Zhx2 Promotes NAFLD By Enhancing Pro-inflammatory Responses Of Macrophage

BackgroundNonalcoholic fatty liver disease(NAFLD),also known as metabolism-related fatty liver disease(MAFLD),including simple steatosis and steatohepatitis,can develop to liver fibrosis,irreversible cirrhosis and hepatocellular carcinoma(HCC).Multiple factors contribute to the progress of NAFLD.Large amount of lipid deposits promotes oxidative stress,endoplasmic reticulum stress and metabolic disorders in hepatocytes,leading to enhanced release of cytokines,such as IL-1β,IL-6,IL-18 and TNF-α,which in turn mediate liver inflammation and hepatocyte injury.On the other hand,due to the changes of liver microenvironment,a variety of hepatic immune cells are activated and secrete numerous cytokines which induce aggravation of liver inflammation and further development of NAFLD.Macrophages are the most abundant non-parenchymal cells in the liver and play an indispensable role in liver inflammatory injury and progression of NAFLD.As the most plastic immune cells,macrophage show different polarization states in different environments,which include classical activated pro-inflammatory(M1)macrophage,alternative activated anti-inflammatory(M2)macrophage and many other excessive states.Inhibition of pro-inflammatory function of macrophage significantly protect mice from high-fat diet(HFD)and Methionine and choline-deficient diet(MCD)induced NAFLD.Drugs targeting macrophage have become potential approaches for the treatment of NAFLD.Anti-CD 163-1gG dexamethasone delivery of corticosteroid dexamethasone to macrophage through CD 163 receptors significantly reduces liver inflammation and fibrosis caused by high lipids.Macrophage polarization is affected by multiple factors,such as cytokines and transcription factors.Identification of novel key factors regulating macrophage is of great significance for NAFLD intervention.Transcription factor Zhx2,a member of zinc finger protein and homeobox protein family,inhibits the occurrence and development of HCC by transcriptional regulation of growth-and metabolism-related genes in hepatocytes.Bioinformatics studies have shown that Zhx2 is a key regulator in the transcriptional regulatory network of macrophage.Recently,our and another laboratory’s studies have shown that macrophage-specific knockout of Zhx2 significantly protect mice from atherosclerosis and LPS-induced sepsis.However,it has not been reported whether Zhx2 participate in NAFLD progression by modulation of macrophages.To address this,myeloid Zhx2 knockout mice(LysMcre+,Zhx2f/f;MKO)and control mice(LysMcre-,Zhx2f/f;WT)were used to establish mouse NAFLD models through MCD,HFD and high-fat and high-cholesterol diet(HFHC).Methods and results1.Lipid promotes the expression of Zhx2 in macrophageWe first evaluated Zhx2 expression in RAW264.7 cells treated with oleic acid(OA)and triglyceride(TG).The results of western blot showed that both OA and TG significantly promoted the expression of Zhx2 in RAW264.7 cells.Similarly,OA increased the expression of Zhx2 in mouse bone marrow-derived macrophage(BMDM)and human THP-1 cells.In accordance,western blot and quantitative real-time PCR(RT-qPCR)showed that the expression of Zhx2 in BMDM,peritoneal macrophage(PEMs)and liver-derived macrophage from HFD mice were significantly increased,compared to those from normal diet mice.More importantly,Opal staining with 5 pairs paracancerous liver tissues from HCC patients showed that Zhx2 expression was significantly higher in hepatic macrophages from steatosis liver tissues than that in non-steatosis liver tissues.These results suggest that lipids significantly promote the expression of Zhx2 in macrophage.2.Zhx2 regulates macrophage to promote the progression of NASH in mice2.1 Deficiency of Zhx2 in myeloid cells attenuates MCD-induced NASH6-week-old male MKO mice and WT mice were fed with MCD for 3 weeks to induce NASH model.HE staining showed less vacuolization in liver tissues of MKO mice.Oil red O(ORO)staining displayed less lipid deposition in liver of MKO mice than that of WT mice,Constantly,lower TG and total cholesterol(T-CHO)were detected in supernatant of liver homogenate fromMKO mice.RT-qPCR results showed that the expression of pro-inflammatory cytokinesIl-1b,Il-6,Tnf a and chemokines Ccl2,Ccl5 and Cxcl10 decreased significantly in liver tissues of MKO mice.And serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were significantly lower in MKO mice than those in WT mice.2.2 Myeloid cells deficiency of Zhx2 attenuates HFD/HFHC-induced NASH2.2.1 Myeloid cells deficiency of Zhx2 reduces HFD/HFHC-induced hepatic lipid deposition and insulin resistance6-week-old male MKO and WT mice were fed with HFD and HFHC for 17 weeks to establish NASH model.HE and ORO staining were performed for the liver tissue sections,and the TG and T-CHO in the supernatant of liver homogenate were detected.The results showed that the lipid deposition in the liver of MKO mice was less than that of WT mice.Cocurrently,RT-qPCR results confirmed that the mRNA levels of fatty acid uptake and synthesis related genes(Cd36,Scdl and Pparg)and metabolism-related gene Ppara in liver of MKO mice were significantly lower than those in WT mice.In addition,compared to WT mice,MKO mice displayed ameliorated glucose tolerance and relieved insulin resistance.2.2.2 Deficiency of Zhx2 in myeloid cells decreases HFD/HFHC-induced inflammation in mouse liverHepatic macrophages were accessed by histoimmunochemical staining(CD68),immunofluorescence staining(F4/80)with mouse liver tissues.Also,F4/80+CD11b+macrophages were detected by flow cytometry with liver mononuclear cells isolated from HFHC diet mice.All results showed that the infiltration of liver macrophage in MKO mice was significantly lower than that in WT mice.RT-qPCR showed that the expression of inflammatory cytokines(Il-1b,Il-6 and Tnf-α)and chemokines(Ccl2,Ccl5 and Cxcl10)was significantly decreased in liver tissues of MKO mice than WT mice.Similarly,results of western blot demonstrated that the expression of p65 was significantly downregulated in MKO mice.Also the phosphorylation levels of p65 and JNK was decreased.These results suggest that myeloid knockout Zhx2 attenuates liver inflammation in NASH mice.2.2.3 Myeloid cells deficiency of Zhx2 ameliorates MCD-induced liver injury and fibrosisIn order to further investigate the progression of NASH in mice,liver fibrosis was analyzed by Sirius red staining,and the levels of α-SMA and Collal in liver tissues were detected by RT-qPCR.It was found that the liver fibrosis in MKO group was significantly diminished compared to that in WT mice.Additionally,the levels of ALT and AST in serum of MKO mice were significantly lower than those in WT mice,suggesting the alleviated liver injury in MKO mice.2.3 Zhx2 promoted NASH in mice via macrophage regulationBMDM isolated from WT or MKO mice were respectively intravenously injected into irradiated WT mice,then the mice were fed with MCD diet for 2 weeks.Results of oil red O staining and the detection of TG and T-CHO in the supernatant of liver homogenate showed that hepatic steatosis was alleviated in mice transfered with MKO BMDM.HE staining confirmed the less inflammation in MKO mouse liver.In addition,serum levels of ALT and AST were lower in mice transferred with MKO-derived BMDM.Further RT-qPCR detection demonstrated that the expression of pro-inflammatory factors(Il-1b,Il-6 and Tnf-α)in the liver tissue of mice transfered with MKO-derived BMDM were significantly lower than that of control mice.These results suggest that macrophage specific knockout of Zhx2 can significantly inhibit development of NASH.3.Zhx2 enhances expression of p65 in macrophage and regulates functions of macrophage.3.1 Zhx2 depletion weakenes pro-inflammatory responses in macrophage.RNA-seq data of BMDM from WT and MKO mice were analyzed by GSEA.The results showed that compared with MKO-derived BMDM,inflammation-related genes were significantly enriched in WT group.Consistently,RT-qPCR results showed that the expression of inflammatory cytokines(Il-1b,11-6 and Tnf-α)and chemokines(Ccl5 and Cxcl10)was significantly lower in BMDM of MKO group than those of WT group after stimulated with LPS or supernatant of mouse fatty liver tissue homogenate.3.2 Zhx2 suppressed pro-inflammatory response of macrophage and mediated in hepatic steatosis via NF-κB signaling pathway.The BMDM from WT and MKO mice were stimulated by LPS,and inflammation-related pathways were detected through western blot.The results showed that the levels of the total protein level of p65,phosphorylated p65 and phosphorylated JNK of BMDM in MKO group were significantly lower than WT group.In order to determine whether Zhx2 regulates the inflammatory response of macrophage through p65,BMDM from WT and MKO mice were pretreated with p65 inhibitor QNZ and then stimulated by LPS.The RT-qPCR results showed that the difference in mRNA expression of macrophage pro-inflammatory cytokines between the two groups was abolished after the addition of p65 inhibitor QNZ.BMDM of two groups were co-cultured with primary hepatocytes,treated with QNZ or DMSO,then treated with OA.The fat deposition of cultured hepatocytes showed that the lipid deposition of primary hepatocytes co-cultured with BMDM in MKO mice was significantly less than that in WT mice,and this difference disappeared after QNZ pretreatment.3.3 Zhx2 affected macrophage migration and phagocytosis.In order to study the effect of Zhx2 on the migration ability of macrophage,macrophage from two groups was detected by tanswell test.The results showed that the migration ability of macrophage in MKO mice was significantly lower than that in WT mice.We test the phagocytosis of macrophage with fluorescent microspheres.Fluorescence microscopy and flow cytometry,the results shows that the phagocytosis of macrophage in MKO mice was stronger than that in WT mice.These informations suggest that Zhx2 regulates multiple functions of macrophage.Conclusion and significanceCollectively,this study found for the first time that Zhx2 inhibits the progression of NASH by regulating macrophage.The expression of Zhx2 in macrophages was increased in high-fat environment.The loss of Zhx2 in macrophage protected mice from NASH.Mechanically,Zhx2 regulates multiple functions of macrophage,including inflammatory response,migration and phagocytosis.This may at least partically be due to that Zhx2 enhances inflammatory response of macrophages by up-regulating p65 experssion and promoting the activation of NF-kappa B.The detail mechanism by which Zhx2 enhances the expression of p65 and regulates macrophage migration and phagocytosis need to be further studied.Our research here reveals a new mechanism of NASH progress and provides a new target for clinical intervention.