D29-18 Targeting Mir-33 Ameliorates Pulmonary Fibrosis Through Metabolic Reprogramming of Macrophages and Improves Lung Function Beyond Nintedanib

Abstract Rationale Pulmonary Fibrosis (PF) is a chronic, progressive, and often fatal lung disease. Growing evidence suggests that changes in macrophage metabolism and mitochondrial dysfunction contribute to the initiation and progression of fibrosis. Micro RNA-33 (miR-33) regulates lipid metabolism and mitochondrial homeostasis in macrophages. We developed a peptide nucleic acid inhibitor of miR-33 (PNA-33) and hypothesized that targeting miR-33 could rebalance macrophage metabolism and reduce profibrotic activation in PF. Methods In situ hybridization (ISH) was performed on lung tissue isolated from IPF patients and healthy controls to determine miR-33 expression. PF was induced in C57BL/6 mice by oropharyngeal (OP) instillation of bleomycin (1.75 U/kg) or saline. On Day 10, mice received PNA33, Nintedanib, or vehicle (OP) every three days until Day 21. Pulmonary function testing and histological analyses were performed. Human precision-cut lung slices (hPCLS) from healthy controls were pretreated with fibrotic or control cocktail for 24 h and subsequently treated with PNA33, Nintedanib, Pirfenidone, or vehicle. Histological analyses were performed, and profibrotic markers were quantified using ELISA and bulk RNA sequencing. RAW 264.7 macrophages were pretreated with PNA33 (24 h) and stimulated with profibrotic cytokines (IFN-γ, TGF-β, or IL-13). Mitochondrial membrane potential was assessed using JC-10 fluorescence and normalized to unstimulated controls. Results ISH assay revealed elevated miR-33 expression in lung tissues from IPF patients compared to controls. In the bleomycin-induced PF model, PNA-33 treatment significantly improved body weight (29.13 ± 2.35 g vs. 24.5 ± 2.81 g; p 0.01) and survival rate (100% vs. 60%; p 0.05) relative to untreated controls. Pulmonary function testing demonstrated marked increases in static compliance, inspiratory capacity, and total respiratory compliance, along with reduced tissue damping (p 0.05). In contrast, no significant improvements were observed in the Nintedanib or vehicle-treated groups. In fibrotic hPCLS, PNA-33 markedly reduced collagen accumulation compared with Nintedanib, pirfenidone, or vehicle treatment. Bulk RNA sequencing revealed that PNA-33 reduces fibrotic genes, increases antifibrotic genes, and alters profibrotic macrophage signature in fibrotic hPCLS. Consistent with these findings, PNA-33 decreased collagen secretion to a similar extent as Nintedanib, and to a greater extent than pirfenidone. In macrophages, PNA-33 prevented mitochondrial hyperpolarization induced by TGF-β, IFN-γ, and IL-13 (p 0.05), suggesting restoration of mitochondrial homeostasis under profibrotic conditions. Conclusion Here we demonstrate that PNA-33 mitigates pulmonary fibrosis, improves lung function, and restores macrophage mitochondrial homeostasis, outperforming current antifibrotic therapies and identifying miR-33 as a promising therapeutic target in IPF. This abstract is funded by: none