue mrophges (Mws) re of interest not only to the fiel of immunology s prt of the innte immune system, ut lso to other fiels euse they perform vrious homeostti funtions tht re ssoite with tissue orgniztion uring orgnogenesis n in mture tissues. 1,2 During the inflmmtory response, tissue Mws re expete to sequentilly evelop into three ifferent supopultions: type-1 (Mw-1), type-2 (Mw-2) n regultory (Reg-Mws). 3 6 During the initition phse of inflmmtion, the pro-inflmmtory Mw-1 sutype is generte in response to two signls (i.e., Interferon gmm (IFN-) n tumor-nerosis ftor lph (TNF-)) or Toll-like reeptor (TLR) lign, suh s lipopolyshrie (LPS). During the resolution phse of inflmmtion, the presene of the type-2 ytokines interleukin 4 (IL-4) n IL-13 les to the genertion of nti-inflmmtory Mw-2, whih re ssoite with the oorintion of tissue repir. The thir sutype (i.e., Reg-Mws) is funmentlly ifferent from Mw-1 n Mw-2, n the genertion of this sutype requires two signls. 3 6 The first signl my inlue IL-1, prostglnins, immune omplexes, enine nuleoties, gluoortiois n 1 Groupe e Reherhe en Onologie et Enorinologie Moléulires, Université u Quée à Trois-Rivières, éprtement e iologie méile, Trois- Rivières, PQ, G9A 5H7, Cn; 2 Centre e reherhe BioMe UQAM-UQTR-INRS, Montrél, PQ, H3C 3P8, Cn; 3 INRS-Institut Armn-Frppier, Université u Quée, Lvl, PQ, H7V 1B7, Cn; 4 Université u Quée à Montrél, éprtement es sienes iologiques, Montrél, QC, H3C 3P8, Cn n 5 Centre Hospitlier Régionl e Trois-Rivières, Trois-Rivières, PQ, G9A 1Y1, Cn Corresponene: Dr C Reyes-Moreno, Déprtement e Biologie Méile, Université u Quée à Trois-Rivières, 3351 oulevr es Forges, C.P. 5, Trois-Rivières, QC G9A 5H7, Cn. E-mil: Reeive: 6 Deemer 213; Revise: 3 My 214; Aepte: 3 My 214

2 2 Tropholst-meite etivtion of mrophges A Dllgi et l poptoti ells, n the seon signl is TLR tivtion. Reg-Mws overproue IL-1 ut not IL-12 n exhiit potent immunosuppressive tivity for moulting the ute inflmmtory response n limiting tissue mge. 3 6 Tissue Mws onstitute one of the most unnt popultions of inflmmtory ells in the uterus, n the numers of these ells remin reltively onstnt in the eiu throughout gesttion. 7,8 In norml pregnnies, the phenotype of uterine Mws is lolly moifie to quire type-2 phenotype, 9 11 whih my ontriute to tropholst ifferentition, support emryo implnttion n supply growth ftors to the plent. 7,8 However, lthough Mws ply importnt roles t the mternl2fetl interfe, errnt tivtion of inflmmtory pthwys in Mws n ffet tropholst survivl n funtion, 12 potentilly leing to pregnny omplitions, suh s reurrent spontneous ortion, pre-elmpsi, fetl growth restrition n intruterine infetion-ssoite preterm lor in humns. 7,8 Wheres gesttion hs long een reognize s n ntiinflmmtory Th2 onition, reent stuies emonstrte tht the proess of lstoyst implnttion requires pro-inflmmtory Th1 environment. 13,14 The trnsitory mintenne of this Th1 environment ppers to e essentil for the growth n survivl of tropholst ells, whih in turn proue lne of pro- n nti-inflmmtory ytokines tht re linke to the ontrol of leukoyte reruitment, to the regultion of inflmmtory tivity, n to the promotion of Th2 immune responses. 14 Among the numerous ytokines proue t the implnttion site, IFN- n TNF- re potent inuers of the pro-inflmmtory type-1 phenotype in tissue Mws; 15 in the ontext of the eveloping emryo, these ytokines hve oth enefiil n hrmful ttriutes. 16 Through the tivtion of the trnsription ftor signl trnsuer n tivtor of trnsription 1 (Stt1), IFN- is known to inue the expression of severl pro-inflmmtory genes in Mws, suh s the ell eth-inuing ytokine TNF- n the ell surfe reeptor lusters of ifferentition 14 n 4 (CD14 n CD4). 15 During erly pregnny, uterine nturl killer (unk) ells serete IFN-, whih plys ritil roles in the initition of enometril vsulture remoeling, in the proess of ngiogenesis t implnttion sites, n in the mintenne of eiul n plentl tissues. 16 On the other hn, inflmmtory stress in pregnnt mie inues higher levels of IFN- n TNF- synthesis y unk ells n Mws, respetively; these ytokines in turn trget uterine enothelil ells to provoke vsulr mge n plentl ishemi. 17,18 For instne, gesttionl omplitions hve een linke to elevte IFN- n/or TNF- expression in the immune-meite, erly ortion mouse moel CBA/J3DBA/2J 17,18 n in the preterm lor n elivery moel tht employs LPS-trete IL-1 knokout (KO) mie. 19,2 Moreover, s oserve uring reurrent spontneous ortion n intruterine infetion-ssoite preterm lor in humns, 7,8 errnt inflmmtory ehvior in Mws is ssoite with spontneous n enotoxin-meite ortion in roents. 21 Thus, even if pro-inflmmtory Th1 environment ppers to e require for suessful lstoyst implnttion, 13,14 the regultion of pro-inflmmtory Mw tivtion within the pregnnt uterus my e vitl to the voine of etrimentl Mw funtions, whih oul ultimtely le to plentl n fetl emise. Emryoni tropholst ells hve een propose to prtiipte intensively in this proess y prouing ftors tht moulte leukoyte funtion. 22 Reently, Mor et l. 14 propose the theory tht tropholst/immune ell intertion involves three stges. During the ttrtion step, tropholst ells serete hemokines tht n reruit immune ells to the implnttion site; uring the eution step, tropholst ells proue regultory ytokines tht moulte the ifferentition proess of immune ells; n finlly, in the response step, immune ells quire tropholst-supporting phenotype in response to signls from the lol miroenvironment. 14 However, the moleulr mehnism y whih uterine Mws re progrmme to opt Th2 phenotype in response to tropholst-erive ftors remins elusive. Mny potentil Mw progrmming ftors hve een esrie in the pregnnt humn uterus. 23,24 One ftor of interest is the pleiotropi ytokine leukemi inhiitory ftor (LIF). LIF plys n importnt role in the estlishment of pregnny y supporting eiul n plentl ifferentition n y influening reproutive trt ells, suh s leukoytes n luminl/glnulr epithelil ells. 25 The est eviene tht LIF my ply role in the regultion of Mws omes from t otine from LIF KO mie, whih present more thn hlf reue perentge of uterine Mws y ys 3 5 of pregnny. 25 The LIF KO mouse moel lso revele the entrl regultory role of LIF in enotoxi shok n host efense. Essentilly, LIF expression following enotoxi shok enhnes the expression of hepti ute-phse proteins n IL-1, whih ownregultes TNF- synthesis n relese in the liver, onferring protetion ginst enotoxemi. 26 In the uterus of pregnnt mie, LIF likely plys similr ut lol role in the protetive effet of progesterone ginst enotoxin-inue fetl emise y reuing nitri oxie levels in utero. 27 Another importnt gesttionl ftor is IL-1, whih is type-2 ytokine tht is expete to ply key role in pregnny immunotolerne through the estlishment of Th2 immune response t the mternl2fetl interfe. 28,29 IL-1 inhiits the proution of severl pro-inflmmtory ytokines, inluing TNF- in Mws n IFN- in NK ells, therey preventing the evelopment of type-1 immune retions tht re eleterious for oth the estlishment n mintenne of pregnny Thus, using the IL-1 KO mie moel to stuy LPS-inue preterm prturition, Murphy et l. 19,2 emonstrte tht in the sene of IL-1, IFN--prouing unk ells my exert ytotoxi funtions through TNF- proution n invsiveness into the plentl zone, leing to fetl emise or intruterine fetl growth restrition. 19,2 Moreover, IL-1 is note for its ilities to inhiit Mw prolifertion y tivting Stt3 33 n to suppress IFN--meite funtions of Mws y loking Stt1 tivtion. 34 In the pregnnt humn uterus, IL-1 is proue in lrge mounts y oth the synytiotropholst n the eiul Cellulr & Moleulr Immunology

3 Tropholst-meite etivtion of mrophges A Dllgi et l 3 Mws, 9,1,28,29 while LIF is highly expresse y eiul Mws n unk ells. 35 On the other hn, the IL-1 reeptor is onstitutively expresse on plentl tropholsts 28,29 n eiul Mws re known to e highly responsive to IL-1. 9,1 In ontrst, the expression level of LIF reeptors is signifintly higher in oth villous n extrvillous tropholsts thn in the eiu, 35 suggesting tht ll ells of the tropholst linege re mjor trgets for the tion of LIF in the plent. However, the possile interply etween LIF, tropholst ells n IL-1 in the moultion of Mw funtionl phenotypes hs never een investigte. Using in vitro Mw n tropholst ifferentition moels tht were previously estlishe in our l, we investigte whether n how synytiotropholst-erive ftors in generl n IL-1 in prtiulr regulte the effets of IFN- on the ehvior of Mws in response to LIF stimultion n esrie the mehnism of tion of these ftors t the moleulr level. MATERIALS AND METHODS Regents n hemils All ell ulture mei, serum n regents were purhse from Wisent (St-Bruno, PQ, CAN). Boyen hmers, ell ulture pltes n flsks were from Corning (Corning, NY, USA). The ytokines LIF, IL-1 n IFN- were purhse from Peproteh (Roky Hill, NJ, USA). The neutrlizing polylonl nti-humn IL-1 ntioy (#IC2172F) ws purhse from R&D Systems (Minnepolis, MN, USA). The hemils methylthizolyliphenyl-tetrzolium romie (MTT), imethyl sulfoxie (DMSO), forskolin (FK), propiium ioie (PI) ye n ll eletrophoresis gre hemils were purhse from Sigm Chemil Compny (Okville, ON, CAN). Trizol regent n PCR primers were purhse from Invitrogen (Crls, CA, USA). Tq DNA polymerse n M-MLV reverse trnsriptse were purhse from New Engln Biols (Whity, ON, USA). Coktils of proteses n phosphtse inhiitors were purhse from Rohe Applie Siene (Quee, PQ, Cn). The speifi inhiitors of Stt1 (i.e., JAK inhiitor I) n Stt3 (i.e., Stt3 inhiitor V) were purhse from Cliohem (Sn Diego, CA, USA). The rit polylonl ntioies (As) ginst phospho (p) Stt1 (py71), Stt1, pstt3 (py75) n Stt3 were purhse from Cell Signling Tehnologies (Pikering, ON, CAN). The monolonl peroxise-onjugte mouse nti--tin ntioy ws purhse from Sigm Chemil Compny, n the horserish peroxise-onjugte got nti-rit IgG ws purhse from Bio-R Lortories (Mississug, ON, CAN). Isoltion n purifition of term villous ytotropholsts This stuy ws pprove y the ethil ommittee of CHUM-St- Lu Hospitl (Montrel, QC, Cn). Humn term plents (i.e., weeks) were otine immeitely fter spontneous vginl eliveries from unomplite pregnnies. Immeitely fter elivery, the plents were immerse in DMEM-HG ontining ntiiotis (i.e., 5 mg/ml of mphoteriin, 5 mg/ml of gentmyin n.12 mg/ml of peniillin), mintine t 4 uc n proesse within 1 h. Villous ytotropholsts (vcts) were isolte n purifie s previously esrie y our group, using the trypsin-dnse/peroll metho Mononuler vcts were purifie vi immunomgneti lelling using n utomacs Myltenyi Biote (Auurn, CA, USA) n n nti-hla-abc ntioy, s esrie previously. 39 The purity of the immunopurifie vcts ws etermine vi flow ytometry using FITC-onjugte monolonl ntioy ginst ytokertin-7. All vct preprtions use in this stuy were t lest 98% pure fter ell sorting. The vcts were entrifuge, suspene in 37 uc ulture meium, whih onsiste of DMEM-High Gluose meium supplemente with 2 mm L-glutmine, 25 mm 4-(2-Hyroxyethyl)piperzine- 1-ethnesulfoni i (HEPES), 5% fetl ovine serum (FBS) n 13 peniillin-streptomyin-neomyin ntiioti mixture, n seee t ensity of ells/well in CellBIND six-well miropltes. After seeing, the ells were ulture for 12 h t 37 uc in humiifie tmosphere with 5% CO 2, the meium ws remove gently, n the ells were wshe twie with prewrme meium. Twenty-four hours lter, the ells were trete with pre-wrme meium ontining 5 ng/ml of humn LIF n susequently ulture for mximum of 48 h Villous ytotropholst-like BeWo ell ifferentition The BeWo humn plentl horiorinom ell line (ATCC numer CCL-98) ws ulture in RPMI-164 ell ulture meium supplemente with 1% het-intivte FBS, 1 mm soium pyruvte, 1 mm HEPES n 5 mg/ml of gentmiin (referre to s 1% FBS-RPMI meium). The humn BeWo ell line is the most extensively use in vitro ellulr moel for stuying villous ytotropholst fusion n funtion. 42 Unifferentite BeWo ells re morphologilly similr to primry ultures of mononuler ytotropholst ells, 43 with low spontneous fusion rte tht n e ooste upon tretment with forskolin (FK). 44 As we esrie previously, FK-stimulte BeWo ells lso express mrkers of typil synytiotropholst ells, suh s humn horioni gonotrophin n mrkely reue E-herin expression. 38 Moreover, BeWo ells express IL-1 t the mrna n the protein levels 45,46 n reent stuy emonstrte tht this ell line respons very well to exogenous IL Thus, these similrities to tropholst ells support the use of BeWo ells s vli n suitle moel for stuying ifferent spets of villous ytotropholst ifferentition n funtion. In this stuy, to inue ytotropholst ifferentition into synytiotropholsts (ST), 1 mm FKwsetoBeWo ell ultures (BW/ST), s previously esrie. 38 Tropholst onitione meium olletion To otin ontrol meium (), fresh 1% FBS-RPMI meium ws ple in the ell inutor for 48 h. Conitione meium (CM) ws prepre from primry ultures of villous ytotropholsts (vcts) n BW/ST ells tht were ulture for 48 h in 5% FBS-RPMI meium in the sene (i.e., CM vct2lif n CM BW/ST2LIF) or the presene of 5 ng/ml of LIF (i.e., CM vct1lif n CM BW/ST1LIF). n CM (5 ml) were entrifuge one t 25g for 5 min to eliminte ells n ell eris n were then entrifuge in entrifugl evie with moleulr weight utoff of 3 kd (Pll Life Siene; Port Wshington, NY, USA) t 32g for 45 min to seprte proteins from low moleulr weight moleules, inluing Cellulr & Moleulr Immunology

4 4 Tropholst-meite etivtion of mrophges A Dllgi et l resiul FK n LIF. The onentrte proteins were ilute with n equl volume of 1% FBS-RPMI meium. Sterile ellfree n CM were snp-frozen, store t 28 uc, n thwe on ie when require. Monoyte n mrophge ifferentition Humn sujets provie written informe onsent for loo ontion t the hemto-onologi servie of the Centre Hospitlier Régionl e Trois-Rivières (Trois-Rivières, PQ, Cn), s pprove y the Institutionl Review Bor of Centre Hospitlier Régionl e Trois-Rivières. Over perio of 1 yer, 35 voluntry loo onors were enrolle in this stuy. The men ge ws yers, n the numer of mle onors (69%) ws muh higher thn the numer of femle onors (31%). Most of the loo onors in the urrent stuy (91%) were oth non-smokers n non-loholis. The exlusion riteri inlue history of hroni immunosuppression, nti-inflmmtory use n history of heptitis B infetion. Bloo monoytes were isolte s previously esrie. 36 Aherent mononuler ells were reovere using Lymphoyte Seprtion Meium (Wisent; St-Bruno, PQ, CAN) n ulture for 5 ys with 1 ng/ml of GM-CSF to inue Mw ifferentition. On y 1, the purity of the monoyte-erive Mws (MDMw) ws pproximtely 98%, s ssesse vi the etetion of CD14 y flow ytometry. 36 The THP1 humn monoyti leukemi ell line (ATCC numer TIB-22) ws ulture in 1%FBS-RPMImeium.TheTHP1elllineisoneofthemost wiely use ell lines for investigtions of the funtion n ifferentition of monoytes n Mws in response to vrious inflmmtory meitors, suh s IFN- n teril LPS Unifferentite THP1 ells resemle primry monoytes/mws isolte from helthy onors or onors with inflmmtory iseses, suh s ietes mellitus n theroslerosis. 5 After tretment with phorol esters, THP1 ells ifferentite into mrophge-like ells, whih mimi ntive monoyte-erive Mws in severl respets. 49,51 Beuse of these hrteristis, the THP-1 ell line ws roly propose s vlule moel for stuying the regultion of Mw-speifi genes, 51 the mehnisms involve in Mw ifferentition 48 n the moleulr mehnisms in monoytes n Mws tht re ssoite with the physiology n pthophysiology of inflmmtory responses. 5 In this stuy, green fluoresent protein (GFP)-expressing-THP1 ells were ulture for 18 h in 5 nm phorol 12- myristte 13-ette to inue monoyte-to-mw ifferentition. To investigte whether tropholst-erive ftors regulte the effets of IFN- on the ehvior of Mws, MDMws ngfp- THP1-erive Mws (TDMws) were ulture in the sene of ytokines to otin ontrol ells (Mws) or in the presene of 5 U/ml of IFN- to inue pro-inflmmtory type-1 phenotype (Mw1IFN-). Migrtory/hemotti ssy Unifferentite GFP-THP1 ells were use to investigte whether tropholst ells re le to regulte the hemotti responsiveness of monoytes (Mo) to tropholst-erive ftors. The trnswell migrtory ssy ws onute in moifie Boyen hmer with memrne inserts with n 8-mm poresize, s esrie previously. 36,37 Briefly, GFP-THP1 ells were ulturefor24hinthepreseneorseneof5u/mlofifn- n susequently wshe, ounte, n seee into the upper well of the hmer t ensity of ells/1 ml. Menwhile, BW/ST ells tht were previously stimulte with phosphte-uffere sline solution (BW/ST2LIF) or LIF (BW/ST1LIF) were seee in the lower well. The GFP- THP1 ells tht migrte own to the lower well in response to tropholst-erive hemotti ftors were visulize using fluoresene mirosopy n ounte from imges pture t t5 h n t548 h. All oservtions were me t 35 mgnifition using ell monolyers. Five fiels were selete rnomly for eh tretment. Motility ssy The in vitro srth woun heling ssy ws performe to stuy the effets of tropholst-erive ftors on Mw ell migrtion. 52 Briefly, MDMws (331 3 ells/ml) n TDMws ( ells/ml) were seee into 24-well tissue ulture pltes to hieve monolyer with out 7% 8% onfluene. The ell monolyers were srpe with p2 pipet tip in stright line in one iretion to rete srth. To otin the sme fiel uring imge quisition, nother stright line ws srthe perpeniulr to the first woun line to rete ross in eh well. The eris ws remove, n the ege of the srth ws smoothe y wshing the ells one with 1 ml of Hnk s uffer. The ells were then ulture in, CM BW/ST2LIF or CM BW/ST1LIF in the presene or sene of 5 U/ml of IFN- for the in vitro srth ssy. To ssess the effets of exogenous IL-1, TDMws were pre-trete for 3 h with 25 ng/ml of IL-1 n susequently wshe n tivte with 5 U/ml of IFN-. Using the ross s referene point, the plte ws ple uner n inverte fluoresene mirosope n imges of the srth were quire t t5 h n t548 h. The numer of motile ells ws etermine using the Jv-se imge proessing progrm ImgeJ (Ntionl Institutes of Helth; Bethes, MD, USA), n the reltive ell motility ws expresse s rtio of motile ells t t548 h/t5h within the initil woun. To stuy the impt of inhiiting IFN--inue signling pthwy tivtion uring the heling of the woun, Mws were plte s esrie ove. After the srth ross line ws rete, the ell monolyers were trete for 6 min with either vehile (i.e.,.1% DMSO) or n optiml ose of the hemil inhiitors JAK inhiitor I (JAK Inh I) (i.e., 1 mm) n Stt3 inhiitor V (Stt3 Inh V) (i.e., 5 mm) efore inution with, CM DMSO, CM FK or CM FK1LIF in the presene or sene of 5 U/ml of IFN-. To etermine the influene of JAK Inh I n Stt3 Inh V on IFN--inue signling pthwy tivtion, pre-trete Mws were stimulte for 15 min n 3 min n ell lystes were prepre n nlyze vi immunolotting, s esrie previously Cell viility/prolifertion ssys To ssess the effets of tropholst-erive ftors on the ell viility of monoytes n Mws, unifferentite GFP-THP1 Cellulr & Moleulr Immunology

5 Tropholst-meite etivtion of mrophges A Dllgi et l 5 ells n TDMw were ulture in, CM BW/ST2LIF or CM BW/ST1LIF in the presene or sene of 5 U/ml of IFN. Cell viility ws ssesse t t5hn48husingmttssys, s previously esrie To evlute the numer of e ells, the ells were wshe n stine for 15 min with 2 mg/ ml of PI solution prior to nlysis vi flow ytometry. To estimte the influene of BW/ST ell-erive ftors on the ytotoxi tivity of Mws, TDMw were ulture for 24 h with, CM BW/ST2LIF or CM BW/ST1LIF in the presene or sene of 5 U/ml of IFN- n susequently wshe n ulture for 24 h in serum-free RPMI-164 ulture meium. Unifferentite BeWo ells were inute with onitione meium from these ell ultures. MTT ssys n PI stining were performe t t5 h,24hn48htoevlutethenumerofvilen e ells, respetively. Eh ssy ws performe in quruplite n represents three inepenent experiments. Protein immunoetetion TDMws(75313 ells/ml) were pre-trete for 24 h with, CM BW/ST2LIF or CM BW/ST1LIF or pre-trete for 3 h with exogenous IL-1 t onentrtions of 5 n 25 ng/ml. The ells were then wshe n tivte with 5 U/ml of IFN- t t5min, 15 min n 3 min. Cell lystes were prepre n nlyze y immunolotting, s esrie previously Briefly, protein smples were resolve y SDS PAGE uner reuing onitions n trnsferre onto polyvinyliene fluorie (PVDF) memrne. The lots were first proe with rit polylonl ntioies ginst pstt1 n pstt3 (oth t ilutions of 1 : 2) overnight t 4 uc. The lots were then inute with horserish peroxise-onjugte got nti-rit IgG A t ilution of 1 : 3 for 1 h t room temperture. The sme lots were strippe n then proe with nti-stt1 n nti-stt3 As, whih were oth use t ilutions of 1 : 1. In oth ses, the proe moleules were visulize using n enhne hemiluminesene etetion kit (Thermo Fisher Sientifi; Wlthm, MA, USA). RNA isoltion n reverse trnsriptse polymerse hin retion (RT-PCR) nlyses Totl RNA extrtion, preprtion of first-strn DNA y RT PCR were performe s previously esrie y our group. 36,37 The PCR retion onitions were hosen to ensure tht the mplifition of mrna ourre in the mile of the exponentil mplifition phse to voi mrna mplifition lose to plteu n sturtion. Glyerlehye 3-phosphte ehyrogense (GAPDH) mrna expression ws use s n internl stnr. The sequenes of the primers use for mplifition were 59-GTCAGTGGTGGACCTGACCT-39 (sense (S)) n 59-TGAGCTTGACAAAGTGGTCG-39 (ntisense (AS)) for G- APDH; 59-CAGAGGGAAGAGTTCCCCAG-39 (S) n 59-CC- TTGGTCTGGTAGGAGACG-39 (AS) for TNF-; n 59-TG- AGAACCAAGACCCAGACA-39 (S) n 59-TCATGGCTTT- GTAGATGCCT-39 (AS) for IL-1. Surfe ntigen expression nlysis To stuy memrne reeptor expression, Mws were pre-trete for 24 h with CM from BW/ST ell ultures n ollete fter 48-hour stimultion perio in the presene or sene of 5 U/ ml of IFN-. The expression levels of CD14 n CD4 were evlute y flow ytometry, s esrie previously. 36,37 IL-1 neutrliztion ssys To etermine the potentil involvement of tropholst-erive IL-1 s etivting meitor of Mw ehvior, we use polylonl ntioy irete ginst IL-1 (#IC2172F; R&D System; Minnepolis, MN, USA) to neutrlize IL-1 in tropholst CM. Western lot nlysis, MTT ssys n in vitro srth woun heling ssys were performe s esrie ove. Neutrlizing nti-il-1 or isotype ontrol ntioies, whih were oth use t onentrtions of 2 mg/ml, were e ily to Mw monolyers. A representtive result from three inepenent experiments is shown. Sttistil nlyses For ll experiments, the vlues were presente s the men6s.. from three inepenent experiments. The t were nlyze using one-wy nlysis of vrine (ANOVA) followe y the Bonferonni post-test using Prism softwre, version 3.3 (GrphP, Sn Diego, CA, USA). P vlues f.5 were onsiere to inite sttistil signifine. RESULTS Villous ytotropholst-erive ftors repress Stt1 tivtion ut inversely sustin Stt3 tivtion in IFN--tivte Mws This set of experiments ws plnne to investigte whether primry ultures of vcts re responsive to LIF n whether LIF-ifferentite vcts hve the ility to regulte the effets of IFN- on the ehvior of MDMws. As shown in Figure 1, the tivtion of the Stt3 signling pthwy ws effiiently inue in vcts; this effet ws mintine t similr levels uring 15 min n 3 min of LIF stimultion. Figure 1 shows the representtive tivtion sttus of Stt1 n Stt3 following stimultion with IFN- in MDMws preonitione for 24 h with, CM vct2lif, CM vct1lif n reominnt humn LIF (rlif). MDMws express low sl levels of pstt1 n pstt3 (t5 min) when preonitione in, in CM vct2lif, in CM vct1lif n in the presene of rlif (Figure 1). Very little or no vrition in totl Stt1 n Stt3 expression ws oserve, irrespetive of tretment. As expete, IFN- tivtes Stt1, with pstt1 levels inresing y 4.3-fol in the presene of t t515 min n y 7.6-fol t t53 min (Figure 1). These inreses in pstt1 levels were effiiently reue when MDMws were preonitione with oth CM vct1lif (1.9-fol t t515 min n 2.8-fol t t53 min) n rlif (no hnge t t515 min n 2.1-fol t t53 min) ut not with CM vct2lif (Figure 1). In ontrst to pstt1, the inution of pstt3 ws signifintly enhne y IFN- only t t515 min when MDMws were preonitione with CM vct2lif (4-fol), CM vct1lif (4.5-fol) n rlif (2.8-fol). Villous ytotropholst-erive IL-1 sustins Stt3 tivtion in Mws In this set of experiments, we investigte the iret effets of vcts-erive ftors on the inution of pstt3 in MDMws n Cellulr & Moleulr Immunology

6 6 Tropholst-meite etivtion of mrophges A Dllgi et l pstt3 Stt3 Pretretment pstt1 rlif (ng/ml) min 3 min 1 Stt3 tivtion (pstt3/stt3 rtio) Ctl 1, ; 2, CM vct-lif; 3, CM vct+lif; 4, rlif rlif 5 ng/ml 15 min 3 min Stt1 pstt3 Stt3 min 15 min 3 min Mj tivtion: IFNg 5 U/ml 1.5 Stt1 tivtion (pstat1/stat1 rtio) 1.5 min 15 min 3 min Stt3 tivtion (pstat3/stat3 rtio) min 15 min 3 min CM vct-lif CM vct+lif rlif Figure 1 () Representtive imges n grphil nlysis showing the immunoetetion of phosphorylte Stt3 in primry ultures of vcts stimulte with 5 rlifs for 15 min n 3 min. () Representtive imges n grphil nlysis showing the immunoetetion of phosphorylte Stt1 n Stt3 in Mws pre-trete for 24 h with, CM vct2lif, CM vct1lif or rlif n susequently tivte with 5 U/ml of IFN- for min, 15 min n 3 min. (, ) The rtio of phosphorylte/unphosphorylte proteins ws lulte using ensitometri nlysis of eh smple to evlute the reltive tivtion of pstt1 or pstt3. Different supersripts enote signifint ifferenes etween tretments (P,.5). CM, onitione meium;, ontrol meium; IFN-, interferon gmm; LIF, leukemi inhiitory ftor; vct, villous ytotropholst; rlif, reominnt humn LIF; Stt1, signl trnsuer n tivtor of trnsription 1. the potentil involvement of IL-1 in this proess. As shown in Figure 2, Stt3 is signifintly inue in MDMws y solule ftors present in CM vct2lif n to greter extent y solule ftors present in CM vct1lif n exogenous IL-1. Cell signling stuies performe in the presene of neutrlizing nti-il-1 ntioies emonstrte tht IL-1 is require for vct1lif ells to trigger enhne Stt3 tivtion in MDMws (Figure 2). For the remining stuies, we opte to use BeWo ell moel ue to its relevne n prtiulrly ue to its vilility n Cellulr & Moleulr Immunology

7 Tropholst-meite etivtion of mrophges A Dllgi et l 7 Pretretment: pstt3 Isotype ontrol Stt3 Pretretment: pstt3 Stt3 Stimultion: nti-il1 A min 3 min 6 min 1, ; 2, CM vct-lif; 3, CM vct+lif; 4, ril1 Stt3 tivtion (pstt3/stt3 rtio) min 3 min 6 min 15 min 3 min 6 min Isotype ontrol e Anti-IL1 A CM vct-lif CM vct+lif ril1 f IL1 GAPDH BW BW/ST-LIF BW/ST+LIF IL1 expression (IL1/GAPDH rtio) BW BW/ST-LIF BW/ST+LIF Figure 2 () Representtive imges n grphil nlysis showing the immunoetetion of phosphorylte Stt3 in Mws pre-trete for 15 min, 3 min n 6 min with, CM vct2lif, CM vct1lif or ril-1 in the presene of isotype ontrol n nti-il-1 As. The rtio of phosphorylte/unphosphorylte proteins ws lulte using ensitometri nlysis of eh smple to evlute the reltive tivtion of pstt1 or pstt3. () Representtive imges n grphil nlysis showing the trnsription level of IL-1 mrna in resting (BW) n ifferentite BW/ST ells (6LIF), s ssesse using RT-PCR. Different supersripts enote signifint ifferenes etween tretments (P,.5). CM, onitione meium;, ontrol meium; A, ntioy; LIF, leukemi inhiitory ftor; Mw, mrophge; RT-PCR, reverse trnsriptse polymerse hin retion; ril-1, reominnt humn IL-1; Stt1, signl trnsuer n tivtor of trnsription 1. reprouiility when use t erly ell ulture pssges. 38 IL-1 mrna expression ws stuie using RT-PCR to vlite the use of BeWo ells s pertinent moel for stuying the influene of villous ytotropholst-erive ftors, suh s IL-1, on the funtionl phenotype of pro-inflmmtory Mws. As expete, 28,29 unifferentite BeWo ells express sl levels of IL-1 mrna. However, we foun tht the IL-1/GAPDH rtio sustntilly inrese from.26.1 to.76.2 in BW/ST2LIF ells n to in BW/ST1LIF ells (Figure 2). Furthermore, ell signling stuies emonstrte tht similr to solule ftors from vct1lif ells, solule ftors from BW/ST1LIF ells inhiit Mw Stt1 tivtion ut inversely sustin Stt3 tivtion in response to IFN- (Supplementry Figure 1). Synytiotropholst-erive ftors ffet ell prolifertion ut enhne the migrtory response of IFN--tivte monoytes/mws To etermine the influene of ST-erive ftors on ell prolifertion, monoytes n Mws were inute for 48 h in, CM BW/ST2LIF or CM BW/ST1LIF in the presene of 5 U/ml IFN- n MTT viility/prolifertion ssys were then performe. As seen in Figure 3, the rtes of prolifertion re onsierly higher in IFN--tivte monoytes (lose squre; reltive ell viility5.982t1.5; R 2 51) thn in MDMws (lose squre; reltive ell viility5.64t1.383; R 2 51). However, strikingly reue rtes of ellulr prolifertion were oserve when IFN--tivte monoytes were ulture in Cellulr & Moleulr Immunology

8 8 Tropholst-meite etivtion of mrophges A Dllgi et l the presene of CM BW/ST2LIF (lose tringle; reltive ell viility5.476t1.924; R 2 51) n to greter extent in the presene of CM BW/ST1LIF (lose irle; reltive ell viility5.159t1.1276; R 2 51). Similr results were oserve when IFN--tivte MDMw were ulture in the presene of CM BW/ ST2LIF (lose tringle; reltive ell viility5.341t1.61; R 2 51) n CM BW/ST1LIF (lose irle; reltive ell viility5.13t1.863; R 2 51). To explore the possiility tht the oserve reution in the numer of vile ells ourre s result of ell eth, monoytes n Mw were reovere t the en of tretment n stine with PI DNA ye n the numer of e ells ws evlute y flow ytometry. The results inite tht the effet ws most likely ytostti rther thn ytotoxi euse very low n similr Monoytes/mrophges, Reltive viility (Asorne units) h 24h h 24h CM BW/ST -LIF CM BW/ST +LIF Mo+IFNg MDM+IFNg Mo t=48h Mo+IFNg t=48h Numer of migrtory monoytes Mono -76% Mo+IFNg -34% -14% e,e -LIF +LIF BW/ST ells (48h) Monoytes Alone Monoytes BW/ST-LIF Monoytes BW/ST+LIF t= h t= 48h t= h t= 48h -LIF +LIF CM BW/ST Reltive numer of motile Ms t t=48h % -38% e -82% -LIF w/o LIF +LIF wt LIF CM BW/ST Figure 3 () The effet of solule ftors from BW/ST ells on the ell prolifertion of tivte monoyte/mws ws ssesse using MTT ssys. IFN--tivte monoytes (Mo1IFN-) n Mws(Mw1IFN-) were ulture for 48 h with, CM BW/ST2LIF or CM BW/ST1LIF. Different supersripts enote signifint ifferenes etween tretments (P,.5). CM, onitione meium;, ontrol meium; GFP, green fluoresent protein; IFN-, interferon gmm; LIF, leukemi inhiitory ftor; MTT, methylthizolyliphenyl-tetrzolium romie; Mw, mrophge. () Trnswell migrtory ssys were performe in moifie Boyen hmer with memrne inserts. Monoytes stimulte in the sene (Mo) or presene of 5 U/ml of IFN- (Mo1IFN-) were seee into the upper well of the hmer, n previously ifferentite BW/ST ells (6LIF) were seee into the lower well. Monoytes tht migrte own to the lower well in response to tropholst-erive hemotti ftors were visulize using fluoresene mirosopy t t5 h n t548 h. GFP-expressing monoytes in the lower well were ounte from the pture imges. () Representtive imges n grphil nlysis showing srth woun heling ssys performe in MDMw monolyers ulture in n in CM from BW/ST ells (6LIF). A totl of 5 U/ml of IFN- ws e to inue pro-inflmmtory type-1 phenotype (Mw1IFN-). Imges of the srth were quire t t5 h n t548 h using fluoresene mirosopy. (, ) Five fiels were hosen rnomly for eh tretment. All oservtions were me t 35 mgnifition. The reltive numer of motile monoytes n Mws ws etermine n expresse s the rtio of motile ells t t548 h/t5 h. Cellulr & Moleulr Immunology

9 Tropholst-meite etivtion of mrophges A Dllgi et l 9 levels of e ells were foun in monoytes n Mws ulture with, CM BW/ST2LIF or CM BW/ST1LIF in the presene or sene of 5 U/ml of IFN- (t not shown). In this set of migrtory/hemotti ssys, unifferentite GFP-THP1 ells were use to investigte whether BW/ST ells effetively regulte the hemotti responsiveness of monoytes to LIF. To hieve this im, monoytes were first ulture for 48 h in the presene n sene of IFN- n susequently ple in the upper well of trnswell migrtory hmers. As shown in Figure 3, the numer of migrtory monoytes pssing to the lower well t t548 h in the sene of tropholst ells ws 3565; this numer inrese sustntilly when the ells were ulture in the presene of BW/ST2LIF ells (19611) n inrese to greter extent when the ells were ulture with BW/ST1LIF ells (127613). In the sene of BW/ST ells, the numer of migrtory monoytes erese from 3565 to 963, whih represents erese to 24% of the ontrol, when the ells were tivte with IFN- (Figure 3). In the presene of BW/ST ells in the lower well, IFN- inhiits monoyte migrtion less effetively (Figure 3). The proportion of migrtory ells tivte y IFN- grully inreses to 66% when the ellsreoulturewithbw/st2lif ells (71613) n to 86% when the ells re oulture with BW/ST1LIF ells (19613). A set of woun heling ssys ws esigne to investigte whether BW/ST ftors lso hnge the reltive numers of motile ells in Mw ultures pre-trete with, CM BW/ ST2LIF or CM BW/ST1LIF in the presene or sene of 5 U/ ml of IFN-. IFN- stimultion is known to e ssoite with reue hemotti responsiveness n motility in humn monoytes/mws vi the seletive inhiition of the expression of CCR2, whih is the reeptor for the hemottrtnt CCL2, whih ws formerly known s mrophge hemottrtnt protein 1 or MCP1. 53 As expete, MDMws re motile in the sene of IFN-. However, fter 48-hour ell ulture perio, the ell motility of MDMws grully inrese in response to CM BW/ST2LIF n CM BW/ST1LIF (Figure 3). The reltive numer of motile ells, whih ws expresse s rtio of motile ells t t548 h/t5 h within the initil woun, ws etermine to e 1261 with, 2562 with CM BW/ ST2LIF n 3262 with CM BW/ST1LIF (Figure 3). MDMws exhiit low motility in the presene of IFN- (i.e., reution to 18% of the ontrol). However, the perentge of motile ells within the woun grully inreses to 62% n to 81% when the ells re ulture in the presene of ftors from BW/ST2LIF n BW/ST1LIF ells, respetively (Figure 3). Similr results were otine with TDMw ultures, for whih ftors from BW/ST1LIF ells reverse the effets of IFN- on Mw motility most effiiently (Supplementry Figure 2). Inhiition of Mw motility is meite y IFN--inue Stt1 tivtion To etermine whether Stt1 n Stt3 meite the inhiitory effets of IFN- on Mw motility, TDMws were seprtely preonitione with n CM BW/ST1LIF for 24 h n susequently pre-trete with.1% DMSO, 1 mm JAK Inh I, or 5 mm Stt3 Inh V for 3 min prior to tivtion with 5 U/ml of IFN-. As shown in Figure 4, the reltive numer of motile ells t t548 h in ws etermine to e when the ells were pre-trete with DMSO, when the ells were pre-tretewithjkinhin when the ells were pretrete with Stt3 Inh V (Figure 4). The results otine using CM BW/ST1LIF emonstrte tht s expete, ftors from BW/ST1LIF ells promote the motility of Mws immoilize y IFN-; the reltive numer of motile ells t t548 h ws fter pre-tretment with Jk Inh I. It is worth noting tht this promoting effet ws signifintly inrese in Mws pre-trete with JAK Inh I ut not with Stt3 Inh V, with respetive rtios of motile ells of n (Figure 4). Cell signling stuies inite tht the IFN--meite rrest of ell motility is meite y Stt1 tivtion, s the inution of pstt1 ut not tht of pstt3 ws effiiently impee y JAK Inh I in response to IFN- (Figure 4). Moreover, s shown in Figure 4, Stt3 tivtion is require for the mintenne of ell motility in IFN--immoilize Mws y BW/ST1LIF ftors; the Mws exhiite poor motility fter pre-tretment with Stt3 Inh V (Figure 4). Synytiotropholst-erive ftors moulte Mw proinflmmtory mrker expression This set of experiments ws esigne to investigte Mw TNF- n IL-1 gene expression using RT-PCR n the expression of CD14 n CD4 using flow ytometry. Consistent with the pro-inflmmtory role of IFN-, the trnsript level of TNF ws grully upregulte in Mws stimulte with IFN-; these levels inrese from to fter 24 h of stimultion (Figure 5, left pnel). The levels of TNF- mrna were ownregulte when the Mws were pre-trete with CM BW/ST1LIF n stimulte with IFN- ( versus.396.4) (Figure 5, left pnel). In ontrst to TNF-, the levels of IL-1 mrna were upregulte when the Mws were pre-trete with CM BW/ST1LIF; the IL-1/GAPDH rtio inrese from to (Figure 5, right pnel). Notly, this rise in IL-1 mrna expression ws mintine even fter Mw tivtion with IFN-, when the IL-1/GAPDH rtio inrese from to (Figure 5, right pnel). In response to INF-,Mw CD14 n CD4 expression were enhne y 2-fol n 3.5-fol, respetively (Figure 5). However, this rise in CD14 n CD4 expression in IFN-tivte Mws ws loke when the ells were preonitione with solule ftors from BW/ST1LIF ells (Figure 5). Thus, in response to LIF, BW/ST ells hve the ility to generte IFN--tivte Mws with enhne expression of nti-inflmmtory IL-1, ut reue expression of pro-inflmmtory TNF-, CD14 n CD4. Synytiotropholst-erive IL-1 regultes proinflmmtory Mw funtion This set of experiments ws esigne to investigte the involvement of ST-erive IL-1 in the regultion of Mw prolifertion, ytotoxiity n motility. The results from MTT ssys emonstrte tht the ntiprolifertive effet of BW/ST1LIF ells on Cellulr & Moleulr Immunology

10 1 Tropholst-meite etivtion of mrophges A Dllgi et l Pretretment: DMSO Jk Inh I Stt3 Inh V Ativtion: IFNg 5 U/ml CM BW/ST +LIF t= h t= 48h t= h t= 48h IFNg CM BW/ST +LIF e Reltive numer of motile Mφs t t=48h DMSO Jk Inh I Stt3 Inh V Pretretment: DMSO JAK Inh I pstt1 Stt1 pstt3 Stt (min) Ativtion: IFNg 5 U/ml CM BW/ST+LIF Pretretment: DMSO Stt3 Inh V pstt1 Stt1 pstt3 Stt (min) Ativtion: IFNg 5 U/ml Reltive Stt1 or Stt3 tivtion Reltive Stt1 or Stt3 tivtion DMSO JAK Inh I pstt1/stt1 pstt3/stt3 DMSO Stt3 Inh V pstt1/stt1 pstt3/stt3 (min) rtio (min) rtio Figure 4 () Mws were seprtely preonitione for 24 h with n CM BW/ST1LIF n susequently pre-trete for 3 min with.1% DMSO, 1 mm JAK Inh I or 5 mm Stt3 Inh V prior to tivtion with 5 U/ml of IFN-. The motility of the Mws ws evlute using n in vitro srth ssy n visulize using fluoresene mirosope t t5 h n t548 h. Five fiels were hosen rnomly for eh tretment. All oservtions were me t 35 mgnifition. The results re presente s the men6s.. from three inepenent experiments. Different supersripts enote signifint ifferenes etween tretments (P,.5). (, ) Mws were preonitione for 24 h with or CM BW/ST1LIF n susequently pretrete with.1% DMSO, 1 mm Jk Inh I or 5 mm Stt3 Inh prior to inution with n 5 U/ml of IFN- for the inite times. The tivtion sttus of Stt1 n Stt3 ws ssesse using immunolotting stuies. The supersript enotes signifint ifferenes etween DMSO n inhiitor tretments (P,.5). CM, onitione meium;, ontrol meium; IFN-, interferon gmm; LIF, leukemi inhiitory ftor; Mw, mrophge; Stt1, signl trnsuer n tivtor of trnsription 1. Mws ws reue when the ells were preonitione with neutrlizing nti-il-1 As. The numer of IFN--tivte Mws ws reue y 38% in the presene of isotype As n y 15% in the presene of nti-il-1 As (Figure 6). To further onfirm tht LIF ts on ST/BW ells to proue IL-1 n ountert the tivting effets of IFN-, Mw-meite ytotoxiity ginst unifferentite BeWo ells ws evlute in the presene of neutrlizing nti-il-1 As. As shown in Figure 6, solule ftors from IFN--tivte Mws preonitione with ompletely loke the prolifertion of BeWo ells. However, the ytotoxi tivity of IFN--tivte Mws ginst BeWo ells ws effiiently neutrlize when the Mws were preonitione with CM BW/ST1LIF. Further, Mw-meite ytotoxiity ws restore in the presene of neutrlizing nti-il-1 As (Figure 6). Moreover, we foun tht IL-1 is require for BW/ST1LIF ells to trigger sustine motility in IFN--tivte Mws (Figure 6). Exogenous IL-1 regultes ell motility in IFN--tivte Mws Srth ssys were performe to further emonstrte the etivting effets of IL-1 on IFN--tivte Mws. We foun tht pre-tretment for 3 h with exogenous IL-1 signifintly enhne Mw motility; the rtio of motile ells inrese Cellulr & Moleulr Immunology

11 Tropholst-meite etivtion of mrophges A Dllgi et l 11 Mφ pretretment CM BW/ST + LIF CM BW/ST + LIF CM BW/ST + LIF CM BW/ST +LIF TNF IL1 GAPDH GAPDH Mφ tivtion: IFNg 5 U/ml Gene expression (Gene/GAPDH rtio) TNFα IL1. Pretretment: Ativtion (IFNg): CM BW/ST+LIF CM BW/ST+LIF Events CM BW/ST+LIF CD14 expression (MFI) CM BW/ST+LIF CD4 expression (MFI) Protein expression (MFI) CD4 CD Ve Série2 BW/ST+LIF Ve IFNg BW/ST+LIF Figure 5 Representtive imges n grphil nlysis showing the expression levels of TNF- n IL-1 () n CD14 n CD4 () in resting n IFN--tivte Mws pre-trete with n CM BW/ST1LIF. The Mws were pre-inute with n CM for 24 h n then tivte for 16 h with 5 U/ml of IFN- to evlute mrna n protein expression using RT-PCR n flow ytometry, respetively. () The supersript enotes signifint ifferenes in TNF- mrna expression etween 2IFN- n 1IFN- (P,.5). The supersript enotes signifint ifferenes in IL-1 mrna expression etween CM BW/ST1LIF n (P,.5). () Different supersripts enote signifint ifferenes etween tretments (P,.5). CM, onitione meium;, ontrol meium; IFN-, interferon gmm; LIF, leukemi inhiitory ftor; Mw, mrophge; RT-PCR, reverse trnsriptse polymerse hin retion; TNF, tumor-nerosis ftor. y up to 38% ompre to phosphte-uffere sline pretretment (Figure 7). However, s oserve with BW/ ST1LIF ftors (Figure 3), the inhiitory effet of IFN- on Mw motility ws signifintly loke when the Mws were pretrete with IL-1; the rtio of motile ells inrese y up to 78% ompre to phosphte-uffere sline pre-tretment (Figure 7). The ifferentil influene of IL-1 on Mw prolifertion n motility my result from the ility of IL-1 to suppress IFN--inue Stt1 tivtion while Stt3 tivtion is mintine (Figure 7). DISCUSSION Although IFN- hs een efine s n importnt gesttionl ftor n potent inuer of the pro-inflmmtory type-1 phenotype in Mws, 15 this ytokine exhiits oth enefiil n hrmful ttriutes in the ontext of the eveloping emryo. 54 The present stuy emonstrtes tht multinulete tropholst ells effiiently etivte pro-inflmmtory Mw- 1. For instne, Mw prolifertion, inflmmtory gene/protein expression, n ytotoxiity were signifintly reue when IFN--tivte Mws were preonitione with solule ftors Cellulr & Moleulr Immunology

12 12 Tropholst-meite etivtion of mrophges A Dllgi et l Mrophges, reltive viility (Asorne units) % -15% CM BW/ST+LIF. Isotype nti-il1 Tropholst, reltive viility (Asorne units) h 24h 48h. Mj Pretretment: BW/ST+LIF; isotype Mj Ativtion (IFNg): BW/ST+LIF; nti-il1 CM BW/ST+LIF Pretretment: Isotype nti-il1 Isotype nti-il1 Ativtion: IFNg IFNg t= h t= 48h IFNg CM BW/ST+LIF -71% Isotype nti-il Reltive numer of motile Mφs t t=48h Figure 6 Representtive imges n grphil nlysis showing the involvement of LIF-ifferentite BW/ST ells n tropholst-erive IL-1 in the regultion of Mw prolifertion (), ytotoxiity () n motility (). () MTT ssys were performe to etermine the ell survivl/prolifertion of IFN--tivte Mws inute for 48 h with n CM BW/ST1LIF in the presene of isotype ontrol n nti-il-1 ntioies. () MTT ssys were performe to ssess the ell survivl/prolifertion of unifferentite BeWo ells inute with CM from IFN--tivte Mws tht were pre-trete for 24 h with n CM BW/ST1LIF in the presene of isotype ontrol n nti-il-1 ntioies. () Srth woun heling ssys were performe to evlute the ell motility of IFN--tivte Mws tht were inute for 24 h with n CM BW/ST1LIF in the presene of isotype ontrol n nti-il-1 ntioies. Cell motility ws visulize using fluoresene mirosope t t5 h n t548 h. Different supersripts enote signifint ifferenes etween tretments (P,.5). CM, onitione meium;, ontrol meium; IFN-, interferon gmm; LIF, leukemi inhiitory ftor; MTT, methylthizolyliphenyl-tetrzolium romie; Mw, mrophge. from synytiotropholst ells. In line with our results suggesting the involvement of IL-1 in this proess, our previous stuies emonstrte tht IL-1 n IL-1-prouing Mw-2 ells inhiit Mw-1 ytotoxi tivity, 36 likely y loking the enogenous proution of TNF-, whih is the most importnt miroiil n tumoriil meitor proue y IFN-tivte Mws. 55 Severl lines of eviene suggest tht IL-1 is n importnt suppressor of tive mternl immunity tht my e involve in the proess of eptne of the fetl llogrft. 19,2,28,29 IL-1 levels inrese mrkely in women uring erly pregnny n remin elevte up to the thir trimester immeitely prior to the onset of lor. Of note, ompre to synytiotropholsts, extrvillous ytotropholsts re intrinsilly poor in IL-1 proution, 28,29 initing tht the synytium might inee ontriute lolly to ountert the eleterious effets of pro-inflmmtory ytokines uring erly pregnny. Cellulr & Moleulr Immunology

13 Tropholst-meite etivtion of mrophges A Dllgi et l 13 Uterine CD14 1 Mws re the most importnt leukoyte popultion in the pregnnt uterus of humns n mie. These ells hve potentil inflmmtory n ntigen-presenting ell funtions tht oul e etrimentl to the fetus if the ells were over-tivte in response to infetion. 7,8,13,14,17,22 CD14 is prt of ognte reeptor omplex, whih inlues TLR4 n myeloi ifferentition ftor 2 n is involve in Mw etetion of Grm-negtive teri vi ining to the outer-wll omponent LPS. 56 IFN--prime Mws proue higher levels of pro-inflmmtory ytokines n hemokines fter stimultion with LPS vi the upregultion of CD14, TLR4 n myeloi ifferentition ftor 2 expression. 57 The physiologil importne of the ownregultion of the expression of the LPS reeptor CD14 y synytiotropholst ells eomes pprent in the ontext of reent stuies tht emonstrte tht LPS my provie the nger signl through whih IFN-meite stress triggers mternl immune tivtion. This tivtion woul susequently use fetl rejetion in the ortion-prone CBA/J3DBA/2J mouse moel. This interprettion is onsistent with results emonstrting tht loking reeptors for LPS or neutrlizing LPS rogtes the loss of emryos in these pregnnt mie. 58 CD4 is ostimultory immune reeptor tht is memer of the TNF reeptor superfmily. 59,6 CD4 expression n tivtion on ntigen-presenting ells, inluing Mws n DCs, is ruil for Th1-ell meite immune responses. 61 Pretretment: PBS IL 1 PBS IL 1 Ativtion: PBS IFNg t= h t= 48h PBS +38% IFNg +78% PBS IL Reltive numer of motile Mφs t t=48h Pretretment IL1 (ng/ml) pstt1 Stt1 pstt3 Stt (min) Mφ tivtion: IFNg 5 U/ml Stt1 tivtion (reltive to t = min) Stt3 tivtion (reltive to t = min) IL1, IL1, 5 IL1, min 3 min 15 min 3 min Figure 7 () Representtive imges n grphil nlysis of srth woun heling ssys showing the involvement of IL-1 in the regultion of ell motility in resting n IFN--tivte Mws. Imges of the srth were quire t t5 h n t548 h vi fluoresene mirosopy. () Representtive imges showing the tivtion sttus of Stt1 n Stt3. The Mws were pre-trete for 3 h with 5 ng/ml n 25 ng/ml of IL-1 n susequently tivte with 5 U/ml of IFN- for min, 15 min or 3 min. Different supersripts enote signifint ifferenes etween tretments (P,.5). IFN-, interferon gmm; Mw, mrophge; Stt1, signl trnsuer n tivtor of trnsription 1. Cellulr & Moleulr Immunology

14 14 Tropholst-meite etivtion of mrophges A Dllgi et l IFN- is the most potent inuer of CD4 expression in Mws, 62 n s we estlishe previously, this inution is ssoite with enhne ytotoxi funtion tht triggers roust Mwmeite estrution of tumor ells. 37 Thus, s we suggeste for TNF- n CD14, the repression of Mw CD4 expression y synytiotropholst ells oul e viewe s mehnism tht les to reue ytotoxi n ostimultory funtions of IFN--tivte Mws. Inee, reent stuies suggeste tht humn eiul Mws oul represent regultory or suppressive type of tissue Mws tht ontriute to the evelopment of pproprite mternl immune responses to the fetus uring erly pregnny. Deiul Mws spontneously proue high levels of IL IL- 1-prouing eiul Mws expresse CD14 n HLA-DR, ut these ells exhiite lower levels of the ostimultory moleules CD4 n CD86 thn peripherl loo monoytes from pregnnt n non-pregnnt women. 11 Furthermore, eiul Mws filto ifferentite into enriti ells uner the influene of IL-4 n GM-CSF. 11 Sym et l. 63 reently emonstrte tht eiul Mws re le to suppress T-ell IFN- proution. This suppression ours vi B7-H1, whih is ostimultory lign in the B7 fmily tht negtively moultes T-ell tivity y ining to the orresponing reeptor PD-1. The Fisher s group propose tht eiul Mws n lso protet fetl ells (i.e., tropholst ells) ginst the eth-inuing tivity of IFN--prouing uterine NK ells through trnsforming growth ftor et 1-epenent mehnism. 64 IFN--meite tivtion of gene trnsription ours primrily through the trnsription ftor Stt1. 15 One Stt1 is phosphorylte y Jk1 n Jk2, Stt1 homoimerizes, trnslotes to the nuleus, n tivtes the trnsription of multiple genes thtontinnifn--tivting sequene in their promoters. 65 As reporte here, IFN- signling in Mws involves Stt1 tivtion, n synytiotropholst-erive ftors, suh s IL-1, hve the ility to sustin Stt3 tivtion ut inversely repress Stt1 tivtion in response to IFN- stimultion. Our results strongly suggestthttheseinverseeffets on Stt1 n Stt3 represent moleulr mehnism tht oul ntgonize the inhiitory effets of IFN- on Mw motility vi synytiotropholst-erive ftors n IL-1. In orne with our premise, previous stuies emonstrte tht Stt1 n Stt1-inue gene prouts meite the inhiitory effets of IFN- on Mw migrtion n tht this inhiitory effet is ompletely rogte in Stt1-efiient Mws. 66 Moreover, these stuies emonstrte tht Stt3 is require to sustin Mw motility. However, Stt3 oes not oppose the effets of IFN- euse this ytokine n still suppress THP1 ell migrtion, even if the THP1 ells express high levels of tivte Stt3. 66 IL-1 n iretly inhiit Stt-epenent gene expression inue y IFN- in Mws y suppressing the tyrosine phosphoryltion of Stt1. 34 Furthermore, similr to IL-1, other immunosuppressive ytokines n growth ftors, suh s trnsforming growth ftor et 1 n peroxisome prolifertortivte reeptor gmm ligns, re lso known to promote Mw etivtion vi the suppression of IFN--inue Stt1 phosphoryltion n tivtion. 67,68 In ition to its effet on Mw motility, we lso emonstrte tht IL-1 meites the inhiitory tion of synytiotropholst ells on Mw prolifertion. These t re onsistent with previous stuy tht emonstrte tht IL-1 inhiits Mw prolifertion n tht this ntiprolifertive signl ws not ssoite with enhne poptosis. 33 Notly, se on the ft tht Stt3 is the primry meitor of the effets of IL-1, the uthors lso emonstrte tht the suppression of Mw prolifertion is Stt3-epenent event; in ontrst, Mw etivtion oul e meite vi Stt3-inepenent pthwy. 33 The fining tht monoytes n Mws re poorly motile in the presene of IFN- is in orne with previous stuies tht emonstrte tht IFN- stimultion is ssoite with reue hemotti responsiveness n motility in humn monoytes/ Mws vi the seletive n rpi inhiition of CCR2 expression. 53 For instne, CCR2 is the reeptor for CCL2, whih is hemotti hemokine tht is essentil for the trffiking n reruitment of monoytes/mws totheeiu. 8,69 The inhiition of CCR2 expression y pro-inflmmtory gents suh s IFN- hs een propose to retin Mws t sites of inflmmtion n to t s negtive feek loop for monoyte reruitment from the loo. 7 However, euse the numer of uterine Mws remins reltively onstnt throughout gesttion, irulting loo monoytes re expete to e ontinuously reruite to the eiu y oth stroml n tropholst ells in response to iverse hemotti ftors. 8,69 Moreover, Mws re expete to e motile within oth the eiu n the fetl memrnes, s they express higher levels of genes tht promote the tivtion of migrtion. 71 One of these genes enoes the nuler repressor protein gluoortioi reeptor DNA ining ftor-1 tht ntgonizes the inhiitory effet of trnsforming growth ftor et 1 on monoyte migrtion. 72 In this ontext, our results suggest tht tropholst ells oul fvor the movement of monoytes n Mws vi similr mehnism, ntgonizing the potentil loking effets of IFN- on monoyte/mw motility. In summry, we emonstrte tht tropholst-erive ftors, suh s IL-1, n inue the ifferentition of Mws into tropholst-supporting phenotype y ontrolling Mw migrtion/motility n the mgnitue of the Mw-meite immune responses tht re inue y pro-inflmmtory ytokines, prtiulrly IFN- n TNF-. Overll, our stuy supports the theory tht immune-moulting moleules expresse y tropholst ells re key plyers in the regultion of Mw inflmmtory tivity n in the promotion of regultory/suppressive phenotype in Mws. 14 ACKNOWLEDGEMENTS This work ws supporte y grnts from the Nturl Sienes n Engineering Reserh Counil of Cn (NSERC), the Fons Quééois e l Reherhe sur l Nture et les Tehnologies (FQRNT) n the Réseu Quééois en Reproution (RQR) to CR-M AD, RD n JH-M were supporte y the RQR-CREATE sholrships progrm. JG hols postotorl fellowship from the Fons e l Reherhe en Snté u Quée (FRSQ). The uthors wish to knowlege Nhl Mr n Rorigo Flores Soto for tehnil ssistne in ell signling n ell motility stuies s well s Dr Cellulr & Moleulr Immunology

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