Wenxia Wang

FibronectinEDA Promotes Chronic Cutaneous Fibrosis Through Toll-Like Receptor Signaling

FibronectinEDA is an endogenous TLR4 ligand in scleroderma. Scleroderma Takes Its Toll Scleroderma is a disease where the immune system attacks the connective tissues of the body, with notable hardening of the skin and fibrosis in multiple organs. However, what causes scleroderma—and the associated persistent fibrosis activation—remains unknown. Bhattacharyya et al. now report that the fibronectin extra domain A (FnEDA)—an endogenous damage-induced TLR4 ligand—may contribute to cutaneous fibrosis. The authors found that FnEDA is elevated in lesions and circulation both of patients with scleroderma and of a mouse model of fibrosis. FnEDA was up-regulated by TGF-β in healthy fibroblasts. In vitro, FnEDA increased mechanical stiffness of human skin equivalents. Indeed, the profibrotic responses induced by FnEDA were dependent on TLR4 signaling and could be blocked by genetic, RNA interference, and pharmacologic TLR4 inhibition. These data suggest that a damage-induced TLR4 signaling may contribute to fibrogenesis in scleroderma. 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 (FnEDA) 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 FnEDA was preferentially stimulated by transforming growth factor–β in normal fibroblasts and was constitutively up-regulated in scleroderma fibroblasts. Exogenous FnEDA 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 FnEDA responses was abrogated by genetic, RNA interference, or pharmacological disruption of TLR4 signaling. Moreover, either genetic loss of FnEDA or TLR4 blockade using a small molecule mitigated experimentally induced cutaneous fibrosis in mice. These observations implicate the FnEDA-TLR4 axis in cutaneous fibrosis and suggest a paradigm in which aberrant FnEDA 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.

A synthetic PPAR-γ agonist triterpenoid ameliorates experimental fibrosis: PPAR-γ-independent suppression of fibrotic responses

Background Persistent fibroblast activation initiated by transforming growth factor β (TGF-β) is a fundamental event in the pathogenesis of systemic sclerosis, and its pharmacological inhibition represents a potential therapeutic strategy. The nuclear receptor, peroxisome proliferator-activated receptor γ (PPAR-γ), exerts potent fibrotic activity. The synthetic oleanane triterpenoid, 2-cyano-3,12-dioxo-olean-1,9-dien-28-oic acid (CDDO), is a PPAR-γ agonist with potential effects on TGF-β signalling and dermal fibrosis. Objective To examine the modulation of fibrogenesis by CDDO in explanted fibroblasts, skin organ cultures and murine models of scleroderma. Material and methods The effects of CDDO on experimental fibrosis induced by bleomycin injection or by overexpression of constitutively active type I TGF-β receptor (TgfbR1ca) were evaluated. Modulation of fibrotic gene expression was examined in human skin organ cultures. To delineate the mechanisms underlying the antifibrotic effects of CDDO, explanted skin fibroblasts cultured in two-dimensional monolayers or in three-dimensional full-thickness human skin equivalents were studied. Results CDDO significantly ameliorated dermal fibrosis in two complementary mouse models of scleroderma, as well as in human skin organ cultures and in three-dimensional human skin equivalents. In two-dimensional monolayer cultures of explanted normal fibroblasts, CDDO abrogated fibrogenic responses induced by TGF-β. These CDDO effects occurred via disruption of Smad-dependent transcription and were associated with inhibition of Akt activation. In scleroderma fibroblasts, CDDO attenuated the elevated synthesis of collagen. Remarkably, the in vitro antifibrotic effects of CDDO were independent of PPAR-γ. Conclusions The PPAR-γ agonist triterpenoid CDDO attenuates fibrogenesis by antagonistically targeting canonical TGF-β/Smad and Akt signalling in a PPAR-γ-independent manner. These findings identify this synthetic triterpenoid as a potential new therapy for the control of fibrosis.

Tenascin-C drives persistence of organ fibrosis

The factors responsible for maintaining persistent organ fibrosis in systemic sclerosis (SSc) are not known but emerging evidence implicates toll-like receptors (TLRs) in the pathogenesis of SSc. Here we show the expression, mechanism of action and pathogenic role of endogenous TLR activators in skin from patients with SSc, skin fibroblasts, and in mouse models of organ fibrosis. Levels of tenascin-C are elevated in SSc skin biopsy samples, and serum and SSc fibroblasts, and in fibrotic skin tissues from mice. Exogenous tenascin-C stimulates collagen gene expression and myofibroblast transformation via TLR4 signalling. Mice lacking tenascin-C show attenuation of skin and lung fibrosis, and accelerated fibrosis resolution. These results identify tenascin-C as an endogenous danger signal that is upregulated in SSc and drives TLR4-dependent fibroblast activation, and by its persistence impedes fibrosis resolution. Disrupting this fibrosis amplification loop might be a viable strategy for the treatment of SSc.

Early Growth Response 3 (Egr-3) Is Induced by Transforming Growth Factor-β and Regulates Fibrogenic Responses

Members of the early growth response (Egr) gene family of transcription factors have nonredundant biological functions. Although Egr-3 is implicated primarily in neuromuscular development and immunity, its regulation and role in tissue repair and fibrosis has not been studied. We now show that in normal skin fibroblasts, Egr-3 was potently induced by transforming growth factor-β via canonical Smad3. Moreover, transient Egr-3 overexpression was sufficient to stimulate fibrotic gene expression, whereas deletion of Egr-3 resulted in substantially attenuated transforming growth factor-β responses. Genome-wide expression profiling in fibroblasts showed that genes associated with tissue remodeling and wound healing were prominently up-regulated by Egr-3. Notably, <5% of fibroblast genes regulated by Egr-1 or Egr-2 were found to be coregulated by Egr-3, revealing substantial functional divergence among these Egr family members. In a mouse model of scleroderma, development of dermal fibrosis was accompanied by accumulation of Egr-3-positive myofibroblasts in the lesional tissue. Moreover, skin biopsy samples from patients with scleroderma showed elevated Egr-3 levels in the dermis, and Egr-3 mRNA levels correlated with the extent of skin involvement. These results provide the first evidence that Egr-3, a functionally distinct member of the Egr family with potent effects on inflammation and immunity, is up-regulated in scleroderma and is necessary and sufficient for profibrotic responses, suggesting important and distinct roles in the pathogenesis of fibrosis.

Toll-like Receptor 9 Signaling Is Augmented in Systemic Sclerosis and Elicits Transforming Growth Factor β-Dependent Fibroblast Activation.

Although transforming growth factor β (TGFβ) is recognized as being a key trigger of fibroblast activation in systemic sclerosis (SSc), prominent innate immunity suggests that additional pathways contribute to disease persistence. Toll‐like receptor 9 (TLR9) is implicated in autoimmunity and fibrosis; however, the expression, mechanism of action, and pathogenic role of TLR9 signaling in SSc remain uncharacterized. The aim of this study was to explore the expression, activity, and potential pathogenic role of TLR9 in the context of skin fibrosis in SSc and in mouse models of experimental fibrosis.

TLR9 signaling is augmented in systemic sclerosis and elicits TGF‐β‐dependent fibroblast activation

Although transforming growth factor β (TGFβ) is recognized as being a key trigger of fibroblast activation in systemic sclerosis (SSc), prominent innate immunity suggests that additional pathways contribute to disease persistence. Toll‐like receptor 9 (TLR9) is implicated in autoimmunity and fibrosis; however, the expression, mechanism of action, and pathogenic role of TLR9 signaling in SSc remain uncharacterized. The aim of this study was to explore the expression, activity, and potential pathogenic role of TLR9 in the context of skin fibrosis in SSc and in mouse models of experimental fibrosis.

A20 suppresses canonical Smad-dependent fibroblast activation: novel function for an endogenous inflammatory modulator

BackgroundThe ubiquitin-editing cytosolic enzyme A20, the major negative regulator of toll-like receptor (TLR)-mediated cellular inflammatory responses, has tight genetic linkage with systemic sclerosis (SSc). Because recent studies implicate endogenous ligand-driven TLR signaling in SSc pathogenesis, we sought to investigate the regulation, role and mechanism of action of A20 in skin fibroblasts.MethodA20 expression and the effects of forced A20 expression or siRNA-mediated A20 knockdown on fibrotic responses induced by transforming growth factor-ß (TGF-ß) were evaluated was evaluated in explanted human skin fibroblasts. Additionally, A20 regulation by TGF-ß, and by adiponectin, a pleiotropic adipokine with anti-fibrotic activity, was evaluated.ResultsIn normal fibroblasts, TGF-ß induced sustained downregulation of A20, and abrogated its TLR4-dependent induction. Forced expression of A20 aborted the stimulation of collagen gene expression and myofibroblast transformation induced by TGF-ß, and disrupted canonical Smad signaling and Smad-dependent transcriptional responses. Conversely, siRNA-mediated knockdown of A20 enhanced the amplitude of fibrotic responses elicited by TGF-ß. Adiponectin, previously shown to block TLR-dependent fibrotic responses, elicited rapid and sustained increase in A20 accumulation in fibroblasts.ConclusionThese results identify the ubiquitin-editing enzyme A20 as a novel endogenous mechanism for negative regulation of fibrotic response intensity. Systemic sclerosis-associated genetic variants of A20 that cause impaired A20 expression or function, combined with direct suppression of A20 by TGF-ß within the fibrotic milieu, might play a significant functional role in persistence of fibrotic responses, while pharmacological augmentation of A20 inhibitory pathway activity might represent a novel therapeutic strategy.

TLR4-dependent fibroblast activation drives persistent organ fibrosis in skin and lung

Persistent fibrosis in multiple organs is the hallmark of systemic sclerosis (SSc). Recent genetic and genomic studies implicate TLRs and their damage-associated molecular pattern (DAMP) endogenous ligands in fibrosis. To test the hypothesis that TLR4 and its coreceptor myeloid differentiation 2 (MD2) drive fibrosis persistence, we measured MD2/TLR4 signaling in tissues from patients with fibrotic SSc, and we examined the impact of MD2 targeting using a potentially novel small molecule. Levels of MD2 and TLR4, and a TLR4-responsive gene signature, were enhanced in SSc skin biopsies. We developed a small molecule that selectively blocks MD2, which is uniquely required for TLR4 signaling. Targeting MD2/TLR4 abrogated inducible and constitutive myofibroblast transformation and matrix remodeling in fibroblast monolayers, as well as in 3-D scleroderma skin equivalents and human skin explants. Moreover, the selective TLR4 inhibitor prevented organ fibrosis in several preclinical disease models and mouse strains, and it reversed preexisting fibrosis. Fibroblast-specific deletion of TLR4 in mice afforded substantial protection from skin and lung fibrosis. By comparing experimentally generated fibroblast TLR4 gene signatures with SSc skin biopsy gene expression datasets, we identified a subset of SSc patients displaying an activated TLR4 signature. Together, results from these human and mouse studies implicate MD2/TLR4-dependent fibroblast activation as a key driver of persistent organ fibrosis. The results suggest that SSc patients with high TLR4 activity might show optimal therapeutic response to selective inhibitors of MD2/TLR4 complex formation.

Fibronectin EDA forms the chronic fibrotic scar after contusive spinal cord injury

Gliosis and fibrosis after spinal cord injury (SCI) lead to formation of a scar that is an impediment to axonal regeneration. Fibrotic scarring is characterized by the accumulation of fibronectin, collagen, and fibroblasts at the lesion site. The mechanisms regulating fibrotic scarring after SCI and its effects on axonal elongation and functional recovery are not well understood. In this study, we examined the effects of eliminating an isoform of fibronectin containing the Extra Domain A domain (FnEDA) on both fibrosis and on functional recovery after contusion SCI using male and female FnEDA-null mice. Eliminating FnEDA did not reduce the acute fibrotic response but markedly diminished chronic fibrotic scarring after SCI. Glial scarring was unchanged after SCI in FnEDA-null mice. We found that FnEDA was important for the long-term stability of the assembled fibronectin matrix during both the subacute and chronic phases of SCI. Motor functional recovery was significantly improved, and there were increased numbers of axons in the lesion site compared to wildtype mice, suggesting that the chronic fibrotic response is detrimental to recovery. Our data provide insight into the mechanisms of fibrosis after SCI and suggest that disruption of fibronectin matrix stability by targeting FnEDA represents a potential therapeutic strategy for promoting recovery after SCI.

Pharmacological inhibition of Toll-like receptor-4 signaling by TAK242 prevents and induces regression of experimental organ fibrosis

Systemic sclerosis (SSc) is a poorly understood heterogeneous condition with progressive multi-organ fibrosis. Recent genetic and genomic evidence suggest a pathogenic role for dysregulated innate immunity and toll-like receptor (TLR) activity in SSc. Levels of both TLR4, as well as certain endogenous TLR ligands, are elevated in skin and lung tissues from patients with SSc and correlate with clinical disease parameters. Conversely, genetic targeting of TLR4 or its endogenous “damage-associated” ligands ameliorates progressive tissue fibrosis. Targeting TLR4 signaling therefore represents a pharmacological strategy to prevent intractable fibrosis. We examined the effect of TAK242, a small molecule TLR4 inhibitor, in preclinical fibrosis models and in SSc fibroblasts. TAK242 treatment prevented, promoted regression of, bleomycin-induced dermal and pulmonary fibrosis, and reduced the expression of several pro-fibrotic mediators. Furthermore, TAK242 ameliorated peritoneal fibrosis and reduced spontaneous hypodermal thickness in TSK/+ mice. Importantly, TAK242 abrogated collagen synthesis and myofibroblasts differentiation in explanted constitutively active SSc fibroblast. Altogether, these findings identify TAK242 as an anti-fibrotic agent in preclinical models of organ fibrosis. TAK242 might potentially represent a novel strategy for the treatment of SSc and other fibrotic diseases.