Role of the protease-activated receptor 2 in multi-walled carbon nanotube-induced macrophage polarization ex vivo and airway fibrosis in murine allergic lung disease in vivo.

Our previous work demonstrated that co-exposure to multi-walled carbon nanotubes (MWCNTs) exacerbates allergic lung disease induced by house dust mite (HDM) extract, and that mice deficient in protease-activated receptor 2 (PAR2) show less airway fibrosis following co-exposure. In this study, we examined the role of PAR2 in mediating macrophage polarization in the presence of MWCNTs under Th2-like conditions and the subsequent effects on fibroblast activation in vitro and collagen deposition in vivo. Bone marrow-derived macrophages (BMDMs), murine ex vivo alveolar macrophages (mexAMs), and mouse lung fibroblasts (MLFs) were isolated from wild-type (WT) and PAR2 knockout (KO) mice. Macrophages were pretreated with IL-4/IL-13 before exposure to MWCNTs, and polarization markers were analyzed through flow cytometry and Western blot analysis. Conditioned media from treated macrophages were applied to MLFs to assess fibroblast activation. In vivo, WT and myeloid-specific PAR2 KO mice were co-exposed to MWCNTs and HDM extract over 21 days, followed by analysis of bronchoalveolar lavage fluid (BALF) and lung tissue for markers of mucous cell metaplasia and airway fibrosis. MWCNTs exacerbated IL-4/IL-13-induced M2 polarization, increasing Arg-1 and phosphorylated STAT6 levels in both BMDMs and mexAMs. This enhancement was attenuated in PAR2-deficient macrophages. Conditioned media from M2-polarized WT macrophages induced significantly higher expression of profibrotic genes, including Col1a1 and Col1a2, in MLFs compared to conditioned media from PAR2 KO macrophages. In vivo, myeloid-specific PAR2 deletion significantly decreased lung collagen deposition and mucus hypersecretion induced by MWCNT and HDM extract co-exposure. MWCNT exposure exacerbates Th2-driven M2 macrophage polarization in a PAR2-dependent manner, leading to increased fibroblast activation and collagen deposition. Myeloid PAR2 is a critical driver of fibrotic remodeling in allergic lung disease, representing a potential therapeutic target for mitigating fibrosis in environmentally exacerbated asthma.