ASXL1 in Proinflammatory Macrophages Has a Protective Role in AKI by Inhibiting Inflammatory Response

Background: Acute kidney injury (AKI) is triggered by various events including ischemia and inflammation. In this study, we aimed to elucidate the effect of AKI-induced proinflammatory (M1) macrophages on the renal tubular cells from an epigenetic perspective. ASXL1, the histone modifier, regulates both H3K27me3, which suppresses gene expression, and H3K4me3, which activates gene expression. However, the role of ASXL1 in M1 macrophages in AKI is unknown. Methods: Cultured macrophages RAW 264.7 were differentiated into M1 macrophages by stimulation of LPS, and we knocked down the expression of Asxl1 in RAW 264.7 by siRNA. Downstream target genes regulated by Asxl1 in M1 macrophages were identified by RNA-seq. And, mouse tubular cells were co-cultured with the medium of M1 macrophages whose Asxl1 was knocked down, then we evaluated the cytotoxicity and apoptosis of mouse tubular cells. Moreover, we performed chromatin immunoprecipitation (CHIP) using histone modification antibodies in order to clarify the epigenetic role of Asxl1 in M1 macrophages. Results: Gene ontology analysis revealed that pathways such as responses to viruses and innate immune responses were further upregulated by knocking down Asxl1 in M1 macrophages. And these pathways commonly contained inflammatory genes such as Il6 and Il1b. On the other hand, the IL17 signaling pathway, which is involved in host defense against microorganisms and inflammatory diseases, were downregulated by knocking down Asxl1 in M1 macrophages. In this pathway, Csf3 and Lcn2, which have been reported to have anti-apoptotic and renal protective effects on tubular cells, were included. In the co-culture experiment, knockdown of Asxl1 in M1 macrophages enhanced cytotoxicity and apoptosis of mouse tubular cells. Besides, CHIP analysis showed that the H3K27me3 levels in the promoter regions of the inflammatory cytokines (Il6 and Il1b) were further suppressed by knockdown of Asxl1 in M1 macrophages. In addition, the H3K4me3 levels in the promoter regions of Csf3 and Lcn2 were reduced by knockdown of Asxl1 in M1 macrophages. Conclusion: In M1 macrophages, ASXL1 suppresses the excessive expression of inflammation-related genes including Il6 and Il1b by regulating H3K27me3, and promotes the expression of anti-apoptosis-related genes such as Csf3 andLcn2 by regulating H3K4me3. Funding: Government Support – Non-U.S.