The Role Of Neddylation In Macrophage M2 Polarization-driven Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is a kind of chronic and irreversible fatal lung disease.The main pathological features of IPF are the formation of fibrosis foci,deposition of extracellular matrix and destruction of alveolar structures,leading to death from respiratory failure in patients.IPF is more common in middle-aged and older people,and the prognosis is poor.The five-year survival rate of patients is less than 30%,and the median survival time is only three years.Because the cause is unknown,there is a lack of effective treatment for IPF.Research on the pathogenesis of IPF has been a difficult and hot topic in basic research.As a consequence,exploring the pathogenesis of IPF is of great significance.It is generally believed that pulmonary fibrosis is caused by abnormal damage repair.Macrophage,as a highly viable immune cell,can play an important role in the pathogenesis of pulmonary fibrosis through activation.Under different stimuli,macrophages can be activated into two subtypes,namely,classically activated macrophage(M1)and alternatively activated macrophage(M2).M1 type macrophages can secrete inflammatory factors,including TNF?,IL-1?,IL-6,exacerbating the inflammatory environment.M2 type macrophages can inhibit inflammatory reactions,promote fibroblast differentiation into myofibroblasts,and promote tissue repair processes.Neddylation is a novel protein post-translational modification.It is a high-dimensional,complex protein functional complex composed of a variety of protein macromolecules.It is a cascade process(activated enzyme E1,coupled enzyme E2 and ligase E3).NEDD8 is covalently bound to target molecules such as CRL,activates the CRL(Cullin-RING Ligase)ubiquitin ligase superfamily,and controls about 20%of the cell's protein degradation through the proteasome,thereby regulating a variety of physiological and pathological processes.Previous studies have shown that in liver fibrosis,the expression of Neddylation pathway is enhanced,inhibition of Neddylation can reduce the activation of hepatic macrophages,reduce the expression of hepatic macrophage chemokine receptors and the release of cytokines,thereby alleviating liver fibrosis.Organ fibrosis has similarities,suggesting that Neddylation may also play a role in pulmonary fibrosis.Therefore,our subject uses the Neddylation E2 enzyme Ubc12 as an entry point to study the mechanism by which Neddylation participates in pulmonary fibrosis by regulating the function of macrophages.We first detected an increase in Ubc12 expression in lung tissue sections of IPF patients,and detected high expression of Nedd8 and Ubc12 in alveolar macrophages of mice with pulmonary fibrosis,suggesting that Neddylation may be involved in the regulation of macrophage polarization under the circumstance of pulmonary fibrosis.In addition,we constructed a mouse strain of LysM-Cre Ubc12f/f that macrophages specifically knocked out Ubc12,and explored the pathophysiological significance of high expression of macrophage Ubc 12 in pulmonary fibrosis.In the bleomycin-induced pulmonary fibrosis model,compared with the wild type,the pulmonary fibrosis of Ubc12Myko mice was significantly aggravated,and the alveolar macrophages were more inclined to M2 polarization.In the chitin-induced macrophage type 2 polarization model,Ubc 12 knockdown not only increased the infiltration of peritoneal eosinophils,but also increased the expression of M2 marker gene in peritoneal macrophages,indicating that Ubc12 negatively regulates M2 polarization in macrophages.Further studies have found that inhibition of Neddylation in macrophage cell lines promotes phosphorylation of AKT,whereas knockdown of Ubc12 in primary myeloid macrophages enhances basal level of AKT,suggesting that Neddylation may regulate macrophage M2 polarization through the PI3K/AKT signaling pathway.Finally,we found the non-receptor tyrosine phosphatase SHP2 which is an antifibrotic regulator of pulmonary fibrosis interacts with Nedd8 and Cullin-1.Phosphorylation and Neddylation,two post-translational modifications,may have cross-interaction effects in the pathogenesis of pulmonary fibrosis.These results indicate that Ubc12 regulates the type 2 polarization of macrophages through the PI3K/AKT signaling pathway.Ubc12 knockdown can aggravate bleomycin-induced pulmonary fibrosis,and Neddylation and phosphorylation may cooperate to regulate the pathological process of pulmonary fibrosis.The results of this study not only provide new insights into the systematic study of pulmonary fibrosis,but also provide new targets for the treatment of pulmonary fibrosis.