Autocrine IFN Signaling Inducing Profibrotic Fibroblast Responses By

A Synthetic TLR3 Ligand Mitigates Profibrotic Fibroblast Responses by Inducing Autocrine IFN Signaling

This information is current as Feng Fang, Kohtaro Ooka, Xiaoyong Sun, Ruchi Shah, of September 29, 2021. Swati Bhattacharyya, Jun Wei and John Varga J Immunol published online 16 August 2013 http://www.jimmunol.org/content/early/2013/08/16/jimmun ol.1300376 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2013/08/20/jimmunol.130037 Material 6.DC1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 29, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 16, 2013, doi:10.4049/jimmunol.1300376 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: 000–000. 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 29, 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 accession 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 2 POLY(I:C) ABROGATES FIBROTIC RESPONSES

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 passages two and eight then washed with PBS and incubated with secondary Abs at 1:200 dilution (14). At confluence, serum-free media supplemented with 0.1% BSA were (Alexa Fluor 488 and 594; Invitrogen) and viewed under a Nikon C1Si added to the cultures for 24 h prior to addition of TGF-b2 (PeproTech, confocal microscope. Rocky Hill, NJ) or poly(I:C) (InvivoGen, San Diego, CA). In selected experiments, 1 mg/ml neutralizing Ab to IFNAR1 (InvivoGen) was added Western analysis to the cultures 30 min prior to addition of poly(I:C). At the end of each experiment, fibroblasts were harvested and whole-cell RNA isolation and real-time quantitative PCR lysates subjected to Western analysis as described (17). The following At the end of each experiment, cultures were harvested. RNA was isolated Abs were used: phospho-Smad3 (Cell Signaling Technology), type I col- using RNeasy Plus mini kits (Qiagen, Valencia, CA) and examined by real- lagen (SouthernBiotech), TLR3 (Santa Cruz Biotechnology), Smad7 Downloaded from time quantitative PCR (qPCR) (16). (Abcam, Cambridge, MA), and GAPDH (Zymed, San Francisco, CA). Bands were visualized using ECL reagents (Pierce, Rockford, IL). Transient transfection assays ELISAs Fibroblasts at early confluence were transfected with Smad binding element (SBE)4-luc plasmids harboring four copies of a minimal Smad-binding Supernatants from confluent cultures of fibroblasts incubated for 24 h with element using a SuperFect transfection kit (Qiagen) as described (17). poly(I:C) were collected and stored at 280˚C. Samples were thawed at Cultures were incubated in serum-free media containing 0.1% BSA for room temperature and IFN-b levels were determined in triplicate using 24 h, followed by TGF-b2 for a further 24 h. Whole-cell lysates were ELISA kits (R&D Systems, Minneapolis, MN) following the manu- http://www.jimmunol.org/ assayed for their luciferase activities using a dual-luciferase reporter assay facturer’s instructions. system (Promega, Madison, WI). In each experiment, Renilla luciferase pRL-TK (Promega) was cotransfected as a control for transfection effi- Collagen gel contraction assays ciency (18). Transient transfection experiments were performed in triplicate and repeated at least twice with consistent results. To determine the effect of poly(I:C) on fibroblast contractility, in vitro collagen gel contraction assays were performed (19). Briefly, fibroblasts Small interfering RNA–mediated knockdown and adenovirus (106 cells/well) were seeded in 12-well plates in DMEM with 10% FBS infection and type I collagen (3.8 mg/ml; BD Biosciences). Incubation for 60 min resulted in polymerization of the collagen gel. Cultures were then incu- Fibroblasts were transfected with target-specific small interfering RNAs bated with TGF-b (10 ng/ml) in the presence or absence of poly(I:C) (10 by guest on September 29, 2021 (siRNAs) (Dharmacon, Lafayette, CO) or scrambled control siRNA (10 mg/ml) for up to 6 d. Plates were photographed at indicated intervals, the nM). Twenty-four hours following transfection, fresh media containing extent of gel contraction was measured, and the percentage of gel diameter TGF-b (10 ng/ml) or poly(I:C) (10 mg/ml) were added to the cultures, and in poly(I:C)-treated compared with control gels was calculated. Each ex- the incubations were continued for a further 24 h. Knockdown efficiency periment was performed in triplicate.

FIGURE 1. Expression of TLR3 in skin. (A and B) Previously published microarray datasets repre- senting scleroderma skin biopsy–derived genome- wide expression profiles (http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE9285) were interrogated. (A) Fold change in TLR3 mRNA levels normalized with average expression levels across the entire cohort. Biopsies are clustered into intrinsic subsets as described (22). Box plots indicate the ranges of lower and upper quartiles (p , 0.001). (B) Correlation of the fold change in levels of TLR3 and Stat1 mRNA normalized with average expression levels across the entire cohort. (C and D) Explanted normal skin fibroblasts were incubated in media with poly(I:C) (10 mg/ml) for up to 24 h. (C) Whole-cell lysates were examined by Western analysis. Repre- sentative immunoblot. (D) Fibroblasts were immunostained with Abs to TLR3 (red) or stained with DAPI (blue). Representative immunofluorescence photomicrographs are shown. Original magnification 360. The Journal of Immunology 3

FIGURE 2. Poly(I:C) represses collagen and ASMA gene expression. Confluent skin fibroblasts were incubated in media with indicated concentrations of poly(I:C) for 24 h. (A and D) Total RNA was subjected to real- time qPCR. The results represent the means 6 SEM of triplicate determinations from three independent experiments. **p , 0.01, ***p , 0.001. (B) Whole-cell lysates were subjected to Western analysis. Representative immunoblots. (C) Fibroblasts were immunostained with Abs to ASMA (green) or type I collagen (red), or stained with DAPI (blue). Representative immunofluorescence photomicrographs are shown. Original magnification 3100. Downloaded from

Microarray procedures and data analysis We next used explanted normal skin fibroblasts to evaluate TLR http://www.jimmunol.org/ To examine gene expression changes induced by poly(I:C) at the genome- mRNA expression. Real-time qPCR showed that whereas mRNA wide scale, serum-starved confluent fibroblasts were incubated with transcripts for all 10 TLRs were readily detected in these fibro- poly(I:C) (10 mg/ml) or media for up to 24 h. Total RNA was isolated blasts, TLR3 was the most highly expressed. Levels of TLR3 from three independent cultures using RNeasy Mini Plus kits (Qiagen). The integrity of RNA was determined using an Agilent bioanalyzer (Agilent Technologies, Santa Clara, CA). Fluorescent-labeled cDNA was prepared using labeling kits (Ambion, Austin, TX), followed by hybridization to Illumina human HT-12 version 4 microarray chips containing 44,000 probes (Illumina, San Diego, CA). Raw signal intensities for each probe were obtained using Illumina BeadStudio data analysis software and imported to by guest on September 29, 2021 the Bioconductor lumi package for transformation and normalization (20, 21). The data were preprocessed using a variance stabilization transformation method (21) followed by quantile normalization. Data from probes that produced signals near or lower than background levels (based on Illumina negative control probes) with all samples were discarded. Genes with .2-fold up- or downregulation in poly(I:C)-treated fibroblasts compared with control (p , 0.05, false discovery rate [FDR] , 0.05) were subjected to further analysis. Microarray data were deposited to Gene Ex- pression Omnibus (accession no. GSE47616, http://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE47616). Statistical analysis Statistical analysis was performed on Excel (Microsoft, Redmond, WA) using a Student t test or ANOVA. The results are shown as the means 6 SEM. A p value , 0.05 was considered statistically significant.

Results TLR3 expression is elevated in scleroderma in skin biopsies and explanted fibroblasts To evaluate the expression levels of TLR3 mRNA in scleroderma, an extensively characterized genome-wide expression microarray dataset was interrogated (22). Skin biopsies were clustered into previously defined intrinsic subsets based on their mRNA expression profiles, and expression level of each gene was centered on its mean value across all arrays (22). Biopsies mapping to the FIGURE 3. Poly(I:C) inhibition of fibrotic gene expression is TLR3- A inflammatory intrinsic subset were found to have significantly independent. ( ) Fibroblasts were pretreated with chloroquine (10 mM) for 60 min, followed by poly(I:C) (10 mg/ml) for a further 24 h, and RNA was elevated TLR3 expression compared with those in the normal-like, 6 , analyzed by real-time qPCR. The results represent the means SEM of limited, and diffuse intrinsic subsets (p 0.001) (Fig. 1A). Ex- triplicate determinations. (B and C) Fibroblasts were transiently transfected pression of Stat1, an IFN-regulated transcription factor, also with indicated siRNA or scrambled control siRNA (siCTR) in parallel and showed significantly elevated levels in these skin biopsies; more- incubated in media with or without poly(I:C) (10 mg/ml) for a further 24 h, over, Stat1 expression levels were strongly correlated with TLR3 followed by real-time qPCR. Results are the means 6 SEM of triplicate levels (r = 0.7, p , 0.01) (Fig. 1B). determinations. *p , 0.05, **p , 0.01, ***p , 0.001. 4 POLY(I:C) ABROGATES FIBROTIC RESPONSES mRNA were 10-fold higher in fibroblasts compared with human independent manner (Fig. 3). Pretreatment of the fibroblasts with macrophages or epithelial cells studied in parallel (data not shown). the endosomal TLR inhibitors chloroquine or bafilomycin (Fig. 3A Western analysis and immunocytochemistry demonstrated that and data not shown), RNA interference (RNAi)–mediated knock- TLR3 was readily detected in explanted confluent fibroblasts, and down of endogenous TLR3 (Fig. 3B, 3C), and peptide blockade of its expression was markedly upregulated in cultures incubated with TRIF to inactivate TLR3 signaling (data not shown) each failed to the synthetic TLR3 ligand poly(I:C) (Fig. 1C, 1D). Similar results abrogate the downregulation of Col1A1 or ASMA elicited in fibro- were obtained with adult skin and lung fibroblasts (data not shown). blasts by poly(I:C). In contrast, TLR3 inhibitors effectively blocked Examination of skin fibroblasts explanted from scleroderma patients poly(I:C)-induced stimulation of IL-6, as expected (data not shown). (n = 4) showed no significant difference in TLR3 mRNA levels To characterize the changes elicited by poly(I:C) treatment of compared with healthy control fibroblasts (n = 4) studied in parallel fibroblasts at the genome-wide scale, total RNA was isolated from (data not shown). foreskin fibroblasts incubated for 24 h with or without poly(I:C) and hybridized to Illumina cDNA microarrays containing 44,000 a Poly(I:C) inhibits collagen and -smooth muscle actin gene probes. Incubation with poly(I:C) induced broad changes in fi- expression via TLR3- independent pathways broblast gene expression. Approximately 700 transcripts showed Having demonstrated TLR3 expression in explanted fibroblasts, .2-fold change (314 up and 363 down) compared with control we next sought to examine the effects of ligands of TLR3 on cultures (p , 0.05, FDR , 0.05). The genes with the greatest modulating fibroblast function. For this purpose, confluent fibro- increase or decrease in expression are shown in Tables I and II; blasts were incubated in media with poly(I:C) for up to 24 h. Real- a complete list of poly(I:C)-regulated genes is shown in Supple- time qPCR, immunofluorescence, and Western analysis demon- mental Table I. Among the most highly upregulated genes were Downloaded from strated a time- and dose-dependent inhibition of Col1A1 and those encoding IL-6, IL-8, chemokines (CXCL10, CXCL11, CCL5, a-smooth muscle actin (ASMA) mRNA expression and protein and CCL8), IFN response genes (OASL and OAS1), inflammatory levels, with a 60–80% reduction elicited by poly(I:C) at 24 h (Fig. signal transducers (IRF7 and CFB), and IDO1. Downregulated 2). In contrast, the expression of the inflammatory chemokine genes included those encoding several ECM molecules (fibrillin 2, CXCL10 showed a dramatic increase, indicating fully functional laminin, and periostin). Gene ontology analysis of the data revealed

TLR3 pathways in these cells. Moreover, poly(I:C) induced potent multiple biological functions that were significantly enriched with http://www.jimmunol.org/ and selective stimulation of type I IFN, with a .10-fold increase poly(I:C)-regulated genes (Fig. 4A). Real-time qPCR of the same in IFN-b mRNA levels, whereas IFN-a levels were not signifi- RNA preparations that were used for microarray analysis was cantly changed. Comparable responses were seen in adult lung performed to validate poly(I:C)-induced changes in the expression fibroblasts exposed to poly(I:C) (data not shown). of selected genes (Fig. 4B and data not shown). Of note, the ex- To determine the role of TLR3 in mediating the inhibitory repression of all three known poly(I:C) receptors (TLR3, MDA5, and sponse elicited by poly(I:C), multiple complementary strategies RIG-1) was upregulated by poly(I:C). were pursued. Surprisingly, this series of studies firmly indicated that Transcripts with known gene identifiers (HUGO gene symbols) the inhibitory responses elicited by poly(I:C) occurred in a TLR3- and expression levels showing fold change .2(p , 0.05, FDR , by guest on September 29, 2021

Table I. Top 30 genes upregulated by poly(I:C) in ﬁbroblasts

Symbol Description Fold Change CXCL10 Chemokine (C-X-C motif) ligand 10 129 CCL5 Chemokine (C-C motif) ligand 5 118 CCL5 Chemokine (C-C motif) ligand 5 115 OASL 29-59-Oligoadenylate synthetase-like 83.3 CCL8 Chemokine (C-C motif) ligand 8 78.5 IDO1 IDO1 76.3 IFI27 IFN, a-inducible protein 27 70.6 TNFSF10 TNF (ligand) superfamily, member 10 57.2 IDO1 IDO1 54.1 GBP4 Guanylate binding protein 4 48.1 IFIT3 IFN-induced protein with tetratricopeptide repeats 3 45.6 BST2 Bone marrow stromal cell Ag 2 43.4 IL18BP IL-18 binding protein 43.4 TNFSF13B TNF (ligand) superfamily, member 13b 43.1 CFB Complement factor B 41.4 IFIT2 IFN-induced protein with tetratricopeptide repeats 2 41.2 OAS1 29-5 9-Oligoadenylate synthetase 1, 40/46kDa 40.9 HERC5 Hect domain and RLD5 38.8 OAS2 29-5 9-Oligoadenylate synthetase 2, 69/71kDa 37.7 OAS1 29-5 9-Oligoadenylate synthetase 1, 40/46kDa 36.9 IFIT3 IFN-induced protein with tetratricopeptide repeats 3 36 ISG20 IFN-stimulated exonuclease gene of 20kDa 35.6 OASL 29-5 9-Oligoadenylate synthetase-like 33.8 RSAD2 Radical S-adenosyl methionine domain containing 2 30.6 TNFSF13B TNF (ligand) superfamily, member 13b 30.5 CXCL11 Chemokine (C-X-C motif) ligand 11 29.6 CD38 CD38 molecule 28 IFI6 IFN, a-inducible protein 6 27 MX2 Myxovirus (inﬂuenza virus) resistance 2 (mouse) 25.1 IL6 IL-6 (IFN, b2) 24.5 Genes regulated by poly(I:C) were ﬁltered (p , 0.05, FDR , 0.05). Values in third column indicate fold change ranked from highest to lowest. The Journal of Immunology 5

Table II. Top 30 genes downregulated by poly(I:C) in ﬁbroblasts

Symbol Description Fold Change TTC3 Tetratricopeptide repeat domain 3 27.9 PSAT1 Phosphoserine aminotransferase 1 27.3 VAT1 Vesicle amine transport protein 1 homolog (Torpedo californica) 26.6 GSTM2 GST m 2 (muscle) 26.0 FBN2 Fibrillin 2 26.0 SGCD Sarcoglycan, d (35-kDa dystrophin-associated glycoprotein) 26.0 ACO1 Aconitase 1, soluble 25.8 C7orf41 Chromosome 7 open reading frame 41 25.6 APPL2 Adaptor protein, phosphotyrosine interaction, PH domain, 25.6 and leucine zipper containing 2 PHGDH Phosphoglycerate dehydrogenase 25.5 IARS Isoleucyl-tRNA synthetase 25.4 FEZ1 Fasciculation and elongation protein z 1 (zygin I) 25.2 RCN2 Reticulocalbin 2, EF-hand calcium binding domain 25.2 NCRNA00188 Non-protein coding RNA 188 25.1 EIF4B Eukaryotic translation initiation factor 4B 25.1 GSTM1 GST m 1 25.1 DDIT4 DNA-damage-inducible transcript 4 25.1 FAM171A1 Family with sequence similarity 171, member A1 25.0 Downloaded from GALNT11 UDP-N-acetyl-a-D-galactosamine:polypeptide 25.0 N-acetylgalactosaminyltransferase 11 (GalNAc-T11) KDELR3 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein 25.0 retention receptor 3 PCYOX1 Prenylcysteine oxidase 1 24.9 LAMA2 Laminin, a 2 24.8 LTA4H Leukotriene A4 hydrolase 24.8 http://www.jimmunol.org/ EIF4BP7 Eukaryotic translation initiation factor 4B pseudogene 7 24.8 POSTN Periostin, osteoblast-specific factor 24.7 PRKCA Protein kinase C, a 24.7 ASNS Asparagine synthetase (glutamine-hydrolyzing) 24.6 MFAP5 Microfibrillar-associated protein 5 24.6 FAM172A Family with sequence similarity 172, member A 24.6 MAGED1 Melanoma Ag family D, 1 24.6 Genes regulated by poly(I:C) were filtered (p , 0.05, FDR , 0.05). Values in third column indicate fold change ranked from highest to lowest. by guest on September 29, 2021

0.05) were filtered from above and were then entered into the IFNAR1 Abs to block IFNAR1 signaling, yielded comparable results Ingenuity Pathways Knowledge Base to overlay into a global (data not shown). Taken together, these genetic and pharmacological molecular network. Ingenuity Pathway Analysis of genes that approaches firmly establish the indispensable role of endogenous were most significantly changed by poly(I:C) implicated the IFN type I IFN in mediating the antifibrotic effects of poly(I:C). signaling pathway (Supplemental Fig. 1). Comparison of the Poly(I:C) abrogates profibrotic responses induced by TGF-b poly(I:C)-induced genes and the core IFN signature yielded 105 genes shared among the two sets (24) (Supplemental Fig. 2). In light of the fundamental role of TGF-b in orchestrating fibrogenesis, it was of interest to evaluate the modulation of TGF-b Poly(I:C) induces endogenous IFN signaling and responses by poly(I:C). For this purpose, fibroblasts were pre- IFN-mediated antifibrotic effects treated with poly(I:C) followed by incubation with TGF-b for 24 The levels of IFN-b mRNA, as well as secretion of IFN-b, showed h. The results of real-time qPCR and Western analysis showed that a dose-dependent stimulation in fibroblasts incubated with whereas poly(I:C) caused a marked stimulation of CXCL10 and poly(I:C), as has been shown previously in inflammatory cells (Fig. IFN-b mRNA expression both in the absence and presence of 4). We therefore proceeded to examine the potential role of en- TGF-b, the stimulation of collagen and ASMA gene expression dogenous IFN-b in mediating the antifibrotic effect of poly(I:C). by TGF-b were abrogated by poly(I:C) (Fig. 6). Additionally, Incubation of fibroblasts with IFN-b for 24 h resulted in a time- poly(I:C) potently inhibited the increase in collagen gel contraction and dose-dependent inhibition of collagen and ASMA gene ex- induced by TGF-b. Furthermore, in contrast to wild-type MEFs, in pression, whereas at the same time a marked increase in TLR3 IFNAR1-null MEF poly(I:C) was unable to suppress TGF-b–in- mRNA levels was seen (Fig. 5A, 5B and data not shown). Multiple duced stimulation of collagen and ASMA gene expression, indi- complementary approaches to inhibit the IFN-b pathway were cating a critical role for endogenous IFN in mediating poly(I:C) then taken to evaluate the possibility that the inhibitory effects of antagonism for the TGF-b response (Fig. 6D). poly(I:C) are mediated by endogenous IFN-b. The initial set of It is now increasingly recognized that although poly(I:C) serves experiments evaluated poly(I:C) responses in IFNAR1-null mouse as a potent TLR3 ligand, it can also elicit a variety of cellular embryonic fibroblasts (MEFs). The results showed that in contrast to responses via the ubiquitously expressed cytosolic receptors MDA5 wild-type MEFs, in IFNAR1-null MEFs poly(I:C) failed to repress and RIG-1 (26). Because we showed that the antifibrotic effect collagen and ASMA gene expression (Fig. 5C and data not shown). of poly(I:C) appeared to be independent of TLR3, we used RNAi Moreover, pretreatment of normal fibroblasts with LY294002, to investigate the role of the cytosolic poly(I:C) receptors in me- a PI3K inhibitor that blocks IFN-b1 induction (25), attenuated the diating repression of profibrotic genes. The results from a series of antifibrotic effects of poly(I:C) (Fig. 5D). Additional experiments experiments showed that significant and selective downregulation usingRNAitoknockdowncellularIFNAR1,andneutralizing of MDA5 or RIG-1 was achieved by transfection of fibroblasts 6 POLY(I:C) ABROGATES FIBROTIC RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Genome-wide gene expression changes induced by poly(I:C). Fibroblasts were incubated in media with or without poly(I:C) (10 mg/ml unless indicated) for 24 h. RNA was isolated and processed for microarray analysis using Illumina chips (A) or for real-time qPCR (B). (A) The list of genes showing .1.5-fold change was used for Gene Ontology analysis and pathway analysis based on DAVID (http://david.abcc.ncifcrf.gov/) (23). Of the 82 Gene Ontology categories with p , 0.001identified,thetop22areshown(p value from low to high). X-axis is the –log p value of each Gene Ontology category. (B)Real-time qPCR validation for selected poly(I:C)-regulated genes. The results represent the means 6 SEM of triplicate determinations from three independent experiments. (C) Culture supernatants were assayed for IFN-b by ELISA. Results represent the means 6 SEM of triplicate determinations. with appropriate siRNAs (data not shown). Whereas knockdown try and transient transfection assays showed that poly(I:C) mark- of neither MDA5 nor RIG-1 alone resulted in significant attenu- edly attenuated TGF-b–induced Smad3 phosphorylation, as well ation of the inhibitory effect of poly(I:C) on TGF-b responses, as stimulation of Smad-dependent transcription, in normal fibro- simultaneous knockdown of both cytosolic receptors fully rescued blasts (Fig. 7). We therefore focused on Smad7, a TGF-b–in- the TGF-b stimulatory effect on of Col1A1 and ASMA gene ducible endogenous Smad inhibitor known to have a fundamental expression in the presence of poly(I:C) (Fig. 6E). role in modulating the duration/intensity of fibrotic TGF-b responses (27). Remarkably, we found that incubation of fibro- Poly(I:C) disrupts Smad2/3 activation via endogenous type I blasts with poly(I:C) by itself induced a substantial increase of IFN and inhibitory Smad7 Smad7 mRNA and protein (Fig. 8). Stimulation of Smad7 mRNA To characterize the mechanisms underlying the inhibitory effect of peaked between 30 and 90 min, with a magnitude that equaled and poly(I:C) on TGF-b responses, a series of experiments focusing on even exceeded the stimulatory response elicited by TGF-b. The canonical Smad signaling were undertaken. Immunocytochemis- induction of Smad7 by poly(I:C) was not mediated through TLR3, The Journal of Immunology 7

FIGURE 5. IFN-b inhibits fibrotic gene expression and mediates poly(I:C) antifibrotic effects. (A and B) Foreskin fibroblasts were incubated with indicated concentrations of IFN-b for 24 h. (A) Whole-cell lysates were subjected to Western analysis. Representative autographs are shown. (B) RNA was subjected to real-time qPCR. The results represent the means 6 SEM of triplicate determinations from three independent experiments. (C) IFNAR1-null MEFs and wild-type MEFs in parallel were incubated with poly(I:C) (10 ng/ml) for 24 h. Total RNA was analyzed by real-time qPCR. The results represent the means 6 SEM of triplicate determinations. (D) Foreskin fibroblasts were preincubated with LY294002 (10 mM) for 30 min prior to incubation with poly(I:C) Downloaded from (10 ng/ml) for 24 h. *p , 0.05. http://www.jimmunol.org/ because it was significantly (p , 0.05) attenuated in fibroblasts The results demonstrate that TLR3 mRNA expression was elevated with siRNA knockdown of RIG1 and MDA5, but not of TLR3 in a subset of scleroderma skin biopsies, and furthermore that (Fig. 8C). Moreover, in the presence of TGF-b, poly(I:C) induced TLR3 levels were positively correlated with STAT1 in these a further increase in Smad7 levels. To examine the potential role biopsies. Moreover, TLR3 was abundantly expressed and func- of Smad7 in mediating the poly(I:C)-dependent inhibition of fi- tionally competent in explanted fibroblasts. Poly(I:C), a dsRNA brotic TGF-b responses, fibroblasts were transfected with a Smad7- analog, was used as a ligand to examine the effect of TLR3 ac- specific siRNA. Knockdown of Smad7 substantially reduced the tivation in vitro. Incubation of normal skin and lung fibroblasts basal levels of endogenous Smad7 and attenuated induction by with poly(I:C) elicited stereotypic immunostimulatory responses TGF-b and poly(I:C) (Fig. 8D and data not shown). Significantly, with secretion of the inflammatory mediators CXCL10, CCL5, by guest on September 29, 2021 poly(I:C) in the presence of Smad7-specific siRNA was unable to and CCL8 and production of OAS and IDO, accompanied by abrogate the stimulation of collagen and ASMA gene expression marked upregulation of type I IFN. Remarkably, however, we induced by TGF-b. These results demonstrate that poly(I:C) re- simultaneously observed profound downregulation of fibrotic pression of the profibrotic TGF-b response was associated with gene expression in fibroblasts incubated with poly(I:C). Even more disruption of canonical Smad signaling and involved the induction significantly, these fibroblasts failed to mount fibrotic response when of antagonistic Smad7. challenged with TGF-b. We therefore conclude that in fibroblasts TLR3 ligands cause concurrent inflammatory response induction and Discussion fibrotic response blockade. Scleroderma is characterized by inflammation and fibrosis, with Mechanistic studies revealed that despite the presence of TLR3 dermal fibroblasts serving as key effector cells of the latter process. and a fully intact TLR3 signaling pathway in fibroblasts, the The links between inflammation and fibrosis, as well as the nature antifibrotic effects exerted by poly(I:C) in these cells were largely of the inflammatory activation of dermal fibroblasts during the independent of TLR3, and instead appeared to be mediated by fibrotic process, remain incompletely understood. Although TGF-b cytosolic pattern recognition receptors RIG-1 and MDA5. The has long been implicated as a pivotal factor for initiating fibroblast antifibrotic response could be recapitulated by type I IFN, which activation, recent reports focus attention on the presence of a prom- was sufficient and required for the poly(I:C) effect. Taken together, inent IFN-responsive gene signature in lesional skin and peripheral these findings therefore support a mechanistic model to explain the blood cells from patients with scleroderma (28–30), as well as genetic inflammatory regulation of fibroblast function in fibrosis, where associations of scleroderma with Stat4, IRF5, and other medi- endogenous type I IFN production elicited by a bona fide TLR3 ators of innate immune responses (31). Additional studies show ligand establishes a local negative autocrine/paracrine feedback that circulating IFN-inducible chemokines are elevated in sclero- loop with consequent inhibition of fibroblast function and abro- derma, and levels show strong correlation with indices of disease gation of profibrotic responses. The results therefore imply that the severity (32). Taken together, these recent observations suggest that prominent type I IFN signature described in scleroderma lesional innate immunity, pattern recognition receptor signaling, and type I tissues might in fact reflect DAMP-induced in situ activation of IFN play important but poorly understood roles in initiating the fibroblast innate immune responses during fibrogenesis that are fibrotic response in scleroderma. aimed toward restraining fibroblast activation and limiting pro- Because the production of type I IFN and the generation of the gressive fibrosis. Moreover, our findings provide the first evidence, IFN gene signature are closely tied to molecular pattern sensing by to our knowledge, that dsRNA has potent antifibrotic activity and TLR3 and cytosolic receptors, the current study sought to evaluate might potentially represent an innovative approach to therapy. TLR3 expression in scleroderma tissues and normal fibroblasts, as Poly(I:C) has long been recognized as a potent inducer of IFN well as the consequences of its activation in the context of fibrosis. production and signaling (33), a finding reproduced in the present 8 POLY(I:C) ABROGATES FIBROTIC RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Poly(I:C) abrogates profibrotic TGF-b responses. (A–C) Confluent skin fibroblasts were incubated with TGF-b (10 ng/ml) in the presence or absence of poly(I:C) (10 mg/ml) for up to 48 h (A, C) or for 6 d (B). (A) Total RNA was subjected to real-time qPCR. The results represent the means 6 SEM of triplicate determinations. (B) Gel contraction assays. Fibroblasts seeded in type I collagen gels were incubated in media with poly(I:C). Gel diameters were measured at indicated time points. Results are shown as means 6 SEM of triplicate determinations. *p , 0.05. (C) Whole-cell lysates were subjected to Western analysis. Representative autoradiograms are shown. (D) IFNAR1-null MEFs or wild-type controls were pretreated with poly(I:C) (10 mg/ml) for 24 h, followed by TGF-b (10 ng/ml) for 24 h. RNA was isolated for real-time qPCR. The results represent the means 6 SEM of triplicate determinations. (E) Skin fibroblasts were transfected with indicated siRNA or scrambled control siRNA (siCTR) in parallel, followed by incubation with poly(I:C) (10 mg/ml) for a further 24 h prior to RNA isolation and real-time qPCR. *p , 0.05, **p , 0.01. results. TLR3 uniquely senses, and responds to, extracellular poly(I:C)-induced inflammatory disease models is congruent with RNA duplexes as well as synthetic poly(I:C). In rodent models, the hypothesis that although poly(I:C) caused inflammation, en- poly(I:C) has been shown to induce inflammation and accelerate hanced IFN production, and accumulation of alternately activated autoimmunity through mechanisms that are both TLR3-dependent macrophages associated with tissue remodeling and scar forma- and -independent (9, 34). Lupus nephritis in NZW/B mice was tion, its direct effects at the tissue level resulted in attenuated exacerbated by poly(I:C), and the inflammatory effects were me- fibroblast activation and fibrotic signaling. diated by endogenous IFN and accumulation of alternately activated Earlier studies focusing on the modulation of fibrosis by poly(I:C) macrophages in the kidney (34). In contrast, poly(I:C) can also have yielded conflicting results. It was shown that injection of downregulate inflammation in murine arthritis (35) and other poly(I:C) ameliorated lung and liver fibrosis in rodents (12, 37–39). models of inflammation (36). Interestingly, poly(I:C)-induced in- The mechanism for these in vivo antifibrotic effects has not been flammation is generally associated with only a modest degree of clarified. In contrast, Farina et al. (13) recently showed that fibrosis. For instance, in the murine lupus nephritis model, the poly(I:C) delivered by continuous s.c. infusion using implanted kidneys of poly(I:C)-injected NZW/B mice showed only scant osmotic pumps induced epidermal hyperplasia, inflammatory cell collagen accumulation despite extensive inflammation and crescent accumulation in the deep dermis and fat, and increased matrix de- formation. The modest-to-absent fibrotic response in this and other position. Moreover, the expression of the TGF-b–inducible genes The Journal of Immunology 9

FIGURE 7. Poly(I:C) blocks canonical Smad2/3 signaling. Skin fibroblasts (A, B) or NIH-3T3 mouse fibroblasts transiently transfected with Smad binding element (SBE)4-luc (C) were pretreated with poly(I:C) (10 mg/ml) for 24 h, followed by TGF-b (10 ng/ml) for 60 min (A, B)or24h(C). (A) Cultures were fixed and immunostained with Abs to phospho-Smad3 (green), type I collagen (red), or DAPI (blue). Representative immunofluorescence photomicrographs are shown. Original magnification 3100. (B) Whole-cell lysates were subjected to Western analysis. Representative autoradiograms are shown. (C) Cell lysates were assayed for their luciferase activities. The results represent the means 6 SEM of triplicate determinations. Downloaded from *p , 0.05, **p , 0.01. http://www.jimmunol.org/

PAI-1 and COMP was elevated in the lesional skin. Additional signaling. The authors concluded that chronic TLR3 stimulation can experiments showed that the cutaneous inﬂammatory response in- induce cutaneous ﬁbrosis and might play a role in the pathogenesis duced by poly(I:C) was dependent on both TLR3 and type I IFN of scleroderma. It has been pointed out that poly(I:C) elicits by guest on September 29, 2021

FIGURE 8. Poly(I:C) abrogates profibrotic TGF-b responses through Smad7. (A) Fibroblasts were incubated with TGF-b (10 ng/ml) in the presence or absence of poly(I:C) (10 mg/ml) for 24 h or indicated periods. (A) Whole-cell lysates were examined by Western analysis. Representative immunoblots are shown. (B) Total RNA was subjected to real-time qPCR. The results are the means 6 SEM of triplicate determinations. (C–E) Fibroblasts were transfected with indicated siRNA or scrambled control siRNA (siCTR) prior to incubation with poly(I:C) (10 mg/ml) for 24 h. Total RNA was examined by real-time qPCR. Results are means 6 SEM of triplicate determinations. *p , 0.05, **p , 0.001. (F) Proposed mechanisms accounting for the TLR3-independent IFN-dependent antifibrotic activities of poly(I:C) in fibroblasts. See text for explanation. 10 POLY(I:C) ABROGATES FIBROTIC RESPONSES markedly different responses and profiles of cytokines in murine and multaneously inducing dramatic upregulation of inflammatory human cells (40). This differential effect might account for the mediators including chemokines and type I IFN, coupled with profibrotic poly(I:C) responses observed by Farina et al. (13) in the suppression of TGF-b signaling and inhibition of fibrotic respon- mouse, as well as the marked antifibrotic responses in human ses. Because dsRNA is released from damaged or dying cells and fibroblasts in the present study. A potential explanation for these its levels in situ are likely to be elevated in tissue injury and seemingly conflicting observations regarding the pro- or antifibrotic fibrosis, the present results using poly(I:C) as a surrogate for effects of poly(I:C) might lie in the dual effect of poly(I:C): in- endogenous TLR3 ligands are relevant for understanding inducing potent systemic inflammation, mediated in part through flammatory fibroblast regulation during fibrogenesis. As ex- endogenous type I IFN and accumulation of alternately activated pected, poly(I:C) was a potent inducer of type I IFN production macrophages in target tissues; and concurrent downregulation of in fibroblasts, and type I IFN in turn was required for, and suffi- fibrotic gene expression in lesional fibroblasts, as well as abrogation cient to recapitulate, the antifibrotic effects elicited by poly(I:C). of TGF-b–mediated stimulation of fibrotic programs in affected The inhibitory effects of type I IFN appear to be mediated through tissues. The net result of these coupled proinflammatory and anti- induction of Smad7, which blocks canonical Smad signaling (Fig. fibrotic responses then might be attenuation of scar formation and 8). Taken together, these results suggest that the IFN gene sig- organ fibrosis. With persistence of injury and sustained activation of nature in scleroderma patients might be an indication of an on- pattern recognition receptors by endogenous RNA ligands of going DAMP-induced innate immune response designed to TLR3, the proinflammatory responses might come to dominate. restrain and control TGF-b–induced fibroblast activation. In this In common with other dsRNA moieties, poly(I:C) serves as scenario, type I IFN signaling in the lesional skin might play a a surrogate for RNA released during tissue injury and is recognized beneficial role, and therapies aimed at locally enhancing the re- Downloaded from as a damage-associated endogenous TLR3 ligand (8). Our results, sponse, rather than blocking it, might in fact be desirable for the consistent with other studies, indicate that TLR3 is expressed on treatment of fibrosis. Accordingly, poly(I:C) therapy targeting the explanted skin and lung fibroblasts, and its levels are elevated in fibrotic lesion represents a potential treatment strategy in sclero- skin biopsies from patients with scleroderma (13, 41). Because of derma. its ability to respond to RNA released from damaged or necrotic cells, TLR3 has been proposed to serve as an endogenous sensor Acknowledgments http://www.jimmunol.org/ of tissue necrosis during inflammatory states, and it plays an We are grateful to Dr. Michael Whitfield (Dartmouth College) and to mem- important role in amplifying sterile inflammatory responses (42). bers of the Varga Laboratory for helpful discussions. We also thank the However, poly(I:C) triggers activation of multiple pattern recog- help from the Cell Imaging Facility, Skin Disease Research Center, and nition receptors, including RIG-1 and MDA5, and under certain Genomics Core Facility in Northwestern University. conditions the TLR3-independent cellular response predom- inates. For example, inhibition of vascular endothelial growth Disclosures factor–mediated responses in transgenic mice by poly(I:C) in- The authors have no financial conflicts of interest. volved a TLR3-independent RIG-1 recognition mechanism (36). Consistent with these observations, we demonstrate that the by guest on September 29, 2021 antifibrotic effects elicited by poly(I:C) in fibroblasts are largely References TLR3-independent and are mediated through both RIG-1 and 1. Jimenez, S. A., and C. T. Derk. 2004. Following the molecular pathways toward an understanding of the pathogenesis of systemic sclerosis. Ann. Intern. Med. MDA5. 140: 37–50. Poly(I:C) induced robust stimulation of type I IFN in normal 2. Bhattacharyya, S., J. Wei, and J. Varga. 2011. Understanding fibrosis in systemic sclerosis: shifting paradigms, emerging opportunities. Nat. Rev. Rheumatol. 8: fibroblasts. Importantly, we used multiple complementary strate- 42–54. gies to establish that endogenous type I IFN plays an essential role 3. Varga, J., and B. Pasche. 2008. Antitransforming growth factor-b therapy in of in mediating the antifibrotic effects of poly(I:C). The results fibrosis: recent progress and implications for systemic sclerosis. Curr. Opin. Rheumatol. 20: 720–728. indicated that a poly(I:C) response was absent in IFNAR1-null 4. Sozzani, S., D. Bosisio, M. Scarsi, and A. Tincani. 2010. Type I interferons in fibroblasts, and it could be abrogated by Abs to IFNAR1, as systemic autoimmunity. Autoimmunity 43: 196–203. well as RNAi knockdown of the receptor, or by a pharmacological 5. Hall, J. C., and A. Rosen. 2010. Type I interferons: crucial participants in disease amplification in autoimmunity. Nat. Rev. Rheumatol. 6: 40–49. inhibitor of type IFN production. IFN is well known to be a potent 6. Kawai, T., and S. Akira. 2010. The role of pattern-recognition receptors in innate inhibitor of collagen gene expression, and it has been shown to immunity: update on Toll-like receptors. Nat. Immunol. 11: 373–384. 7. Hennessy, E. J., A. E. Parker, and L. A. O’Neill. 2010. Targeting Toll-like attenuate a broad range of fibrotic responses in both normal and receptors: emerging therapeutics? Nat. Rev. Drug Discov. 9: 293–307. scleroderma fibroblasts (43–45). We and others have further 8. Cavassani, K. A., M. Ishii, H. Wen, M. A. Schaller, P. M. Lincoln, N. W. Lukacs, demonstrated that IFN blocks Smad-dependent TGF-b responses C. M. Hogaboam, and S. L. Kunkel. 2008. TLR3 is an endogenous sensor of tissue necrosis during acute inflammatory events. J. Exp. Med. 205: 2609–2621. (46). The present results indicate that poly(I:C) blocked canonical 9. Kato, H., O. Takeuchi, S. Sato, M. Yoneyama, M. Yamamoto, K. Matsui, Smad signaling, and this effect was mediated, at least in part, S. Uematsu, A. Jung, T. Kawai, K. J. Ishii, et al. 2006. Differential roles of through induction of the antagonist Smad7. This inducible Smad MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441: 101– 105. blocks canonical TGF-b signaling by preventing Smad3 interac- 10. Radstake, T. R., O. Gorlova, B. Rueda, J. E. Martin, B. Z. Alizadeh, R. Palomino- tion with TGF-b receptors, and its upregulation has been previ- Morales, M. J. Coenen, M. C. Vonk, A. E. Voskuyl, A. J. Schuerwegh, et al; Spanish Scleroderma Group. 2010. Genome-wide association study of sys- ously shown to abrogate the stimulation of collagen (27). temic sclerosis identifies CD247 as a new susceptibility locus. Nat. Genet. 42: Furthermore, similar to poly(I:C), both type I and type II IFN 426–429. have been previously shown to induce Smad7, which may account 11. Jeong, W. I., O. Park, S. Radaeva, and B. Gao. 2006. STAT1 inhibits liver fibrosis in mice by inhibiting stellate cell proliferation and stimulating NK cell cyto- for their inhibitory effects of fibrotic responses (47–50). The toxicity. Hepatology 44: 1441–1451. mechanism accounting for Smad7 induction by poly(I:C) is likely 12. Wild, J. S., D. M. Hyde, and S. N. Giri. 1994. Dose and regimen effects of poly to involve NF-kB or Stat1 (47, 48). ICLC, an interferon inducer, in a multi-dose bleomycin model of interstitial pulmonary fibrosis. Pharmacol. Toxicol. 75: 42–48. Taken together, the present results demonstrate for the first 13. Farina, G. A., M. R. York, M. Di Marzio, C. A. Collins, S. Meller, B. Homey, time, to our knowledge, that synthetic RNA, a known ligand for I. R. Rifkin, A. Marshak-Rothstein, T. R. Radstake, and R. Lafyatis. 2010. Poly(I:C) drives type I IFN- and TGFb-mediated inflammation and dermal TLR3 as well as the cytosolic receptors RIG-1 and MDA5, acts fibrosis simulating altered gene expression in systemic sclerosis. J. Invest. as a potent and bifunctional stimulus for fibroblast activation, si- Dermatol. 130: 2583–2593. The Journal of Immunology 11

14. Varga, J., D. Brenner, and S. H. Phan. 2005. Fibrosis Research: Methods and synthesis and release of eotaxin-1/CCL11 from human bronchial smooth muscle Protocols. Humana Press, Totowa, N.J. cells stimulated with double-stranded RNA. J. Immunol. 178: 489–495. 15. Stirnweiss, A., A. Ksienzyk, K. Klages, U. Rand, M. Grashoff, H. Hauser, and 34. Triantafyllopoulou, A., C. W. Franzke, S. V. Seshan, G. Perino, G. D. Kalliolias, A. Kro¨ger. 2010. IFN regulatory factor-1 bypasses IFN-mediated antiviral effects M. Ramanujam, N. van Rooijen, A. Davidson, and L. B. Ivashkiv. 2010. Pro- through viperin gene induction. J. Immunol. 184: 5179–5185. liferative lesions and metalloproteinase activity in murine lupus nephritis me- 16. Fang, F., A. J. Flegler, P. Du, S. Lin, and C. V. Clevenger. 2009. Expression of diated by type I interferons and macrophages. Proc. Natl. Acad. Sci. USA 107: cyclophilin B is associated with malignant progression and regulation of genes 3012–3017. implicated in the pathogenesis of breast cancer. Am. J. Pathol. 174: 297–308. 35. Yarilina, A., E. DiCarlo, and L. B. Ivashkiv. 2007. Suppression of the effector 17. Fang, F., K. Ooka, S. Bhattacharyya, J. Wei, M. Wu, P. Du, S. Lin, F. Del Galdo, phase of inflammatory arthritis by double-stranded RNA is mediated by type I C. A. Feghali-Bostwick, and J. Varga. 2011. The early growth response gene IFNs. J. Immunol. 178: 2204–2211. Egr2 (alias Krox20) is a novel transcriptional target of transforming growth 36. Ma, B., C. S. Dela Cruz, D. Hartl, M. J. Kang, S. Takyar, R. J. Homer, C. G. Lee, factor-b that is up-regulated in systemic sclerosis and mediates profibrotic and J. A. Elias. 2011. RIG-like helicase innate immunity inhibits vascular en- responses. Am. J. Pathol. 178: 2077–2090. dothelial growth factor tissue responses via a type I IFN-dependent mechanism. 18. Fang, F., G. Antico, J. Zheng, and C. V. Clevenger. 2008. Quantification of PRL/ Am. J. Respir. Crit. Care Med. 183: 1322–1335. Stat5 signaling with a novel pGL4-CISH reporter. BMC Biotechnol. 8: 11. 37. Wild, J. S., D. M. Hyde, H. R. Hubbell, and S. N. Giri. 1996. Dose-related effects 19. Montesano, R., and L. Orci. 1988. Transforming growth factor beta stimulates of Ampligen (poly(I)·poly(C12U)), a mismatched double-stranded RNA, in collagen-matrix contraction by fibroblasts: implications for wound healing. Proc. a bleomycin-mouse model of pulmonary fibrosis. Exp. Lung Res. 22: 375–391. Natl. Acad. Sci. USA 85: 4894–4897. 38. Hyde, D. M., and S. N. Giri. 1990. Polyinosinic-polycytidylic acid, an interferon 20. Du, P., W. A. Kibbe, and S. M. Lin. 2008. lumi: a pipeline for processing Illu- inducer, ameliorates bleomycin-induced lung fibrosis in mice. Exp. Lung Res. mina microarray. Bioinformatics 24: 1547–1548. 16: 533–546. 21. Lin, S. M., P. Du, W. Huber, and W. A. Kibbe. 2008. Model-based variance- 39. Giri, S. N., and D. M. Hyde. 1988. Ameliorating effect of an interferon inducer stabilizing transformation for Illumina microarray data. Nucleic Acids Res. 36: e11. polyinosinic-polycytidylic acid on bleomycin-induced lung fibrosis in hamsters. 22. Milano, A., S. A. Pendergrass, J. L. Sargent, L. K. George, T. H. McCalmont, Morphologic and biochemical evidence. Am. J. Pathol. 133: 525–536. M. K. Connolly, and M. L. Whitfield. 2008. Molecular subsets in the gene ex- 40. Lundberg, A. M., S. K. Drexler, C. Monaco, L. M. Williams, S. M. Sacre, pression signatures of scleroderma skin. PLoS ONE 3: e2696. M. Feldmann, and B. M. Foxwell. 2007. Key differences in TLR3/poly I:C 23. Dennis, G., Jr., B. T. Sherman, D. A. Hosack, J. Yang, W. Gao, H. C. Lane, and signaling and cytokine induction by human primary cells: a phenomenon absent Downloaded from R. A. Lempicki. 2003. DAVID: database for annotation, visualization, and in- from murine cell systems. Blood 110: 3245–3252. tegrated discovery. Genome Biol. 4: P3. 41. Agarwal, S. K., M. Wu, C. K. Livingston, D. H. Parks, M. D. Mayes, 24. Wong, D., B. Kea, R. Pesich, B. W. Higgs, W. Zhu, P. Brown, Y. Yao, and F. C. Arnett, and F. K. Tan. 2011. Toll-like receptor 3 upregulation by type I D. Fiorentino. 2012. Interferon and biologic signatures in dermatomyositis skin: interferon in healthy and scleroderma dermal fibroblasts. Arthritis Res. Ther. 13: specificity and heterogeneity across diseases. PLoS ONE 7: e29161. R3. 25. Zhao, W., J. Qi, L. Wang, M. Zhang, P. Wang, and C. Gao. 2012. LY294002 42. Yu, M., and S. J. Levine. 2011. Toll-like receptor, RIG-I-like receptors and the inhibits TLR3/4-mediated IFN-b production via inhibition of IRF3 activation NLRP3 inflammasome: key modulators of innate immune responses to double-

with a PI3K-independent mechanism. FEBS Lett. 586: 705–710. stranded RNA viruses. Cytokine Growth Factor Rev. 22: 63–72. http://www.jimmunol.org/ 26. Sen, G. C., and S. N. Sarkar. 2005. Transcriptional signaling by double-stranded 43. Varga, J., A. Olsen, J. Herhal, G. Constantine, J. Rosenbloom, and S. A. Jimenez. RNA: role of TLR3. Cytokine Growth Factor Rev. 16: 1–14. 1990. Interferon-g reverses the stimulation of collagen but not fibronectin gene 27. Mori, Y., S. J. Chen, and J. Varga. 2000. Modulation of endogenous Smad ex- expression by transforming growth factor-b in normal human fibroblasts. Eur. J. pression in normal skin fibroblasts by transforming growth factor-b. Exp. Cell Clin. Invest. 20: 487–493. Res. 258: 374–383. 44. Freundlich, B., S. A. Jimenez, V. D. Steen, T. A. Medsger, Jr., M. Szkolnicki, and 28. Higgs, B. W., Z. Liu, B. White, W. Zhu, W. I. White, C. Morehouse, P. Brohawn, H. S. Jaffe. 1992. Treatment of systemic sclerosis with recombinant interferon-g. P. A. Kiener, L. Richman, D. Fiorentino, et al. 2011. Patients with systemic lupus A phase I/II clinical trial. Arthritis Rheum. 35: 1134–1142. erythematosus, myositis, rheumatoid arthritis and scleroderma share activation 45. Yuan, W., T. Yufit, L. Li, Y. Mori, S. J. Chen, and J. Varga. 1999. Negative of a common type I interferon pathway. Ann. Rheum. Dis. 70: 2029–2036. modulation of alpha1(I) procollagen gene expression in human skin fibroblasts: 29. Duan, H., J. Fleming, D. K. Pritchard, L. M. Amon, J. Xue, H. A. Arnett, transcriptional inhibition by interferon-g. J. Cell. Physiol. 179: 97–108. G. Chen, P. Breen, J. H. Buckner, J. A. Molitor, et al. 2008. Combined analysis 46. Ghosh, A. K., W. Yuan, Y. Mori, Chen Sj, and J. Varga. 2001. Antagonistic

of monocyte and lymphocyte messenger RNA expression with serum protein regulation of type I collagen gene expression by interferon-g and transforming by guest on September 29, 2021 profiles in patients with scleroderma. Arthritis Rheum. 58: 1465–1474. growth factor-b. Integration at the level of p300/CBP transcriptional coac- 30. York, M. R., T. Nagai, A. J. Mangini, R. Lemaire, J. M. van Seventer, and tivators. J. Biol. Chem. 276: 11041–11048. R. Lafyatis. 2007. A macrophage marker, Siglec-1, is increased on circulating 47. Ulloa, L., J. Doody, and J. Massague´. 1999. Inhibition of transforming growth monocytes in patients with systemic sclerosis and induced by type I interferons factor-b/SMAD signalling by the interferon-g/STAT pathway. Nature 397: 710– and Toll-like receptor agonists. Arthritis Rheum. 56: 1010–1020. 713. 31. Dieude´, P., M. Guedj, J. Wipff, B. Ruiz, E. Hachulla, E. Diot, B. Granel, 48. Bitzer, M., G. von Gersdorff, D. Liang, A. Dominguez-Rosales, A. A. Beg, J. Sibilia, K. Tiev, L. Mouthon, et al. 2009. STAT4 is a genetic risk factor for M. Rojkind, and E. P. Bo¨ttinger. 2000. A mechanism of suppression of TGF- systemic sclerosis having additive effects with IRF5 on disease susceptibility and b/SMAD signaling by NF-kB/RelA. Genes Dev. 14: 187–197. related pulmonary fibrosis. Arthritis Rheum. 60: 2472–2479. 49. Seyhan, H., J. Hamzavi, E. Wiercinska, A. M. Gressner, P. R. Mertens, J. Kopp, 32. Liu, X., M. D. Mayes, F. K. Tan, M. Wu, J. D. Reveille, B. E. Harper, R. E. Horch, K. Breitkopf, and S. Dooley. 2006. Liver fibrogenesis due to H. T. Draeger, E. B. Gonzalez, and S. Assassi. 2013. Correlation of interferon- cholestasis is associated with increased Smad7 expression and Smad3 signaling. inducible chemokine plasma levels with disease severity in systemic sclerosis. J. Cell. Mol. Med. 10: 922–932. Arthritis Rheum. 65: 226–235. 50. Chow, E. K., R. M. O’Connell, S. Schilling, X. F. Wang, X. Y. Fu, and G. Cheng. 33. Niimi, K., K. Asano, Y. Shiraishi, T. Nakajima, M. Wakaki, J. Kagyo, 2005. TLR agonists regulate PDGF-B production and cell proliferation through T. Takihara, Y. Suzuki, K. Fukunaga, T. Shiomi, et al. 2007. TLR3-mediated TGF-b/type I IFN crosstalk. EMBO J. 24: 4071–4081.