Tim-4 Reprograms Cholesterol Metabolism To Suppress Antiviral Innate Immunity In Macrophages

Background and aims:Antiviral innate immune response acts as the first line of the host defense against viral infection.Pattern-recognition receptors(PRRs)detect and recognize pathogen-associated molecular patterns(PAMPs),eliciting antiviral innate response.Various PRRs recognize viral nucleic acids,recruit adaptor protein accordingly and trigger a series of signaling cascades leading to type Ⅰ interferon(IFN)expression.Type Ⅰ IFN further activates the JAK-STAT signaling pathway to initiate the expression of IFN-stimulated genes(ISGs),which exerts antiviral effects eventually.Cholesterol is indispensable for viral entry,replication,assembly and release process.Virus infection disturbs cholesterol homeostasis of the host.Furthermore,the cholesterol metabolism is closely related to the antiviral innate immunity.Limiting cholesterol biosynthesis upregulates type Ⅰ IFN and ISG expression.Thus,suppression of cholesterol biosynthesis pathway may be critical for efficient type Ⅰ IFN response and virus elimination,which has been emerged as a promising strategy for antiviral therapy.T-cell immunoglobulin and mucin domain-containing molecule 4(Tim-4)is mainly expressed on antigen presenting cells,including macrophages and dendritic cells.Tim-4 is essential for the maintenance of resident macrophage homeostasis and function.Acting as a phosphatidylserine(PtdSer)receptor,Tim-4 mediates the uptake of apoptotic cell debris and the enveloped virus entry.However,the exact role of Tim-4 in the antiviral immune response and cholesterol metabolism is completely unclear so far.This study aims to explore the role of Tim-4 in antiviral innate immune response and reveal the regulatory mechanism of cholesterol homeostasis in macrophages,which would clarify the novel biological function of Tim-4 and provide new targets for antiviral strategy.Methods and results:I.Tim-4 suppresses type Ⅰ IFN response and promotes virus replication in macrophagesTo investigate whether Tim-4 regulates the antiviral innate immunity,we first set up vesicular stomatitis virus(VSV)and sendai virus(SeV)infection model.The results of western blotting,flow cytometry and qRT-PCR demonstrated that viral infection induced Tim-4 expression in macrophages.Furthermore,we treated wild-type(WT)and Tim-4 knock out(Tim-4 KO)mice derived primary peritoneal macrophages(PEMs)with RNA viruses or nucleic acid analogs.The results indicated that Tim-4 deficiency significantly enhanced the type Ⅰ IFN response and inhibited viral replication.Tim-4 KO mice exhibited a significant increase in IFN-β expression in serum and tissues.Accordingly,VSV load and tissue damage were decreased in Tim-4 KO mice.To exclude the effect of Tim-4 on other immunocytes in vivo,we performed adoptive transfer of WT or Tim-4 KO PEMs following depletion of macrophages in a mouse model of VSV infection.We found that Tim-4 KO PEMs-transferred mice showed enhanced IFN-β expression and impeded VSV replication.Collectively,Tim-4 indeed negatively regulates the type Ⅰ IFN response of macrophages and consequently facilitates viral replication.Ⅱ-Tim-4 inhibits type Ⅰ interferon response by enhancing SREBP2 activation to promote cholesterol synthesis in macrophagesTo explore the mechanism by which Tim-4 regulates the antiviral response in macrophages,RNA sequencing analysis of WT and Tim-4 KO PEMs was performed.Gene set enrichment analysis revealed that cholesterol homeostasis-associated genes were significantly enriched in WT PEMs,indicating the potential relationship between Tim-4 and cholesterol metabolism in macrophages.Filipin Ⅲ and Amplex red staining demonstrated that Tim-4 markedly reduced cholesterol contents in macrophages.Sterol-regulatory element binding protein 2(SREBP2)is a master transcriptional regulator of cholesterol synthesis.We found that Tim-4 markedly promoted SREBP2 activation and nuclear localization.Consistently,the expression of SREBP2 downstream enzymes were decreased in Tim-4 KO PEMs.Moreover,VSV infection magnified the effect of Tim-4 on cholesterol accumulation and SREBP2 activation.We next clarified whether Tim-4 restrains the IFN response by regulating cholesterol biosynthesis.To address this issue,we treated RAW264.7 cells and PEMs with fatostatin,a synthetic inhibitor of SREBPs activation.The results showed that overexpression of Tim-4 in RAW264.7 cells significantly restricted VSV-induced Ifnb mRNA expression under mock conditions,while Tim-4 had no effect on Ifnb expression upon fatostatin addition.Moreover,fatostatin prevented the increased IFN-β production and IRF3 phosphorylation observed in VSV-stimulated Tim-4 KO PEMs.In vivo experiment also suggested that Tim-4 deficiencymediated upregulation of IFN-β production in tissues were greatly suppressed by fatostatin.Fatostatin treatment significantly abolished the difference in VSV load and tissue damage between WT and Tim-4 KO mice.In addition,we found that si-Srebf2 and exogenous cholesterol supplement offset the role of Tim-4 in regulating type Ⅰ IFN response and viral replication.Taken together,these data demonstrate that Tim-4 suppresses the antiviral response in an SREBP2-dependent manner both in vitro and in vivo.Ⅲ.Tim-4 inhibits the binding of Insigl to SCAP to promote SREBP2 activation and cholesterol synthesis in macrophagesTo elucidate the underlying mechanisms by which Tim-4 promotes SREBP2 activation,HEK293T and THP-1 cells were transfected with overexpression constructs encoding the Nterminal Flag-tagged full-length SREBP2 or the Flag-tagged mature form of SREBP2 together with or without Tim-4 plasmid.We found that Tim-4 promoted the activation of exogenous full-length SREBP2,but no effect was found in exogenous mature SREBP2.These results suggest that Tim-4 might affect SREBP2 at the posttranscriptional level and before the nuclear entry step,which includes the endoplasmic reticulum(ER)-to-Golgi trafficking process.Further,confocal microscopy and subcellular fraction strategy demonstrated that Tim-4 reduced ER-located SREBP2 while increasing SREBP2 localization in the Golgi apparatus,suggesting that Tim-4 may promote SREBP2 trafficking from the ER to the Golgi.Since disassociation of inhibitory protein Insigs with SCAP in the ER triggers SREBP2 translocation and Tim-4 was found to be localized in the ER where Insigs and SCAP are anchored,we used co-immunoprecipitation(Co-IP)and confocal assay to determine the relationship of Tim-4 with Insigs or SCAP.The results demonstrated that Tim-4 interacted with Insigl or SCAP.Tim-4 was able to disrupt the Insigl-SCAP interaction in a dosedependent manner.Moreover,a series of domain deletion mutants of Tim-4 were included in this study.Co-IP assays showed that Tim-4 Δcyto resulted in the loss of the ability to interact with Insigl or SCAP,indicating that the cytoplasmic domain was essential for Tim-4 binding to Insigl or SCAP.Tim-4 Δcyto lost the ability to regulate SREBP2 activation and antiviral response.In conclusion,Tim-4 promotes SREBP2 activation and cholesterol biosynthesis by regulating SREBP2 trafficking from the ER to the Golgi,which is responsible for Tim-4mediated negative regulation on antiviral innate immunity in macrophages.Conclusion and significance:This study elucidates the undescribed role and mechanism of Tim-4 in regulating the type Ⅰ interferon signaling and cholesterol metabolism in macrophages.Tim-4 disrupts the interaction between Insigl and SCAP,promoting SREBP2 activation and de novo cholesterol synthesis,thereby inhibiting the antiviral innate immune response.In conclusion,this study provides a novel target for the antiviral drug design and identifies Tim-4 as a new regulator of cholesterol homeostasis and macrophage function remodeling.