A Synthetic TLR3 Ligand Mitigates Profibrotic Fibroblast Responses by Inducing Autocrine IFN Signaling

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.

Estradiol‐regulated innate antiviral responses of human endometrial stromal fibroblasts

“…Responsiveness to PRR ligands is a characteristic feature of fibroblasts throughout the body. Ovarian, skin, lung, intestinal and foreskin fibroblasts respond to poly (I:C) by upregulating the expression of interferon (IFN) β, cytokines and interferon-stimulated genes (ISG) 34,40–42 . Our findings indicate that following exposure to poly (I:C), a dsRNA analogue that is a common intermediate in viral replication, FRT fibroblasts upregulate the expression and secretion of ISGs, cytokines and chemokines.…”

Poly(I:C) induces expressions of MMP-1, -2, and -3 through various signaling pathways including IRF3 in human skin fibroblasts

“…Poly(I:C) is a synthetic dsRNA analog and has been regarded as a representative RNA ligand not only for TLR3 but also for the cytosolic RNA helicases, such as retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated gene 5 (MDA5) and extensively used instead of both exogenous and endogenous RNA ligands in many studies [19,25]. It is very well known that TLR3 is a receptor for dsRNA [4].…”

“…It is very well known that TLR3 is a receptor for dsRNA [4]. However, in many different cell types, dsRNA can also be recognized by RIG-1 and MDA5 [19,[25][26]. 15 In certain cases, dsRNA-induced effects can be TLR3-independent.…”

“…For example, by using siRNAs of TLR3, MDA5 and RIG-I, it has been demonstrated that poly(I:C)-induced IFN-β expression is TLR3-independent and MDA5/RIG-I-dependent in skin keratinocytes [25]. It has been also observed that poly(I:C) suppresses procollagen expression in skin fibroblasts through TLR3-independent and MDA5/RIG-Idependent pathways [19]. Thus, we wondered whether or not poly(I:C)-induced MMP-1, -2, and -3 expressions are TLR3-dependent in human skin fibroblasts.…”

“…In 7 treatment, human skin fibroblasts were first serum-starved for 24 h in DMEM containing 0.1% FBS, and then treated with poly(I:C) at 1, 10, or 20 μg/ml. The concentrations of poly(I:C) used in this study were screened and determined using the information from many published studies [6,10,[18][19]. Control samples were treated with the vehicle, PBS.…”