B33-12 Cholesterol 25Hydroxylase Drives Collagen Synthesis Through RORa Signaling

Abstract Rationale Pulmonary fibrosis is a chronic and progressive lung disease characterized by excessive collagen deposition and tissue remodeling, leading to irreversible respiratory failure. Its pathogenesis involves complex profibrotic signaling and interactions among multiple cell types, yet the underlying molecular mechanisms remain incompletely understood. Recent studies suggest that dysregulated lipid metabolism contributes to fibrosis development, but the precise pathways remain unclear. Cholesterol 25-hydroxylase (CH25H) is an endoplasmic reticulum-resident enzyme that converts cholesterol to 25-hydroxycholesterol (25-HC), a key regulator of lipid metabolism and immune responses. However, its role in fibroblast activation and pulmonary fibrosis has not been fully defined. Methods Wild-type (WT) and Ch25h-/- mice received four intranasal doses of 12.5 µg of Alternaria alternata extract on days 0, 3, 7, and 10, or a single intranasal dose of 1.75U/kg of bleomycin on day 0. Lung collagen deposition was assessed on day 12 by Trichrome staining. In vitro, normal human lung fibroblasts (NHLFs) were treated with 2 ng/ml TGF-β or 15 µM 25-HC ± 5 µM of an RORα antagonist to assess collagen synthesis. RORα siRNA was used to confirm the role of RORα in regulating collagen synthesis. Bulk RNA-seq and weighted gene co-expression network analysis (WGCNA) were performed on NHLFs treated with 25-HC or an RORα antagonist. Results Compared with WT mice, Ch25h-/- mice exhibited markedly reduced collagen deposition following either Alternaria exposure or bleomycin-induced injury. In vitro, treatment of NHLFs with 25-HC significantly increased collagen synthesis, that was suppressed with an RORα antagonist or with RORα siRNA, confirming that 25-HC drives collagen production through RORα-dependent signaling. Stimulation of fibroblasts with TGF-β, IL-4/13, Alternaria or bleomycin upregulated CH25H expression, establishing a positive feedback loop between profibrotic signaling and cholesterol metabolism. WGCNA revealed that 25-HC treatment enriched gene modules associated with collagen metabolism, extracellular matrix organization, and biosynthesis pathways. Key profibrotic genes including CCN1, CCN2, CYGB, FBLN1, and TNXB were upregulated, further supporting a central role for 25-HC in matrix production. Analysis of the Idiopathic Pulmonary Fibrosis (IPF) Cell Atlas demonstrated that CH25H is predominantly expressed in alveolar fibroblasts, where it is co-expressed with CCN1 and CCN2, highlighting its relevance to human disease. Conclusion In summary, this study reveals that CH25H-derived 25-HC promotes collagen synthesis in NHLFs via an RORα-dependent mechanism. Genetic deletion or pharmacologic inhibition of this pathway may attenuate pulmonary fibrosis. This abstract is funded by: 1K24HL179901