Abstract
Activation of Toll-like receptor-3 (TLR3) by exogenous microbial ligands or endogenous injury-associated ligands leads to production of type I interferon (IFN). Scleroderma patients with progressive skin fibrosis display an IFN-regulated gene signature, implicating TLR3 signaling in the disease. We now 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 double-stranded RNA analog, caused dose- and time-dependent stimulation of IFN-β production and generation of an IFN-response gene signature that was accompanied by substantial down-regulation of collagen and alpha-smooth muscle actin gene expression. Furthermore, Poly I:C abrogated transforming growth factor-β (TGF-β)-induced fibrotic responses and blocked canonical Smad signaling via up-regulation 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 (RIG1) and melanoma differentiation associated gene 5 (MDA5), and involved signaling via the IFN receptor. Taken together, these results demonstrate that induction of a fibroblast IFN response gene signature triggered by double-stranded RNA 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.
Keywords: Poly I:C, TLR, TGF-β, fibrosis, scleroderma, fibroblast, Smad7
Introduction
Progressive fibrosis of the skin and internal organs accounts for the intractable nature and the high mortality of scleroderma (1). As the principal effector cells responsible for fibrosis, stromal fibroblasts and myofibroblasts contribute to excessive deposition of collagens and other extracellular matrix proteins (2). Transforming growth factor β (TGF-β) which stimulates collagen synthesis, myofibroblast differentiation and epithelial-mesenchymal transition (EMT), is implicated as a key initiating factor in both physiological and pathological tissue remodeling (3). However, the mechanism responsible for the persistence of the fibrotic process associated with pathological repair remains poorly understood.
Recent studies have detected an interferon (IFN)-response gene signature, characterized by up-regulation of type I IFN-regulated genes, in both circulating blood cells and in lesional skin from patients with scleroderma (4). The production of type I IFN in plasmacytoid dendritic cells as well as stromal cells is controlled by Toll-like receptors (TLR) (5). These conserved pattern recognition receptors trigger immune responses to both microbial pathogens and to tissue injury-associated endogenous danger signals collectively referred to a damage-associated molecular patterns (DAMPs) (6). In contrast to TLR2 and TLR4 which reside at the cell surface and recognize microbial ligands, TLR3 is normally endosomal in its location and recognizes viral double-stranded RNA, as well as the synthetic double-stranded RNA analog polyinosinic: polycytidylic acid (Poly I:C) (6, 7). Upon injury, double-stranded RNA generated at sites of tissue damage in situ serves as an endogenous ligand for TLR3 (8). In most cell types, double-stranded nucleic acids and their analogues are recognized as DAMPs not only by TLR3, but also by the cytosolic RNA helicases retinoic acid-inducible gene I (RIG1) and melanoma differentiation associated gene 5 (MDA5) (9).
The IFN signature detected in scleroderma lesional tissue suggests potential roles for TLR3 signaling, type I IFN and innate immunity in pathogenesis. The recent demonstration that IRF3 and IRF5, which are downstream of TLR3, are risk alleles for scleroderma provides further evidence implicating type I IFN (10). However, the precise role of innate immunity and of endogenous TLR ligands in the pathogenesis of fibrosis remains poorly understood. We therefore sought to investigate TLR expression in scleroderma, and the modulation of fibroblast function by the synthetic TLR3 ligand Poly I:C, which was shown previously to abrogate (11, 12) or induce (13) fibrosis. The results showed that while Poly I:C treatment served as a potent stimulus for production of Type I IFN and generation of an IFN-response gene signature in explanted skin fibroblasts, it potently reduced fibrotic responses, blocked Smad-dependent TGF-β signaling, and prevented TGF-β stimulation of these cells through endogenous IFN. The results therefore identify the TLR3 ligand Poly I:C as a novel inhibitor of profibrotic TGF-β activity, and suggest the possibility that the IFN-response gene signature seen in scleroderma lesional tissue may in fact represent a cell-autonomous attempt to restrain fibroblast activation and mitigate aberrant fibrogenesis.
MATERIALS AND METHODS
Cell culture and reagents
Primary fibroblast cultures were established by explantation from adult lungs, from biopsies from the distal forearm of patients with scleroderma and healthy adults, and from neonatal foreskin (14). Biopsy protocols were approved by the Institutional Review Board at Northwestern University. Fibroblasts derived from type I IFN receptor-null (IFNAR1−/−) mouse embryos (A. Kroger, Helmholtz Centre for Infection Research, Braunschweig, Germany (15)) or from wild-type control mouse embryos were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Lonza, Basel, Switzerland), 50 µg/ml penicillin, and 50 µg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C, and studied between passages 2–8 (14). At confluence, serum-free media supplemented with 0.1% bovine serum albumin (BSA) were added to the cultures for 24 h prior to TGF-β2 (Peprotech, Rocky Hill, NJ), or Poly I:C (Invivogen, San Diego, CA). In selected experiments, 1 µg/ml of neutralizing antibody to IFNAR1 (Invivogen) was added to the cultures 30 min prior to Poly I:C.
RNA isolation and real-time quantitative PCR
At the end of each experiment, cultures were harvested. RNA was isolated using RNeasy Plus mini kits (Qiagen, Valencia, CA) and examined by real-time quantitative PCR (16).
Transient transfection assays
Fibroblasts at early confluence were transfected with [SBE]4-luc plasmids harboring four copies of a minimal Smad-binding elements using SuperFect Transfection kit (Qiagen, Valencia, CA) as described (17). Cultures were incubated in serum-free media containing 0.1% BSA for 24 h, followed by TGF-β2 for a further 24 h. Whole cell lysates were assayed for their luciferase activities using a dual-luciferase reporter assay system (Promega, Madison, WI). In each experiment, Renilla luciferase pRL-TK (Promega) was cotransfected as control for transfection efficiency (18). Transient transfection experiments were performed in triplicate and repeated at least twice with consistent results.
Short interfering RNA-mediated knockdown and adenovirus infection
Fibroblasts were transfected with target-specific short interfering RNAs (siRNA) (Dharmacon, Lafayette, CO) or scrambled control siRNA (10 nM). Twenty-four h following transfection, fresh media containing TGF-β (10 ng/ml) or Poly I:C (10 µg/ml) were added to the cultures, and the incubations were continued for a further 24 h. Knockdown efficiency was evaluated by determining levels of endogenous protein and mRNA by Western analysis and real-time qPCR.
Confocal immunofluorescence microscopy
Fibroblasts (10,000 cells/well) were seeded onto 8-well Lab-Tek II chamber glass slides (Nalge Nunc International, Naperville, IL) and incubated in serum-free EMEM with Poly I:C (10 µg/ml) or TGF-β2 (10 ng/ml) for 1–24 h. At the end of the experiments, cells were fixed, permeabilized, and incubated with primary antibodies to phospho-Smad3 at 1:200 dilution (Cell Signaling, Beverly, MA), TLR3 (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100 dilution or to Type I collagen at 1:100 dilution (Southern Biotech, Birmingham, AL). Cells were then washed with PBS and incubated with secondary antibodies at 1:200 dilution (Alexa Fluor 488 and 594, Invitrogen) and viewed under a Nikon C1Si confocal microscope.
Western analysis
At the end of each experiment, fibroblasts were harvested and whole cell lysates subjected to Western analysis as described (17). The following antibodies were used: phospho-Smad3 (Cell Signaling), Type I collagen (Southern Biotech), TLR3 (Santa Cruz Biotechnology), Smad7 (Abcam, Cambridge, MA) and GAPDH (Zymed, San Francisco, CA). Bands were visualized using ECL reagents (Pierce, Rockford, IL).
Enzyme-linked immunosorbent assays (ELISA)
Supernatants from confluent cultures of fibroblasts incubated for 24 h with Poly I:C were collected and stored at −80°C. Samples were thawed at room temperature and IFNβ levels were determined in triplicate using ELISA kits (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions.
Collagen gel contraction assays
To determine the effect of Poly I:C on fibroblast contractility, in vitro collagen gel contraction assays were performed (19). Briefly, fibroblasts (106 cells/well) were seeded in 12-well plates in DMEM with 10% FBS and Type I collagen (3.8 mg/ml, from BD Biosciences). Incubation for 60 min resulted in polymerization of the collagen gel. Cultures were then incubated with TGF-β (10 ng/ml) in the presence or absence of Poly I:C (10 µg/ml) for up to 6 days. Plates were photographed at indicated intervals, the extent of gel contraction was measured, and the percent of gel diameter in Poly I:C-treated compared to control gels was calculated. Each experiment was performed in triplicate.
Microarray procedures and data analysis
To examine gene expression changes induced by Poly I:C at the genome-wide scale, serum-starved confluent fibroblasts were incubated with Poly I:C (10 µg/ml) or media for up to 24 h. Total RNA was isolated from three independent cultures using RNeasy Mini Plus Kits (Qiagen, Valencia, CA). The integrity of RNA was determined using Agilent Bioanalyzer (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 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 down-regulation in Poly I:C-treated fibroblasts compared to control (p<0.05, FDR<0.05) were subjected for further analysis. Microarray data was deposited to GEO (accession number 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 Student t-test or ANOVA. The results are shown as the means ± SEM. p < 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 genomewide 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 inflammatory intrinsic subset were found to have significantly elevated TLR3 expression compared to those in the normal-like, limited and diffuse intrinsic subsets (p<0.001) (Fig. 1A). Expression of Stat1, an IFN-regulated transcription factor, also showed significantly elevated levels in these skin biopsies; moreover, Stat1 expression levels were strongly correlated with TLR3 levels (R=0.7, p<0.01) (Fig 1B).
Figure 1. Expression of TLR3 in skin.
A, B. Previously published microarray datasets representing 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 Stat1mRNA normalized with average expression levels across the entire cohort. C, D. Explanted normal skin fibroblasts were incubated in media with Poly I:C (10 µg/ml) for up to 24 h. C. Whole cell lysates were examined by Western analysis. Representative immunoblot. D. Fibroblasts were immunostained with antibodies to TLR3 (red) or stained with DAPI (blue). Representative immunofluorescence photomicrographs. Original magnification x60.
We next used explanted normal skin fibroblasts to evaluate TLR mRNA expression. Real-time qPCR showed that while mRNA transcripts for all ten TLRs were readily detected in these fibroblasts, TLR3 was the most highly expressed. Levels of TLR3 mRNA were 10-fold higher in fibroblasts compared to human macrophages or epithelial cells studied in parallel (data not shown). Western analysis and immunocytochemistry demonstrated that TLR3 was readily detected in explanted confluent fibroblasts, and its expression was markedly up-regulated in cultures incubated with the synthetic TLR3 ligand Poly I:C (Figs. 1C and D). Similar results were obtained with adult skin and lung fibroblasts (data not shown). Examination of skin fibroblasts explanted from scleroderma patients (n=4) showed no significant difference in TLR3 mRNA levels compared to healthy control fibroblasts (n=4) studies in parallel (data not shown).
Poly I:C inhibits collagen and α-smooth muscle actin gene expression via TLR3-independent pathways
Having demonstrated TLR3 expression in explanted fibroblasts, we next sought to examine the effects of ligands of TLR3 on modulating fibroblast function. For this purpose, confluent fibroblasts were incubated in media with Poly I:C for up to 24 h. Real-time qPCR, immunofluorescence and Western analysis demonstrated a time- and dose-dependent inhibition of Col1A1 and ASMA mRNA expression and protein levels, with a 60–80% reduction elicited by Poly I:C at 24 h (Fig. 2). In contrast, the expression of the inflammatory chemokine CXCL10 showed a dramatic increase, indicating fully functional TLR3 pathways in these cells. Moreover, Poly I:C induced potent and selective stimulation of Type I IFN, with a >10-fold increase in IFN-β mRNA levels, whereas IFN-α levels were not significantly changed. Comparable responses were seen in adult lung fibroblasts exposed to Poly I:C (data not shown).
Figure 2. Poly I:C represses collagen and α-smooth muscle actin gene expression.
Confluent skin fibroblasts were incubated in media with indicated concentrations of Poly I:C for 24 h. A, D. Total RNA was subjected to real-time qPCR. The results represent the means ± 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 antibodies to α-smooth muscle actin (green) or Type I collagen (red), or stained with DAPI (blue). Representative immunofluorescence photomicrographs. Original magnification x100.
To determine the role of TLR3 in mediating the inhibitory response elicited by Poly I:C, multiple complementary strategies were pursued. Surprisingly, this series of studies firmly indicated that the inhibitory responses elicited by Poly I:C occurred in a TLR3-independent manner (Fig. 3). Pretreatment of the fibroblasts with the endosomal TLR inhibitors chloroquine or bafilomycin (Fig. 3A and data not shown), RNAi-mediated knockdown of endogenous TLR3 (Fig. 3B and C), and peptide blockade of TRIF to inactivate TLR3 signaling (data not shown), each failed to abrogate the down-regulation of Col1A1 or ASMA elicited in fibroblasts by Poly I:C. In contrast, TLR3 inhibitors effectively blocked Poly I:C-induced stimulation of IL-6, as expected (data not shown).
Figure 3. Poly I:C inhibition of fibrotic gene expression is TLR3-independent.
A. Fibroblasts were pre-treated with chloroquine (10 µM) for 60 min, followed by Poly I:C (10 µg/ml) for a further 24 h, and RNA was analyzed by real-time qPCR. The results represent the means ± SEM of triplicate determinations. B, C. Fibroblasts were transiently transfected with indicated siRNA or scrambled control siRNA (siCTR) in parallel, and incubated in media with or without Poly I:C (10 µg/ml) for a further 24 h, followed by real-time qPCR. Results are the means ± SEM of triplicate determinations. *p<0.05, **p<0.001
To characterize the changes elicited by Poly I:C treatment of fibroblasts at the genome-wide scale, total RNA was isolated from foreskin fibroblasts incubated for 24 h with or without Poly I:C and hybridized to Illumina cDNA microarrays containing 44,000 probes. Incubation with Poly I:C induced broad changes in fibroblast gene expression. Approximately 700 transcripts showed >2-fold change (314 up and 363 down) compared to control cultures (p<0.05, FDR<0.05). The genes with the greatest increase or decrease in expression are shown in Tables 1 and 2; a complete list of Poly I:C-regulated genes is shown in Supplemental Table S1. Among the most highly up-regulated genes were those encoding IL-6, IL-8, chemokines (CXCL10, 11 and CCL5, 8), interferon response genes (OASL and OAS1), inflammatory signal transducers (IRF7 and CFB) and indoleamine 2,3-dioxygenase 1 (IDO1). Down-regulated genes included those encoding several ECM molecules (fibrillin 2, laminin and periostin). Gene Ontology (GO) analysis of the data revealed multiple biological functions that were significantly enriched with Poly I:C-regulated genes (Fig. 4A). Real-time qPCR of the same RNA preparations that were used for microarray analysis was performed to validate Poly I:C-induced changes in the expression of selected genes (Fig. 4B and data not shown). Of note, the expression of all three known Poly I:C receptors (TLR3, MDA5 and RIG-1) was up-regulated by Poly I:C.
Table 1.
Top 30 genes up-regulated 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 | 2'–5'-oligoadenylate synthetase-like | 83.3 |
| CCL8 | chemokine (C-C motif) ligand 8 | 78.5 |
| IDO1 | indoleamine 2,3-dioxygenase 1 | 76.3 |
| IFI27 | interferon, alpha-inducible protein 27 | 70.6 |
| TNFSF10 | tumor necrosis factor (ligand) superfamily, member 10 | 57.2 |
| IDO1 | indoleamine 2,3-dioxygenase 1 | 54.1 |
| GBP4 | guanylate binding protein 4 | 48.1 |
| IFIT3 | interferon-induced protein with tetratricopeptide repeats 3 |
45.6 |
| BST2 | bone marrow stromal cell antigen 2 | 43.4 |
| IL18BP | interleukin 18 binding protein | 43.4 |
| TNFSF13B | tumor necrosis factor (ligand) superfamily, member 13b | 43.1 |
| CFB | complement factor B | 41.4 |
| IFIT2 | interferon-induced protein with tetratricopeptide repeats 2 |
41.2 |
| OAS1 | 2',5'-oligoadenylate synthetase 1, 40/46kDa | 40.9 |
| HERC5 | hect domain and RLD 5 | 38.8 |
| OAS2 | 2'–5'-oligoadenylate synthetase 2, 69/71kDa | 37.7 |
| OAS1 | 2',5'-oligoadenylate synthetase 1, 40/46kDa | 36.9 |
| IFIT3 | interferon-induced protein with tetratricopeptide repeats 3 |
36 |
| ISG20 | interferon stimulated exonuclease gene 20kDa | 35.6 |
| OASL | 2'–5'-oligoadenylate synthetase-like | 33.8 |
| RSAD2 | radical S-adenosyl methionine domain containing 2 | 30.6 |
| TNFSF13B | tumor necrosis factor (ligand) superfamily, member 13b | 30.5 |
| CXCL11 | chemokine (C-X-C motif) ligand 11 | 29.6 |
| CD38 | CD38 molecule | 28 |
| IFI6 | interferon, alpha-inducible protein 6 | 27 |
| MX2 | myxovirus (influenza virus) resistance 2 (mouse) | 25.1 |
| IL6 | interleukin 6 (interferon, beta 2) | 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
Table 2.
Top 30 genes down-regulated by Poly I:C in fibroblasts.
| Symbol | Description | Fold change |
|---|---|---|
| TTC3 | tetratricopeptide repeat domain 3 | −7.9 |
| PSAT1 | phosphoserine aminotransferase 1 | −7.3 |
| VAT1 | vesicle amine transport protein 1 homolog (T. californica) |
−6.6 |
| GSTM2 | glutathione S-transferase mu 2 (muscle) | −6.0 |
| FBN2 | fibrillin 2 | −6.0 |
| SGCD | sarcoglycan, delta (35kDa dystrophin-associated glycoprotein) |
−6.0 |
| ACO1 | aconitase 1, soluble | −5.8 |
| C7orf41 | chromosome 7 open reading frame 41 | −5.6 |
| APPL2 | adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 |
−5.6 |
| PHGDH | phosphoglycerate dehydrogenase | −5.5 |
| IARS | isoleucyl-tRNA synthetase | −5.4 |
| FEZ1 | fasciculation and elongation protein zeta 1 (zygin I) | −5.2 |
| RCN2 | reticulocalbin 2, EF-hand calcium binding domain | −5.2 |
| NCRNA00188 | non-protein coding RNA 188 | −5.1 |
| EIF4B | eukaryotic translation initiation factor 4B | −5.1 |
| GSTM1 | glutathione S-transferase mu 1 | −5.1 |
| DDIT4 | DNA-damage-inducible transcript 4 | −5.1 |
| FAM171A1 | family with sequence similarity 171, member A1 | −5.0 |
| GALNT11 | UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase 11 (GalNAc-T11) |
−5.0 |
| KDELR3 | KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 3 |
−5.0 |
| PCYOX1 | prenylcysteine oxidase 1 | −4.9 |
| LAMA2 | laminin, alpha 2 | −4.8 |
| LTA4H | leukotriene A4 hydrolase | −4.8 |
| EIF4BP7 | eukaryotic translation initiation factor 4B pseudogene 7 | −4.8 |
| POSTN | periostin, osteoblast specific factor | −4.7 |
| PRKCA | protein kinase C, alpha | −4.7 |
| ASNS | asparagine synthetase (glutamine-hydrolyzing) | −4.6 |
| MFAP5 | microfibrillar associated protein 5 | −4.6 |
| FAM172A | family with sequence similarity 172, member A | −4.6 |
| MAGED1 | melanoma antigen family D, 1 | −4.6 |
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
Figure 4. Genome-wide gene expression changes induced by Poly I:C.
Fibroblasts were incubated in media with or without Poly I:C (10 µg/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/) (50). Of the 82 Gene Ontology (GO) categories with p<0.001 identified, the top 22 are shown (p-value from low to high). X-axis is the –log p value of each GO category. B. Real-time qPCR validation for selected Poly I:C-regulated genes. The results represent the means ± SEM of triplicate determinations from three independent experiments. C. Culture supernatants were assayed for IFN-β by ELISA. Results represent the means ± SEM of triplicate determinations.
Transcripts with known gene identifiers (HUGO gene symbols) and expression levels showing fold change > 2 (p < 0.05, FDR <0.05) were filtered from above, and were then entered into the Ingenuity Pathways Knowledge Base IPA to overlay into a global molecular network. Ingenuity Pathway Analysis of genes that were most significantly changed by Poly I:C implicated the IFN signaling pathway ( Supplemental Fig S1). Comparison of the Poly I:C-induced genes and the core IFN signature yielded 105 genes shared among the two sets (23) (Fig S2).
Poly I:C induces endogenous IFN signaling and IFN-mediated anti-fibrotic effects
The levels of IFN-β mRNA, and secretion of IFN-β, showed a dose-dependent stimulation in fibroblasts incubated with Poly I:C, as has been shown previously in inflammatory cells (Fig. 4). We therefore proceeded to examine the potential role of endogenous IFN-β in mediating the anti-fibrotic effect of Poly I:C. Incubation of fibroblasts with IFN-β for 24 h resulted in a time- and dose-dependent inhibition of collagen and ASMA gene expression, while at the same time a marked increase in TLR3 mRNA levels was seen (Fig. 5A, B and data not shown). Multiple complementary approaches to inhibit the IFN-β pathway were then taken to evaluate the possibility that the inhibitory effects of Poly I:C are mediated by endogenous IFN-β. The initial set of experiments evaluated Poly I:C responses in IFNAR1-null mouse embryonic fibroblasts (MEF). The results showed that in contrast to wild-type MEFs, in IFNAR1-null MEFs Poly I:C failed to repress collagen and ASMA gene expression (Fig. 5C and data not shown). Moreover, pretreatment of normal fibroblasts with LY294002, a PI3K inhibitor that blocks IFNβ1 induction (24), attenuated the anti-fibrotic effects of Poly I:C (Fig. 5D). Additional experiments using RNAi to knockdown cellular IFNAR1, and neutralizing IFNAR1antibodies to block IFNAR1 signaling, yielded comparable results (data not shown). Together, these genetic and pharmacological approaches firmly establish the indispensable role of endogenous type I IFN in mediating the anti-fibrotic effects of Poly I:C.
Figure 5. IFN-β inhibits fibrotic gene expression and mediates Poly I:C anti-fibrotic effects.
A, B. Foreskin fibroblasts were incubated with indicated concentrations of IFN-β for 24 h. A. Whole cell lysates were subjected to Western analysis. Representative autographs. B. RNA was subjected to real-time qPCR. The results represent the means ± SEM of triplicate determinations from three independent experiments. C. IFNAR1-null MEFs and wildtype MEF 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 ± SEM of triplicate determinations. D. Foreskin fibroblasts were pre-incubated with LY294002 (10 µM) for 30 min prior to incubation with Poly I:C (10 ng/ml) for 24 h. * p<0.05.
Poly I:C abrogates profibrotic responses induced by TGF-β
In light of the fundamental role of TGF-β in orchestrating fibrogenesis, it was of interest to evaluate the modulation of TGF-β responses by Poly I:C. For this purpose, fibroblasts were pretreated with Poly I:C followed by incubation with TGF-β for 24 h. The results of real-time qPCR and Western analysis showed that while Poly I:C caused a marked stimulation of CXCL10 and IFN-β mRNA expression both in the absence and presence of TGF-β, the stimulation of collagen and ASMA gene expression by TGF-β were abrogated by Poly I:C (Fig. 6). Additionally, Poly I:C potently inhibited the increase in collagen gel contraction induced by TGF-β. Furthermore, in contrast to wild-type MEFs, in IFNAR1-null MEFs Poly I:C was unable to suppress TGF-β-induced stimulation of collagen and ASMA gene expression, indicating a critical role for endogenous IFN in mediating Poly I:C antagonism for the TGF-β response (Fig. 6D).
Figure 6. Poly I:C abrogates profibrotic TGF-β responses.
A-C. Confluent skin fibroblasts were incubated with TGF-β (10 ng/ml) in the presence or absence of Poly I:C (10 µg/ml) for up to 48 h (A, C) or for 6 days (B). A. Total RNA was subjected to real-time qPCR. The results represent the means ± 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 ± SEM of triplicate determinations. * p<0.05. C. Whole cell lysates were subjected to Western analysis. Representative autoradiograms. D. IFNAR1-null MEFs or wild-type controls were pre-treated with Poly I:C (10 µg/ml) for 24 h, followed by TGF-β (10 ng/ml) for 24 h. RNA was isolated for real-time qPCR. The results represent the means ± 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 µg/ml) for a further 24 h prior to RNA isolation and real-time qPCR. * p<0.05.
It is now increasingly recognized that although Poly I:C serves as a potent TLR3 ligand, it can also elicit a variety of cellular responses via the ubiquitously expressed cytosolic receptors MDA5 and RIG-1 (25). Since we showed that the anti-fibrotic effect of Poly I:C appeared to be independent of TLR3, we used RNAi to investigate the role of the cytosolic Poly I:C receptors in mediating repression of profibrotic genes. The results from a series of experiments showed that significant and selective down-regulation of MDA5 or RIG-1 was achieved by transfection of fibroblasts with appropriate siRNAs (data not shown). While knockdown of neither MDA5 nor RIG-1 alone resulted in significant attenuation of the inhibitory effect of Poly I:C on TGF-β responses, simultaneous knockdown of both cytosolic receptors fully rescued the TGF-β stimulatory effect on of Col1A1 and ASMA gene expression in the presence of Poly I:C (Fig. 6E).
Poly I:C disrupts Smad2/3 activation via endogenous Type I IFN and inhibitory Smad7
In order to characterize the mechanisms underlying the inhibitory effect of Poly I:C on TGF-β responses, a series of experiments focusing on canonical Smad signaling were undertaken. Immunocytochemistry and transient transfection assays showed that Poly I:C markedly attenuated TGF-β-induced Smad3 phosphorylation, as well as stimulation of Smad-dependent transcription, in normal fibroblasts (Fig. 7). We therefore focused on Smad7, a TGF-β-inducible endogenous Smad inhibitor known to have a fundamental role in modulating the duration/intensity of fibrotic TGF-β-responses (26). Remarkably, we found that incubation of fibroblasts with Poly I:C by itself induced a substantial increase of Smad7 mRNA and protein (Fig. 8). Stimulation of Smad7 mRNA peaked between 30 and 90 min, with a magnitude that equaled and even exceeded the stimulatory response elicited by TGF-β. The induction of Smad7 by Poly I:C was not mediated through TLR3, since it was significantly (p<0.05) attenuated in fibroblasts with siRNA knockdown of RIG1 and MDA5, but not of TLR3 (Fig. 8C). Moreover, in the presence of TGF-β, Poly I:C induced a further increase in Smad7 levels. To examine the potential role of Smad7 in mediating the Poly I:C-dependent inhibition of fibrotic TGF-β responses, fibroblasts were transfected with a Smad7-specific siRNA. Knockdown of Smad7 substantially reduced the basal levels of endogenous Smad7, and attenuated induction by TGF-β and Poly I:C (Fig. 8D and data not shown). Significantly, Poly I:C in the presence of Smad7-specific siRNA was unable to abrogate the stimulation of collagen and ASMA gene expression induced by TGF-β. These results demonstrate that Poly I:C repression of the profibrotic TGF-β response was associated with disruption of canonical Smad signaling and involved the induction of antagonistic Smad7.
Figure 7. Poly I:C blocks canonical Smad2/3 signaling.
Skin fibroblasts (A, B) or NIH-3T3 mouse fibroblasts transiently transfected with [SBE]4-luc (C) were pre-treated with Poly I:C (10 µg/ml) for 24 h, followed by TGF-β (10 ng/ml) for 60 min (A, B) or 24 h (C). A. Cultures were fixed and immunostained with antibodies to phospho-Smad3 (green), Type I collagen (red), or DAPI (blue). Representative immunofluorescence photomicrographs. Original magnification x100. B. Whole cell lysates were subjected to Western analysis. Representative autoradiograms. C. Cell lysates were assayed for their luciferase activities. The results represent the means ± SEM of triplicate determinations.
Figure 8. Poly I:C abrogates profibrotic TGF-β responses through Smad7.
A. Fibroblasts were incubated with TGF-β (10 ng/ml) in the presence or absence of Poly I:C (10 µg/ml) for 24 h or indicated periods. A. Whole cell lysates were examined by Western analysis. Representative immunoblots. B. Total RNA was subjected to real-time qPCR. The results are the means ± 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 µg/ml) for 24 h. Total RNA was examined by real-time qPCR. Results are means ± SEM of triplicate determinations. *p<0.05, **p<0.001. F. Proposed mechanisms accounting for the TLR3-independent IFN-dependent anti-fibrotic activities of Poly I:C in fibroblasts. See text for explanation.
Discussion
Scleroderma is characterized by inflammation and fibrosis, with dermal fibroblasts serving as key effector cells of the latter process. The links between inflammation and fibrosis, and the nature of the inflammatory activation of dermal fibroblasts during the fibrotic process, remain incompletely understood. While TGF-β has long been implicated as a pivotal factor for initiating fibroblast activation, recent reports focus attention on the presence of a prominent IFN-responsive gene signature in lesional skin and peripheral blood cells from patients with scleroderma (27–29), as well as genetic associations of scleroderma with Stat4, IRF5 and other mediators of innate immune responses (30). Additional studies show that circulating IFN-inducible chemokines are elevated in scleroderma, and levels show strong correlation with indices of disease severity (31). Together, these recent observations suggest that innate immunity, pattern recognition receptor signaling and Type I IFN play important but poorly understood roles in initiating the fibrotic response in scleroderma.
Since the production of Type I IFN and the generation of the IFN gene signature are closely tied to molecular pattern sensing by TLR3 and cytosolic receptors, the present study sought to evaluate TLR3 expression in scleroderma tissues and normal fibroblasts, and the consequences of its activation in the context of fibrosis. The results demonstrate that TLR3 mRNA expression was elevated in a subset of scleroderma skin biopsies, and furthermore, TLR3 levels were positively correlated with STAT1 in these biopsies. Moreover, TLR3 was abundantly expressed and functionally competent in explanted fibroblasts. Poly I:C, a double-stranded RNA analogue, was used as a ligand to examine the effect of TLR3 activation in vitro. Incubation of normal skin and lung fibroblasts with Poly I:C elicited stereotypic immunostimulatory responses with secretion of the inflammatory mediators CXCL10, CCL5 and CCL8, and production of OAS and IDO, accompanied by marked up-regulation of Type I IFN. Remarkably, however, we simultaneously observed profound down-regulation of fibrotic gene expression in fibroblasts incubated with Poly I:C. Even more significantly, these fibroblasts failed to mount fibrotic response when challenged by TGF-β. We therefore conclude that in fibroblasts TLR3 ligands cause concurrent inflammatory response induction and fibrotic response blockade.
Mechanistic studies revealed that despite the presence of TLR3 and a fully intact TLR3 signaling pathway in fibroblasts, the anti-fibrotic effects exerted by Poly I:C in these cells were largely independent of TLR3, and instead appeared to be mediated by cytosolic pattern recognition receptors RIG-1 and MDA5. The anti-fibrotic response could be recapitulated by Type I IFN, which was sufficient, and required for the Poly I:C effect. Taken together, these findings therefore support a mechanistic model to explain the inflammatory regulation of fibroblast function in fibrosis, where endogenous Type I IFN production elicited by a bona fide TLR3 ligand establishes a local negative autocrine/paracrine feedback loop with consequent inhibition of fibroblast function and abrogation of profibrotic responses. The results therefore imply that the prominent Type I IFN signature described in scleroderma lesional tissues might in fact reflect DAMP-induced in situ activation of fibroblast innate immune responses during fibrogenesis that are aimed toward restraining fibroblast activation and limiting progressive fibrosis. Moreover, our findings provide the first evidence that double-stranded RNA has potent anti-fibrotic activity and might potentially represent an innovative approach to therapy.
Poly I:C has long been recognized as a potent inducer of IFN production and signaling (32), a finding reproduced in the present results. TLR3 uniquely senses, and responds to, extracellular RNA duplexes as well as synthetic Poly I:C. In rodent models, Poly I:C has been shown to induce inflammation and accelerate autoimmunity through mechanisms that are both TLR3-dependent and -independent (9, 33). Lupus nephritis in the NZW/B mice was exacerbated by Poly I:C, and the inflammatory effects were mediated by endogenous IFN, and accumulation of alternately activated macrophages in the kidney (33). In contrast, Poly I:C can also down-regulate inflammation in murine arthritis (34) and other models of inflammation (35). Interestingly, Poly I:C-induced inflammation is generally associated with only a modest degree of fibrosis. For instance, in the murine lupus nephritis model, the kidneys of Poly I:C-injected NZW/B mice showed only scant collagen accumulation despite extensive inflammation and crescent formation. The modest-to-absent fibrotic response in this and other Poly I:C-induced inflammatory disease models is congruent with the hypothesis that while Poly I:C caused inflammation, enhanced IFN production and accumulation of alternately activated macrophages associated with tissue remodeling and scar formation, its direct effects at the tissue level resulted in attenuated fibroblast activation and fibrotic signaling.
Earlier studies focusing on the modulation of fibrosis by Poly I:C have yielded conflicting results. It was shown that injection of Poly I:C ameliorate lung and liver fibrosis in rodents (12, 36–38). The mechanism for these in vivo anti-fibrotic effects has not been clarified. In contrast, Farina et al. recently showed that PolyI:C delivered by continuous subcutaneous infusion using implanted osmotic pumps induced epidermal hyperplasia, inflammatory cell accumulation in the deep dermis and fat, and increased matrix deposition (13). Moreover, the expression of the TGF-β-inducible genes PAI-1 and COMP was elevated in the lesional skin. Additional experiments showed that the cutaneous inflammatory response induced by Poly I:C was dependent on both TLR3 and Type I IFN signaling. The authors concluded that chronic TLR3 stimulation can induce cutaneous fibrosis and might play a role in the pathogenesis of scleroderma. It has been pointed out that Poly I:C elicits markedly different responses and profiles of cytokines in murine and human cells (39). This differential effect might account for the profibrotic Poly I:C responses observed by Farina et al. in the mouse (13), and the marked anti-fibrotic responses in human fibroblasts described in the present report. A potential explanation for these seemingly conflicting observations regarding the pro- or anti-fibrotic effects of Poly I:C might lie in Poly I:C’s dual effect: inducing potent systemic inflammation, mediated in part through endogenous Type I IFN and accumulation of alternately activated macrophages in target tissues; and concurrent down-regulation of fibrotic gene expression in lesional fibroblasts, and abrogation of TGF-β-mediated stimulation of fibrotic programs in affected tissues. The net result of these coupled proinflammatory and anti-fibrotic responses then might be attenuation of scar formation and organ fibrosis. With persistence of injury and sustained activation of pattern recognition receptors by endogenous RNA ligands of TLR3, the proinflammatory responses might come to dominate.
In common with other double-stranded RNA moieties, Poly I:C serves as a surrogate for RNA released during tissue injury and is recognized as a damage-associated endogenous TLR3 ligand (8). Our results, consistent with other studies, indicate that TLR3 is expressed on explanted skin and lung fibroblasts, and its levels are elevated in skin biopsies from patients with scleroderma (13, 40). Because of its ability to respond to RNA released from damaged or necrotic cells, TLR3 has been proposed to serve as an endogenous sensor of tissue necrosis during inflammatory states, and plays an important role in amplifying sterile inflammatory responses (41). However, Poly I:C triggers activation of multiple pattern recognition receptors, including RIG-1 and MDA5, and under certain conditions the TLR3-independent cellular response predominates. For example, inhibition of VEGF-mediated responses in transgenic mice by Poly I:C involved a TLR3-independent RIG-1 recognition mechanism (35). Consistent with these observations, we demonstrate that the anti-fibrotic effects elicited by Poly I:C in fibroblasts are largely TLR3-independent and are mediated through both RIG-1 and MDA5.
Poly I:C induced robust stimulation of Type I IFN in normal fibroblasts. Importantly, we used multiple complementary strategies to establish that endogenous Type I IFN plays an essential role of in mediating the anti-fibrotic effects of Poly I:C. The results indicated that Poly I:C response was absent in IFNAR1-null fibroblasts, and could be abrogated by antibodies to IFNAR1, as well as RNAi knockdown of the receptor, or by a pharmacological inhibitor of Type IFN production. IFN is well known to be a potent inhibitor of collagen gene expression, and has been shown to attenuate a broad range of fibrotic responses in both normal and scleroderma fibroblasts (42–44). We and others have further demonstrated that IFN blocks Smad-dependent TGF-β responses (45). The present results indicate that Poly I:C blocked canonical Smad signaling, and this effect was mediated, at least in part, through induction of antagonist Smad7. This inducible Smad blocks canonical TGF-β signaling by preventing Smad3 interaction with TGF-β receptors, and its up-regulation has been previously shown to abrogate the stimulation of collagen (26). Furthermore, like Poly I:C, both Type I and Type II IFN have been previously shown to induce Smad7, which may account for their inhibitory effects of fibrotic responses (46–49). The mechanism accounting for Smad7 induction by Poly I:C is likely to involve NF-kappa β or Stat1 (46, 47).
Taken together, the present results demonstrate for the first time that synthetic RNA, a known ligand for TLR3 as well as the cytosolic receptors RIG1 and MDA5, acts as a potent and bifunctional stimulus for fibroblast activation, simultaneously inducing dramatic up-regulation of inflammatory mediators including chemokines and Type I IFN, coupled with suppression of TGF-β signaling and inhibition of fibrotic responses. Because double-stranded RNA is released from damaged or dying cells and its levels in situ are likely to be elevated in tissue injury and fibrosis, the present results using Poly I:C as a surrogate for endogenous TLR3 ligands are relevant for understanding inflammatory fibroblast regulation during fibrogenesis. As expected Poly I:C was a potent inducer of Type I IFN production in fibroblasts, and Type I IFN in turn was required for, and sufficient to recapitulate, the anti-fibrotic effects elicited by Poly I:C. The inhibitory effects of Type I IFN appear to be mediated through induction of Smad7, which blocks canonical Smad signaling (Fig. 8). Taken together, these results suggest that the IFN gene signature in scleroderma patients might be an indication of an on-going DAMP-induced innate immune response designed to restrain and control TGF-β-induced fibroblast activation. In this scenario, Type I IFN signaling in the lesional skin might play a beneficial role, and therapies aimed at enhance locally the response, rather than block it, might in fact be desirable for the treatment of fibrosis. Accordingly, Poly I:C therapy targeting the fibrotic lesion represents a potential treatment strategy in scleroderma.
Supplementary Material
ACKNOWLEDGEMENTS
We are grateful to Dr Michael Whitfield (Dartmouth College) and to members of the Varga lab for helpful discussions.
Financial support
Supported by a grant from the NIH (AR-42309).
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