ASGR1 Promotes Sepsis-induced Liver Injury By Modulating Monocyte-to-macrophage Differentiation Via NF-κB/ATF5 Pathway

Background: Sepsis is a syndrome of inflammatory response induced by dysregulated host responses to infection.Liver is an important organ susceptible to damage of sepsis.Liver injury is one of the most common complications of sepsis and is characterized by high mortality and poor prognosis.The imbalance of inflammatory response caused by the release of massive inflammatory mediators,is one of the most important pathogeneses in liver injury of sepsis.Upon damage and inflammation stimulation,classical circulating monocytes migrate and infiltrate into the inflamed tissues,followed by differentiate into macrophages,even replace tissue macrophages,and promotion of inflammatory mediators.The release of a series of inflammatory mediators induces the target tissues damage or even dysfunction.Therefore,regulating monocyte-tomacrophage differentiation is a crucial treatment for ameliorating inflammatory diseases,such as sepsis.Asialoglycoprotein receptor 1(ASGR1)is a main member of ASGR,belongs to the C-type lectin receptor.Recent studies have found that ASGR1 is highly expressed in classical monocytes of peripheral blood mononuclear cells(PBMC),and maybe induce monocyte-mediated immune inflammatory response.However,whether ASGR1 regulates monocyte-to-macrophage differentiation or participates in the pathological process of sepsis by affecting the inflammatory responses remains unclear.Objective: Above all,our study attempts to reveal the roles of ASGR1 on immune regulating of macrophage in sepsis-induced liver injury and its regulatory mechanism,and providing a new strategy for the prevention and treatment of immune dysfunction in sepsis.Methods: 1.In vivo: LPS was used to construct a mouse model of sepsis,and the expression levels of ASGR1 and monocyte-to-macrophages differentiation markers(CD68 and CD86)ASGR1 in peripheral blood and different tissues of sepsis mice were detected by Western Blot.Then,establishment of sepsis model with ASGR1 knockdown mice(ASGR1-KD)was used to explore the role and mechanism of ASGR1 on sepsis.The symptoms and survival of mice were observed within 144 h,followed by drawing survival curves and analyzing the survival rate among the groups.H&E staining was performed to evaluate the liver morphology.The levels of TNF-α,IL-6 and IL-1β in plasma were detected by ELISA kit.Plasma concentrations of ALT and AST were assessed by kit.The proportion of classical monocytes in peripheral blood,as well as the number of monocytes-derived macrophages and inflammatory macrophages in the liver tissues were detected by flow cytometry.The protein and m RNA levels were detected by Western Blot and q PCR,respectively.The expression and co-localization of ASGR1 and F4/80 in the liver tissues was observed by immunofluorescence staining.Co-immunoprecipitation was performed to verify protein interaction between ASGR1 and ATF5 in livers.2.Bioinformatics analysis: GEO was used to screen differentially expressed genes(DEGs)for monocyte-to-macrophage differentiation in Mus musculus and Homo sapiens and to verify the change of ASGR1 in monocyte-to-macrophage differentiation.In addition,RNA sequencing(RNA-seq)analysis was used to investigate the DEGs of monocyte-tomacrophage differentiation in ASGR1 overexpressed THP-1 cells.Meanwhile,the mechanism of ASGR1 on monocyte-to-macrophages differentiation was explored by RNA-seq.3.In vitro:(1)THP-1 cells were transfected with lentivirus to construct ASGR1 overexpressed and silenced cells,and then PMA and LPS were challenged after 96 h.Western Blot,q PCR,flow cytometry were used and validated the regulatory effect of ASGR1 on monocyte-tomacrophages differentiation.Then,THP-1 cells were treated with 100ng/ml PMA for 72 h and 1 μg/ml LPS for 24 h to induce differentiation and LPS stimulated inflammatory cell models.Primary bone marrow mononuclear cells(BMMC)of mice were treated with 30% L929-conditioned medium and differentiated into BMDM.ASGR1 and ATF5-overexpressed or-deficient lentivirus and inhibitors of NF-κB were used.Using Western Blot,q PCR,flow cytometry to clarify the role and mechanism of ASGR1 on monocyte-to-macrophages differentiation.Results: 1.ASGR1 increases significantly in peripheral blood and in the liver tissues of sepsis mice.2.Loss of ASGR1 increases the survival rate of ASGR1-KD mice,attenuates inflammatory response,and alleviates sepsis-induced liver injury.3.Bioinformatics analysis uncovers ASGR1-mediated monocyte-to-macrophage differentiation.4.ASGR1 induces monocyte-to-macrophage differentiation in vivo and in vitro.The percentage of inflammatory monocytes in peripheral blood and the monocyte-derived macrophages and inflammatory macrophages dramatically elevated in liver of sepsis mice.However,monocyte-tomacrophage differentiation significantly reduced in ASGR1-KD sepsis mice.Accordingly,ASGR1 significantly increased in both PMAchallenged THP-1 cells and BMDM.Overexpression of ASGR1 increased the monocyte-to-macrophage differentiation,while the monocyte differentiation indicators were inhibited in ASGR1-deficient THP-1 cells.5.ASGR1 regulates monocyte-to-macrophage differentiation by NF-κB/ATF5 signaling pathway in vivo and in vitro.Conclusion: 1.Monocyte-to-macrophage differentiation regulates liver injury in sepsis.2.ASGR1 induces monocyte-macrophage differentiation.3.ASGR1 knockdown attenuates inflammatory response and alleviates sepsis-induced liver injury in mice.4.ASGR1 promotes sepsis-induced liver injury by NF-κB/ATF5 in regulating monocyte-tomacrophage differentiation.