PD-L2 deficiency in Alveolar macrophages drives fibrosis, apoptosis, and ferroptosis via M1 polarization in connective tissue disease-associated interstitial lung disease

Abstract Macrophages play a pivotal role in modulating immune responses in connective tissue disease-associated interstitial lung disease (CTD-ILD). The role of programmed death ligand-2 (PD-L2), an immune checkpoint molecule expressed on macrophages, in macrophage polarization in CTD-ILD remains poorly understood. Serum PD-L2 levels were measured in CTD-ILD patients, CTD-nonILD patients, and healthy controls. Alveolar macrophages (AMs) were transfected with PD-L2-targeting shRNA or control constructs, and their effects on macrophage phenotype and fibroblast fate were evaluated. M1/M2 polarization was assessed by RT-PCR, Western blotting, and flow cytometry. Human embryonic lung fibroblasts (HELFs) were co-cultured or treated with macrophage-conditioned media, and assays for cell viability, apoptosis, fibrosis, and ferroptosis were performed. A bleomycin (BLM)-induced CTD-ILD mouse model was used to evaluate the effects of PD-L2 knockout on lung fibrosis and ferroptosis markers. Serum PD-L2 levels were significantly lower in CTD-ILD patients compared with CTD-nonILD patients and healthy controls, and negatively correlated with the extent of lung fibrosis. In vitro, PD-L2 knockdown in AMs promoted M1 polarization, suppressed M2 related markers, and induced fibroblast apoptosis, fibrosis, and ferroptosis. Conditioned media from PD-L2-deficient macrophages produced similar effects. In vivo, PD-L2 knockout mice exhibited decreased numbers of CD206 + macrophages and regulated ferroptosis markers (ACSL4 upregulation, GPX4 and FTH1 downregulation) in lung tissues following BLM treatment. This study identifies PD-L2 as an important regulator of macrophage polarization and fibroblast responses in CTD-ILD. Our findings suggest that serum PD-L2 levels may reflect disease severity, and that restoring PD-L2 function could represent a potential therapeutic direction warranting further investigation in preclinical models.