Introduction: Kupffer cells are the special macrophages in the liver, which play an important role in the process of chemotaxis to other immune cells in liver Ischemia-reperfusion injury (IRI). Macrophage SETDB1, a histone methyltransferase involved in gene silencing, plays a vital role in regulating the expression of proinflammatory factors in macrophages through epigenetic regulation.
Method: Using CRISPR/Cas9 technique to construct specific macrophage SETDB1 knockout mice, male SETDB1-KO mice and wild-type C57 mice, 70% partial liver IRI models were established respectively. After 40 minutes of ischemia and 6 hours of reperfusion, serum samples were taken to detect biochemical parameters and inflammatory factors. Liver tissue was taken for HE staining and immunofluorescence. Flow cytometry was used to detect the infiltration ratio of various macrophages. The liver macrophages were sorted out for RNA-seq analysis after liver IRI model.
Results: After mouse liver IRI model, Compared with C57 group, the activity of ALT, AST and the expression of IL-1β, IL-6 and TNF-α were increased in SETDB1-KO group. HE results showed that pathological congestion, vacuolation and hepatocellular necrosis were more serious in SETDB1-KO group. Immunofluorescence and flow showed that the infiltration of macrophages increased and the proportion of mononuclear macrophages increased and Kupffer cells decreased in SETDB1-KO group. RNA-seq demonstrated that the expression of P2rx7 in infiltrating macrophages in SETDB1-KO group was significantly higher. Bone marrow-derived macrophages from SETDB1-KO mice and C57 mice were extracted in vitro and treated with ATP, a P2rx7 receptor agonist. Compared with C57 mice, the expression of P2rx7, IL-1β, IL6, TNF-α, as well as CXCL1 and CXCL2 were also increased in SETDB1-KO group. Injecting P2rx7 receptor inhibitor OATP and normal saline respectively in SETDB1-KO mice before hepatic IRI model. Compared with normal saline-treatment group, the activity of serum ALT and AST was decreased, and the expression of IL-1β, IL-6 and TNF-α was decreased in OATP-treated group. HE showed that the pathological congestion of liver in the OATP-treated group was less than normal saline-treated group. Immunofluorescence showed a decrease in liver infiltrating macrophages and flow showed a decrease in the proportion of macrophages and an increase in Kupffer cells in OATP-treated group.
Conclusion: Macrophage SETDB1 suppresses the expression of P2rx7 gene, thus reducing the infiltration of inflammatory macrophages and the secretion of inflammatory factors during hepatic IRI, and finally reducing the hepatic IRI.