Tim-3 Alleviates Liver Injury Induced By NASH By Regulating Macrophage Activation

BackgroundAs a multisystem disease,Nonalcoholic fatty liver disease(NAFLD)severely affects the health of human beings.In China,NAFLD has become a most common chronic liver disease besides HBV.While most NAFLD patients remain asymptomatic,roughly 20%of them develop into a severer condition which is evidenced by steatosis and inflammation and known as non-alcoholic steatohepatitis(NASH).Some NASH patients can progress to cirrhosis or even hepatocellular carcinoma(HCC).So far,multiple hits theory is the dominating pathogenesis explanation of NAFLD.Hepatic lipid deposition appears in the early stage of NAFLD and massive lipid accumulation leads to severer mitochondria damage and more Reactive oxygen species(ROS)production,resulting in inflammatory cytokines release and hepatotoxicity.More and more evidence approves that accumulated ROS is one of the key risk factors for NASH development.Therefore,to identify a critical regulator interfering with ROS-mediated hepatic inflammation in NASH mice is strongly suggested.In liver,there exist numerous innate immune cells participating in the pathogenesis of multiple liver diseases.Accumulated evidence proves that key innate immune cells in liver,especially kupffer cells,play important roles in NASH development.According to the reports,amounts of activated macrophages(kupffer cells,KCs)appear in NASH liver tissue.Activated KCs aggravate liver injury by producing mediators such as ROS and inflammatory cytokines in MCD(methionine-and cystine deficient diet)induced NASH model.According to Baeck C,blocking MCP-1 can efficiently inhibit hepatic macrophages infiltration and improve MCD induced NASH liver injury.Thus,targeting liver macrophages and ROS mediated inflammation has a constructive meaning for NASH therapy.Tim-3(T cell immunoglobulin and mucin-domain containing-3)was first recognized as T cell immune regulator.However,in recent years,more and more evidence confirm that Tim-3 as well is expressed on NK,NKT and macrophages.Tim-3 is highly constitutively expressed on monocytes/macrophages and is regulated by different microenvironment.By affecting the function of monocytes/macrophages,Tim-3 participates in the process of diverse disease.In tumor microenvironment,elevated TGF? plays roles in enhancing Tim-3 expression on tumor associated macrophages and increased Tim-3 can then promote HCC development by regulating macrophage polarization.According to another report,Tim-3+CD14+ macrophages has a positive correlation with serum ALT in CHB(Chronic hepatitis B)patients.However,there are no reports yet to show whether Tim-3 participates in NASH by regulating hepatic macrophages.Also,whether Tim-3 is involved in macrophage activation or ROS mediated inflammation in NASH still need further verification.Herein,we applied MCD induced NASH model to discuss the potential regulatory roles of Tim-3 on macrophages in NASH.Methods and Results1.Tim-3 is increased significantly in NASH liver and Tim-3KO promotes NASH progressTo discuss the potential roles and possible mechanism of Tim-3 in NASH mice,we fed WT C57BL/6 mice with MCD or HFD(High-Fat Diet)diet to establish NASH animal models.Liver tissues were collected from MCD 2 weeks and HFD 24 weeks mice to analyze Tim-3 expression by IHC and western blot techniques.The data showed that,compared to ND(normal diet)group,Tim-3 was enhanced significantly in livers of MCD and HFD mice,indicating that Tim-3 may be involved in the pathogenesis of NASH.To furtherly determine whether Tim-3 affects NASH induced liver injury,we successfully constructed Tim-3KO mice model by TALEN technique.Firstly,we separated liver monocytes and analyzed the knockdown efficiency of Tim-3 in hepatic CD45+ monocytes by performing FCM.Then,we used WT and Tim-3KO mice to establish MCD-induced NASH model and evaluated the exact roles of Tim-3 in NASH mice.In vivo results displayed that,compared to WT MCD mice:serum ALT was elevated,liver TG was increased,hepatic vacuolization was enlarged and liver fibrosis was severer in Tim-3KO mice.The above data suggested Tim-3 as a protector in MCD-induced NASH mice.2.Tim-3 on different hepatic macrophage subpopulations is enhanced in NASH liverLivers macrophages,NK and NKT cells occupy the majority of liver innate immune cells,with high Tim-3 surface expression.The mentioned hepatic innate immune cells play key roles in NASH progress.To check the exact cell source of increased Tim-3 expression in NASH liver,we collected liver tissues from MCD 2 weeks mice and separated liver monocytes by 40%percoll.And we then performed FCM to detect the proportion of hepatic macrophages,NK and NKT cells.The results showed that the percentage of NKT was decreased significantly,while NK and macrophages were increased in NASH livers.We furtherly detected Tim-3 expression in hepatic NK cells and adherent liver macrophages.We found that Tim-3 expression on macrophages was increased but showed no significant difference in hepatic NK.Hepatic macrophages are heterogeneous,consisting of F4/80+CDllb+ liver motile macrophages,F4/80+CD68+and F4/80+CD169+ residential liver macrophages.FCM(Flow Cytometry)results displayed that Tim-3 expression was enhanced on all detected liver macrophages in MCD mice.Meanwhile,FMO(Fluorescence Minus One)test strongly approved the specificity of FCM results.In HFD 24W mice,the same tendency was discovered with significantly enhanced Tim-3 expression on F4/80+CD11 b+ and F4/80+CD68+hepatic macrophages.To analyze Tim-3 level in human fatty livers,we collected paracancerous tissues with or without vacuolation from livers of HCC patients and conducted fluorescent immunohistochemistry.Compared to groups without hepatic vacuolation(as normal controls),Tim-3 expression on CD68+ hepatic macrophages was increased in vacuolated liver tissues(as a reminder of fatty liver).Taken together,Tim-3 expression on diverse macrophages subgroups from different NASH models and human vacuolated livers was remarkably increased.The above results strongly suggested that hepatic macrophages were the main cell source of increased Tim-3 and played important roles in NASH development.3.MCD liver homogenate and OA contribute to Tim-3 overexpression in macrophagesTo identify the possible inducer or microenvironment factor which leads to Tim-3 overexpression in NASH liver,we employed liver homogenate from ND and MCD mice to stimulate PM(peritoneal macrophages)and analyzed Tim-3 levels by qPCR technique.Compared to ND group,Tim-3 expression was significantly boosted by MCD liver homogenate.Next,we detected massive lipid deposition and high Tim-3 expression in PM after unsaturated fatty acid OA(oil acid,OA)stimulation.Our results indicated that OA and NASH hepatic microenvironment could be potential inducers for Tim-3 overexpression in macrophages.4.Tim-3 inhibits ROS and downstream cytokines release in macrophagesROS from macrophages plays important roles in NASH.Firstly,we collected adherent hepatic macrophages from MCD and HFD mice and detect ROS levels.Our result was coincident with previous report,showing higher ROS levels in adherent hepatic macrophages from NASH livers and pointing macrophage derived ROS as a key player in NASH.Furtherly,to determine whether Tim-3 regulated macrophages derived ROS,we collected livers of WT and Tim-3KO MCD 2W mice and detected NOX2 expression by western blot.Compared to WT group,NOX2 expression was much higher in Tim-3KO MCD liver,suggesting Tim-3 act as a potential regulator upon ROS production.To certify the above hypothesis,we applied LPS or PA to stimulate anti-Tim-3 pretreated BMDM(bone marrow-derived macrophages).Then,we detected ROS levels by FCM analysis.Our data showed that Tim-3 negatively regulated ROS generation in BMDM.Consistently,we got the same conclusion in siTim-3 pretreated PM.Consistent with the above results,in Tim-3KO derived BMDM and PM,we observed the inhibitory function of Tim-3 on ROS and Mitochondrial ROS production.Additionally,higher ROS levels were observed in Tim-3KO hepatic macrophages.Taken together,Tim-3 acts as an inhibitor upon ROS generation in macrophages.According to the report,ROS could trigger inflammasome activation and enhance related inflammatory cytokines release.Hence,we furtherly studied whether Tim-3 had the capability to inhibit ROS triggered inflammasome activation.Firstly,we compared levels of inflammasome and related cytokines in livers of WT and Tim-3KO MCD mice.Compared to WT group,significantly elevated caspase-1 p20,IL-1? and IL-18 was observed in Tim-3KO MCD livers,however,TNF-? level showed no difference in Tim-3KO mice.Also,results from anti-Tim-3 pretreated BMDM is with the same trend.Furtherly,we pretreated WT and Tim-3KO derived PM with NAC(a small molecular inhibitor for ROS)and then stimulated them with HFD liver homogenate.Interestingly,the difference of IL-1? in WT and Tim-3KO group was reversed after NAC treatment.The above data indicated Tim-3 as a potential regulator on ROS/inflammasome axis and downstream cytokine release in macrophages.5.The aggravated liver injury of Tim-3KO MCD mice is alleviated by inhibiting ROSTo furtherly testify the Tim-3 function of regulating ROS generation in NASH livers.We intervened WT and Tim-3KO MCD mice with NAC.150mg/kg NAC was given to WT and Tim-3KO MCD mice every other day.Serum and liver tissues were obtained after 2 weeks to observe liver inflammation index.Serum was separated for ALT detection;liver homogenate was prepared for TQ IL-1? and IL-18 analysis;liver protein was extracted for caspase-1 p20 detection;while liver paraffin section was obtained for vacuolization analysis.The above data showed no significant difference between WT and Tim-3 KO MCD group,suggesting NAC pretreatment could improve liver injury induced by Tim-3KO treatment.Taken together,the aggravating impact of Tim-3KO on MCD mice is ROS dependent.Conclusion:NASH liver microenvironment enhances Tim-3 expression on diverse hepatic macrophages;Tim-3 inhibits ROS and downstream inflammatory cytokines release in macrophage,protecting liver damage induced by NASH.Innovation and significance:For the first time,our study showed that Tim-3 was highly expressed on different macrophages subpopulations in NASH livers and enhanced Tim-3 could inhibit ROS and downstream response,reducing NASH mediated liver injury.Above-mentioned studies not only highlighted the roles of Tim-3 in innate immune response in NASH livers,but also offered new target for NASH treatment.