Deficiency of the TLR4 inhibitory homolog RP105 exacerbates fibrosis

The pathogenic networks of immune, vascular, and fibrotic processes underlying non-resolving fibrosis in SSc remain poorly understood. Our previous studies demonstrated that while TLR4 and its cognate damage-associated endogenous ligands (DAMPs) in SSc elicited potent profibrotic effects and myofibroblasts activation, genetic targeting of TLR4 or its DAMPs in mice accelerated fibrosis resolution. To prevent aberrant DAMP-TLR4 signaling, a variety of negative regulators evolved to dampen the magnitude and duration of the signaling. Radioprotective 105 KDa (RP105), a TLR4 homolog, competitively inhibits DAMP recognition of TLR4 ,and block TLR4 signaling in immune cells. However, the role of RP105 in TLR4-depedent fibrotic responses in SSc is unknown. Using unbiased transcriptome analysis of skin biopsies, we found that both TLR4 and its adaptor MD2 were elevated in SSc and significantly correlated with each other (r=-0.54, p=0.0062). In contrast, levels of RP105 and its adaptor MD1 failed to show significant elevation and association (r=-0.35, p=0.11). Notably, RP105 expression was negatively associated with the myofibroblasts marker alpha-smooth muscle actin (ASMA) in SSc fibroblasts (r=-0.53). In vitro, exogenous RP105 abrogated DAMP-induced fibrotic responses, while RP105-depleted fibroblasts showed exaggerated responses. Importantly, in vivo ablation of RP105 in mice aggravated skin fibrosis in complementary disease models and was associated with augmented TLR4 signaling. Thus, we identify RP105-MD1 as a novel cell-intrinsic negative regulator of TLR4-MD1-driven sustained fibroblast activation, constituting a critical regulatory network governing the fibrotic process. Moreover, we propose that impaired RP105 function in SSc might contribute to progression of the disease.