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An Endosomal LAPF Is Required for Macrophage Endocytosis and Elimination of Bacteria

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An Endosomal LAPF Is Required for Macrophage Endocytosis and Elimination of Bacteria An endosomal LAPF is required for macrophage endocytosis and elimination of bacteria Tianliang Lia, Kewei Qina, Nan Lia, Chaofeng Hana,1, and Xuetao Caoa,b,c,1 aNational Key Laboratory of Medical Immunology, Institute of Immunology, Second Military Medical University, 200433 Shanghai, China; bDepartment of Immunology, Center for Immunotherapy, Institute of Basic Medical Sciences, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100005, China; and cCollege of Life Sciences, Nankai University, 300071 Tianjin, China Edited by Attila Mocsai, Semmelweis University, Budapest, Hungary, and accepted by Editorial Board Member Carl F. Nathan May 22, 2019 (received for review March 6, 2019) Macrophages can internalize the invading pathogens by raft/ Caveolae, a kind of specialized lipid rafts, are bulb-shaped caveolae and/or clathrin-dependent endocytosis and elicit an immune plasma membrane invaginations first described in the 1950s (9). response against infection. However, the molecular mechanism for Since then, caveolae have been reported to be broadly involved in macrophage endocytosis remains elusive. Here we report that LAPF many cell processes, such as endocytosis, transcytosis (a specialized (lysosome-associated and apoptosis-inducing protein containing PH form of endocytosis), lipid homeostasis, and signal transduction and FYVE domains) is required for caveolae-mediated endocytosis. (10, 11). Pathogens that are internalized via caveolae-mediated Lapf-deficient macrophages have impaired capacity to endocytose endocytosis include FimH-expressing Escherichia coli,SimianVi- Lapf and eliminate bacteria. Macrophage-specific -deficient mice are rus 40 (SV-40), Group A streptococci, and Brucella abortus (12). Escherichia coli E. coli more susceptible to ( ) infection with higher Caveolae are also involved in the endocytosis of receptors, such Lapf bacterial loads. Moreover, deficiency impairs TLR4 endocytosis, as TLR4 (13, 14), and this is an essential regulatory mechanism resulting in attenuated production of TLR-triggered proinflamma- for innate immune responses and signal transduction. The main tory cytokines. LAPF is localized to early endosomes and interacts component proteins of caveolae are caveolins and cavins. Caveolin with caveolin-1. Phosphorylation of LAPF by the tyrosine kinase Src family consists of three members, namely, caveolin-1 (Cav1), is required for LAPF-Src-Caveolin complex formation and endocyto- sis and elimination of bacteria. Collectively, our work demonstrates caveolin-2 (Cav2), and caveolin-3 (Cav3). Cav1 and Cav2 are INFLAMMATION that LAPF is critical for endocytosis of bacteria and induction of in- expressed in most cell types (15, 16). Cav3 is specifically expressed IMMUNOLOGY AND flammatory responses, suggesting that LAPF and Src could be po- in muscle cells (17). Cav1 in caveolae binds TLR4, endothelial tential targets for the control of infectious diseases. nitric oxide synthase (eNOS), MAPK, cyclooxygenase (COX), and integrin signaling molecules to initiate different signaling pathways – LAPF | Caveolin-1 | endocytosis | macrophage | inflammatory response (18 21). However, the innate function of Cav1 in bacterial in- fection and the underlying mechanism are yet to be determined. In this study, we identified LAPF (lysosome-associated and nnate immune cells detect invading pathogens and launch Iappropriate inflammatory responses to eliminate infections. apoptosis-inducing protein containing PH and FYVE domains), These cells express many kinds of pattern recognition receptors which was cloned by our laboratory (22, 23), as a Cav1-interacting (PRRs), such as Toll-like receptors (TLRs), which can recognize protein by mass spectrometry. LAPF has been reported to act as pathogen-associated molecular patterns (PAMPs) and activate an adaptor protein that recruits phosphorylated p53 to lysosomes downstream signaling cascades to induce inflammation (1). TLR4, to trigger lysosomal destabilization during apoptosis (22, 23). the PRR that specifically recognizes lipopolysaccharide (LPS) of We now find that LAPF is critically involved in inducing innate im- Gram-negative bacteria, activates nuclear factor kappa light-chain mune responses and in enhancing bacterial endocytosis and the enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) through myeloid differentiation factor Significance 88 (MyD88) to induce the expression of proinflammatory cyto- kines (2, 3). In addition, TLR4 is internalized into endosomes Host innate immune cells, such as macrophages, can endocytose upon recognizing LPS to induce IRF3-dependent type-I IFN the invading pathogens and induce inflammatory innate re- production through the adaptor proteins Toll-IL-1 resistance sponses to fight infection. In this study, we show that a Cav1- domain-containing adaptor-inducing IFN-β (TRIF) and TRAM interacting protein, LAPF, promotes the endocytosis of bacteria (TRIF-related adaptor molecule, refs. 4 and 5). However, the and production of proinflammatory cytokines by inducing Src- molecular mechanism by which innate signaling induces efficient LAPF-Caveolin complex formation in response to innate stimuli. innate responses needs further investigation. Our results reveal a host defense strategy against bacterial in- Innate immune cells, such as macrophages, dendritic cells (DCs), fection by increasing macrophage endocytosis and induction of monocytes, and neutrophils, can internalize the invading pathogens innate response. We propose that pharmacological activation by endocytosis. After being internalized, pathogens become targets of LAPF and Src could potentially be applied to the control of of a series of vesicular trafficking at organelles ranging from early bacterial infections. endosomes to lysosomes, where they are killed by Mst1-Mst2-Rac Author contributions: X.C. designed research; T.L., K.Q., N.L., and C.H. performed re- signaling-induced reactive oxygen species (ROS) and subsequently search; T.L., C.H., and X.C. analyzed data; and T.L., C.H., and X.C. wrote the paper. degraded by hydrolytic enzymes. The antigens derived from those The authors declare no conflict of interest. pathogens are then presented on major histocompatibility complex This article is a PNAS Direct Submission. A.M. is a guest editor invited by the Editorial (MHC) molecules, which are subsequently recognized by T cell Board. + + receptors and activate CD4 and CD8 T cells of the adaptive Published under the PNAS license. immune system (6). Endocytosis occurs via a variety of mechanisms, 1To whom correspondence may be addressed. Email: [email protected] or caoxt@ specifically clathrin-mediated endocytosis, caveolae-mediated en- immunol.org. docytosis, macropinocytosis, and phagocytosis (7, 8). However, the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mechanisms linking the endocytosis of invading pathogens and 1073/pnas.1903896116/-/DCSupplemental. activation of innate signaling remain to be fully explored. www.pnas.org/cgi/doi/10.1073/pnas.1903896116 PNAS Latest Articles | 1of6 Downloaded by guest on September 27, 2021 − − fluorescence intensity of Lapf / macrophages was significantly + − lower than that of Lapf / macrophages (Fig. 1 E and F). These results indicate that Lapf deficiency impairs the endocytosis of var- ious pathogen particles by macrophages. To directly evaluate the microbicidal capacity of macrophages, we exposed macrophages to E. coli for6handsetthatastheinitial internalization phase (shown as 0 h in Fig. 1G). We then counted the number of surviving E. coli inside the cells 3 or 6 h later by CFU assay(shownas3and6hinFig.1G). The percentage of live E. coli counts to initially internalized counts was significantly higher in − − + − Lapf / macrophages than in Lapf / macrophages (Fig. 1G), sug- gesting that Lapf deficiency impairs bacterial endocytosis and com- promises the bactericide ability of macrophages. Macrophage-Specific Lapf Deficiency Impairs the Elimination of Bacteria in Vivo. We further investigated whether LAPF was re- − − quired for the elimination of bacteria in vivo. Lapf / mice were + − more susceptible to E. coli infection than Lapf / mice (Fig. 2A). − − Remarkably, the bacterial loads in spleen and liver of Lapf / mice were significantly higher compared with control mice (Fig. 2 B and C). Concomitantly, we also measured the production of E. coli- induced inflammatory cytokines in vivo. Productions of TNFα, − − IFN-β, and IL-6 in serum significantly decreased in Lapf / mice Fig. 1. Lapf-deficient macrophages endocytose less E. coli and S. aureus and eliminate less endocytosed E. coli.(A) Confocal microscopy of immunofluorescence + − −− staining of heat-killed PI-labeled E. coli (red) endocytosed by Lapf / and Lapf / macrophages 1 h after incubation. (Scale bars, 25 μm.) (B) Counts of E. coli engulfed + − −− per Lapf / or Lapf / macrophages as in A.(C and D) CFU assays of E. coli (C)andS. aureus (D) engulfed by Lapf+/− and Lapf−/− macrophages 1 h after infection. CFU, colony-forming units. (E and F) MFI of BMDMs (bone marrow-derived macro- phages) after incubation with Zymosan (E) and latex beads (F). MFI, mean fluo- + − −− rescence intensity. (G) Relative percent of live E. coli counts in Lapf / or Lapf / macrophages at indicated times compare with initial endocytosed E. coli (0 h) counts. Data are presented as mean ± SD of three independent experiments (B–G) or shown for one representative experiment from three independent experiments with similar
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