Attenuation of Fibroblast Activation and Fibrosis by Adropin in Systemic Sclerosis (SSc)
Fibrotic diseases impose a major socioeconomic challenge on modern societies with limited treatment options. Adropin, a peptide hormone encoded by the energy-homeostasis-associated (ENHO) gene, is implicated in metabolism and vascular homeostasis, but its role in the pathogenesis of fibrosis remains enigmatic. Here, we used machine learning approaches in combination with functional in vitro and in vivo experiments to characterize Adropin/ENHO as a potential regulator involved in fibroblast activation and tissue fibrosis in systemic sclerosis (SSc). We demonstrated consistent downregulation of Adropin/ENHO in SSc skin across different SSc cohorts. The prototypical profibrotic cytokine TGFß reduced Adropin/ENHO expression in a JNK-dependent manner. Restoration of Adropin signaling by therapeutic application of bioactive Adropin34-76 peptides in turn inhibited TGFß-induced fibroblast activation and fibrotic tissue remodeling in primary human dermal fibroblasts, three-dimensional full-thickness skin equivalents, the mouse models of bleomycin-induced pulmonary fibrosis and sclerodermatous chronic graft-versus-host-disease (sclGvHD), and precision-cut human skin slices (PCSS). Knockdown of GPR19, receptor of Adropin, abrogated the antifibrotic effects of Adropin in fibroblasts. RNA-seq demonstrated that the antifibrotic effects of Adropin34-76 were functionally linked to deactivation of GLI1 dependent profibrotic transcriptional networks, which was experimentally confirmed in vitro, in vivo and ex vivo. ChIP-seq confirmed Adropin34-76-induced changes in TGFß/GLI1 signaling. Our study thus characterizes the TGFß-induced downregulation of Adropin/ENHO expression as a potential pathomechanism of SSc as a prototypical systemic fibrotic disease that unleashes uncontrolled activation of profibrotic GLI1 signaling. We also provide evidence in multiple preclinical models that Adropin34-76 might offer potential for the treatment of fibrosis. Overall design: In this study, we aimed to characterize the role of Adropin/ENHO in fibroblast activation and fibrotic tissue remodeling in SSc as a prototypic multisystem fibrotic disorder. Machine learning based evaluation of bulk RNA-seq datasets identified ENHO as a signature gene in SSc. Furthermore, these machine learning-derived results have been corroborated by various approaches, including DEG analysis. Screening of two large external cohorts, the PRESS and the GENISOS (15, 16), as well as our internal cohort with 133 SSc patients and 92 controls in total, demonstrated downregulation of Adropin/ENHO in dermal fibroblasts of SSc patients. Screening of different profibrotic mediators revealed that the reduction of Adropin/ENHO was mediated by profibrotic cytokine TGFß, which was confirmed by overexpression of TßRICA or pharmacological inhibition of TGFß signaling by the TßRI selective inhibitor SD-208 in mice. Screening using inhibitors targeting TGFß mediated signaling pathways identified the suppression of ENHO expression was dependent on JNK signaling, which was also confirmed by siRNA mediated knockdown. Studies on cultured human dermal fibroblasts, 3D full-thickness human skin equivalents, complementary murine models with pre-established fibrosis and PCSS were used to demonstrate that administration of bioactive peptides Adropin34-76 ameliorated fibroblast-to-myofibroblast differentiation, collagen release and tissue fibrosis. Study designs and timeline diagrams were shown in Fig. 4A, 5A, 5D and 7A respectively. Transcriptional profiling was used to reveal that the antifibrotic effects of Adropin34-76 were functionally linked to Hedgehog signaling and GLI1 deactivation, which was experimentally confirmed in vitro, in vivo and ex vivo. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to further investigate the intricate interactions between TGFß and GLI1 signaling in response to Adropin treatment. Knockdown of GPR19, a putative receptor of Adropin, abrogated the antifibrotic effects of Adropin in fibroblasts.