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Therapeutic interleukin-6 blockade reverses transforming growth factor-beta pathway activation in dermal fibroblasts: insights from the faSScinate clinical trial in systemic sclerosis.

ABSTRACT: OBJECTIVES:Skin fibrosis mediated by activated dermal fibroblasts is a hallmark of systemic sclerosis (SSc), especially in the subset of patients with diffuse disease. Transforming growth factor-beta (TGF?) and interleukin-6 (IL-6) are key candidate mediators in SSc. Our aim was to elucidate the specific effect of IL-6 pathway blockade on the biology of SSc fibroblasts in vivo by using samples from a unique clinical experiment-the faSScinate study-in which patients with SSc were treated for 24 weeks with tocilizumab (TCZ), an IL-6 receptor-? inhibitor. METHODS:We analysed the molecular, functional and genomic characteristics of explant fibroblasts cultured from matched skin biopsy samples collected at baseline and at week 24 from 12 patients receiving placebo (n=6) or TCZ (n=6) and compared these with matched healthy control fibroblast strains. RESULTS:The hallmark functional and molecular-activated phenotype was defined in SSc samples and was stable over 24 weeks in placebo-treated cases. RNA sequencing analysis robustly defined key dysregulated pathways likely to drive SSc fibroblast activation in vivo. Treatment with TCZ for 24 weeks profoundly altered the biological characteristics of explant dermal fibroblasts by normalising functional properties and reversing gene expression profiles dominated by TGF?-regulated genes and molecular pathways. CONCLUSIONS:We demonstrated the exceptional value of using explant dermal fibroblast cultures from a well-designed trial in SSc to provide a molecular framework linking IL-6 to key profibrotic pathways. The profound impact of IL-6R blockade on the activated fibroblast phenotype highlights the potential of IL-6 as a therapeutic target in SSc and other fibrotic diseases. TRIAL REGISTRATION NUMBER:NCT01532869; Post-results.

SUBMITTER: Denton CP

PROVIDER: S-EPMC6104680 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Therapeutic interleukin-6 blockade reverses transforming growth factor-beta pathway activation in dermal fibroblasts: insights from the faSScinate clinical trial in systemic sclerosis.

Denton Christopher P CP Ong Voon H VH Xu Shiwen S Chen-Harris Haiyin H Modrusan Zora Z Lafyatis Robert R Khanna Dinesh D Jahreis Angelika A Siegel Jeffrey J Sornasse Thierry T

Annals of the rheumatic diseases 20180531 9

<h4>Objectives</h4>Skin fibrosis mediated by activated dermal fibroblasts is a hallmark of systemic sclerosis (SSc), especially in the subset of patients with diffuse disease. Transforming growth factor-beta (TGFβ) and interleukin-6 (IL-6) are key candidate mediators in SSc. Our aim was to elucidate the specific effect of IL-6 pathway blockade on the biology of SSc fibroblasts in vivo by using samples from a unique clinical experiment-the faSScinate study-in which patients with SSc were treated ...[more]

PMID: 29853453

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Project description:Systemic sclerosis (SSc) is characterized by severe and often progressive fibrosis of the skin and multiple internal organs. The mechanisms responsible for these alterations remain obscure, although excessive reactive oxygen species (ROS)-mediated oxidative stress has been implicated. NOX-4 is 1 of 7 isoforms of NADPH oxidase responsible for the generation of ROS. The purpose of this study was to examine NOX-4 expression in skin and cultured dermal fibroblasts from SSc patients and to examine its regulation by transforming growth factor ?1 (TGF?1).NOX-4 was assessed in normal and SSc skin by immunohistologic analysis and in normal and SSc cultured dermal fibroblasts by quantitative polymerase chain reaction analysis, fluorescence microscopy, and Western blotting. ROS levels were assessed by fluorescence measurement of H2 O2 production. Specific kinase inhibitors were used to study the TGF?1 signaling involved in NOX-4 stimulation. NOX-4 inhibition/down-regulation was induced with a selective NOX-4 small-molecule inhibitor and NOX-4 small interfering RNA (siRNA).In contrast with normal skin fibroblasts, those from SSc skin showed intense NOX-4 staining. Cultured SSc fibroblasts displayed increased NOX-4 expression. TGF?1 caused potent NOX-4 protein and messenger RNA stimulation in normal and SSc fibroblasts, which was mediated by the protein kinase C? (PKC?) and Smad2/3 pathways. NOX-4 knockdown in SSc fibroblasts reduced the production of ROS and lowered the expression of type I collagen.NOX-4 expression and production were found to be constitutively elevated in SSc skin and cultured SSc dermal fibroblasts. TGF?1 stimulated NOX-4 expression in normal and SSc fibroblasts through PKC? and Smad2/3 signaling pathways. A small-molecule NOX-4 inhibitor decreased collagen and fibronectin production by normal and SSc fibroblasts, and NOX-4 siRNA knockdown reduced ROS and collagen production by SSc fibroblasts. These results demonstrate the involvement of NOX-4 in SSc-associated fibrosis and indicate NOX-4 inhibitors as novel therapeutic approaches for SSc.

Project description:Functional cytokine networks have been poorly characterized in systemic sclerosis (SSc). While interleukin-17A (IL-17A) is increased in SSc skin and other organs, its role is still debated, particularly considering fibrogenesis. We uncover here a dual function of IL-17A in the presence of transforming growth factor-? 1 (TGF-?), the master pro-fibrotic cytokine. In the one hand, we report an unexpected synergic activity resulting in enhanced production of IL-6 by dermal fibroblasts; in the other hand, a substantial inhibition of type I collagen (col-I) production. IL-17A or TGF-? enhanced the production of IL-6 by 8- to 16-folds when compared to control in healthy donors (HD) and SSc cultures. However, the joint presence of IL-17A and TGF-? resulted in robustly exuberant responses with levels of IL-6 up to 100-folds higher than those observed in untreated cells. Inhibition of NF?B signaling pathway preferentially inhibited the production of IL-6 driven by IL-17A in HD fibroblasts, while inhibition of PI3K preferentially inhibited the production of IL-6 driven by TGF-?. Interestingly, when p38 MAPK was inhibited, substantial reduction of IL-6 production was observed for both IL-17A and TGF-?. Consistently with the inhibition experiments, the combined stimulation of fibroblasts by IL-17A and TGF-? resulted in 1.8-fold increase in p38 MAPK phosphorylation (P?=?0.025), when compared to levels of phosphorylated p38 MAPK induced by IL-17A alone. Furthermore, the enhanced phosphorylation of p38 MAPK in the joint presence of IL-17A and TGF-? was unique among the signaling molecules we examined. As expected, TGF-? induced SMAD2 phosphorylation and col-I production. However, in fibroblasts cultured in the joint presence of TGF-? and IL-17A, SMAD2 phosphorylation was decreased by 0.6-folds (P?=?0.022) when compared to that induced by TGF-? alone. Remarkably, in this condition, the production of col-I and fibronectin was significantly decreased in both HD and SSc. Thus, IL-17A and TGF-? reciprocally influence each other effector functions in fibroblasts. Intracellular molecular switches may favor synergic or antagonistic activities, which are revealed by specific readouts. The implications of these data in the context of SSc are far reaching, particularly in terms of therapeutic approaches since IL-6, IL-17A, and TGF-? are all putative targets of treatment.

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Therapeutic interleukin-6 blockade reverses transforming growth factor-beta pathway activation in dermal fibroblasts: insights from the faSScinate clinical trial in systemic sclerosis.

Publications

Therapeutic interleukin-6 blockade reverses transforming growth factor-beta pathway activation in dermal fibroblasts: insights from the faSScinate clinical trial in systemic sclerosis.

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