FibronectinEDA promotes chronic cutaneous fibrosis through Toll-like receptor signaling

Abstract

Scleroderma is a progressive autoimmune disease affecting multiple organs. Fibrosis, the hallmark of scleroderma, represents transformation of self-limited wound healing into a deregulated self-sustaining process. The factors responsible for maintaining persistent fibroblast activation in scleroderma and other conditions with chronic fibrosis are not well understood. Toll-like receptor 4 (TLR4) and its damage-associated endogenous ligands are implicated in immune and fibrotic responses. We now show that fibronectin extra domain A (Fn(EDA)) is an endogenous TLR4 ligand markedly elevated in the circulation and lesional skin biopsies from patients with scleroderma, as well as in mice with experimentally induced cutaneous fibrosis. Synthesis of Fn(EDA) was preferentially stimulated by transforming growth factor-β in normal fibroblasts and was constitutively up-regulated in scleroderma fibroblasts. Exogenous Fn(EDA) was a potent stimulus for collagen production, myofibroblast differentiation, and wound healing in vitro and increased the mechanical stiffness of human organotypic skin equivalents. Each of these profibrotic Fn(EDA) responses was abrogated by genetic, RNA interference, or pharmacological disruption of TLR4 signaling. Moreover, either genetic loss of Fn(EDA) or TLR4 blockade using a small molecule mitigated experimentally induced cutaneous fibrosis in mice. These observations implicate the Fn(EDA)-TLR4 axis in cutaneous fibrosis and suggest a paradigm in which aberrant Fn(EDA) accumulation in the fibrotic milieu drives sustained fibroblast activation via TLR4. This model explains how a damage-associated endogenous TLR4 ligand might contribute to converting self-limited tissue repair responses into intractable fibrogenesis in chronic conditions such as scleroderma. Disrupting sustained TLR4 signaling therefore represents a potential strategy for the treatment of fibrosis in scleroderma.

Fig. 1. Fn^EDA is elevated in scleroderma, and its expression is stimulated by TGF-β

(A) Fn^EDA serum levels in scleroderma patients (n = 48) and healthy adults (n = 16) were determined by enzyme-linked immunosorbent assay (ELISA). Each data point is the mean ± SD of trip-licate determinations from a single subject. *P < 0.0001, Mann-Whitney U test. (B) RNA isolated from skin biopsies from scleroderma patients (n = 20) and healthy adults (n = 8) was analyzed by real-time qPCR. Results, expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are the means ± SD of duplicate determinations. *P = 0.0002, Mann-Whitney U test. (C) Immunofluorescence analysis. Skin biopsies from scleroderma patients (n = 13) and healthy controls (n = 6) in parallel were immunostained with antibodies to Fn^EDA and examined by immunofluorescence confocal microscopy. Upper panels: Representative images. Scale bar, 25 μm. Lower panels: 3D plots of fluorescence intensity (see Table 3). P = 0.0159, Mann-Whitney U test. (D and E) Confluent human skin fibroblasts were incubated for 24 hours with TGF-β (1 and 10 ng/ml) alone (D) or in the presence or absence of SB431542 or U0126 (E). (D) Upper panel: Levels of mRNA were determined by real-time quantitative PCR. Results, normalized with GAPDH, are means ± SD of triplicate determinations. *P < 0.0001, one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. Lower panel: Whole-cell lysates were subjected to Western analysis. Representative immunoblots.

Fig. 2. Skin fibrosis is attenuated in mice lacking Fn^EDA

Fn^EDA-null mice and wild-type (WT) mice in parallel received daily subcutaneous injections of phosphate-buffered saline (PBS) or bleomycin (Bleo) for 14 days and sacrificed at day 24 (A to C), or two subcutaneous injections of AdTGF-β1 or empty vector 14 days apart and sacrificed at 28 days (D). Lesional skin was harvested for analysis. (A) Left panel: Masson's trichrome stain. Representative images. Arrows indicate dermis. Scale bar, 100 μm. Right panel: Dermal thickness (distance from dermal-epidermal junction to adipose layer), shown as the means ± SD of triplicate determinations per hpf from five mice per group. Ovals, PBS; inverted triangles, bleomycin. *P < 0.0001, PBS versus bleomycin; P < 0.001, WT versus Fn^EDA-null bleomycin, one-way ANOVA followed by Bonferroni's multiple comparison test. (B) mRNA levels were determined by real-time quantitative PCR. The results, normalized with GAPDH, represent the means ± SD of triplicate determinations from four mice per group. *P = 0.014, Mann-Whitney U test. (C) Immunofluorescence using antibodies against αSMA (green) and 4′,6′-diamidino-2-phenylindole (DAPI) (blue). Left panel: Representative images. Scale bar, 50 μm. Red arrows indicate αSMA-positive cells in the dermis. Right panel: The proportion of αSMA-immunopositive cells in the lesional dermis was determined in five randomly selected hpf per slide from four mice per group. Open bars, PBS; closed bars, bleomycin. PBS versus bleomycin. *P < 0.0001, WT bleomycin versus null bleomycin; P < 0.001, one-way ANOVA followed by Bonferroni's multiple comparison test. (D) Masson's trichrome stain. Left panel: Representative images. Scale bar, 100 μm. Right panel: Dermal thickness, shown as the means ± SD of triplicate determinations per hpf from four mice per group. Open bars, PBS; closed bars, AdTGF-β1. *P = 0.02, PBS versus bleomycin; P = 0.03, WT versus Fn^EDA-null bleomycin, Sidak's multiple comparison test.

Fig. 3. Fn^EDA elicits profibrotic responses in normal fibroblasts

(A to C) Normal skin fibroblasts were incubated in medium with Fn^EDA (5 and 10 μg/ml) for 72 hours. (A) Left panel: mRNA levels were determined by real-time qPCR. Results, normalized with GAPDH, are the means ± SD of triplicate determinations from three independent experiments (P < 0.01, one-way ANOVA followed by Bonferroni's multiple comparison test). Open bars, COL1A1; closed bars, αSMA. Right panel: Whole-cell lysates were examined by Western analysis. Representative immunoblots. Cgn I, type I collagen. (B) Left panel: Representative immunoblots. Values below indicate fold induction (means from three independent experiments corrected for tubulin in each lane). Right panel: Slides were immunostained with antibodies to αSMA (green). Nuclei are identified by DAPI (blue). Representative images. Scale bar, 50 μm. Values indicate fold induction (means from three independent experiments). (C) Left panel: Fibroblasts were seeded on plates coated with Fn^EDA or left untreated, and at confluence, scratch wounds were created. Fibroblast migration was determined by measuring gap width at 24 and 48 hours. Results are means ± SD of triplicate determinations at three randomly selected locations from triplicate determinations (at 24 hours, P = 0.0043; at 48 hours, P = 0.0450, Mann-Whitney U test). Right panel: Collagen gel contraction assays. Fibroblasts were seeded in type I collagen gels and were incubated in medium in the presence or absence of TGF-β and Fn^EDA (10 μg/ml). At 6 and 24 hours, gel diameters were determined. Results, expressed as percentage of gel area compared to controls (time 0), are the means ± SD of triplicate determinations (at 24 hours: control versus TGF-β, P < 0.0001; control versus Fn^EDA, P < 0.0001, one-way ANOVA followed by Bonferroni's multiple comparison test). (D) 3D human skin equivalents constructed without or with Fn^EDA (10 μg/ml) were seeded with fibroblasts. After 18 days, rafts were stained with Picrosirius Red. Left panel: Representative images. Scale bars, 25 μm (a and b) or 10 μm (c and d). Right panel: Immunofluorescence intensity. Bars represent the means ± SD from six randomly selected hpf per raft from three independent raft experiments (P = 0.022, Mann-Whitney U test). (E) Total protein from dermal compartments was analyzed. Representative Western blot. Numbers below indicate relative band intensities (means from two independent experiments corrected for GAPDH in each lane). (F) Stiffness of the dermal compartment was determined as described in Materials and Methods. The results represent the means ± SD from three independent rafts (P < 0.0001, control versus Fn^EDA low; P < 0.0001, control versus Fn^EDA high, oneway ANOVA followed by Bonferroni's multiple comparison test).

Fig. 4. Fn^EDA elicits TLR4-dependent fibroblast responses

(A) Fn^EDA interacts directly with TLR4. Whole-cell lysates (WCL) from normal skin fibroblasts incubated in the presence or absence of TGF-β for 24 hours were immunoprecipitated (IP) with antibodies against TLR4 or Fn^EDA or IgG and immuno-blotted using antibodies to Fn^EDA or TLR4. Representative immunoblots. (B) Fibroblasts transiently transfected with NF-κB-luc were incubated in medium with endotoxin-stripped Fn^EDA in the presence or absence of neutralizing antibodies to Fn^EDA (anti-EDA) for 24 hours. Whole-cell lysates were assayed for their luciferase activities. Results are means ± SD from three independent experiments. *P = 0.002, control versus Fn^EDA; *P = 0.04, Fn^EDA versus anti-EDA–treated, Sidak's multiple comparison test. ns, not significant. (C to E) Human skin fibroblasts (C and D) or skin fibroblasts (E) from TLR4-mutant and WT mouse were incubated in medium with Fn^EDA (10 μg/ml) in the presence or absence of CLI-095 for 72 hours. (C) Left panels: Whole-cell lysates were subjected to Western analysis. Numbers below indicate relative band intensities corrected for tubulin (means from three independent experiments). Right panels: Fibroblasts were immunostained with antibodies to αSMA. Representative immunofluorescence confocal images. Scale bar, 50 μm. Numbers represent fluorescence intensity determined in four randomly selected locations per hpf for each sample (means from three independent experiments). (D) Human skin fibroblasts were transfected with TLR4-specific siRNA or scrambled (Scr) siRNA, followed by incubation in medium with Fn^EDA for 72 hours. Left upper panels: mRNA levels were determined by real-time qPCR. Results, normalized with GAPDH mRNA, are means ± SD of triplicate determinations from an experiment representative of two independent experiments. Left lower panels: Immunofluorescence using antibodies to αSMA (red). Representative images. Scale bar, 50 μm. Right panels: Whole-cell lysates were examined by Western analysis. Representative immunoblots. (E) Skin fibroblasts from WT and TLR4-mutant mice in parallel were incubated in medium with Fn^EDA for 72 hours. Whole-cell lysates were examined by Western analysis. Representative immunoblots. Numbers below indicate relative band intensities corrected for tubulin (means from two independent experiments).

Fig. 5. Pharmacological TLR4 blockade both prevents and reverses bleomycin-induced skin fibrosis

(A to D) C57BL/6J mice received daily subcutaneous injections of bleomycin or PBS. Mice also received CLI-095 (2 mg/kg) starting on day 0 (A and B) or on day 15 (C and D) and sacrificed at day 24 (A and B) or day 28 (C and D), and lesional skin was harvested for analysis. (A and C) Masson's trichrome stain. Representative images (left panels). Scale bars, 100 μm. Right panel: Dermal thickness, determined in four random locations per hpf, is shown as means ± SD from five mice per group. Ovals, PBS; inverted triangles, bleomycin. *P < 0.0001, PBS versus bleomycin; *P < 0.001, bleomycin versus CLI-095, one-way ANOVA followed by Bonferroni's multiple comparison test for (A). *P = 0.0005, PBS versus bleomycin; *P = 0.007, bleomycin versus CLI-095, Sidak's multiple comparison test for (C). (B and D) Levels of mRNA were determined by real-time qPCR. The results, normalized for GAPDH, represent the means ± SD of triplicate determinations from four (A and B) or five (C and D) mice per group. *P = 0.002, WT versus bleomycin; *P = 0.014, bleomycin versus bleomycin + CLI-095, one-way ANOVA followed by Bonferroni's multiple comparison test. (E) Cartoon depicting contribution of TLR4-mediated fibroblast activation elicited by Fn^EDA to persistent fibrogenesis. By triggering fibroblast TLR4 signaling, Fn^EDA serves as a switch converting self-limited tissue repair into sustained fibrogenesis. Persistent injury leads to fibroblast activation with generation and extracellular accumulation of Fn^EDA that in turn triggers TLR4-dependent cellular signaling, resulting in sustained fibroblast activation with production of Fn^EDA and other ECM molecules. A self-amplifying vicious cycle of fibrogenesis ensues.