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

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 endocytosis 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 infection 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 cytokines (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), suggesting that Lapf deficiency impairs bacterial endocytosis and compromises 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 macrophages) 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 results (A). *P < 0.05.

bactericide capacity of macrophages by inducing Src-LAPF-Caveolin complex formation. Results Lapf Deficiency Impairs Bacterial Endocytosis and Compromises the Bactericidal Ability of Macrophages. To explore the molecular mechanism of caveolae-medicated endocytosis, we first screened for Cav1-interacting proteins by mass spectrometry (MS). LAPF was identified in the assay as a possible Cav1-interacting protein (SI − − Appendix,Fig.S1). We generated Lapf fl/fl -LysMcre (Lapf / )mice, in which Lapf was deleted conditionally and efficiently in macro- + + − phages, using the littermate Lapf fl/ -LysMcre (Lapf / )miceasa control group (SI Appendix,Figs.S2andS3). We stimulated + − − − Lapf / and Lapf / peritoneal macrophages with heat-killed and PI (propidium iodide)-labeled E. coli for 1 h and visualized the internalization of bacteria particles using confocal microscopy (Fig. 1A Fig. 2. Lapf-deficient mice are more susceptible to E. coli infection with less + − − − and SI Appendix,Fig.S4). Lapf deficiency significantly impaired inflammatory cytokines. (A) Survival of Lapf / and Lapf / mice (n = 10 per E. coli B genotype), monitored daily after i.p. injection with E. coli (1 × 109/kg body endocytosis of by macrophages (Fig. 1 ). We incubated + − E. coli Staphylococcus aureus S. au- weight). (B and C) Bacterial load in spleen (B) and liver (C)ofLapf / and macrophages with viable and ( − − − − / α β reus). Lapf / macrophages showed significantly decreased endocy- Lapf mice after injection as in A.(D) ELISA of TNF- , IL-6, and IFN- in +/− serum collected 4 or 8 h after the injection as in A.(E) Hematoxylin-and- tosis of E. coli or S. aureus in comparison with Lapf macrophages, +/− −/− C D eosin staining of lung sections from Lapf and Lapf mice after injection as measured by colony-forming units (CFUs) (Fig. 1 and ). We as in A. (Scale bars, 400 μm.) Data are presented as mean ± SD of three in- +/− −/− then incubated Lapf and Lapf macrophages with Zymosan dependent experiments (B–D) or shown for one representative experiment and latex beads for 1 h, respectively. We found that the mean from three independent experiments with similar results (A and E). *P < 0.05.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1903896116 Li et al. Downloaded by guest on September 27, 2021 Lapf deficiency affects endocytosis of TLR4. Indeed, TLR4 ex- − − pression on Lapf / macrophages surface upon LPS stimulation was significantly higher than that on control macrophages surface (Fig. 3C and SI Appendix, Fig. S8). We next investigated whether LAPF was involved in MyD88/ TIRAP-dependent signaling. Neither Lapf deficiency nor LAPF overexpression affected the interaction of Myd88 with TLR4 or TRAF6 (SI Appendix,Fig.S9). Collectively, these results demonstrate that Lapf deficiency impairs TLR4-triggered signal transduction and inflammatory response by decreasing TLR4 endocytosis.

LAPF Promotes Internalization of E.coli by Caveolae-Mediated Endocytosis. To investigate the underlying mechanism of LAPF in promoting bacterial internalization, we decided to examine the potential interaction of LAPF with Cav1 and Cav2 in more detail as LAPF was identified as a possible Cav1-binding protein in our MS experiment (SI Appendix, Fig. S1). Ectopically overexpressed LAPF colocalized with both Cav1 and Cav2 in A549 human adenocarcinoma cells (Fig. 4A). Likewise, overexpressed LAPF interacted with endogenous Cav1 and Cav2 in HEK293T human embryonic kidney cells, as detected by coimmunopreci- pitation and immunoblotting analysis (Fig. 4B). Considering that caveolae-mediated endocytosis is critical for E. coli internalization (26), we investigated whether Lapf deficiency would impact the caveolae-dependent endocytosis +/− −/− of E. coli. We incubated Lapf or Lapf macrophages with INFLAMMATION IMMUNOLOGY AND

Fig. 3. Lapf deficiency impairs LPS-activated endocytosis of TLR4 and production of TNF-α, IL-6, and IFN-β.(A) ELISA of TNF-α, IL-6, and IFN-β in the supernatant of Lapf+/− and Lapf−/− macrophages stimulated with LPS (100 ng/mL) for the indicated times. (B) ELISA of TNF-α, IL-6, and IFN-β in serum of + − − − Lapf / and Lapf / mice challenged by LPS (10 mg/kg) for 3 h. (C) Percent- + − − − age of surface TLR4 on Lapf / and Lapf / BMDMs (bone marrow-derived macrophages) upon LPS (100 ng/mL) stimulation. Data are presented as mean ± SD of three independent experiments. *P < 0.05. + − in comparison with Lapf / mice (Fig. 2D). Infiltration of in- − − flammatory cells into lungs of Lapf / mice also decreased (Fig. 2E). These results indicate that LAPF is essential for the clearance of bacterial infection and the induction of inflammatory response in vivo.

Lapf Deficiency Impairs TLR-Triggered Cytokine Productions by Decreasing TLR4 Endocytosis. We further investigated how Lapf deficiency in macrophages affects the inflammatory response to bacterial infection. We found that the cytokine productions (TNF-α, IL-6, and IFN-β)ofLapf-deficient macrophages significantly decreased upon E. coli stimulation compared with control macrophages (SI Appendix, Fig. S5). To rule out the ef- fect of bacterial endocytosis on cytokines production, we stimu- + − − − lated Lapf / and Lapf / macrophages with LPS. LPS-induced productions of TNF-α, IL-6, and IFN-β were significantly lower − − + − in Lapf / macrophages than in Lapf / macrophages (Fig. 3A). Likewise, the expression of Tnfa, Ifnb, and Il6 mRNA was also − − significantly decreased in Lapf / macrophages (SI Appendix, Fig. S6). We further examined whether Lapf deficiency affected TLR4-triggered signal transduction and found that Lapf deficiency decreased TLR4-triggered activation of TBK1, IRF3, Fig. 4. LAPF interacts with Cav1/2 and promotes caveolae-dependent endocy- IKKα/β, p65, JNK, ERK1/2, and p38 (SI Appendix, Fig. S7). tosis. (A) Confocal microscopy of the immunofluorescence staining of overex- When mice were challenged with LPS, we found that in- pressed LAPF and Cav1/2 in A549 cells. (Magnification, 252×.) (B) Immunoblot Lapf −/− analysis of anti-V5 immunoprecipitated lysates of 293T cells. (C) Confocal mi- flammatory cytokines in serum of mice were significantly +/− +/− croscopy of the immunofluorescence staining of Cav1 and E. coli in Lapf and dampened compared with Lapf mice (Fig. 3B). −− Lapf / macrophages. (Magnification, 252×.) (D) CFU assay after E. coli infection in We previously showed that endocytosis and membrane trans- μ μ γ macrophages pretreated with Filipin (3 M) or CPZ (10 M). Data are presented as location of cytoplasmic of TLR4 and IFN R2 were critical for mean ± SD of three independent experiments (D), or shown for one represen- the activation of innate response against intracellular bacterial tative experiment from three independent experiments with similar results (A–C). infection in macrophages (24, 25). We thus evaluated whether *P < 0.05. ns., no significant differences.

Li et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 27, 2021 Phosphorylated LAPF Is Critical for Caveolae Formation and Bacterial Endocytosis. We next examined the role of LAPF in caveolae formation. It has been reported that Cav1 can be phosphorylated by the tyrosine kinase Src and that activated Cav1 promotes caveolae-mediated endocytosis (27, 28). On the other hand, Cav2 could not be phosphorylated by Src (SI Appendix, Fig. S12). We found that overexpressed LAPF colocalized with Src (Fig. 6A and SI Appendix, Fig. S13). Furthermore, Src promoted tyrosine phosphorylation of LAPF (Fig. 6B), as well as oligomerization of LAPF (SI Appendix, Fig. S14). LAPF contains four tyrosine residues that can be potential Src phosphorylation sites. We replaced each tyrosine in LAPF with phenylalanine (Y64F, Y74F, Y208F, or Y268F point mutation) and cooverexpressed each mutant together with Src. Interaction of LAPF with Src was impaired by tyrosine mutation of LAPF (Fig. 6C). Phosphorylation of LAPF mutants was substantially decreased compared with wild-type LAPF, especially of the Y208F and Y268F mutants (Fig. 6D). LAPF mutants were also unable to increase the aggregation of Cav1 and Cav2 (Fig. 6E). LAPF-Y208F and LAPF-Y268F still colocalized with early endosomal marker EEA1 (Fig. 6F and SI Appendix,Fig.S15), but no longer colocalized with Cav1 (Fig. 6G). These results indicate that phosphorylation of LAPF is required for its colocalization with Src and Cav1, as well as its function of promoting caveolae formation. Next, we investigated the function of LAPF phosphorylation in bacterial endocytosis. We detected Src-LAPF-Cav1 formation in macrophages upon E. coli stimulation by immunoprecipitation and found that Src-LAPF-Cav1 complex formation peaked at 1 h after + − − − E. coli infection (Fig. 6H). We challenged the Lapf / and Lapf / Fig. 5. LAPF locates on early endosomes and promotes aggregation of macrophages with E. coli and found that Lapf deficiency impaired Cav1/2. (A) Confocal microscopy of the immunofluorescence staining of I EEA1, RAB7, LAMP1, and overexpressed LAPF. (Magnification, 126×.) (B) the interaction between Src and Cav1 (Fig. 6 ). Overexpression of Immunoblot analysis of anti-myc immunoprecipitated lysates of 293T cells LAPF-WT in RAW264.7 cells increased E. coli endocytosis, cotransfected with V5-tagged LAPF and Flag-tagged Cav1. (C) Immunoblot whereas overexpression of LAPF-Y268F did not (Fig. 6J). These analysis of anti-myc immunoprecipitated lysates of 293T cells cotransfected results indicate that phosphorylation of LAPF by Src is required for with V5-tagged LAPF and Flag-tagged Cav2. (D) Immunofluorescence anal- Src-Cav1 complex formation and bacterial endocytosis. ysis of overexpressed Cav1, Cav2, and LAPF in A549 cells. (Magnification, × 252 .) Data are shown for one representative experiment from three in- Src-LAPF-Cav1 Complex Is Critical for Endocytosis of Bacteria in Vivo dependent experiments with similar results. and in Vitro. Finally, we investigated whether the Src-LAPF-Cav1 complex is required for bacterial endocytosis. Overexpression of heat-killed PI-labeled E. coli for indicated times and found that +/− Src or LAPF-WT alone increased the endocytosis of bacteria in endocytosed E. coli colocalized with Cav1 in Lapf macro- A549 cells, which was further increased by cooverexpression of Src −/− phages, but not in Lapf macrophages (Fig. 4C and SI Ap- and LAPF-WT together, but not by cooverexpression of Src and pendix,Fig.S10). We treated LAPF-overexpressed RAW264.7 LAPF-Y268F (Fig. 7A). These results suggest that Src-LAPF-Cav1 cells with Filipin (caveolae-mediated endocytosis inhibitor) or complex was required for the endocytosis of bacteria in vitro. Src Chlorpromazine (CPZ, clathrin-mediated endocytosis inhibitor) inhibitor dasatinib markedly inhibited the interaction of Src with before E. coli infection. The endocytosis of E. coli by LAPF- LAPF and Cav1 (Fig. 7B), as well as phosphorylation of Cav1 overexpressed RAW264.7 cells was significantly inhibited by (Fig. 7C). Moreover, dasatinib decreased bacteria endocytosis in +/− −/− Filipin, but not by CPZ (Fig. 4D). These data demonstrate that Lapf macrophages, but not in Lapf macrophages (Fig. 7D). LAPF promotes the endocytosis of bacteria by a caveolae- Furthermore, dasatinib-treated mice were more susceptible to E. dependent mechanism. coli infection compared with control-treated mice (Fig. 7E). These data indicate that Src-mediated LAPF phosphorylation and for- LAPF Promotes Caveolae Formation. To investigate the mechanism mation of Src-LAPF-Cav1 complex is critical for endocytosis of of LAPF in caveolae-mediated endocytosis, we first examined the bacteria in vivo and in vitro. subcellular localization of LAPF. Overexpressed LAPF colocalized Discussion with EEA1, a marker of early endosomes, but not RAB7 (markers Innate immune cells can eliminate pathogens by internalizing of late endosomes) or LAMP1 (markers of lysosomes) (Fig. 5A SI Appendix them and inducing inflammatory innate responses. In this study, and ,Fig.S11). Since there was no obvious difference we found that LAPF functioned as a Cav1-interacting protein Lapf +/− Lapf −/− between the expression of Cav1/Cav2 in and and promoted the endocytosis and elimination of bacteria and SI Appendix macrophages ( ,Fig.S3), we examined whether LAPF the production of proinflammatory cytokines. Lapf-deficient influenced caveolae formation, which was indicated by caveolins mice were susceptible to E. coli infection in vivo with higher aggregation (27). Indeed, LAPF overexpression promoted both the bacteria load and less lung inflammation, highlighting the func- oligomerization of Cav1 and the aggregation of Cav1/2 (Fig. 5 B and tional significance of LAPF-mediated balance of endocytosis and C), and LAPF overexpression substantially increased the colocali- inflammation in host defense. Our results uncover a host defense zation of Cav1/2 (Fig. 5D). These results suggest that LAPF can strategy against bacterial infection both by increasing endocytosis interact with Cav1 and Cav2 and increase caveolae-mediated en- and elimination of bacteria and by amplifying the innate immune docytosis by promoting caveolae formation. response.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1903896116 Li et al. Downloaded by guest on September 27, 2021 Fig. 7. Disruption of Src-LAPF-Cav1 complex formation impairs endocytosis of bacteria. (A) CFU analysis of E. coli in A549 cells overexpressing Src, LAPF-WT, or

LAPF-268 as indicated. (B) Immunoblot analysis of Cav1 and LAPF in anti-Src INFLAMMATION IMMUNOLOGY AND immunoprecipitated lysates of macrophages pretreated with dasatinib as indicated and then stimulated with E. coli, with Src as a loading control and IgG as an immunoprecipitation control. (C) Immunoblot analysis of pTyr in anti-Cav1 immunoprecipitated lysates of dasatinib (1 μM) treated macrophages stimulated with E. coli, with Src as a loading control and IgG as an immunoprecipitation control. (D) CFU analysis of E. coli in Lapf+/− and Lapf−/− macrophages pretreated with dasatinib (1 μM). (E) Survival of mice (n = 10 per group) pretreated with DMSO or dasatinib (15 mg/kg body weight) and then in- traperitoneally injected with E. coli (1 × 109/kg body weight). Data are presented as mean ± SD of three independent experiments (A and D), or shown for one representative experiment from three independent experiments with similar results (B, C,andE). ns, not significant. *P < 0.05.

Caveolins, the main proteins of caveolae, broadly participate in both endocytosis of bacteria and the signaling of inflammation. Cav1 deficiency suppressed LPS-induced lung inflammation by decreasing eNOS expression (29), and Cav1 inhibited the upstream regulators of antioxidant defense enzymes to further exacerbate the inflammatory response in nonimmune cells, such as endothelial cells (30). Deletion Fig. 6. LAPF induces Src-Cav1 complex upon E. coli stimulation. (A) Confocal mi- of Cav1 attenuated phagocytosis in macrophages by decreasing the croscopy of the immunofluorescence staining of overexpressed-LAPF and constitu- expression of CD36 and TLR4 (31). These results indicate that Cav1 tively active Src (CA-Src). (Magnification, 252×.) (B) Immunoblot analysis of anti-Flag may promote the inflammatory responses. Recently, ZNRF1 was immunoprecipitated lysates of 293T cells cotransfected with LAPF-Flag and myc- shown to promote LPS and bacterial-induced proinflammatory cy- tagged CA-Src with HA-tagged Akt as tyrosine phosphorylation negative control. tokine production by ubiquitinating and degrading Cav1 (32), sug- (C) Immunoblot analysis of anti-myc immunoprecipitated lysates of 293T cells gesting that Cav1 also has a negative role in TLR4 signaling. Thus, cotransfected with V5-tagged LAPF wild type (WT) or LAPF point mutant (Y64F, the role of caveolae and caveolins in inflammation or infection re- Y74F,Y208F,andY268F).(D) Immunoblot analysis of anti-V5 immunoprecipitated quires further clarification in different contexts of cell types and/or lysates of 293T cells cotransfected with myc-tagged CA-Src. (E) Immunoblot analysis of anti-myc immunoprecipitated lysates of 293T cells cotransfected with Flag-tagged infection models. We discovered that Src-LAPF-Caveolin complex Cav2, V5-tagged LAPF wild type (WT), or LAPF point mutant (Y64F, Y74F, Y208F, was required for endocytosis and elimination of bacteria in both and Y268F). (F) Confocal microscopy of the immunofluorescence staining of EEA1 epithelia cells and macrophages, suggesting a positive role of Cav1 in and overexpressed-LAPF (Y208F or Y268F). (Magnification, 252×.) (G) Confocal mi- regulating inflammatory response. croscopy of the immunofluorescence staining of overexpressed-Cav1 and LAPF-WT It is known that TLR4 translocates to early endosomes where and LAPF mutants (Y208F or Y268F). (Magnification, 252×.) (H) Immunoblot analysis it activates the TRAM/TRIF pathway. Subsequently, TLR4 is of LAPF and Cav1 in anti-Src immunoprecipitated lysates of macrophages stimulated sorted either to lysosomes for degradation to terminate the sig- with E. coli, with Src as a loading control and IgG as an immunoprecipitation control. + − naling or to the plasma membrane for receptor recycling (33). (I) Immunoblot analysis of Cav1 in anti-Src immunoprecipitated lysates of Lapf / −/− Endocytosis of TLR4 is required to amplify inflammatory re- and Lapf macrophages stimulated with E. coli, with Src as a loading control and Lapf IgG as an immunoprecipitation control. (J) CFU assays of LAPF-WT or LAPF-Y268F sponses (33). Our current work showed deficiency signifi- overexpressed RAW264.7 cells 1 h after E. coli infection. Data are presented as cantly impaired the endocytosis of TLR4 and suggested that the mean ± SD of three independent experiments (J) or shown for one representative LAPF-Src-Cav complex is a molecular mediator involved in the experiment from three independent experiments with similar results (A–I). *P < 0.05. signaling transduction of TLR4. Further research to reveal

Li et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 27, 2021 the function of LAPF in the recycling of TLR4 from the endo- changed our strategy to conditional knockout of Lapf instead of some or lysosome to cell membrane and oligomerization of whole genome knockout because of the embryonic lethality. TLR4 on membrane is needed. However, taking into account that LAPF plays a critical role in Src-family kinases have been broadly studied and shown to cell apoptosis (22, 23), the functions of LAPF in other cells or modulate a wide range of cellular processes and regulators, such organs should be further determined using different conditional as the regulation of immunoreceptors, C-type lectins, integrins, G gene knockout strategies. protein-coupled receptors, and many others through phosphory- In conclusion, LAPF is required for endocytosis and elimina- lating of immunoreceptor tyrosine-based inhibitory motifs (ITIMs), tion of bacteria, which provides host resistance to the invading immunoreceptor tyrosine-based activation motifs (ITAM), or others (34). Src was also found to induce “inhibitory” signaling bacteria. Our results reveal a molecular mechanism for how the pathway on TLR-induced inflammatory responses from low avidity innate immune cells exert their function against bacterial in- of integrin CD11b, DAP12, or FcRg (35). In the present study, a fection and propose that activation of LAPF and Src may be a Src-LAPF-Caveolin complex is found to be critical for bactericide potential approach to the control of bacterial infection. and inducing inflammatory responses. Moreover, inhibition of Src in vivo and in vitro by dasatinib aggravates bacterial infection and Materials and Methods mortality. These results further suggest that activation of Src could Full details of materials and methods, including the generation and genotype be a therapeutic strategy for infectious diseases. identification of mice, cell culture, ELISA, plasmid constructs and transfection, Our study suggests that LAPF is located in early endosomes. RNA quantification, bacterial infection and CFU assay, immunoprecipitation Considering that both LAPF and Cav1 have lipid-binding do- and immunoblot analysis, immunofluorescent confocal microscopy, flow mains in sequence, further studies are needed to reveal which cytometry, and statistical analysis are provided in SI Appendix. kind of lipid or membrane components, such as phosphatidyli- nositol, are required for their interactions. Our study found that ACKNOWLEDGMENTS. This study was supported by grants from the National Lapf Key Research and Development Program of China (2018YFA0507401); National deficiency impaired the bactericidal efficiency of macro- Natural Science Foundation of China (31522019, 81471568, 80178101, and phages. We speculate that early endosome-located LAPF might 31770945); and the Chinese Academy of Medical Sciences Innovation Fund for promote macrophages to kill intracellular bacteria by increasing Medical Sciences (2016-12M-1-003). We thank Ms. Xiaofei Li and Dr. Xingguang acidification of endosome and lysosome (36). In addition, we Liu for technical assistance.

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