Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021 Inducing is online at: average * The Journal of Immunology , 11 of which you can access for free at: 2013; 191:2956-2966; Prepublished online 16 from submission to initial decision 4 weeks from acceptance to publication August 2013; doi: 10.4049/jimmunol.1300376 http://www.jimmunol.org/content/191/6/2956 A Synthetic TLR3 Ligand Mitigates Profibrotic Fibroblast Responses by Autocrine IFN Signaling Feng Fang, Kohtaro Ooka, Xiaoyong Sun, Ruchi Shah, Swati Bhattacharyya, Jun Wei and John Varga J Immunol cites 49 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. 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The Journal of Immunology A Synthetic TLR3 Ligand Mitigates Profibrotic Fibroblast Responses by Inducing Autocrine IFN Signaling Feng Fang,* Kohtaro Ooka,* Xiaoyong Sun,† Ruchi Shah,* Swati Bhattacharyya,* Jun Wei,* and John Varga* Activation of TLR3 by exogenous microbial ligands or endogenous injury-associated ligands leads to production of type I IFN. Scleroderma patients with progressive skin fibrosis display an IFN-regulated gene signature, implicating TLR3 signaling in the disease. In this study, we show that TLR3 expression was detected on foreskin, adult skin, and lung fibroblasts, and TLR3 levels were significantly elevated in a subset of scleroderma skin biopsies. In explanted skin and lung fibroblasts, the synthetic TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)), a dsRNA analog, caused dose- and time-dependent stimulation of IFN-b production and generation of an IFN-response gene signature that was accompanied by substantial downregulation of collagen and a-smooth muscle actin gene expression. Furthermore, poly(I:C) abrogated TGF-b–induced fibrotic responses and blocked canonical Smad Downloaded from signaling via upregulation of inhibitory Smad7. Surprisingly, the inhibitory effects of poly(I:C) in fibroblasts were independent of TLR3 and were mediated by the cytosolic receptors retinoic acid–inducible gene 1 and melanoma differentiation-associated gene 5, and involved signaling via the IFN receptor. Taken together, these results demonstrate that induction of a fibroblast IFN response gene signature triggered by dsRNA is associated with potent TLR3-independent anti-fibrotic effects. The characteristic IFN response gene signature seen in scleroderma lesions might therefore signify a tissue-autonomous protective attempt to restrict fibroblast activation during injury. The Journal of Immunology, 2013, 191: 2956–2966. http://www.jimmunol.org/ rogressive fibrosis of the skin and internal organs accounts responses to both microbial pathogens and to tissue injury–as- for the intractable nature and the high mortality of sclero- sociated endogenous danger signals collectively referred to as P derma (1). As the principal effector cells responsible for damage-associated molecular patterns (DAMPs) (6). In contrast to fibrosis, stromal fibroblasts and myofibroblasts contribute to exces- TLR2 and TLR4, which reside at the cell surface and recognize sive deposition of collagens and other extracellular matrix proteins microbial ligands, TLR3 is normally endosomal in its location and (2). TGF-b, which stimulates collagen synthesis, myofibroblast dif- recognizes viral dsRNA, as well as the synthetic dsRNA analog ferentiation, and epithelial–mesenchymal transition, is implicated as polyinosinic-polycytidylic acid (poly(I:C)) (6, 7). Upon injury, by guest on September 23, 2021 a key initiating factor in both physiological and pathological tissue dsRNA generated at sites of tissue damage in situ serves as an remodeling (3). However, the mechanism responsible for the per- endogenous ligand for TLR3 (8). In most cell types, double-stranded sistence of the fibrotic process associated with pathological repair nucleic acids and their analogs are recognized as DAMPs not only remains poorly understood. by TLR3, but also by the cytosolic RNA helicases retinoic acid– Recent studies have detected an IFN-response gene signature, inducible gene 1 (RIG-1) and melanoma differentiation-associated characterized by upregulation of type I IFN–regulated genes, in gene 5 (MDA5) (9). both circulating blood cells and in lesional skin from patients with The IFN signature detected in scleroderma lesional tissue scleroderma (4). The production of type I IFN in plasmacytoid suggests potential roles for TLR3 signaling, type I IFN, and innate dendritic cells as well as in stromal cells is controlled by TLRs immunity in pathogenesis. The recent demonstration that IFN (5). These conserved pattern recognition receptors trigger immune regulatory factor (IRF) 3 and IRF5, which are downstream of TLR3, are risk alleles for scleroderma provides further evidence *Division of Rheumatology, Northwestern University Feinberg School of Medicine, implicating type I IFN (10). However, the precise role of innate Chicago, IL 60611; and †McDermott Center for Human Growth and Development, immunity and of endogenous TLR ligands in the pathogenesis University of Texas Southwestern Medical Center, Dallas, TX 75390 of fibrosis remains poorly understood. We therefore sought to in- Received for publication February 6, 2013. Accepted for publication July 15, 2013. vestigate TLR expression in scleroderma and the modulation of This work was supported by National Institutes of Health Grant AR-42309. fibroblast function by the synthetic TLR3 ligand poly(I:C), which The sequences presented in this article have been submitted to the Gene Expression was shown previously to abrogate (11, 12) or induce (13) fibrosis. Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47616) under ac- cession number GSE47616. The results showed that whereas poly(I:C) treatment served as Address correspondence and reprint requests to Dr. John Varga, Division of Rheuma- a potent stimulus for production of type I IFN and generation of an tology, Feinberg School of Medicine, Northwestern University, McGaw Pavillion M230, IFN-response gene signature in explanted skin fibroblasts, it po- 240 East Huron Street, Chicago, IL, 60611. E-mail address: [email protected] tently reduced fibrotic responses, blocked Smad-dependent TGF-b The online version of this article contains supplemental material. signaling, and prevented TGF-b stimulation of these cells through Abbreviations used in this article: ASMA, a-smooth muscle actin; DAMP, damage- endogenous IFN. The results therefore identify the TLR3 ligand associated molecular pattern; FDR, false discovery rate; IRF, IFN regulatory factor; MDA5, melanoma differentiation-associated gene 5; MEF, mouse embryonic fibro- poly(I:C) as a novel inhibitor of profibrotic TGF-b activity, and blast; poly(I:C), polyinosinic-polycytidylic acid; qPCR, quantitative PCR; RIG-1, they suggest the possibility that the IFN-response gene signature retinoic acid–inducible gene 1; RNAi, RNA interference; siRNA, small interfering seen in scleroderma lesional tissue may in fact represent a cell- RNA. autonomous attempt to restrain fibroblast activation and mitigate Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 aberrant fibrogenesis. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300376 The Journal of Immunology 2957 Materials and Methods was evaluated by determining levels of endogenous protein and mRNA Cell culture and reagents by Western analysis and real-time qPCR. Primary fibroblast cultures were established by explantation from adult Confocal immunofluorescence microscopy lungs, from biopsies from the distal forearm of patients with scleroderma Fibroblasts (10,000 cells/well) were seeded onto eight-well Lab-Tek II and healthy adults, and from neonatal foreskin (14). Biopsy protocols were chamber glass slides (Nalge Nunc International, Naperville, IL) and in- approved by the Institutional Review Board at Northwestern University. 2/2 cubated in serum-free Eagle’s MEM with poly(I:C) (10 mg/ml) or TGF-b2 Fibroblasts derived from type I IFN receptor-null (IFNAR1 ) mouse (10 ng/ml) for 1–24 h. At the end of the experiments, cells were fixed, embryos (A. Kroger, Helmholtz Center for Infection Research, Braunschweig, permeabilized, and incubated with primary Abs to phospho-Smad3 at Germany) (15) or from wild-type control mouse embryos were main- 1:200 dilution (Cell Signaling Technology, Beverly, MA), TLR3 (Santa tained in DMEM supplemented with 10% FBS (Lonza, Basel, Switzerland), Cruz Biotechnology, Santa Cruz, CA) at 1:100 dilution, or to type I col- 50 mg/ml penicillin, and 50 mg/ml streptomycin in a humidified atmo- lagen at 1:100 dilution (SouthernBiotech, Birmingham, AL). Cells were sphere of 5% CO2 at 37˚C, and studied between