Modulation Of Liver Macrophages In Mouse By CD24-siglec G Signaling Pathway

Object and contents:The immune system recognizes the"self"and"non-self"and molecular mechanism is one of the important issues in immunologic research.It is now well accepted that the innate immune system recognizes both damage?or danger?and pathogen associated molecular patterns?DAMP and PAMP,respectively?through pattern recognition receptors,such as Toll-like receptors.However,Less clear are whether and how the response to PAMP and DAMP are regulated differentially.Recent studies have revealed that some members in the sialic acid-binding Ig-like lectin?Siglecs?family can reduce the inflammation in host through the sialic acid glycoside-based pattern recognition,especially the selective interactions of CD24with Siglec 10?in human?/Siglec G?in mouse?can inhibit the host inflammatory reaction induced by DAMPs but not PAMPs.Macrophages are an essential component of innate immunity and play a central role in inflammation and host defense.However,the role of CD24-Siglec G signal loop in macrophages has not been defined.This study aims to investigate the expression of CD24 and Siglec G in macrophages,the response of CD24 and Siglec G to DAMPs and PAMPs and the regulation of CD24-Siglec G signaling pathway in inflammatory responses.Methods and results:The project is divided into three parts:Part one:Regulation of CD24-Siglec G signal loop in mouse peritoneal macroph-ages,including the following experiments.We harvested and purified peritoneal macrophages from wild type mice?WT mice?,CD24 knockout mice(CD24^-/-mice)and Siglec G knockout mice(Siglec G^-/-mice)respectively.These macrophages were stimulated with high mobility group protein Bl?HMGB1?and lipopolysaccharide?LPS?for 24 h.The expression of CD24 and Siglec G were determined by quantitative real-time PCR.Flow cytometry assays were employed to identify the correlation of CD24 and Siglec G.ELISA was used to detect the secretion of TNF-?and IL-6 in peritoneal macrophages.Results show that increased expression of CD24 and Siglec G was observed in peritoneal macrophages stimulated with HMGB1 but not with LPS,and the expression of CD24 and Siglec G was related.Also secretion of TNF-?and IL-6 was markedly increased in the supernatant of peritoneal macrophages treated with HMGB1.Part two:Regulation of CD24-Siglec G signal loop in mouse hepatic macrophag-es,including the following experiments.Liver macrophages were isolated by percoll density centrifugation and then stimulated with HMGB1,LPS and Poly I:C for 24 h,respectively.The expression of CD24 and Siglec G was detected by real-time PCR.TNF-?and IL-6 levels in liver macrophage supernatant were quantified by ELISA.We used concanavalin A?Con A?-induced liver injury to explore the role of CD24-Siglec G signaling,histological examination of liver sections from Con A-treated mice was assessed by H&E,immunofluorescence staining and western blot.The macrophages in Con A-induced liver injury were analyzed by flow cytometry.Acute liver injury in WT,CD24^-/-and Siglec G^-/-mice was evaluated by H&E,serum levels of ALT and AST,and real-time PCR.Results show that increased expression of CD24 and Siglec G was also observed in liver macrophages stimulated with HMGB1 but not with LPS or Poly I:C.And there is a correlation between the expression of CD24 and Siglec G.Consistent with the previous study,secretion of TNF-?and IL-6 was markedly increased in the supernatant of hepatic macrophages treated with HMGB1.Accordingly,a large amount of HMGB1 was released in WT Con A–induced hepatitis as compared to controls,and flow cytometry analysis showed that the percentage of hepatic macrophages was increased after mice modeling.The hepatic damage in CD24^-/-and Siglec G^-/-mice was more severe than in WT mice.Part three:The molecular mechanism of CD24-Siglec G loop in inflammatory regulation,including the following experiments.To examine the possible role for SHP-1 in CD24-Siglec G signaling cascades in macrophages,peritoneal and hepatic macrophages were extracted and purified from WT and Siglec-G^-/-mice.The expression of SHP-1 and SHP-2 in macrophages was analyzed by western blot.Results show that western blot analysis demonstrated the SHP-1 expression was decreased in peritoneal and hepatic macrophages treated with HMGB1 in Siglec G^-/-mice as compared to in WT mice.By contrast,there was no difference in the expression of SHP-2 between WT mice and Siglec G^-/-mice.Conclusion:CD24 and Siglec G can specifically recognize HMGB1 but not LPS in peritoneal and hepatic macrophages,and there is a correlation between the expression of CD24 and Siglec G.Moreover,the secretion of inflammatory cytokines is significantly increased in peritoneal and hepatic macrophages from CD24-KO mice and Siglec G-KO mice relative to those observed in WT.In Con A-induced acute liver hepatitis model,similar secretion characteristics of inflammatory cytokines can also be observed,and the secretion of inflammatory cytokines is associated with an increase in the number of intrahepatic macrophages after acute liver injury in mice and associated with severe liver dysfunction,and CD24-Siglec G pathway mainly exerts its immunosuppressive effect through activation of SHP-1.