Tim-4 Participates In The Development Of NAFLD By Regulating Macrophages

As a chronic liver disease,fatty liver disease is a clinicopathologic syndrome characterized by excessive deposition of triglycerides in the liver,and now it has become the second most common liver disease inferior to viral hepatitis in China.Based on etiology,fatty liver disease is classified into two categories:alcoholic fatty liver disease and non-alcoholic fatty liver disease(NAFLD).The global prevalence of NAFLD is estimated to be 24%,growing in the recent years and showing an increased risk in youngers.Without exterior intervene,NAFLD may progress from simple steatosis to non-alcoholic steatohepatitis(NASH),advanced liver fibrosis,cirrhosis and even hepatocellular carcinoma(HCC)eventually.NAFLD would become the leading cause of liver transplantation in future,which represents a substantial public health concern.Currently,there is no effective pharmacological treatment targeted NAFLD in humans to date.Thus,it is significant and urgent to understand and explore the pathogenesis of NAFLD,which may supply new immune targets for NAFLD prevention.NAFLD displays complicated pathogenesis and is characterized by excessive lipid deposition and inflammation in liver,including the imbalance of lipid metabolism in hepatic parenchyma cells and the excessive secretion of pro-inflammatory cytokines by immune cells to promote the progress of NAFLD.A growing evidence shows that a reasonable number of liver parenchyma cells(the largest cell proportion in liver,accounting for 60%)and Kupffer cells(KCs,the largest group of immune cells in liver,accounting for 15%)play important roles in the progress of NAFLD.Discovering the novel regulatory molecules negatively regulating inflammation and lipid metabolism will provide new targets for treating NAFLDT cell immunoglobulin mucin domain protein-4(Tim-4),as the natural ligand of Tim-1,belongs to type I transmembrane glycoprotein,and contains IgV domain,mucin domain,transmembrane domain and intracellular domain.Tim-4 is selectively and highly expressed in antigen presenting cells,especially in activated macrophages and mature dendritic cells,and also in B1 cells and iNKT cells,but not in T cells.Tim-4,as the receptor of phosphatidylserine(PS),binding PS exposed on the cell membrane surfaces undergoing apoptosis through the FG-CC’ pocket of IgV region,which mediates the phagocytosis of apoptotic cells by macrophages and maintains the immune homeostasis.In recent years,studies confirmed that macrophage Tim-4 inhibited inflammation under various conditions of immune activation,such as hepatitis induced by ConA and sepsis induced by LPS,and it was also found that the negative correlation of Tim-4 in peripheral blood mononuclear cells(PBMCs)with serum IL-1β and IL-18 in type II diabetes.These results suggest that Tim-4 in macrophages may be a promising target for the treatment of inflammatory related diseases.Recently,genome-wide association studies have identified that the relative expression of Tim-4 in liver tissues of rats exposed to a high-fat diet(HFD)is 3.10-fold higher than observed in normal diet(ND)controls,and certain single nucleotide polymorphisms in Tim-4 show associations with lipid traits,such as triglyceride(TG)and low density lipoprotein(LDL).However,whether Tim-4 is involved in abnormal lipid metabolism induced diseases such as NAFLD remains unaddressed.Here,in this study we aim to explore the novel biological functions of hepatic Tim-4 in NAFLD and unveil the pathogenesis of NAFLD,which would shed new lights on diagnosis and treatment of NAFLD or inflammatory liver diseases by targeting hepatic Tim-4.In summary,the research is aimed to mainly solve the following problems:(1)What is the role of Tim-4 in NAFLD?(2)Which kind of cells do NAFLD microenvironments induce the expression of Tim-4 in?(3)How does Tim-4 regulate macrophages to participate in NAFLD?Part 1 The role of Tim-4 in NAFLDIn order to uncover the role of Tim-4 in NAFLD,clinical samples of healthy subjects and NAFLD patients were collected to detect the expression level of Tim-4 in plasma and PBMCs.Paracancerous tissues of HCC were used to measure Tim-4 expression in liver tissues with or without steatosis.In addition,Tim-4 expression was further confirmed in liver tissues from NAFLD mice models induced by high-fat diet(HFD)or methionine choline deficiency(MCD)diet.In order to further elucidate the role of Tim-4 in liver tissues,the NAFLD model was induced with MCD diet in Tim-4 knockout(Tim-4-/-)mice and wild type(Tim-4+/+)mice.The role of Tim-4 in NAFLD was determined by detecting indicators of lipid deposition and hepatic inflammation.1.1 Clinical data showed the changes of Tim-4 expression in patients with NAFLD1.1.1 Elevated secretory Tim-4(sTim-4)in plasma from patients with NAFLDIn order to determine the relationship of Tim-4 and NAFLD in clinical samples,we collected plasma of 117 patients with NAFLD and 60 healthy subjects,and sTim-4 level was detected by ELISA.The results showed that the plasma sTim-4 in patients with NAFLD was significantly higher than that in healthy controls.1.1.2 Decreased mRNA level of Tim-4 in PBMCs from NAFLD patientsPBMCs were isolated by Ficoll reagent from peripheral blood of 68 NAFLD patients and 44 healthy subjects,and Tim-4 expression was detected by Real-time quantitative PCR(qPCR).The results indicated that the expression of Tim-4 mRNA was decreased in PBMCs from NAFLD patients.1.1.3 Increased Tim-4 in liver tissues with steatosisImmunohistochemical staining was performed to further detect Tim-4 expression in liver tissues on HCC tissues chip,and Tim-4 expression level in all paracancerous tissues were analyzed.The results showed that the expression of Tim-4 in liver tissues with ballooning vacuolation was higher than that in tissues without steatosis.Then,immunofluorescence(IF)staining was carried out in paracancerous tissues of HCC with or without steatosis.The results were consistent with that of IHC.1.2 Enhanced Tim-4 in liver tissues of NAFLD mice modelsIn order to clarify Tim-4 expression level in liver tissues of NAFLD mice,NAFLD mice models were established with HFD diet for 6 months or MCD diet for 2 weeks respectively and successfully,evidenced by body weight,Hematoxylin-eosin(HE)and oil red O(ORO)staining,TG,cholesterol(CHO)and LDL in liver homogenate supernatants(LHSs)and alanine aminotransferase(ALT)in serum.We found that the expression level of Tim-4 in liver tissues from HFD and MCD fed mice was significantly higher than that in ND fed mice,certified by western blot(WB),qPCR,IHC,and IF.These results suggested that Tim-4 in liver tissue might be involved in the regulation of NAFLD progress.1.3 Tim-4 knockout aggravates MCD-induced NAFLDTo address the roles of enhanced Tim-4 expression in liver tissues of NAFLD models,we conducted MCD fed mice model in Tim-4 knockout(Tim-4-/-)mice and wild type(Tim-4+/+)mice.Results displayed that Tim-4-/-mice with MCD diet exhibited more obvious hepatic inflammation and severer lipid accumulation verified by accelerated body weight loss,higher ALT and AST levels in serum,more ballooning vacuolation in HE and ORO staining,higher TG,CHO and LDL in LHSs relative to corresponding controls.These data indicated that Tim-4 exerted a protective role in NAFLD progression and targeting Tim-4 might be an effective strategy for intervention of NAFLD.Part 2 The effects of NAFLD microenvironments on Tim-4 expression in immune cells and hepatic parenchymal cellsNAFLD is characterized by the excessive lipids accumulation in cytoplasmic presented as ballooning vacuolation of different sizes in hepatocytes and increased inflammatory cell infiltration in the absence of alcohol abuse.Thus,abnormal lipid metabolism in hepatocytes and increased inflammation caused by activation of hepatic macrophages are the typical manifestations of NAFLD.As a group of innate immune cells in the liver,KCs account for 80%-90%in resident macrophages of human body,which are abundant and responsible for the innate immune response in liver.Under physiological conditions,macrophages,polarized to be an anti-inflammatory M2-like phenotype maintain liver homeostasis by clearing apoptotic cells via scavenger receptors and activating T cells to generate cytotoxic T lymphocytes via Toll-like receptors.In the HFD/MCD diet-induced NAFLD mice models,monocytes tend to be a pro-inflammatory M1-like phenotype.LPS,reactive oxygen species,saturated fatty acids and cholesterol crystals activate macrophages through NF-κB and JNK pathways.Activated KCs play a central role in NAFLD by up-regulating the synthesis and release of multiple inflammatory cytokines including IL-1β,TNF-α,IL-6,TGF-β,and TNF-related apoptosis-inducing ligand,CCL2,CCL3-5,CCL7,CCL8,and further promoting infiltration of monocytes and apoptosis of hepatocytes.Eventually,all these factors aggravate the progress of NAFLD.Eliminating macrophages by clodronate-liposome alleviates the inflammation and lipid accumulation of NAFLD.These studies confirm that macrophages activation aggravates NAFLD,and plays an important role in the progression from NAFLD to NASH.Tim-4 was initially found to be highly expressed in macrophages.Our previous studies also showed that LPS,IFN-y and ConA could enhance Tim-4 expression in macrophages.It was also reported that damage-associated molecular patterns released from chemotherapy-damaged tumor cells also induced Tim-4 expression in tumor-associated macrophages and dendritic cells.So,we would like to make clear whether NAFLD microenvironments could up-regulate Tim-4 expression in KCs.Hepatic parenchyma cells,as epithelial cells source,accounting for the largest volume(80%)and quantity(60%)in liver,are responsible for the physiological functions of liver,including glucose,lipid,protein and vitamin metabolism,key protein synthesis,detoxification,and bile secretion.Under NAFLD microenvironments,the balance of lipid metabolism in hepatic parenchyma cells is disturbed,and macrophages also contribute to dysfunction of lipid metabolism.Increasing evidences show that IL-1(3 secreted from activated macrophages indirectly regulate lipid metabolism in hepatocytes by promoting the synthesis of TG and inhibiting fatty acid oxidation in hepatic parenchyma cells.In this part,we also wonder whether NAFLD microenvironments lead to the ectopic expression of Tim-4 in liver parenchyma cells.2.1 Expression of Tim-4 in NK and NKT cells from liver tissues of NAFLD miceFirstly,we detected the expression of Tim-4 in liver innate immune cells-NK and NKT cells from isolated liver mononuclear cells of NAFLD mice induced by ND and HFD by FCM.The results showed that there were no significant differences of Tim-4 expression in CD3-NK1.1+CD49a+DX5-NK cells and CD3+NK1.1+NKT cells between NAFLD and control mice,and both NK and NKT cells only showed a relatively low endogenous expression of Tim-4.2.2 Increased Tim-4 expression in KCs from liver tissues with NAFLDIn view of the constitutive expression of Tim-4 in macrophages,we detected the expression of Tim-4 in KCs of NAFLD mice and human liver tissues with steatosis.FCM demonstrated that Tim-4 was significantly increased in liver CD11b+F4/80+macrophages from isolated liver mononuclear cells of HFD and MCD fed mice,and MCD model mice exhibited the most robust increase of Tim-4 in liver macrophages.12-week-old db/db mice were used as a natural NAFLD model and the increased proportion of Tim-4 in CD11b+F4/80+ macrophages from isolated liver monocytes was also confirmed in db/db mice by FCM.We further confirmed enhanced Tim-4 in hepatic macrophages by IF.The results showed more co-localization of Tim-4 and macrophage marker CD68 in liver tissues of NAFLD mice relative to controls,and enhanced Tim-4 was also observed in CD68 hepatic macrophages from human HCC adjacent tissues with ballooning vacuolation.In summary,the results in both NAFLD mice models and human liver tissues with steatosis confirmed that the elevated expression of Tim-4 in KCs of NAFLD than controls.Thus,we wonder whether NAFLD microenvironments elevate Tim-4 expression in macrophages in vitro.2.3 NAFLD microenvironments up-regulated the expression of Tim-4 in macrophagesLHSs of liver tissues from NAFLD mice were prepared to stimulate KCs,PEMs and BMDMs to mimic the NAFLD microenvironments in vitro.FCM,WB and qPCR analysis demonstrated that Tim-4 expression was increased significantly in macrophages following stimulation with LHSs from HFD and MCD fed mice respectively.We further stimulated PEMs with small molecular lipid catabolic metabolites-unsaturated fatty acid-oleic acid(OA)in vitro,and the consistent results were obtained in PEMs by WB and qPCR.2.4 Elevated Tim-4 expression in hepatic parenchyma cells with NAFLDBesides immunocytes,we also detected the expression level of Tim-4 in liver parenchyma cells of NAFLD mice.The expression of Tim-4 in hepatic parenchyma cells from liver tissues of HFD mice was significantly higher than that of ND mice by WB and qPCR analysis.IF assay showed that more co-expression of Tim-4 and hepatocytes marker Cytokeratin-18(CK18)was observed in liver tissues of NAFLD models including HFD,MCD fed mice and db/db mice relative to controls.We also detected augmented expression of Tim-4 in CK18+ hepatocytes from human paracancerous tissues of HCC with steatosis by IF.2.5 NAFLD microenvironments up-regulated the expression of Tim-4 in hepatocytesCytokines,OA and OX-LDL were used to stimulate human HCC cell lines to mimic the NAFLD microenvironments in vitro.Results demonstrated that Tim-4 expression was increased in HepG2 cells with OA,OX-LDL or cytokines stimulation respectively,as well as HuH7 cells stimulated with OA.We further stimulated Hepal-6 cells with LHSs from HFD fed mice,and the consistent results were confirmed by qPCR.Part 3 Tim-4 inhibits NLRP3 inflammasome via LKB1/AMPKα pathway in macrophagesSubsequently,we further explored the possible mechanisms of Tim-4 regulating macrophages in NAFLD.The previous studies show that Tim-4 inhibits macrophages mediated inflammation under multiple conditions,indicating the potential negative regulation of Tim-4 on IL-1β production,the secretion of which is mainly determined by inflammasome activation.Emerging evidences support that NLRP3 inflammasome is the best known inflammasome and has become the hot topic in multiple liver diseases including NAFLD.NLRP3 inflammasome is activated both in murine models and in humans with NAFLD.NLRP3 inflammasome related gene-knockout or specific inhibitor could alleviate hepatic steatosis and inflammation.Therefore,we addressed the role of Tim-4 in NLRP3 inflammasome activation and related mechanisms.3.1 Activation of NLRP3 inflammasome in liver tissues of NAFLD miceFirst co-staining of NLRP3 and ASC was tested in liver tissues of NAFLD mice by IF,and cytokines IL-1β and IL-18 were assayed in LHSs from NAFLD mice by ELISA.Consistent with the references,more co-localization of NLRP3 and ASC was found in liver tissues of HFD mice and MCD mice compared with corresponding controls,accompanied by increased release of IL-1β and IL-18 in LHSs from HFD and MCD fed mice,indicating the NLRP3 inflammasome activation in livers under conditions of NAFLD.3.2 Tim-4 knockout promotes activation of inflammasome in NAFLD micePreviously,we found that increased expression of Tim-4 in PBMCs negatively correlated with IL-1β and IL-18 levels in type 2 diabetes.Since NLRP3 inflammasome major resides in macrophages,above results impel us to consider whether NAFLD environments induced Tim-4 expression in macrophages contributes to NLRP3 inflammasome activation in livers.We subsequently evaluated the regulation of Tim-4 on NLRP3 inflammasome in macrophages and HEK293 cells with reconstitution system of NLRP3 inflammasome in vitro.Interestingly,after stimulation with LPS/ATP or LPS/Nigericin(NIG),IL-1β and IL-18 levels in culture supernatants of PEMs and BMDMs from Tim-4-/-mice were significantly higher than those from Tim-4+/+mice.The effect of Tim-4 inhibiting NLRP3 inflammasome activation was further verified in purified KCs and HEK293 cells using NLPR3 inflammasome reconstitution system together with pTim-4 in vitro.We found that Caspase-1 activation and IL-1βconcentration decreased gradually with Tim-4 increasing in a dose dependent manner both in purified KCs and HEK293 cells.3.3 PS binding motif is required for Tim-4 mediated inhibition of NLRP3 inflammasome activationIn order to identify the key domain involved in Tim-4 mediated NLRP3 inflammasome inhibition,we successfully constructed mutant vectors with deletion of Tim-4 different domains.These deletions were named △IgV,AMucin,and △Cyto respectively.Then the reconstitution system of NLRP3 inflammasome was set up together with these recombinants.We found that the inhibitory effect was obviously lost in the △IgV group compared with WT Tim-4,AMucin and ACyto groups.Similar tendency was observed in measurements of IL-1β level from culture supernatants.The PS binding site and RGD motif,two functional domains in the IgV region of Tim-4,were further assayed with mutation constructs(Tim-4 mutPS and Tim-4 mutRGD).The inhibitory effect of Tim-4 on NLRP3 inflammasome activation was almost abolished in the Tim-4 mutPS group but not in mutRGD group.In summary,Tim-4 inhibited NLRP3 inflammasome activation depending on the PS binding site.3.4 Tim-4 inhibits NLRP3 inflammasome through activating autophagyAs a transmembrane protein,Tim-4 inhibiting NLRP3 inflammasome in the cytoplasm through extracellular domain amazed us greatly.Interestingly,we observed that Tim-4 decreased the protein level of NLRP3 gradually in a concentration dependent manner in Tim-4 overexpressed HEK293 cells,but the effect was not obvious at mRNA level.Accordingly,Tim-4 appeared to promote NLRP3 inflammasome components degradation.To assay Tim-4 mediated protein degradation pattern of NLRP3 inflammasome components,HEK293 cells co-transfected with pTim-4 and pcDNA3-NLRP3-Flag plasmids were treated with proteasome inhibitor MG-132,PI3K class III inhibitor 3-methyladenine(3-MA)or autophagosome-lysosome fusion inhibitor chloroquine(CQ).The results showed that CQ and 3-MA treatment significantly increased NLRP3 accumulation,suggesting that autophagy was probably involved in Tim-4-mediated NLRP3 protein degradation.To further validate the potential role of autophagy pathway in Tim-4 suppressing NLRP3 inflammasome,NLRP3 inflammasome reconstitution system was performed in HEK293 cells with or without CQ treatment.Without CQ treatment,Tim-4 over-expression significantly attenuated NLRP3 inflammasome components and increased autophagic flux as assessed by increased in the LC3-II/GAPDH ratio and decreased in p62 level.After treatment with CQ,the protein levels of NLRP3 inflammasome components were increased,and the corresponding autophagic flux was decreased as assessed by increases in both LC3-II/GAPDH ratio and p62 level,which were even more significant in the pTim-4 group.3.5 Tim-4 induces autophagy by activating AMPKa,which is PS binding domain dependentAMPKa is a key energy sensor in maintaining cellular homeostasis by regulating both anabolic and catabolic processes,including autophagy.It has been reported that Tim-4 activates autophagy by interacting with AMPKα1 during tumor chemotherapy.In this study,we further demonstrated that Tim-4 interacted with endogenous AMPKa in HEK293 cells and PEMs under starvation through IP(Immunoprecipitation).Furthermore,we found that Tim-4 overexpression increased the phosphorylation of AMPKa(pAMPKa)and autophagic flux significantly in HEK293 cells.Above data indicated that Tim-4 activated autophagy via the AMPKa pathway.We next sought to verify the key domain of Tim-4 responsible for promoting phosphorylation of AMPKa.Similar to the effect of Tim-4 on NLRP3 inflammasome activation,we found that AMPKa phosphorylation and autophagic flux were not impaired in △IgV and Tim-4 mutPS groups.To further validate the key role of the Tim-4 PS binding site in activating AMPKa,PS was introduced to stimulate HEK293 cells cotransfected with Tim-4 and NLRP3 inflammasome components.With PS treatment,pAMPKa was further increased in pTim-4 group relative to controls,however,the pAMPKa remained at a very low level in Tim-4 mutPS group with or without PS treatment.Altogether,PS binding site was necessary for Tim-4 induced pAMPKa and the subsequent formation of autophagic flux.3.6 Tim-4 promotes AMPKa phosphorylation by interacting with LKB1To date,three main upstream kinases are essential for the phosphorylation of Thr172 in AMPKα:calcium(Ca2+)/calmodulin-depedent protein kinase kinase β(CaMKKP),TGF-β-activated kinase-1(TAK1),and LKB1.In the following,siRNA of CaMKKβ,TAK1 or LKB1 was used to verify the major upstream kinase responsible for AMPKa phosphorylation induced by Tim-4.The results showed that Tim-4 induced pAMPKa was reduced significantly only after LKB1 knockdown,but no significant decrease was observed in siCaMKKβ or siTAK1 groups compared with scrambled controls,indicating that LKB1 was essential for Tim-4 induced AMPKa phosphorylation.Furthermore,regardless of siLKB1 transfection,pAMPKa levels remained at a very low level in Tim-4 mutPS group.To further elucidate the relationship of Tim-4 with LKB1 and AMPKa,Co-IP assay was performed in HEK293 cells transfected pTim-4 or pTim-4 mutPS with HA Tag.We found that Tim-4 could immunoprecipitate enhanced AMPKa and LKB1 with Tim-4 plasmid concentration increasing,but decreased in the Tim-4 mutPS group.Consistently,AMPKa also immunoprecipitated increased amounts of Tim-4 and LKB1 with increasing pTim-4,but this effect was weakened in Tim-4 mutPS group.Further,LKB1 could immunoprecipitate Tim-4 and AMPKα in wild type Tim-4 transfected HEK293 cells excepting the Tim-4 mutPS group.3.7 Co-localization of Tim-4,LKB1 and AMPKα in liver tissues of NAFLD miceTo further confirm the co-expression of Tim-4,AMPKα and LKB1 in hepatic macrophages,multiplex fluorescent IHC was performed in liver tissues from NAFLD mice.The staining results confirmed that more infiltration of spindle and crowned macrophages,and more co-localization of Tim-4(pink),AMPKa(red),and LKB1(green)was observed in liver tissues from HFD or MCD diet induced NAFLD mice than controls.In summary,it is evidenced for the first time that NAFLD microenvironments significantly increase Tim-4 expression in macrophages and hepatic parenchyma cells of liver tissues.Tim-4-/-mice display more obvious inflammation and severe steatosis with MCD diet than Tim-4+/+mice.In macrophages,we confirm that enhanced Tim-4 inhibits the activation of NLRP3 inflammasome in vitro,which is depending on PS binding domain.Mechanistically,Tim-4 overexpression promotes interaction between LKB1 and AMPKα,and Tim-4/LKB 1/AMPKα interaction promotes AMPKαphosphorylation,which is responsible for activation of the autophagy signaling pathway,and PS binding motif is required in this process.Taken together,Tim-4 might be a potential target for treatment of NAFLD or inflammatory related diseases in future.However,the specific mechanism of Tim-4 involvement in NAFLD remains to be further investigated in hepatic parenchyma cells.