Inhibition of HIF-1α ameliorates pulmonary fibrosis by suppressing M2 macrophage polarization through PRMT1/STAT6 signals

Abstract

Objective: Pulmonary fibrosis (PF) is a chronic, progressive, and irreversible lung interstitial disease of unknown etiology with a fatal outcome. M2 macrophages have been recognized to play a significant role in PF pathogenesis. The role of protein hypoxia-inducible factor 1-α (HIF-1α) in M2 macrophage polarization in PF is largely unknown. This study aimed to investigate the role of macrophage HIF-1α in the regulation of PF.

Methods: PF was induced in C57BL/6 mice by the intratracheal injection of bleomycin (BLM), and small hairpin RNA (shRNA) lentiviral construct specifically targeting HIF-1α were designed for in vitro and in vivo experiments. In the in vitro experiment, bone marrow-derived macrophages (BMDMs) were used to explore molecular mechanism analysis. In the in vivo experiment, mice were administered BLM intratracheally on day 0, treated with shRNA on day 7, and sacrificed on day 21. Histopathological techniques (H&E and Masson's trichrome staining) were used to evaluate PF severity. Western blot, immunofluorescence, quantitative real-time PCR, and flow cytometry were performed to explore the underlying mechanisms.

Results: HIF-1α was upregulated and macrophages polarized toward M2 phenotype in BLM-induced mouse pulmonary fibrosis models. By constructing HIF-1α knockdown shRNA lentiviral construct, we found that the knockdown of HIF-1α in macrophages significantly suppressed M2-type polarization in vitro, hence alleviating fibrosis in lung epithelial cells. Further results revealed that HIF-1α in macrophages promoted M2-type polarization by mediating the signal transducer and activator of transcription 6 (STAT6) arginine methylation. Meanwhile, its arginine methylation modification site is at position Arg27. Further experiments indicated that the regulation of STAT6 arginine methylation by HIF-1α mainly depended on the protein arginine methyltransferase 1 (PRMT1). Finally, animal experiments demonstrated that Knockdown of HIF-1α, PRMT1, and STAT6 relieved the BLM-induced pulmonary fibrosis of mice.

Conclusion: HIF-1α may act as a novel factor to promote macrophage of the M2 program. Therapeutic approaches to target macrophage HIF-1α may act as a new therapeutic strategy to combat PF in the future.

Keywords: HIF-1α; Macrophage; PRMT1; Polarization; Pulmonary fibrosis; STAT6.