LAPF Promotes Anti-bacterial Innate Immune Response By Activating Cav1 Through Src

Host eliminates invading pathogens by inducing innate and adaptive immune response.Innate immune cells recognize invading pathogens and induce immune response to defense infection.Pattern recognition receptors?PPRs?,such as Toll-like receptors?TLRs?,RIG-I like receptors?RLRs?,NOD like receptors?NLRs?recognize pathogen-associated molecular patterns?PAMPs?and consequently activate downstream signaling to mediate host defense against pathogens.TLR4,as the receptor that specifically recognizes lipopolysaccharide?LPS?of gram-negative bacteria,activates nuclear factor kappa light-chain-enhancer of activated B cells?NF-?B?and other signaling pathways to induce production of inflammatory cytokines.In addition,TLR4 is internalized into the cytoplasm to trigger production of IRF3-dependent Type-I interferon?IFN?.Phagocytes such as macrophages,dendritic cells?DCs?,monocytes and neutrophils recognize and internalize the invading pathogens by endocytosis.After a series of vesicular trafficking events from endosomes to lysosomes,the internalized pathogens are endocytosed into phagosomes,where they are degraded by hydrolytic enzymes.The resultant pathogen peptides are presented within major histocompatibility complex?MHC?molecules,which are subsequently recognized by T cell receptors and activate CD4⁺and CD8⁺T cells of the adaptive immune system.Caveolae,the specialized lipid rafts,are bulb-shaped plasma membrane invaginations.Caveolae have been reported to be broadly involved in many physiological and pathological processes such as endocytosis of bacteria,lipid homeostasis and signal transduction of cells,mechanoprotection of cell membrane and even the development of cancer.The main component proteins of caveolae are caveolins and cavins.The caveolin protein family consists of three members,namely caveolin-1?Cav1?,caveolin-2?Cav2?and caveolin-3?Cav3?.Cav1 is required for formation of caveolae and is expressed in most cell types.Cav2 promotes the formation of caveolae.We investigated the role of Cav1-interacting protein by mass-spectrometry to identify the key regulator of endocytosis and elimination of bacteria.We found that LAPF?lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains,or PLEKHF1?,which was first cloned by our laboratory,might play important role by interacting with Cav1.LAPF has been reported by us to induce caspase-independent apoptosis via the lysosomal-mitochondrial pathway and act as an adaptor protein that recruits phosphorylated P53 to lysosomes to trigger lysosomal destabilization during apoptosis.However,function of LAPF in innate immune,especially in endocytosis and elimination of bacteria,has not been reported.To study the function of LAPF further,we generated Lapf^fl/fl-LysMcre(Lapf^-/-)mice in which Lapf was deleted conditionally and efficiently in macrophages,with the Lapf ^fl/+-LysMcre(Lapf^+/-)littermate as a control group.We stimulated Lapf^+/-and Lapf^-/-peritoneal macrophages with heat-killed PI-labeled E.coli,viable E.coli and S.aureus to examine the capacity of macrophage to internalize bacteria.Lapf^-/-macrophages showed a significant decrease of bacterial endocytosis compared with Lapf^+/-macrophages.Moreover,the endocytosis of Zymosan paticles and latex beads in macrophages was impaired by Lapf deficiency.We exposed macrophages to E.coli for indicated times as initial internalization and then measured the survived E.coli counts inside the cells 3h or6h later.The percentage of the survived E.coli counts to the initially internalized counts in Lapf^-/-macrophages was significantly higher than that in Lapf^+/-macrophages.These results indicate that LAPF is critical for endocytosis and the subsequent bactericidal ability in macrophages.In order to investigate the role of LAPF in bacterial infection in vivo,we challenged Lapf^+/-and Lapf^-/-mice with E.coli by intraperitoneal injection and found that Lapf^-/-mice were more susceptible to E.coli infection compared with Lapf^+/-mice.The bacterial loads in spleen and liver of Lapf^-/-mice were significantly higher than those in control mice.Serum cytokine of TNF?,IFN-?,and IL-6 decreased significantly in Lapf^-/-mice compared with Lapf^+/-mice.Moreover,infiltration of inflammatory cells into the lung of Lapf^-/-mice was also decreased compared with Lapf^+/-mice.These results indicate that inflammation in Lapf-deficient mice is decreased upon E.coli infection.Next,we investigated how Lapf deficiency in macrophages affected the inflammatory response to bacterial infection.Cytokine production?TNF-?,IL-6 and IFN-??in Lapf-deficient macrophages was significantly decreased upon LPS or E.coli stimulation compared with control macrophages.The expression of Tnfa,Ifnb and Il6mRNA were also significantly decreased in Lapf^-/-macrophages compared with Lapf^+/-macrophages.Lapf deficiency decreased the TLR4-triggered activation of TBK1,IRF3,IKK?/?,P65,JNK,ERK1/2 and P38.We challenged the mice with LPS and found that Lapf^-/-mice produced significantly less inflammatory cytokines in serum than Lapf^+/-mice did.The percentage of TLR4 on the surface of Lapf^-/-macrophages was higher than that on control macrophages.Collectively,these results demonstrate that Lapf deficiency impairs the TLR4-induced signal transduction and inflammatory response by decreasing endocytosis of TLR4.We further inquired how LAPF regulated the endocytosis of bacteria.Overexpressed LAPF co-localized with both Cav1 and Cav2 in A549 cells and interacted with endogenous Cav1 and Cav2 in HEK293T cells.Endocytosis of E.coli in LAPF overexpressed RAW264.7 cells was significantly inhibited by Filipin,a caveolin-mediated endocytosis inhibitor.These data demonstrates that LAPF promotes the internalization of bacteria by caveolae-dependent mechanism.To further investigate the underlying mechanism of LAPF in promoting bacterial internalization and TLR4 endocytosis,we examined the subcellular localization of LAPF.Overexpressed LAPF co-localized with EEA1,a marker of early endosomes.There was no obvious difference in the expression levels of Cav1 and Cav2 in Lapf^+/-and Lapf^-/-macrophages.So we then examined whether LAPF affected caveolae formation,which was indicated by the caveolin oligomerization or multiple caveolins aggregation.LAPF overexpression promoted both the oligomerization of Cav1 and the aggregation of Cav1/2.These results suggest that LAPF interact with Cav1 and Cav2 and increase their aggregation to promote caveolae formation.We next investigated the underlying mechanism that LAPF increase aggregation of Cav1 and Cav2.Cav1 can be phosphorylated by tyrosine kinase Src,which promotes caveolae-mediated endocytosis.Overexpressed LAPF co-localize with Src.we replaced each tyrosine in LAPF with phenylalanine?Y64F,Y74F,Y208F or Y268F point mutation?,and expressed each of the mutants together with Src.All four mutants impaired the interaction of LAPF with Src.The phosphorylation of the LAPF mutants was substantially decreased compared with wild-type LAPF,especially of the Y208F and Y268F mutants.The LAPF mutants were also unable to increase the aggregation of Cav1and Cav2.Overexpression of Src or LAPF-WT alone increased the endocytosis of bacteria,which was further increased by co-expression of Src and LAPF-WT,but not by co-expression of Src and LAPF-Y268F.We detected Src-LAPF-Cav1 formation by immunoprecipitation in macrophages upon viable E.coli stimulation and found that Src-LAPF-Cav1 complex formation increased following infection.We challenged the Lapf^+/-and Lapf^-/-macrophages with E.coli and found that Lapf deficiency impaired the interaction between Src and Cav1.These results indicate that phosphorylation of LAPF by Src is required for Src-LAPF-Cav1 complex formation,which promotes bacterial endocytosis.The above data indicate that phosphorylation of LAPF by Src is required for Src-Cav1 complex formation.Therefore,we predicted that a Src inhibitor could decrease cell endocytosis and elimination of bacteria.Indeed,dasatinib pretreatment significantly decreased the number of bacteria endocytosed in Lapf^+/-macrophages,but not in Lapf^-/-macrophages.Dasatinib inhibited the interaction of Src with LAPF and Cav1 and the phosphorylation of Cav1.Furthermore,dasatinib-treated mice were more susceptible to E.coli infection compared with control-treated mice.These data suggest that Src-mediated LAPF phosphorylation is required for Cav1-mediated endocytosis.In conclusion,we demonstrate a new function for LAPF in promoting bacterial endocytosis and the subsequent bactericide capacity of macrophages by inducing formation of a Src-LAPF-caveolin complex.Our results suggest that activating LAPF and its partner Src may be a promising strategy for treating bacterial infections.