Toll-like receptor 4 signaling augments transforming growth factor-β responses: a novel mechanism for maintaining and amplifying fibrosis in scleroderma

Abstract

Because recent studies implicate Toll-like receptors (TLRs) in the pathogenesis of fibrosis, we sought to investigate the in vitro and in vivo role and mechanism of TLR4-mediated fibroblast responses in fibrogenesis. We found that TLR4 was constitutively expressed, and accumulation of endogenous TLR4 ligands significantly elevated, in lesional skin and lung tissues from patients with scleroderma. Activation of TLR4 signaling in explanted fibroblasts resulted in enhanced collagen synthesis and increased expression of multiple genes involved in tissue remodeling and extracellular matrix homeostasis. Moreover, TLR4 dramatically enhanced the sensitivity of fibroblasts to the stimulatory effect of transforming growth factor-β1. These profibrotic responses were abrogated by both genetic and pharmacological disruption of TLR4 signaling in vitro, and skin fibrosis induced by bleomycin in vivo was attenuated in mice harboring a mutated TLR4. Activation of TLR4 in fibroblasts augmented the intensity of canonical Smad signaling, and was accompanied by suppression of anti-fibrotic microRNA expression. Together, these results suggest a novel model to account for persistent fibrogenesis in scleroderma, in which activation of fibroblast TLR4 signaling, triggered by damage-associated endogenous TLR4 ligands, results in augmented transforming growth factor-β1 sensitivity with increased matrix production and progressive connective tissue remodeling. Under these conditions, fibroblast TLR4 serves as the switch for converting self-limited tissue repair into intractable fibrosis.

Figure 1

Elevated TLR4 expression in scleroderma skin. A: Skin biopsy specimens from the lesional forearm of patients with scleroderma (n = 19) and forearms of healthy controls (n = 8) were examined by IHC. Left panels: Representative images. Brown staining indicates TLR4-positive cells. Scale bars: 200 μm (top panels); 50 μm (bottom panels). Blue, hematoxylin counterstain. Right panel: Quantitation of dermal cell TLR4 staining. Each dot, number (mean) of immune-positive spindle-shaped interstitial cells (fibroblast-like) from four separate microscopic fields per biopsy. B: Double-immunofluorescence labeling using antibodies to TLR4 and CD31 or α-SMA, or stained with DAPI. Yellow, colocalization of two antibodies. Arrows, colocalization. Scale bar = 50 μm. C: Cell lysates from explanted skin fibroblasts from healthy controls (n = 3) and patients with scleroderma (n = 4) were subjected to Western blot analysis using antibodies against TLR4 and type I collagen (Cgn I). Left panel: Representative immunoblots. Right panel: Quantitation of TLR4 levels. Values represent the mean ± SD, corrected for tubulin in each lane. D: Explanted healthy control (n = 3) and scleroderma (n = 3) fibroblasts were incubated with CLI-095 (3 μmol/L) for 24 hours. RNA and protein were isolated and subjected to qPCR. Left panel: Mean ± SD of triplicate determinations relative to levels in untreated fibroblasts. Right panel: Western blot analysis. Values shown indicate relative levels corrected for tubulin in each lane. E: Immunofluorescence. Antibodies to Fn-EDA or tenascin-C, or HA-binding protein, were used on skin biopsy specimens from healthy controls (n = 3) and patients with scleroderma (n = 5). Scale bar = 50 μm. *P < 0.05.

Figure 2

Elevated TLR4 expression in scleroderma lungs. A: IHC of lung biopsy specimens from patients with scleroderma and pulmonary fibrosis (n = 4) and normal donor lungs (n = 4). Left panels: Brown staining indicates TLR4-positive cells. Blue, hematoxylin counterstain. Scale bars: 200 μm (top panels); 50 μm (bottom panels). Right panel: Quantitation of TLR4 staining. Each dot, number (mean) of immunopositive fibroblasts from four separate microscopic fields per biopsy. B: Red, immunofluorescence using antibodies to TLR4; green, α-SMA; yellow, colocalization of the two antibodies (white arrows). Mouse isotype antibodies were used as a negative control, and a Hoechst stain was used to detect nuclei. Scale bars: 200 μm (top panels); 50 μm (bottom panels). *P < 0.05.

Figure 3

LPS sensitizes fibroblasts to TGF-β1 stimulation of profibrotic gene expression. Fibroblasts were incubated with TGF-β1 (10 ng/mL) in the presence or absence of LPS for 24 hours. A: Total RNA was examined by qPCR. Results, normalized with GAPDH mRNA, are shown as the mean ± SD of triplicate determinations. B: Whole cell lysates and culture supernatants were subjected to Western blot analysis. Representative immunoblots are shown. C: Fibroblasts were immunostained with antibodies to type I collagen (Cgn I) or α-SMA or stained with DAPI, and viewed by immunofluorescence microscopy (top panels) or laser-scanning confocal microscopy (bottom panels). Original magnification, ×400. D: Right panel: Mean immunofluorescence intensity of individual fibroblasts stained with α-SMA was determined in five different microscopic fields. Results are given as the mean ± SD. *P < 0.05.

Figure 4

TLR4 stimulates collagen gene expression. Fibroblasts were transiently transfected with constitutively active TLR4 (CD4TLR4) or inactive TLR4 (P-H CD4TLR4), 772COL1A2-luc, or empty vector and incubated with TGF-β1 for 24 hours. A: Total RNA was examined by qPCR. Results, normalized with GAPDH, are the mean ± SD of triplicate determinations. Inset: Secreted type I collagen (Cgn I). Whole cell lysates were assayed for their luciferase activities (B) or examined by using Western blot analysis (C). The results of luciferase assays are normalized with Renilla luciferase. mut, mutant.

Figure 5

LPS synergy with TGF-β1 is TLR4 dependent. A: Normal skin fibroblasts were incubated with LPS and TGF-β1 in the absence or presence of TLR4 inhibitors. Total RNA was examined by qPCR. B: Whole cell lysates and secreted proteins in the culture media were subjected to Western blot analysis. Type I collagen (Cgn I) protein levels were quantitated. Values shown indicate relative levels corrected for GAPDH in each lane. C: Normal skin fibroblasts were transfected with TLR4-specific siRNA or scrambled (Scr) control siRNA and incubated with LPS and TGF-β1 for 24 hours. Whole cell lysates were examined by using Western blot analysis. Representative immunoblots. Cgn I levels were quantitated. Values shown indicate relative levels corrected for tubulin in each lane. D and E: Skin fibroblasts from WT mice and TLR4-mutant (C3H/HeJ) mice were incubated in parallel with LPS and TGF-β1 for 24 hours. Whole cell lysates were subjected to Western blot analysis (D). Fibroblasts were fixed, incubated with antibodies to α-SMA, and examined by immunofluorescence microscopy (E). Blue, nuclei were identified by DAPI. Original magnification, ×100. *P < 0.05.

Figure 6

TLR4 mediates fibrotic responses via enhanced Smad signaling. A: Normal skin fibroblasts transfected with TLR4-specific siRNA or scrambled (Scr) control siRNA were incubated in media with LPS and TGF-β1 for 24 hours. Smad2/3 expression and phosphorylation were examined by using Western blot analysis. Representative immunoblots are shown. Human (B) or WT (C3H/HeOuJ) and TLR4-mutant (C3H/HeJ) mouse skin fibroblasts (C) were transfected with [SBE]₄-luc in the presence of LPS and TGF-β1 for 24 hours, and cell lysates were assayed for their luciferase activities, normalized with Renilla luciferase. Results, normalized with GAPDH, are shown as the mean ± SD from triplicate determinations.

Figure 7

TLR4 down-regulates antifibrotic miR-29. A: miRNA was isolated and subjected to qPCR analysis. Results, expressed relative to RNU24, are the mean ± SD of triplicate determinations. B: Normal skin fibroblasts were transfected with pre-miR-29b and incubated with LPS and TGF-β1 for 24 hours. Cell lysates were examined by using Western blot analysis. Representative immunoblots are shown. Cgn I, type I collagen.

Figure 8

Attenuated skin fibrosis in TLR4-mutant mice. C3H/HeJ and WT mice, in parallel, received daily s.c. injections of PBS or bleomycin (Bleo) for up to 21 days. Mice were sacrificed, and lesional skin was examined. A: Left panel: H&E stain. Scale bar = 200 μm. Middle panel: Masson’s Trichrome stain. Scale bar = 200 μm. Right panel: Dermal thickness. Results are the mean ± SD of triplicate determination from five mice per group. Open bars, PBS; closed bars, Bleo. B: Picrosirius red stain, viewed under polarized light. Original magnification, ×100. C: Immunofluorescence images are shown. Red arrows, α-SMA–positive cells. *P < 0.05. Original magnification, ×400.

Figure 9

Reduced fibrotic gene expression in TLR4-mutant mice. C3H/HeJ and WT mice received daily s.c. injections of PBS or bleomycin (Bleo) for 21 days. Lesional skin was harvested and analyzed. A: RNA was quantitated by qPCR. The results, normalized for 36B4, represent the mean ± SD of triplicate determinations from four mice per group. Open bars, PBS; closed bars, Bleo. B: Collagen content determined by colorimetric assays. Results represent the mean ± SD of triplicate determinations from at least three mice per group. Open bars, PBS; closed bars, Bleo. C: Protein was examined by using Western blot analysis. Cgn I, type I collagen. *P < 0.05.

Figure 10

A: Bleomycin (Bleo)-induced skin fibrosis is associated with accumulation of endogenous TLR4 ligands. TLR4-mutant mice and WT mice received daily s.c. injections of PBS or Bleo for 21 days. Lesional skin was harvested and examined by immunofluorescence. Representative images are shown. Original magnification, ×400. B: Cycle of fibrogenesis. Innate immune signaling via TLR4 switches self-limited repair into sustained fibrogenesis. Prolonged injury leads to tissue damage, and generation of endogenous TLR4 ligands, with activation of TLR4 and sustained fibroblast activation.