IFNλ Stimulates MxA Production in Human Dermal Fibroblasts via a MAPK-Dependent STAT1-Independent Mechanism

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1 ORIGINAL ARTICLE in Humn Derml Firolsts vi MAPK-Dependent STAT1-Independent Mechnism Adewonuol A. Alse 1, Ysser M. El-Sheriny 2,3, Edwrd M. Vitl 2, Desmond J. Toin 1, Neil A. Turner 4 nd Mirim Wittmnn 1,2,5,6 IFNλ is importnt for epiderml defense ginst viruses. It is produced y, nd cts on, kertinocytes, wheres firolsts were previously considered to e unresponsive to this type III IFN. Herein we report findings reveling cell type-specific differences in IFNλ signling nd function in skin resident cells. In derml firolsts, IFNλ induced the expression of myxovirus protein A (MxA), potent ntivirl fctor, ut not other IFN signture genes s it does in primry kertinocytes. In contrst to its effect on kertinocytes, IFNλ did not phosphorylte signl trnsducer nd ctivtor of trnscription 1 in firolsts, ut insted ctivted mitogen ctivted protein kinses (MAPK). Accordingly, inhiition of MAPK ctivtion (p38 nd p42/44) locked the expression of MxA protein in firolsts ut not in kertinocytes. Functionlly, IFNλ inhiited prolifertion in kertinocytes ut not in firolsts. Moreover, IFNλ upregulted the expression of Tumor growth fctor et 1 ()-induced collgens in firolsts. Tken together, our findings identify primry humn derml firolsts s responder cells to IFNλ. Our study shows cutneous cell type-specific IFN signling nd suggests tht IFNλ, lthough importnt for epiderml ntivirl competence, my lso hve regultory role in the derml comprtment lncing type I IFN-induced inhiition of tissue repir processes. Journl of Investigtive Dermtology (215) 135, ; doi:1.138/jid ; pulished online 24 Septemer 215 INTRODCTION Firolsts nd kertinocytes re the min tissue cell types found in the dermis nd epidermis respectively. Although mny recent studies hve identified the immunologicl role of kertinocytes, the role of derml firolsts in host defense nd inflmmtory diseses is only eginning to emerge. Furthermore, derml firolsts synthesize extrcellulr mtrix including collgens (Xu et l., 29) nd thus hve n importnt role in mintining skin structure nd wound 1 Centre for Skin Sciences, Fculty of Life Sciences, niversity of Brdford, Brdford, K; 2 Leeds Institute of Rheumtic nd Musculoskeletl Medicine (LIRMM), Fculty of Medicine nd Helth, niversity of Leeds, Leeds, K; 3 Clinicl Pthology Deprtment, Fculty of Medicine, Mnsour niversity, Mnsour, Egypt; 4 Division of Crdiovsculr nd Dietes Reserch, Leeds Institute for Crdiovsculr nd Dietes Reserch (LICAMM), Fculty of Medicine nd Helth, niversity of Leeds, Leeds, K; 5 Deprtment of Dermtology, Brdford Teching Hospitls NHS Foundtion Trust, St Luke s Hospitl, Brdford, K nd 6 Leeds Musculoskeletl Biomedicl Reserch nit, Ntionl Institute of Helth Reserch (NIHR), Chpel Allerton Hospitl, Leeds, K Correspondence: Adewonuol Alse, Centre for Skin Sciences, Fculty of Life Sciences, niversity of Brdford, Room H37, Richmond Building, Brdford, BD7 1DP, K. E-mil:..lse@student.rdford.c.uk Arevitions: GBP-1, gunulte inding protein 1; MAPK, mitogen ctivted protein kinse; MxA, myxovirus protein A; STAT1, signl trnsducer nd ctivtor of trnscription Received 15 Jnury 215; revised 17 June 215; ccepted 11 July 215; ccepted rticle preview online 19 August 215; pulished online 24 Septemer 215 heling. IFNλ is type III IFN comprising four memers, (IL-29), IFNλ2 (IL-28A), IFNλ3 (IL-28B), nd IFNλ4 (Kotenko et l., 23; Shepprd et l., 23; Prokunin- Olsson et l., 213). IFNλ elongs to the IL-1 fmily of cytokines long with IL-1, IL-19, IL-2, IL-22, IL-24, nd IL-26. IFNλ uses the heterologous receptor complex comprising (lso known s IL-28Rα1) nd the uiquitously expressed IL-1 receptor 2 (IL-1Rβ) which is lso used y IL-1, IL-22, nd IL-26 s second receptor suunit (Kotenko et l., 23; Shepprd et l., 23). is the signling suunit nd so its presence determines cellulr responsiveness. Although IFNλ is structurlly relted to IL-1, it resemles type I IFNs in its ntivirl nd ntiprolifertive functions (Miknis et l., 21). They oth signl minly through the recruitment nd ctivtion of memers of the Jnus fmily of kinses, JAK1/TYK2 leding to the phosphoryltion of signl trnsducer nd ctivtor of trnscription (STAT) 1/2 nd susequently to induction of set of downstrem genes known s IFN-stimulted genes (ISGs) coding for proteins, such s Myxovirus protein A (MxA), Gunylte inding protein 1 (GBP-1), 2,5-oligodenylte synthetse (2,5-OAS), nd IFN inducile protein 16 (IFI-16) with potent ntivirl ctivities (Ank et l., 28; Sommereyns et l., 28). Beyond JAK/STAT ctivtion, type I IFNs hve lso een found to ctivte mitogen ctivted protein kinse (MAPK; p38 nd Extrcellulr signl-regulted kinses ERK (p42/p44) MAPK), which llows for full ctivtion of 215 The Society for Investigtive Dermtology

2 downstrem ISGs (Nguyen et l., 2; ddin et l., 2); nd possily downstrem phosphoryltion of STAT1 on serine 727 (Goh et l., 1999). Type I IFNs signl through memrne ssocited IFNAR1 nd IFNAR2 (Lutfll et l., 1995). Different from the two suunits of IFNAR which re present on ll nucleted cell types, is selectively expressed nd functionl in certin cell types only (Kotenko et l., 23; Witte et l., 29; Zhn et l., 21; Dickensheets et l., 213). Witte et l. (29) descried the presence of IL-28Rα1 mrna expression in humn derml firolsts. However, it ws oserved tht these cells were unresponsive to IFNλ, nd this ws ttriuted to the very low expression level of the receptor in these cells (Lsfr et l., 211). IFNλ is the min IFN produced y kertinocytes nd production of vrying mounts of this cytokine hs een reported lso in other cell types (Zhn et l., 21; Yin et l., 212; Wolk et l., 213). Kertinocytes re susceptile to nd this cytokine sustntilly contriutes to the ntivirl competence of the humn epidermis (Wolk et l., 213). Aprt from ntivirl properties, oth types I nd III IFNs re known for their propoptotic nd ntiprolifertive ctivities (Mher et l., 28; Aushh et l., 21; Steen nd Gmero, 21). These ctivities hve een linked to the ility of IFNs to phosphorylte STAT1 (Zitzmnn et l., 26). High expression of ISGs, such s CXCL9, CXCL1, MxA, nd GBP-1 hs een reported in interfce dermtitis conditions including cutneous lupus erythemtosus (CLE) nd here incresed expression is linked with disese severity (Wenzel et l., 25; Nscherger et l., 21). In this study, we sought to understnd if humn derml firolsts re responsive to through the ctivtion of STAT1 nd susequent downstrem induction of ISGs. We lso wnted to understnd the role of this cytokine in mintennce of skin integrity with regrd to collgen expression nd prolifertion of skin resident cells. RESLTS is expressed y humn derml firolsts To confirm the expression of in firolsts nd to investigte its regultion, firolsts were stimulted for 4 or 16 hours for nlysis of mrna nd protein expression, respectively. mrna expression ws mesured y quntittive rel time polymerse chin rection (qrt-pcr) nd protein expression ws mesured y western lotting nd flow cytometry. We found tht ws expressed in derml firolsts nd ws upregulted in the presence of (Figure 1e nd Supplementry Figure S1 online). IFNλ induces significnt expression of MxA in humn derml firolsts To investigte if is functionl in humn derml firolsts, cells were treted with rhifnλ or rh (positive control) for 4 or 24 hours. mrna expression of ISGs ws quntified using qrtpcr. We found significnt expression of MxA ut not GBP-1 (Figure 2), OAS2 (Supplementry Figure S2 online) or other nlyzed ISGs (CXCL9 nd IFI-16; not shown) fter 4 nd 24 hours of tretment. As expected, when compred with, induced significntly higher expression of MxA nd GBP-1 fter 4 hours of tretment (Figure 2d). However, while the expression of MxA remined high fter 24 hours of tretment with, GBP-1 expression dropped significntly over this time period (Figure 2). In firolsts, ppered to induce higher upregultion of MxA s compred with GBP-1 (Figure 2). ws shown to hve dose-dependent effect on MxA expression in derml firolsts (Supplementry Figure S3 online). nd IFNλ induce ISGs expression in humn primry kertinocytes We compred our findings on ISGs expression in firolsts with the expression pttern in humn primry kertinocytes. The responsiveness of kertinocytes to oth type I nd III IFNs is well documented (Zhn et l., 21, 211; Bchmnn et l., 213). We found tht oth nd induced significnt mrna expression of MxA, OAS2, nd GBP-1 with higher induction of MxA s compred with OAS2 nd GBP-1 (Figure 2 nd Supplementry Figure S2 online). Interestingly, we oserved different time kinetic for the ISG response to s compred with. showed strong upregultion of MxA nd GBP-1 t 6 hours, wheres stimultion resulted in strong increse in MxA nd c e % of mximum K Isotype PE 6 d Protien nd intensity MxA/ Figure 1. expression nd production in humn derml firolsts. Cultured derml firolsts were either untreted or treted with 1 ng ml 1 for 4 or 16 hours in serum-free Dulecco's Modified Egle's medium (DMEM) for mrna nd protein expression, respectively. mrna expression ws mesured using qrt-pcr nd protein y western lotting nd flow cytometry. () qpcr products for nd housekeeping gene 6snRNA on 2% grose gel re shown s representtive of five independent experiments. (). (c) A representtive western lot result for protein nd (d) protein nd intensity nlysis re depicted; n = 3. (e) A representtive (n = 3) histogrm of flow cytometric nlysis of nd pproprite isotype control. For sttisticl nlysis, Mnn Whitney test ws used. Vlues represent men ± SEM. Po.1. K, kertinocytes;, untreted Journl of Investigtive Dermtology (215), Volume 135

3 Firolsts MxA 4 h MxA 24 h GBP-1 4 h GBP-1 24 h 1 15 * Kertinocytes MxA 6 h MxA 24 h GBP-1 6 h GBP-1 24 h Figure 2. Interferon stimulted genes (ISGs) expression in humn skin resident cells. Cultured derml firolsts were either untreted or treted with 1 ng ml 1 or 1 ng ml 1 for 4 or 24 hours in serum-free medium. Primry kertinocytes were treted with the sme concentrtion of cytokines for 6 or 24 hours in KGM ( / ). mrna expression of ISGs ws mesured using qrtpcr. Top pnel shows expression levels of MxA, 4 hours; MxA, 24 hours; GBP-1, 4 hours, nd GBP-1, 24 hours in firolsts. Bottom pnel shows expression levels of MxA, 6 hours; MxA, 24 hours; GBP-1, 6 hours, nd GBP-1, 24 hours in kertinocytes. For sttisticl nlysis, MnnWhitney test ws used; n = 5, vlues represent men ± SEM. *Po.5, Po.1. GBP, gunulte inding protein; KGM, kertinocyte growth medium; MxA, myxovirus protein A;, untreted GBP-1 expression fter 24 hours. This suggests tht stimultion my result in more delyed nd/or longer lsting effects (Figure 2) s previously oserved in heptocytes (Bolen et l., 214). IFNλ ctivtes p38 nd ERK MAPKs ut not STAT1 (Tyr71) in humn derml firolsts Hving estlished tht derml firolsts respond to, we were interested in understnding the mechnism of ction. To chieve this, derml firolsts were treted with either or for different time periods nd protein phosphoryltion ws determined using western lotting. As expected, -induced STAT1 phosphoryltion with mximl phosphoryltion detected fter 3 minutes of stimultion (Figure 3). However, we filed to oserve ny STAT1 phosphoryltion in -treted cells in ny of the time points investigted. Pulished studies hve reported tht phosphoryltion of p38 my e necessry for full ctivtion of STAT1-dependent genes (Goh et l., 1999; ddin et l., 2; Pltnis, 23). We therefore investigted the ility of to induce the phosphoryltion of p38 in derml firolsts nd clerly found ctivtion of this MAPK (Figure 3). We confirmed tht tretment resulted in p38 phosphoryltion in ddition to STAT1 phosphoryltion (Figure 3). Furthermore, we found tht oth nd induced phosphoryltion of ERK (Figure 3), ut not the Akt (p6; dt not shown) pthwy in derml firolsts. As expected, we found tht oth nd induced STAT1 phosphoryltion in primry kertinocytes (Figure 3). However, we filed to oserve cler ctivtion of either the p38 or ERK pthwy upon tretment with IFNs (Figure 3). P38 nd ERK inhiitors ttenute -induced MxA protein expression We sought to understnd if the ctivtion of p38 nd ERK ws responsile for the induction of MxA expression in derml firolsts. To investigte this, firolsts were pretreted with p38 (1 μm 2358) or ERK (3 μm PD9859 or 1 μm 126) inhiitors for 1 hour efore tretment with or. mrna nd protein expression levels were mesured y qrtpcr nd western lotting, respectively. Interestingly, our results showed tht the expression of MxA ws not regulted t the mrna level y the MAPK inhiitors (Figure 4 nd ). However, the presence of either the p38 or ERK inhiitor rogted -induced MxA protein production (Figure 4c). We did not oserve decrese in -induced MxA protein production in firolsts in the presence of 2358, PD9859 (not shown), or 126 (Figure 4d). We lso nlyzed whether MAPKs hd role in -induced MxA protein expression in humn primry kertinocytes. We filed to find significnt effect of p38 or ERK inhiition on nd -induced MxA protein production in kertinocytes (Figure 4e). IFNλ enhnces collgen expression in humn derml firolsts As conditions with high type I IFN expression cn e linked to impired heling responses, we investigted whether hd ny influence on collgen expression. Firolsts were

4 AA Alse et l. Time min suggests tht the p38 pthwy my e responsile for the synergistic effect oserved on collgen expression. IFNλ hs potent ntiprolifertive ctivity in primry kertinocytes ut not in derml firolsts pstat1 STAT1 p-p38 P38 perk ERK Time min pstat1 STAT1 p-p38 P38 perk Figure 3. induced mitogen ctivted protein kinse (MAPK) nd signl trnsducer nd ctivtor of trnscription 1 (STAT1) phosphoryltion in firolsts nd kertinocytes, respectively. Cultured humn derml firolsts () or primry kertinocytes () were either non-treted or treted with 1 ng ml 1 or 1 ng ml 1 for 1, 3, or 6 minutes, respectively. Totl nd phosphorylted p38, ERK, nd STAT1 long with were detected y western lot. A representtive out of three independent experiments is depicted. ERK, extrcellulr signlregulted kinse;, untreted. treted with,,, or comintion of IFN nd for 6 or 24 hours. ws used s control, given its well descried stimultion of firolst prolifertion nd collgen synthesis. We mesured Col1A1, Col3A1, Col4A2, nd Col7A1 using qrtpcr. There ws no difference in collgen expression fter 6 hours of stimultion with these cytokines (dt not shown). However, fter 24 hours of tretment, significntly induced the collgen expression. Comined tretment of cells with nd resulted in significntly higher collgen expression (except for Col3A1) thn with either tretment lone (Figure 5d). To investigte which of the MAPK pthwys my e responsile for the synergy, we treted cultured derml firolsts with either or or oth together for 3 minutes nd nlyzed the phosphoryltion of p38 nd ERK y western lotting. We found tht oth nd ctivted the p38 nd ERK pthwys independently (Supplementry Figure S4 online). Interestingly, we oserved cler synergistic effect etween nd on the ctivtion of the p38 pthwy. The effect is not very cler with the ERK pthwy (Supplementry Figure S4 online). This 2938 Journl of Investigtive Dermtology (215), Volume 135 The ntiprolifertive effect of on HCT kertinocyte cell lines hs een descried (Mher et l., 28). We imed to compre the effect of oth nd on primry skin cells. Kertinocytes nd firolsts were stined with cell trcer dye nd susequently stimulted with either IFNλ or for 72 hours. As expected, oth IFNs clerly reduced kertinocyte prolifertion (Figure 6 nd ). In contrst, we oserved tht ut not inhiited derml firolst prolifertion (Figure 6c nd d)., known to induce firolst prolifertion, ws used s positive control (Figure 6c nd d). We lso investigted the effects of nd on gp closure in monolyer cultures of derml firolsts. We oserved the trend tht enhnced gp closure in firolsts while delyed gp closure fter 48 hours of stimultion in comprison with the untreted cells (Supplementry Figure S5 online). DISCSSION Type I nd III innte IFNs were initilly identified for their potent ntivirl ctivities (Sdler nd Willims, 28; McMicking, 212). In ddition, IFNs lso hve immunoregultory, ntiprolifertive, nd propoptotic properties; they hve een identified s potentil nticncer drugs (Aushh et l., 21; Witte et l., 21). However, they re lso importnt meditors in the pthogenesis of utoimmune diseses, such s CLE (Meyer, 29; Ronnlom nd Elornt, 213). MxA is highly inducile ISG nd one of the most potent ntivirl fctors cple of locking erly repliction events of different RNA nd DNA viruses (Sdler nd Willims, 28). Type III IFN is the most recently descried group of IFNs. Expression of their specific receptor ws thought to e restricted minly to epithelil cells (kertinocytes) nd heptocytes. There is, however, emerging evidence tht is lso present nd functionl on hemtopoietic cells (Mennechet nd ze, 26; Di et l., 29; Liu et l., 211; Yin et l., 212). There re reports identifying MAPKs s regultors of IFN downstrem ctivities (ddin et l., 2; vn Boxel-Dezire et l., 26; Gough et l., 28) nd the importnce of different (non JAK) kinses ctivted in response to IFNs hs een speculted efore to contriute to cell-type nd IFN sutype specific responses (vn BoxelDezire et l., 26). Our work shows distinct signling nd lso functionl response of primry humn firolsts to IFNλ, which is different from the response of these cells to type I IFN nd clerly different from the response of epiderml cells. Indeed, kertinocytes show similr signling nd functionl response to oth type I nd type III IFNs which is chrcterized y STAT1-dependent upregultion of rod rnge of ISGs. Derml firolsts were considered unresponsive to IFNλ (Aushh et l., 21). We here show tht humn firolsts indeed respond to tretment with significnt expression of MxA mrna nd protein. In line with our

5 findings, recent study hs reported the responsiveness of cytomeglovirus infected foreskin firolst to IFNλ3 (Egli et l., 214). It is well documented tht type I nd III IFNs induce similr sets of ISGs in mny cell types (Ank et l., 28; Sommereyns et l., 28). However, in firolsts, we filed to oserve significnt expression of GBP-1, OAS2, or other ISGs upon tretment. The inility of to ctivte STAT1 (Tyr71) in firolsts ws unexpected s this c MxA 4 h MxA 1 1 MxA 24 h % Protien nd intensity MxA/ Firolsts * * d 1 Firolsts MxA e MxA % protien nd intensity MxA/ % Protien nd intensity MxA/ Kertinocytes Figure 4. Inhiition of myxovirus protein A (MxA) production y p38 nd extrcellulr signlregulted kinse (ERK) inhiitors. Cultured derml firolsts or primry kertinocytes were either untreted or treted with MAPK inhiitors or IFNs for 4 nd 24 hours nd MxA ws detected on the mrna level nd y western lot (24 hours). () MxA mrna expression, 4 hours; n = 3; () MxA mrna expression, 24 hours; n = 3; (c) MxA protein expression y in the presence or sence of MAPK inhiitors in firolsts; (d) MxA protein expression y in the presence or sence of MAPK inhiitors in firolsts; (e) MxA protein expression y nd in humn primry kertinocytes in the presence or sence of MAPK inhiitors. Figures re representtive of three independent experiments. For sttisticl nlysis, unpired t test ws used; vlues represent men± SEM. *po.5. : 2358 (p38 inhiitor); : 126 (MEK/ERK inhiitors). MAPK, mitogen ctivted protein kinse

6 c Col1A1 24 h Col3A1 24 h Col4A2 24 h Col7A1 24 h * 2 * is the known cnonicl pthwy for the induction of downstrem ISG genes. However, our findings on - induced ctivtion of oth p38 nd ERK suggest tht the MAPK pthwy my e n lterntive for the induction of MxA y IFNλ in firolsts. It hs een suggested tht -induced phosphoryltion of MAPKs nd STAT1 re independent of ech other; however, they work in tndem to ensure full ctivtion of ISGs (Li et l., 24). It is interesting tht the inhiition of p38 nd ERK in -treted derml firolsts did not result in significnt downregultion of MxA protein expression; lthough we oserved slight reduction in MxA production y primry kertinocytes following inhiition of ERK ut not p38. Activtion of PI3K-AKT nd Rf-MEK-ERK pthwys y nd in HepG cell lines hs een reported (Chi et l., 211), ut we could not oserve AKT pthwy ctivtion in our experiments. Our proposed mechnism of nd signling in derml firolsts is shown in Supplementry Figure S6 online. To our surprise, we filed to oserve ny significnt regultory effects of the MAPK inhiitors t the trnscriptionl level suggesting tht p38 nd ERK inhiition results in posttrnsciptionl regultion of MxA. Mechnisms of mrna trnsltion of ISGs hve recently een reviewed (Joshi et l., 21). Both the p38 nd d Figure 5. Effect of IFNλ on collgen expression in humn derml firolsts. Cultured derml firolsts were either untreted or treted with 1 ng ml 1, 1 ng ml 1, 5 ng ml 1 or comintion of nd for 24 hours. mrna expression of ISGs ws mesured using qrtpcr. Depicted is Col1A1 (), Col3A1 (), Col4A2 (c), nd Col7A1 (d) expression. For sttisticl nlysis, MnnWhitney test ws used; n = 5, vlues represent men ± SEM. *Po.5, Po.1. ISG, Interferon stimulted gene. Prolifertion index Prolifertion index % kertinocytes prolifertion d Dy Dy BV 421 % firolsts prolifertion BV 421 c Figure 6. Effects of IFNs on prolifertion of skin resident cells. Derml firolsts or primry kertinocytes were leled with cell trce violet in DMEM contining 1% fetl ovine serum nd kertinocyte growth medium, respectively. Dughter cell popultions (highlighted y different colors) of dividing cells were visulized y flow cytometry fter 72 hours of stimultion. Incresed prolifertion results in shift to the left end of the histogrm. () Fluorescent signl in kertinocytes t dy nd fter exposure to / /medium for 72 hours. () Percentge prolifertion of kertinocytes (n = 3). (c) Percentge prolifertion of firolsts (n = 3). (d) Fluorescent signl in firolsts t dy nd 72 hours fter stimultion with TGFβ/// medium. For sttisticl nlysis, unpired t-test ws used; n = 3, vlues represent men ± SEM. Po.1., untreted. p42/44 MAPKs cn control the ctivtion of eif4e nd other sustrtes of MAPK intercting protein kinses 1 nd 2 (Mnk1 nd 2) which influence trnsltion initition (Joshi et l., 21). Posttrnsriptionl regultion of TNFα gene expression in the presence of p38 inhiitor hs een descried (Clrk et l., 23). Future experiments will need to further dissect the underlying events of the posttrnscriptionl regultion oserved for MxA in derml firolsts. In this study, we hve confirmed previous report tht humn derml firolsts express (Witte et l., 29) nd tht cn upregulte its expression. The ility of to enhnce the expression levels of is evidence of cross-tlk etween type I nd type III IFNs (Duong et l., 214). is well known to induce collgen expression in firolsts. Types I, III, IV, nd VII collgens re the min components of extrcellulr mtrix (ECM), sement memrne zone, nd the nchoring firils t the dermlepiderml Journl of Investigtive Dermtology (215), Volume 135

7 junction (Heino, 27). We found tht potentited the TGFβ-ssocited induction of collgen I, IV, nd VII most proly y synergistic effect with on p38 ctivtion. This effect ws not oserved for collgen type III expression. IFNγ, type II IFN exhiited n inhiitory effect on - induced ECM or collgen deposition in primry humn lung (Eickelerg et l., 21) nd in humn foreskin firolsts (Ghosh et l., 21). Of interest, keloid-derived derml firolsts seem non-responsive to this IFNγ-dependent type I collgen regultion (Hsegw et l., 23). Excessive secretion of type I collgen is linked with hypertrophic scr formtion, wheres sufficient mount of type III collgen my prevent scr formtion (Oliveir et l., 29). The net effect of on cutneous repir nd heling responses is not cler yet nd needs to e further investigted. The ility of to upregulte the expression of type IV (Col4A2) nd VII collgens (Col7A1) suggests tht it my support sement memrne integrity (Nystrom et l., 213). Our results on prolifertion of primry kertinocytes re consistent with the roles of nd s ntiprolifertive gents. However, we show tht does not hve n inhiitory effect on derml firolsts prolifertion nd this my e due to its inility to induce STAT1 phosphoryltion in these cells. In ddition, the ntiprolifertive ility of GBP-1 on intestinl epithelil cells hs een descried (Cpldo et l., 212) nd filed to induce GBP-1 expression in derml firolsts. In summry, this study hs shown tht is ctive in derml firolsts through the ctivtion of p38 nd ERK pthwys. We hve shown tht MxA induction y occurs through STAT1-independent pthwy nd this my lso explin the role of this cytokine in enhncement of collgen expression nd lck of ntiprolifertive ctivity. We suggest tht the physiologic role of IFNλ ctivity in the skin orgn is certinly linked with virl defense. Kertinocytes s outer rrier cells re min producers of IFNλ. An infection eyond the epiderml comprtment will led to incresed expression of ntivirl type I IFNs which in turn increse the susceptiility of firolsts to epiderml type III IFNs. Derml firolsts will contriute to the ntivirl competence of the skin tissue y upregultion of MxA protein. In the context of dmge cused y virl infection, they my however hve n importnt prt in mintining tissue integrity nd llowing repir processes involving sement memrne nd type I collgens; s well s mesenchyml cell prolifertion. This repir phenotype my mnifest with some dely in the inflmmtory ntivirl response once the more potent, ut shorter signling effect of type I IFNs decreses. This suggested regultory role of type III IFNs on derml tissue cells is supported y findings from Egli et l. (214) who descried tht in cytomeglovirus-treted foreskin derived firolst cell lines, IFNλ exerts n inhiitory ction on induced ctivity. We propose tht the derml response to could counterct type I IFN-induced impirment of repir mechnisms. With regrd to CLE where IFNλ is highly expressed, our current dt do not support prominent role of IFNλ in repir filure nd ntiprolifertive ctivity seen in discoid CLE (Nyerg et l., 2). However, further experiments regrding functionl nlysis of skin lesionderived cells would e necessry to verify this ssumption. MATERIALS AND METHODS Cytokine nd ntiodies Recominnt humn (rh) (1 ng ml 1 ) ws purchsed from eioscience (Htfield, K); rh2 (1 ng ml 1 ; Merck Millipore, Merck Serono, Middlesex, K), rh (5ngml 1 ; Peprotech EC, London, K). Anti-humn (rit phospho p38, mouse phospho ERK, mouse ERK, rit p38, rit phospho STAT1, nd rit STAT1) ntiodies were purchsed from Cell signling Technology (Leiden, The Netherlnds); nti-humn (rit MxA, rit, nd mouse ) ntiodies were purchsed from Acm (Cmridge, K) nd Snt Cruz (Insight Biotechnology, Middlesex, K), respectively. Horserdish peroxidse (HRP) conjugted donkey nti-rit nd donkey nti-mouse secondry ntiodies were from Snt Cruz. PD9859, 126 (MEK/ERK inhiitors), nd 2358 (p38 inhiitor) were from Cell Signling Technology. Cell isoltion, culture, nd ethics Kertinocytes nd derml firolsts were derived from skin tissue from nonymized helthy volunteers undergoing cosmetic surgery. Smples were collected following written informed consent y ptients nd locl reserch ethicl pprovl (Ethicl Tissue, niversity of Brdford). Skin smples were processed on the dy of collection. Firolsts were isolted y plcing the trypsinized dermis in T75 culture flsk, ensuring the upper lyer just elow the epidermis dhered firmly to the flsk. A volume of 1 ml DMEM contining 1% FBS ws crefully dded to the flsk so s not to llow the dermis to flot. Firolsts explnted fter 5 to 7 dys. Full detils re given in Supplementry Informtion. Cell stimultion Kertinocytes or firolsts were seeded into 24-well or 6-well pltes nd llowed to grow to 8% confluency. For kertinocytes, culture medium ws replced with KGM without humn epiderml growth fctor nd hydrocortisone (KGM / ) prior to cell tretment with cytokines. Derml firolsts were strved of serum for 24 hours efore tretment in serum-free DMEM. For inhiition experiments, cells were pre-treted for 1 hour with 1 μm 2358 (Cell Signling), 3 μm PD9859 or 1 μm 126 (Cell Signling) efore tretment with or. Controls included untreted cells nd cells treted with, 2358, or PD9859/126 lone for 24 hours. Quntittive RTPCR Mx1, GBP-1, OAS2, IFI-16, CXCL9,, Col1A1, Col3A1, Col4A2, nd Col7A1 QuntiTect primer ssys were otined from Qigen (Hilden, Germny), wheres 6snRNA primer (forwrd 5 -CTCGCTTCGGCAGCACA-3 ; reverse-5 -AACGCTTCACGAATTT GC-3 ) ws purchsed from Sigm-Aldrich, Poole, K. The following prmeters were used: initil het ctivtion, 95 C for 5 minutes; denturtion, 95 C for 1 seconds; comined nneling nd elongtion, 6 C for 3 seconds for 4 cycle run. Dt were nlyzed using the deltdelt ct method. mrna expression of ech gene of interest ws normlized to 6snRNA housekeeping gene

8 Agrose gel electrophoresis qrt-pcr products were run t 4 ma for 3 minutes on 2% grose gel. Imge ws tken under V light. Full detils re given in Supplementry Informtion. Western lotting Cells were lysed with CelLytic M lysis uffer (Sigm-Aldrich) contining protese inhiitor cocktil (Roche Applied Bioscience, Rotkreuz, Switzerlnd) nd phosphtse inhiitor (Thermo Scientific, Loughorough, K). 3 μg of totl protein ws seprted on ny kd mini proten gel (Bio-Rd) nd proteins were lotted onto.2 μm PVDF trns-lot pck (Bio-Rd, Hemel Hempsted, K). Memrnes were proed with rit nti-humn pstat1 (1:1), STAT1 (1:1), phospho p38 (1:1), mouse nti-humn perk (1:2), p38 (1:1), or (1:3) in Tris-uffered sline/.1% Tween-2 (TBST) contining 5% BSA overnight t 4 C. Rit ntihumn MxA (1:1), (1:1), nd were used in 5% milk phosphte uffered sline/.1% Tween-2 (PBST) overnight t 4 C. Donkey nti-rit nd donkey nti-mouse HRP-conjugted secondry ntiodies were used t 1:5 nd 1:3 respectively for 1 hour t room temperture. For repet Western, memrnes were stripped, locked, nd re-proed with primry ntiody. CellTrce prolifertion ssy Humn primry kertinocytes nd derml firolsts were stined in kertinocyte growth medium or serum-free DMEM, respectively, with CellTrce violet (Moleculr proes/life technologies) wy from the source of light (2 μl cell trce to 1 million cells in 1 ml medi). Cells were plted in six-well pltes nd were either treted with cytokines or untreted for 72 hours. Some cells were used for the dy nlysis. For flow cytometric nlysis, BD LSRFortess (BD Bioscience, Oxford, K) mchine ws used. Cell prolifertion ws clculted using ModFit softwre version 3.2 (Verity Softwre House, Topshm, ME). Full detils re given in Supplementry Informtion. Sttisticl nlysis Sttisticl significnce ws determined using MnnWhitney test or unpired t-test. Anlysis ws performed using GrphPd Prism softwre (GrphPd Softwre L Joll, CA). All dt re expressed s mens ± SEM nd vlues of Po.5 were considered significnt. n represents independent experiments; *Po.5, Po.1. CONFLICT OF INTEREST The uthors stte no conflict of interest. ACKNOWLEDGMENTS We thnk The niversity of Brdford nd the Centre for Skin Sciences for PhD funding grnted to AA. Dr Vitl is funded y n NIHR Clinicin Scientist Fellowship CS SPPLEMENTARY MATERIAL Supplementry Mteril is linked to the online version of the pper t REFERENCES Aushh W, Bln M, Cstned I et l. (21) Antitumor ctivity of type I nd type III interferons in BNL heptom model. Cncer Immunol Immunother 59:15971 Ank N, Iversen MB, Brtholdy C et l. (28) An importnt role for type III interferon (IFN-lmd/IL-28) in TLR-induced ntivirl ctivity. JImmunol 18: Bchmnn M, lziit S, Hrdle L et l. (213) IFNlph converts IL-22 into cytokine efficiently ctivting STAT1 nd its downstrem trgets. Biochem Phrmcol 85:39643 Bolen CR, Ding S, Roek MD et l. 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