BATF regulates collagen-induced arthritis by regulating T helper cell differentiation

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1 Prk et l. Arthritis Reserh & Therpy (218) 2:161 RESEARCH ARTICLE Open Aess BATF regultes ollgen-indued rthritis y regulting T helper ell differentition Sng-Heon Prk 1, Jinseol Rhee 2, Seul-Ki Kim 1, Jung-Ah Kng 1, Ji-Sun Kwk 1, Young-Ok Son 1, Wn-Su Choi 1, Sung-Gyoo Prk 1 nd Jng-Soo Chun 1 Astrt Bkground: We reently demonstrted tht BATF, memer of the tivtor protein-1 (AP-1) fmily, regultes osteorthriti rtilge destrution. Here, we explored the roles nd regultory mehnisms of BATF in ollgenindued rthritis () in mie. Methods: nd K/BxN serum trnsfer were used to generte inflmmtory rthritis models in wild-type () nd Btf / mie. RA mnifesttions were determined y exmining inidene, linil sore, synovitis, synovil hyperplsi, ngiogenesis in inflmed synovium, pnnus formtion, one erosion, nd rtilge destrution. Immune fetures in RA were nlyzed y exmining immune ell popultions nd ytokine prodution. Results: BATF ws upregulted in the synovil tissues of joints in whih inflmmtory rthritis hd een used y or K/BxN serum trnsfer. The inreses in inidene, linil sore, nd utontiody prodution in -indued mie were ompletely rogted in the orresponding Btf / DBA/1 J mie. Geneti ltion of Btf lso inhiited -indued synovitis, synovil hyperplsi, ngiogenesis in synovil tissues, pnnus formtion, one erosion, nd rtilge destrution. Btf knokout inhiited the differentition of T helper (Th)17 ells nd the onversion of CD4 + Foxp3 + ells to CD4 + IL-17 + ells. However, BATF did not modulte the funtions of firolst-like synovioytes (FLS), inluding the expressions of hemokines, mtrixdegrding enzymes, vsulr endothelil growth ftor, nd reeptor tivtor of NF-κB lignd (RANKL). Conlusion: Our findings indite tht BATF ruilly medites y regulting Th ell differentition without diretly ffeting the funtions of FLS. Keywords: BATF (si leuine zipper trnsription ftor, ATF-like), Th ells, Collgen-indued rthritis, Firolst-like synovioytes (FLS), Crtilge, Bone Bkground BATF (si leuine zipper trnsription ftor, ATF-like) is memer of the tivtor protein-1 (AP-1) fmily whose memers regulte vrious iologil funtions [1 3]. BATF, whih lks trnstivtion domin, heterodimerizes with JUN to ind the AP-1 site for trnsriptionl regultion [3, 4]. We reently demonstrted tht BATF regultes osteorthritis (OA) in mie y modulting noli nd toli gene expression in hondroytes [4]. We found tht overexpression of BATF upregulted mtrix-degrding enzymes nd downregulted rtilge Correspondene: jshun@gist..kr Sng-Heon Prk nd Jinseol Rhee ontriuted eqully to this work. 1 Shool of Life Sienes, Gwngju Institute of Siene nd Tehnology, Gwngju 615, Repuli of Kore Full list of uthor informtion is ville t the end of the rtile mtrix moleules; we lso found tht BATF expression in mouse joint tissues promoted OA rtilge destrution nd tht, onversely, knokout of Btf in mie (Btf / ) suppressed experimentl OA [4]. OA nd rheumtoid rthritis (RA), whih re the most ommon types of joint rthritis, shre ertin phenotypi fetures, suh s rtilge destrution [5]. However, these diseses lerly differ in their etiologies, pthogeni mehnisms, nd the ell types ssoited with eh pthogenesis. OA is degenertive joint disese tht egins with the destrution of surfe rtiulr rtilge [6, 7]. Mehnil stresses (i.e., joint instility) nd ftors tht predispose towrd OA (i.e., ging) re importnt uses of OA pthogenesis [6, 7]. In ontrst, RA is n inflmmtory utoimmune disese tht minly trgets The Author(s). 218 Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( pplies to the dt mde ville in this rtile, unless otherwise stted.

2 Prk et l. Arthritis Reserh & Therpy (218) 2:161 Pge 2 of 11 the synovium, resulting in destrution of the joint rhiteture. Vrious ell types of joint tissues re ssoited with RA pthogenesis, inluding T ells, B ells, mrophges, synovioytes, hondroytes, nd osteolsts [8 1]. T ell-medited utoimmune responses ply ritil role in the RA pthogenesis, in whih interleukin (IL)-17-produing T helper (Th) ells t s ruil effetors [8, 11, 12]. RA is hrterized y synovitis with infiltrtion of immune ells, synovil hyperplsi tht rises vi prolifertion of synovil ells, suh s mrophge-like synovioytes (MLS) nd firolst-like synovioytes (FLS), nd ngiogenesis in the hyperplsti synovium [8 1]. Synovil ells express numerous ytokines tht hve een implited in mny of the immune proesses involved in RA [13]. RA mnifesttions lso inlude erosion of the one nd rtilge whih is used y the formtion of the pnnus, whih is n ggressive front of hyperplsti synovium. The pnnus invdes nd destroys minerlized rtilge nd one through the tion of osteolsts [8 1]. BATF is known to regulte OA rtilge destrution, nd we previously showed tht BATF overexpression in joint tissues uses synovil inflmmtion [4] suggesting tht BATF ould ontriute to inflmmtory rthritis. This notion is supported y reports tht proinflmmtory ytokines, suh s IL-1β nd IL-6, inrese BATF expression in nive CD4 + T ells [14, 15], BATF diretly regultes IL-17 expression, nd Btf-defiient mie show resistne to experimentl utoimmune enephlomyelitis [16]. BATF lso ontrols the development of folliulr Th ells (Tfh) nd lss-swith reomintion in B ells [17]. Additionlly, inhiition of the trnsriptionl tivity of -Fos/AP-1 suppresses rthriti joint destrution in mouse RA model [18]. However, while these previous reports suggest tht BATF my e involved in RA pthogenesis, the role of BATF nd its regultory mehnisms re not yet well understood. In this study, we exmined whether BATF is required for ollgen-indued rthritis (), whih is ommonly used experimentl model of inflmmtory rthritis used y T ell-dependent, ntiody-medited utoimmune response direted ginst rtilge type II ollgen [19]. Here, we show tht geneti ltion of Btf in mie suppresses the mnifesttions of, inluding synovitis, synovil hyperplsi, ngiogenesis in the inflmed synovium, nd rtilge/one erosion in the joint tissues. We lso revel tht BATF regultes y regulting Th ell differentition without diretly ffeting the funtions of FLS. Methods Mie nd experimentl RA Mle wild-type () nd Btf / DBA/1 J mie were used to generte the models. C57BL/6-kground Btf / mie [4] were krossed with DBA/1 J mie to generte Btf / DBA/1 J mie. All mie were used in ordne with protools pproved y the Animl Cre nd Ethis Committees of the Gwngju Institute of Siene nd Tehnology. ws indued y stndrd protool [19, 2]. Mie were intrdermlly injeted with inomplete Freund s djuvnt lone (nonimmunized; ) or Freund s djuvnt ontining 1 μg ollgen type II (). A ooster injetion ws given 21 dys lter. The inidene nd severity of rthritis were evluted on the indited dys fter the first immuniztion. Severity ws evluted using linil sore (grde 4) of pw swelling [19, 2]. Joint tissues were fixed, delified with.5 M EDTA, emedded in prffin, nd setioned t 5-μm thikness. Synovitis ws evluted y hemtoxylin nd eosin (H&E) stining, nd synovil inflmmtion (grde 4) ws sored s previously desried [19, 2]. The pnnus ws visulized y H&E stining nd quntified y soring (grde 4) [19, 2]. Crtilge destrution ws exmined y sfrnin-o stining nd sored using the OARSI (Osteorthritis Reserh Soiety Interntionl) grding system [4, 2]. Inflmmtory rthritis ws lso indued y K/BxN serum trnsfer [21] inndbtf / C57BL/6 mie. Arthriti trnsgeni mie (K/BxN) nd nontrnsgeni littermtes (BxN) were generted y rossing KRN T ell reeptor (TCR)-trnsgeni (K/B) mie with nonoese dieti (NOD) mie. K/BxN nd ontrol ser were olleted from K/BxN nd BxN mie, respetively, nd dministered intrperitonelly to reipient mie on dys nd 2. Mie were srified on dy 14 fter serum trnsfer. Immunohistohemistry, immunofluoresene mirosopy, nd trtrte-resistnt id phosphtse (TRAP) stining Antigens were retrieved y inuting joint setions t 6 C overnight with sodium itrte uffer (1 mm sodium itrte,.5% Tween 2, ph 6.). The setions were loked with 2% ovine serum lumin in phosphte-uffered sline (PBS), nd then inuted with primry ntiodies, inluding rit nti-batf (Brookwood Biomedil), rit nti-rankl (reeptor tivtor of NF-κB lignd) (Am), got nti-il-6 (R&D Systems), rit nti-tnf-α (tumor nerosis ftor lph) (Novus Biologils), nd rit nti-ki67 (Am). The Dko REAL Envision Detetion system ws used for hromogeni olor development. BATF-expressing ells in synovil tissues were identified y doule immunofluoresene leling of vimentin for FLS, CD11 for mrophges, CD4 for T ells, nd B22 for B ells. The following primry ntiodies were used: mouse nti-cd4, mouse nti-b22, rt nti-cd11 (Am), rit nti-batf (ThermoFisher Sientifi), nd mouse nti-vimentin (BD Phrmingen). Blood vessels in synovil tissues were deteted with mouse nti-cd31 (Dinov). TRAP tivity ws determined in joint setions s previously desried [2, 22], nd the numers of TRAP-positive osteolsts were ounted in

3 Prk et l. Arthritis Reserh & Therpy (218) 2:161 Pge 3 of 11 regions ontining pnnus-rtilge nd pnnus-one interfes. FLS ulture nd prolifertion ssys FLS were isolted from or Btf knokout () mie nd ultured s desried y Zho et l. [23]. FLS of pssges 4 8 were used for further nlysis. Pure FLS (> 9% CD9 + /< 1% CD14 + ) were identified y flow ytometry using ntiodies ginst CD9 nd CD14 (Am). FLS prolifertion in ulture ws quntified y mesuring romodeoxyuridine (BrdU) inorportion [2]. Briefly, FLS ultured in 96-well plte were treted with or without tumor nerosis ftor (TNF)-α (1 ng/ml), nd BrdU leling ws deteted using the ell prolifertion enzyme-linked immunosorent ssy (ELISA) BrdU kit (Rohe). Proliferting ells in synovil setions were identified y deteting Ki67 using n ntiody otined from Am. The empty denovirus (Ad-C) nd BATF-expressing denovirus (Ad-Btf) were s previously desried [4]. FLS were infeted with Ad-Btf nd Ad-C t the indited multipliities of infetion (MOI) for 2 h, wshed, nd mintined for 48 h efore nlysis. ELISA of utontiody prodution Collgen type II-speifi ntiodies were mesured y ELISA [2]. Ser from nd mie were loded to type II ollgen-oted 96-well pltes, inuted overnight t 4 C, wshed, nd inuted for 1 h with lkline phosphtse-leled monolonl ntiodies ginst mouse IgG1, IgG2, or IgG2 (Immunology Consultnts L). p-nitrophenyl phosphte ws used s sustrte for hromogeni retions, nd the resulting olor retion ws quntified using n ELISA plte reder. RT-PCR, qrt-pcr, nd Western lotting Totl RNA ws isolted from ultured FLS using the TRI regent. The isolted RNA ws reverse-trnsried, nd the resulting DNA ws used for RT-PCR. The PCR primers nd experimentl onditions were s previously desried for BATF, mtrix metlloproteinse (MMP)3, nd MMP13 [4], s well s CCL2, CCL5, CXCL1, CXCL5, CXCL1, GAPDH, RANKL, nd vsulr endothelil growth ftor (VEGF) [2]. For Western lotting, FLS ells were inuted on ie for 3 min with rdioimmune preipittion ssy uffer (1 mm sodium phosphte, ph 7.2, 15 mm NCl, 1% SDS, 1% deoxyholte, 1% Nonidet P-4). Whole-ell lystes were frtionted y polyrylmide gel eletrophoresis nd immunolotted using rit nti-batf (Brookwood Biomedil) nd got nti-lmin B (Snt Cruz). Flow ytometri nlysis Thymoytes, splenoytes, nd lymphoytes were isolted from 6- to 8-week-old nd Btf / mie s previously desried [2]. For detetion of ell surfe ntigens, ells (1 1 6 ) were leled with fluorohrome-onjugted primry ntiodies. For detetion of intrellulr ntigens, surfe-stined ells were fixed nd permeilized with permeiliztion uffer (ebiosiene) or Foxp3/Trnsription Ftor Stining Buffer (ebiosiene). The following ntiodies were purhsed from ebiosiene for ell stining: Alex Fluor 488 -onjugted nti-mouse CD4 nd nti-mouse/rt Foxp3; FITC-onjugted nti-mouse TCRβ nd nti-mouse CD8; PerCP Cy5.5-onjugted nti-mouse CD25, nti-mouse CD62L, nd nti-mouse interferon (IFN)-γ; APC-onjugted nti-mouse CD4, nti-mouse B22, nd nti-mouse IL-17A; PE-onjugted nti-humn/mouse CD44, nti-mouse IL-4, nti-mouse CD25, nd nti-mouse/rt Foxp3; nd efluor 45 -onjugted nti-mouse CD4, nd PE-Cynine7-onjugted nti-mouse CD4. In-vitro differentition of Th ells CD4 + T ell were isolted from the spleens of nd Btf / mie using n EsySep Mouse CD4 + T ell Isoltion Kit (Stem Cell). The ells ( ) were ultured for 12 h under Th ell-differentiting onditions [23]. Briefly, for in-vitro differentition of CD4 + T ells into Th1 ells, isolted ells were ultured with nti-mouse CD3 (5 μg/ml), nti-mouse CD28 (5 μg/ ml), IL-12 (2 ng/ml), IL-2 (2 ng/ml), nd nti-il-4 (1 μg/ml). For in-vitro differentition of CD4 + Tells into Th2 ells, isolted ells were ultured with nti-mouse CD3 (5 μg/ml), nti-mouse CD28 (5 μg/ ml), IL-4 (25 ng/ml), IL-2 (2 ng/ml), nti-ifn-γ (1 μg/ml), nd nti-il-12r (1 μg/ml). For in-vitro differentition of CD4 + T ells into Th17 ells, isolted ells were ultured with nti-mouse CD3 (5 μg/ml), nti-mouse CD28 (5 μg/ml), IL-6 (1 ng/ml), mouse-trnsforming growth ftor (TGF)-β (5 ng/ml), nti-ifn-γ (1 μg/ml), nd nti-il-4 (1 μg/ml). For in-vitro differentition of CD4 + T ells into Treg ells, isolted ells were ultured with nti-mouse CD3 (5 μg/ml), nti-mouse CD28 (5 μg/ml), mouse-tgf-β (5 ng/ml), nd IL-2 (2 ng/ml). After the Th ell differentition, ytokine prodution ws nlyzed y flow ytometry nlysis fter tivtion with phorol-12-myristte 13-ette (PMA; 5 ng/ml), ionomyin (5 ng/ml), nd Brefeldin A solution (ebiosiene) for n dditionl 4 h. To test the plstiity of Treg ells to Th17 ells in vitro, isolted ells were ultured with nti-mouse CD3 (5 μg/ml), nti-mouse CD28 (5 μg/ml), mouse-tgf-β (5 ng/ml), nd IL-2 (2 ng/ml) for 2 dys. Then, the ells were wshed nd further inuted with nti-mouse CD3 (5 μg/ml), nti-mouse CD28 (5 μg/ml), IL-6 (1 ng/ml), mouse-tgf-β (5 ng/ml), nti-ifn-γ (1 μg/ml), nd nti-il-4 (1 μg/ml) for 3 dys.

4 Prk et l. Arthritis Reserh & Therpy (218) 2:161 Cytokine nlysis For mesurement of sereted ytokine levels, lymphoytes were isolted from the lymph nodes of mie. Isolted lymphoytes (1 16) were ultured for 4 h with PMA (5 ng/ml) nd ionomyin (5 ng/ml) [24]. The LEGEND MAX mouse IL-6 ELISA kit nd mouse IL-13 Pltinum ELISA kit (Invitrogen) were used to detet IL-6 nd IL-1, respetively. The BD Cytometri Bed Arry solution (BD Biosienes) nd FACS Cnto II flow ytometer (BD Biosienes) were used to mesure sereted ytokines suh s IL-2, IL-4, IL-1, IL-17A, IFN-γ, nd TNF-α. Sttistil nlysis The nonprmetri Mnn-Whitney U test ws used for the nlysis of dt sed on n ordinl grding system, suh s the synovitis, pnnus, nd OARSI grdes. For results otined from ELISA nd nlyses of joint thikness, TRAP-positive ells, nd BrdU inorportion, the dt were first tested for onformtion to norml distriution using the Shpiro-Wilk test. The dt were nlyzed y the Student s t test (pir-wise omprisons) or nlysis of vrine (ANOVA) with post-ho tests (multi-omprisons) s pproprite. Signifine ws epted t the.5 level of proility (P <.5). Results BATF is upregulted in synovil tissues of rthriti joints To explore the possile funtions of BATF in inflmmtory rthritis, we first exmined the expression ptterns Pge 4 of 11 of BATF in the rthriti joints of DBA/1 J mie treted with. Immunostining reveled tht BATF expression ws mrkedly inresed in the inflmed synovil tissues of (Fig. 1). In ontrst, BATF ws not deteted (i.e., there ws no ovious synovitis or synovil hyperplsi) in the synovil tissues of Btf / mie sujeted to onditions (Fig. 1). As expeted, TNF-α nd IL-6 were lso mrkedly inresed in the synovil tissues (Fig. 1). To identify ell types expressing BATF in synovil tissues, we performed doule immunofluoresene stining of BATF with mrkers of vrious synovil ell types, inluding vimentin for FLS, CD4 for T ells, B22 for B ells, nd CD11 for mrophges. BATF ws deteted in susets of CD4+ T ells (> 8%), CD11+ mrophges (~ 4%), nd vimentin-expressing FLS (< 5%), ut not in B22-expressing B ells (Fig. 1). Geneti ltion of Btf inhiits We next exmined whether BATF regultes. The mnifesttions oserved in mie, inluding inidene, linil sores, nd pw swelling, were ompletely rogted in Btf / DBA/1 J littermtes (Fig. 2, ). The prodution of IgG utontiodies ginst type II ollgen is key pthologil hnge in RA pthogenesis, nd the IgG2 utontiody is prtiulrly predominnt in the model [25]. Consistent with the ove results, IgG2 prodution ws mrkedly inresed in the ser of -indued mie, ut this ws ompletely suppressed in Btf / littermtes (Fig. 2). These results olletively indite tht Fig. 1 Upregultion of BATF in rthriti synovil tissues. Representtive imges (n = 1) of BATF immunostining in synovil tissue setions of nonimmunized () nd ollgen-indued rthritis () wild-type () DBA/1 J mie nd Btf / littermtes (knokout ()). Representtive immunostining imges (n = 8) of tumor nerosis ftor (TNF)-α nd interleukin (IL)-6 in synovil tissues determined y immunohistohemil stining nd immunofluoresene mirosopy, respetively. Representtive triple-immunofluoresene mirosopi imges of mouse synovil setions (n = 12) immunostined for BATF, ell type-speifi mrkers, nd DAPI. Sle rs = 5 μm

5 Prk et l. Arthritis Reserh & Therpy (218) 2:161 Pge 5 of 11 inidene (%) Pw thikness (mm) 12 1 Autontiody (optil density) X Dys fter immuniztion 3 IgG IgG1 IgG2 2 IgG2 1 BATF is required for the pthogenesis of in DBA/1 J mie. Geneti ltion of Btf inhiits -indued synovitis, synovil hyperplsi, nd ngiogenesis in synovil tissues is hrterized y synovil hyperplsi tht rises through the prolifertion of synovioytes, synovitis with infiltrtion of immune ells, nd ngiogenesis in synovil tissues [8 11]. Here, we found tht -indued synovitis ws signifintly loked in Btf / mie, s determined y H&E stining nd inflmmtion soring (Fig. 3, ). To exmine synovil hyperplsi, we determined synovil ell prolifertion y Ki67 stining [26]. Ki67 ws highly expressed in the synovil tissues of mie sujeted to -induing onditions, wheres it ws ompletely sent from those of the orresponding Btf / mie (Fig. 3). The inhiition of synovil ell prolifertion in Btf / mie ws further onfirmed y BrdU inorportion ssys Clinil sore (-4) X Dys fter immuniztion Fig. 2 Btf inhiits in DBA/1 J mie. Inidene nd severity of ollgen-indued rthritis () symptoms in nonimmunized () nd wild-type () nd Btf / (knokout ()) DBA/1 J mie (n =2miepergroup). Typil pw imges t 3 dys fter the first immuniztion, nd pw thikness mesured with digitl thikness liper (n =2miepergroup). Type II ollgen-speifi utontiody prodution in nd nd Btf / DBA/1 J mie (n = 1 mie per group). Vlues re mens ± SEM; P <.1, P <.1, P <.1 performed using dissoited FLS otined from nd Btf / mie. Compred with FLS, Btf-defiient FLS showed signifintly less prolifertion in response to TNF-α (Fig. 3d). Inflmmtory synovil tissues express VEGF, whih stimultes ngiogenesis to mintin the hroni inflmmtory sttus [27]. Consistent with the ove results, neovsulriztion (s determined y CD31 stining) ws oserved in -indued mie ut not in Btf / mie (Fig. 3e). Previous studies showed tht FLS ontriute to y produing ngiogeni ftors ssoited with lood vessel formtion [1, 2, 27, 28], nd our group reported tht hypoxi-induile ftor (HIF)-2α, whih uses RA pthogenesis y modulting FLS funtions, stimultes VEGF expression in FLS [2]. Here, we found tht overexpression of BATF in FLS did not use detetle expression of VEGF (Fig. 3f). Additionlly, lthough BATF ws upregulted in vimentin-expressing FLS of synovium (Fig. 1), tretment of FLS with IL-6 or TNF-α, ytokines tht ritilly regulte RA pthogenesis [13], did not indue BATF expression (Fig. 3f). Furthermore, TNF-α- or IL-6-indued upregultion of mtrix-degrding enzymes (MMP3 nd MMP13), hemokines (CCL2 nd CCL5), nd hemokine reeptors (CXCL1, CXCL5, nd CXCL1) in FLS were not ffeted y Btf (Fig. 3g). Geneti ltion of Btf inhiits -indued one nd rtilge erosion As involves the formtion of pnnus, whih invdes nd destroys minerlized rtilge nd one [8 11], we next exmined the possile role of BATF in pnnus formtion nd susequent one nd rtilge destrution. Indeed, we found tht the -indued pnnus formtion found in mie ws ompletely rogted in Btf / mie (Fig. 4). Next, we exmined the expression of RANKL, whih promotes osteolst differentition to stimulte one erosion [29, 3]. The indution of in mie inresed the expression of RANKL t the one/pnnus interfe, ut this ws ompletely rogted in Btf / littermtes (Fig. 4). Although FLS re known to produe RANKL [2], BATF overexpression in FLS did not trigger ny upregultion of RANKL (Fig. 3f), suggesting tht the upregultion of BATF in the FLS of synovium is not diretly ssoited with RANKL expression. In ddition, TRAP-positive osteolsts, whih were highly inresed t the one/pnnus interfe of mie, were not oserved in Btf / littermtes (Fig. 4). These results olletively suggest tht Btf inhiits one erosion under -induing onditions y loking RANKL expression nd osteolst differentition.

6 Prk et l. Arthritis Reserh & Therpy (218) 2:161 Pge 6 of 11 d e f g Fig. 3 Btf inhiits -indued synovitis, synovil hyperplsi, nd ngiogenesis. H&E stining () nd soring of synovil inflmmtion (n = 11) () in knee nd nkle joints of nonimmunized () nd ollgen-indued rthritis () wild-type () nd Btf / (knokout ()) DBA/1 J mie. Detetion of ell prolifertion y Ki67 stining in knee joint setions of nd nd Btf / DBA/1 J mie (n = 5). d Bromodeoxyuridine (BrdU) inorportion ssys (n = 6) in FLS from nd Btf / DBA/1 J mie treted with vehile or tumor nerosis ftor (TNF)-α (1 ng/ml for 24 h). e Representtive imges (n = 6) of immunofluoresene mirosopy of CD31, whih ws used to detet lood vessels in the nkle joints of nd nd Btf / DBA/1 J mie. f mrna levels of the indited moleules, s deteted y RT-PCR in FLS isolted from synovium (n = 6). FLS were treted with or without the indited onentrtions of TNF-α or IL-6 (left), or with or without 4 MOI (multipliity of infetion) of empty denovirus (Ad-C) or the indited MOI of Ad-Btf (middle nd right). BATF ws lso deteted y Western lotting (middle). Lmin B ws deteted s loding ontrol. g nd Btf FLS were treted with interleukin (IL)-6 or TNF-α. Indited moleules were deteted y RT-PCR (n > 5). Vlues re mens ± SEM. Sle rs = 5 μm in mie used rtilge destrution, s determined y sfrnin-o stining nd OARSI grding (Fig. 4d). Similr to the se of one erosion, rtilge destrution ws ompletely rogted in Btf / littermtes (Fig. 4d). Crtilge destrution during n e used y pnnus, whih invdes nd destroys minerlized rtilge, or mtrix-degrding enzymes produed y synovioytes nd hondroytes [31, 32]. Although the mtrix-degrding enzymes in inflmmtory joint tissues re primrily produed y synovioytes, suh s FLS [31, 32], BATF overexpression in FLS did not use ny upregultion of the tested mtrix-degrding enzymes (MMP3 nd MMP13) or hemokines nd hemokine reeptors (CCL2, CCL5, CXCL1, CXCL5, nd CXCL1) (Fig. 3f). Notly, however, we previously showed tht BATF overexpression uses hondroytes to produe MMP3 nd MMP13 [4]. These results olletively suggest tht mtrix-degrding enzymes produed y hondroytes, not FLS, my ontriute to rtilge destrution during. Geneti ltion of Btf does not ffet thymi T ell development Given tht T ells, suh s Th17 ells, ply ruil roles in inflmmtory rthritis [33], we exmined whether Btf defiieny ffeted T ell development in DBA/1 J mie. Flow ytometri nlysis reveled tht thymi T ell development ws not ffeted y Btf deletion in DBA/1 J mie (Fig. 5). However, onsistent with previous report [34], Btf / mie exhiited ltered T ell popultions in peripherl orgns, suh s the spleen nd lymph nodes (Fig. 5, ). Compred with mie, T ells were slightly redued in the spleen (Fig. 5) nd lymph nodes (Fig. 5) of Btf / mie. CD4 + effetor/memory T ells were lso slightly redued in the spleen (Fig. 5) ndlymphnodes(fig.5) of Btf / mie. Overll, lthough the popultions of T ell susets in the periphery were ltered y Btf deletion, the degree of ltertion ws smll (i.e., less thn 1%) (Fig. 5, ).

7 Prk et l. Arthritis Reserh & Therpy (218) 2:161 Pge 7 of 11 Pnnus (knee) p p TRAP Pnnus (nkle) p TRAP positive ells/mm Pnnus formtion (1-4) p= d p=.1 Knee Knee rtilge p=.1 Ankle Ankle rtilge OARSI grde (1-6) RANKL p<.1 Knee p<.1 Ankle Fig. 4 Btf inhiits -indued one erosion nd rtilge destrution. Representtive imges of pnnus sujeted to H&E nd Sfrnin-O stining in nd nd Btf / DBA/1 J mie (left). Soring of pnnus formtion (right; n = 9). Representtive imges (n = 9) of reeptor tivtor of NF-κB lignd (RANKL) immunostining in nkle synovi of nonimmunized () nd ollgen-indued rthritis () wild-type () nd Btf / (knokout ()) DBA/1 J mie. Trtse-resistnt id phosphtse (TRAP) stining nd ounting of TRAP-positive multinuleted ells (n = 6 mie per group) t the pnnus-one interfe in nkle joints of nd nd Btf / DBA/1 J mie. d Crtilge destrution ws deteted y Sfrnin-O stining in nd nd Btf / DBA/1 J mie (left) nd sored y OARSI grding (right; n = 12). Vlues re mens ± SEM. Sle rs = 5 μm. one, rtilge, p pnnus p Btf modultes Th ell differentition To further eluidte the funtions of BATF, we exmined whether Btf defiieny ffets the in-vitro differentition of CD4 + T ells into Th ells, inluding Th1 (CD4 + IFNγ + ), Th2 (CD4 + IL-4 + ), Th17 (CD4 + IL-17A + ), nd Treg ells (CD4 + Foxp3 + ). Interestingly, Btf deletion signifintly inresed Th2 ell differentition in Btf / mie, while in-vitro Th1 nd Treg ell differentitions were not ffeted y Btf gene deletion (Fig. 6). Btf gene deletion lso signifintly redued Th17 ell differentition in Btf / DBA/ 1 J mie ompred with ontrol mie (Fig. 6). Consistent with this finding, the Th17 ell popultion ws signifintly redued in the lymph nodes of Btf / mie sujeted to -induing onditions (Fig. 6). Moreover, the CD4 + Foxp3 + IL-17 + ell popultion ws drmtilly redued in the peripherl lymph nodes of -indued Btf / mie (Fig. 6). As the trnsonversion of Treg ells to Th17 ells n reportedly exerte Th17-medited inflmmtion [35], we exmined whether BATF ffets the onversion of Treg ells to Th17 ells in vitro. Indeed, BATF defiieny drmtilly rogted the trnsonversion of Treg ells to Th17 ells (Fig. 6d), inditing tht BATF regultes this onversion. Finlly, we nlyzed ytokine prodution in the lymph nodes of Btf / mie nd littermtes under -induing onditions. Consistent with the inrese in Th2 ell differentition, the prodution of the Th2 ytokines IL-4, IL-1, nd IL-13 were inresed in -indued Btf / mie ompred with littermtes (Fig. 6e). Additionlly, IL-6 ws lso inresed in -indued Btf / mie (Fig. 6e). In ontrst, the prodution of the inflmmtory ytokines IL-2, IL-17, nd TNF-α were redued in Btf / mie under -induing onditions (Fig. 6e). Geneti ltion of Btf does not ffet inflmmtory rthritis used y K/BxN serum trnsfer Finlly, we exmined possile role of BATF in T ell-independent inflmmtory rthritis using C57BL/6 mie sujeted to K/BxN serum trnsfer [21]. Immunostining reveled tht BATF expression ws mrkedly inresed in the inflmed synovil tissues used y K/BxN serum trnsfer (Fig. 7). However, ll exmined mnifesttions of inflmmtory rthritis used y K/BxN serum trnsfer in mie (i.e., pw thikness, synovil inflmmtion, nd rtilge erosion) were not mrkedly inhiited in Btf mie (Fig. 7, ). Our results suggest tht BATF is not essentil for T ell-independent inflmmtory rthritis. Disussion We herein show tht BATF is required for sine Btf / mie exhiit omplete suppression of the mnifesttions of. We further demonstrte tht BATF regultes Th

8 Prk et l. Arthritis Reserh & Therpy (218) 2:161 Pge 8 of 11 Fig. 5 Btf does not ffet thymi T ell development. Representtive flow ytometri nlysis (n = 5 per group) nd quntittion of immune ell popultions. T ell development in thymus () nd the popultions of immune ells in the spleens () nd lymph nodes () of wildtype () nd Btf / (knokout ()) DBA/1 J mie were nlyzed y flow ytometry. Vlues re presented s mens ± SD; P <.1, P <.1, P <.1. ns not signifint ell differentition during. The pthogenesis of RA is ssoited with enrihment of Th17 ells in the inflmed synovium nd enhnement of IL-17 prodution [8, 9, 36, 37]. A reent report indited tht the onversion of Treg ells to Th17 ells ontriutes to one destrution in mie [35], suggesting tht n imlne etween these ell popultions ontriutes to. BATF is known to regulte Th17 ell differentition y modulting the expression of the trnsription ftor RORγt [16], nd to ontrol lss-swith reomintion in T nd B ells [17]. Thus, BATF ws previously known to funtion t multiple hierrhil levels in two ell types to glolly regulte swithed-ntiody responses. Here, we demonstrted tht BATF defiieny dereses Th17 ell differentition oth in vivo nd in vitro. It ws previously reported tht portion of Foxp3-positive ells lso expresses IL-17, nd tht CD4 + Foxp3 + IL-17 + ells n e found in mie [35]. However, we found here tht Btf defiieny ompletely olished CD4 + Foxp3 + IL-17 + ell genertion under -induing onditions. An dditionl interesting finding of our urrent study is tht BATF regultes Th2 ell differentition; we demonstrted tht IL-4-produing CD4 + T ells were inresed in -indued Btf / mie ompred with ontrol mie. This is onsistent with previous report tht IL-4 negtively regultes the indution nd progression of [38]. In ddition, BATF is not deteted in the infiltrted B ells of synovil tissue. Although this does not rule out expression nd the possile role of BATF in B ells of peripherl lymphti tissues, our results indite tht upregultion of BATF in the infiltrted B ells is not essentil for pthogenesis of. Furthermore, we found tht T ell-independent inflmmtory rthritis used y K/BxN serum trnsfer ws not ffeted y Btf / mie. Bsed on these findings, we propose tht BATF defiieny lters Th ell differentition, enling Btf / mie to resist. Thus, BATF inhiition ould e usefulstrtegyforthetretmentofra. We lso report tht FLS re not diretly ssoited with the BATF-medited regultion of, lthough BATF ppers to regulte the prolifertion of FLS in

9 Prk et l. Arthritis Reserh & Therpy (218) 2:161 Pge 9 of 11 e d Fig. 6 Btf redues Th17 differentition in. Popultions of Th1, Th2, Th17, nd Treg ells differentited from unommitted CD4 + T ells of wild-type () nd Btf / (knokout ()) DBA/1 J mie (n = 5). Anlysis of interferon (IFN)-γ, interleukin (IL)-17A, nd IL-4 produing CD4 + T ells in spleens (SP; top pnel) nd lymph nodes (LN; ottom pnel) of nd Btf / DBA/1 J mie 4 to 5 weeks lter from indution (n=5 mie per group). Flow ytometri nlysis of the produtions of Foxp3 nd IL-17A in CD4 + T ells from lymph nodes of nd Btf / DBA/1 J mie 4 to 5 weeks lter from indution (n=5 mie per group) under onditions. d Popultions of Foxp3 + nd IL-17A + ells in CD4 + T ells ultured under Treg differentition ondition to Th17 differentition ondition (n = 5). e Cytokine prodution of totl lymph node ells from nd Btf / DBA/1 J mie 4 to 5 weeks lter from indution (n 5 mie per group). Vlues re presented s mens ± SD; P <.1, P <.1, P <.1. ns not signifint response to TNF-α. Aumulting evidene indites tht FLS re key plyers in RA pthogenesis [1, 28]. Cytokines nd hemokines produed y FLS ttrt T ells to RA synovium, nd the intertion of FLS with T ells results in the tivtion of oth ell types. FLS lso produe mtrix-degrding enzymes involved in rtilge destrution, ngiogeni ftors ssoited with neovsulriztion, nd RANKL [1, 28]. The ltter ftor regultes osteolstogenesis, whih requires physil ontt of preursor ells with RANKL-expressing FLS or T ells [29, 3, 39]. We showed previously tht HIF-2α modultes vrious funtions of FLS, inluding prolifertion, the expressions of RANKL nd vrious toli ftors, nd osteolstogeni potentil [2]. Here, we found tht, unlike HIF-2α, TNF-α nd IL-6 do not use BATF expression in FLS, nd BATF overexpression in FLS does not modulte the expressions of vrious mtrix-degrding enzymes or hemokines. These results olletively support our notion tht the ility of BATF to regulte is not due to diret BATF-medited modultion of FLS funtions.

10 Prk et l. Arthritis Reserh & Therpy (218) 2:161 Pge 1 of 11 Control serum K/BxN serum BATF Control serum K/BxN serum Pw thikness (mm) KBxN -KBxN -Con -Con Dys fter serum trnsfer KBxN Control Fig. 7 Btf does not ffet inflmmtory rthritis used y K/BxN serum trnsfer. Representtive immunostining imges (n = 6) of BATF immunostining in synovil tissue setions of C57BL/6 mie trnsferred with ontrol or K/BxN serum. Pw thikness mesured with digitl thikness liper in wild-type () or Btf knokout () mie trnsferred with ontrol (Con) or K/BxN serum (n = 12 mie per group). Typil imges of rtilge destrution nd synovitis deteted y Sfrnin-O nd H&E stining in or Btf mie trnsferred with ontrol or K/BxN serum (n = 12 mie per group). Vlues re mens ± SEM. Sle rs = 5 μm It hs proven diffiult to eluidte the role of hondroytes in rtilge destrution during RA pthogenesis [5]. Suh destrution ours primrily t the interfe of the pnnus nd lified rtilge [5, 4]. There is evidene tht proteoglyns re lost from the superfiil zone, where rtilge ontts with synovil fluid, ut not with the pnnus [5]. Beuse RA synovium produes vrious mtrix-degrding enzymes [5, 8, 9], rtilge destrution t the superfiil zone my e due to synovil ell funtions. However, proteoglyns n lso e lost from the middle nd deep zones of the rtilge [5], suggesting tht hondroyte my help degrde its own mtrix y relesing mtrix-degrding enzymes. Indeed, we reently demonstrted tht BATF upregultes MMP3 nd MMP13 in hondroytes, leding to rtilge destrution during OA pthogenesis [4]. Therefore, our urrent nd previous [4] results suggest tht the BATF-medited regultion of MMP3 nd MMP13 expression in hondroytes is ssoited with rtilge destrution during. The results of the present study olletively indite tht BATF regultes in mie, nd our previous work showed tht BATF funtions s toli regultor of OA rtilge destrution y upregulting toli enzymes (e.g., MMP3 nd MMP13) in hondroytes [4]. Thus, despite their different etiologies nd pthogeneses, oth RA nd OA re regulted y BATF. However, different mehnisms re involved; BATF regultes OA pthogenesis y upregulting mtrix-degrding enzymes in hondroytes, wheres it ppers to regulte RA pthogenesis y regulting Th ell differentition. Thus, BATF ould e useful trget for the regultion of RA. Conlusions In summry, we demonstrted here tht BATF regultes, inluding synovitis, synovil hyperplsi, ngiogenesis in the inflmed synovium, rtilge destrution, nd one erosion in the joint tissues. We lso revel tht BATF regultes y regulting Th ell differentition without diretly ffeting the funtions of FLS. Arevitions AP-1: Ativtor protein-1; BATF: Bsi leuine zipper trnsription ftor, ATF-like; BrdU: Bromodeoxyuridine; : Collgen-indued rthritis; ELISA: Enzyme-linked immunosorent ssy; FLS: Firolst-like synovioytes; H&E: Hemtoxylin nd eosin; HIF: Hypoxi-induile ftor; IFN: Interferon; IL: Interleukin; : Knokout; MLS: Mrophge-like synovioytes; MMP: Mtrix metlloproteinse; : Nonimmunized; OA: Osteorthritis; PMA: Phorol-12-myristte 13-ette; RA: Rheumtoid rthritis; RANKL: Reeptor tivtor of NF-κB lignd; TCR: T ell reeptor; TGF: Trnsforming growth ftor; Th: T helper; TNF: Tumor nerosis ftor; TRAP: Trtse-resistnt id phosphtse; VEGF: Vsulr endothelil growth ftor; : Wild-type Funding This work ws supported y grnts from the Ntionl Reserh Foundtion of Kore (216R1A3B1969, 216R1A5A17318, nd 216R1A2B48819), the Kore Helthre Tehnology R&D projet through the Kore Helth Industry Development Institute (HI16C287 nd HI14C3484), nd the GIST Reserh Institute (217). Avilility of dt nd mterils The dt supporting the onlusions of this rtile re inluded within the rtile. Authors ontriutions SGP nd JSC ontriuted eqully s orresponding uthors y oneiving nd designing the experiments. SHP, JR, nd SKK performed the experiments. JAK, JSK, YS, nd WSC nlyzed the dt. SHP, JR, SGP, nd JSC wrote the mnusript. All uthors red nd pproved the finl mnusript. Ethis pprovl nd onsent to prtiipte Not pplile.

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