LIMP-2 Links Late Phagosomal Trafficking with the Onset of the Innate Immune Response to Listeria monocytogenes
2010; Elsevier BV; Volume: 286; Issue: 5 Linguagem: Inglês
10.1074/jbc.m110.146761
ISSN1083-351X
AutoresEugenio Carrasco-Marı́n, Lorena Fernández-Prieto, Estela Rodríguez‐Del Río, Fidel Madrazo‐Toca, Thomas Reinheckel, Paul Säftig, Carmen Álvarez-Domínguez,
Tópico(s)Galectins and Cancer Biology
ResumoThe innate immune response to Listeria monocytogenes depends on phagosomal bacterial degradation by macrophages. Here, we describe the role of LIMP-2, a lysosomal type III transmembrane glycoprotein and scavenger-like protein, in Listeria phagocytosis. LIMP-2-deficient mice display a macrophage-related defect in Listeria innate immunity. They produce less acute phase pro-inflammatory cytokines/chemokines, MCP-1, TNF-α, and IL-6 but normal levels of IL-12, IL-10, and IFN-γ and a 25-fold increase in susceptibility to Listeria infection. This macrophage defect results in a low listericidal potential, poor response to TNF-α activation signals, impaired phago-lysosome transformation into antigen-processing compartments, and uncontrolled LM cytosolic growth that fails to induce normal levels of acute phase pro-inflammatory cytokines. LIMP-2 transfection of CHO cells confirmed that LIMP-2 participates in the degradation of Listeria within phagosomes, controls the late endosomal/lysosomal fusion machinery, and is linked to the activation of Rab5a. Therefore, the role of LIMP-2 appears to be connected to the TNF-α-dependent and early activation of Listeria macrophages through internal signals linking the regulation of late trafficking events with the onset of the innate Listeria immune response. The innate immune response to Listeria monocytogenes depends on phagosomal bacterial degradation by macrophages. Here, we describe the role of LIMP-2, a lysosomal type III transmembrane glycoprotein and scavenger-like protein, in Listeria phagocytosis. LIMP-2-deficient mice display a macrophage-related defect in Listeria innate immunity. They produce less acute phase pro-inflammatory cytokines/chemokines, MCP-1, TNF-α, and IL-6 but normal levels of IL-12, IL-10, and IFN-γ and a 25-fold increase in susceptibility to Listeria infection. This macrophage defect results in a low listericidal potential, poor response to TNF-α activation signals, impaired phago-lysosome transformation into antigen-processing compartments, and uncontrolled LM cytosolic growth that fails to induce normal levels of acute phase pro-inflammatory cytokines. LIMP-2 transfection of CHO cells confirmed that LIMP-2 participates in the degradation of Listeria within phagosomes, controls the late endosomal/lysosomal fusion machinery, and is linked to the activation of Rab5a. Therefore, the role of LIMP-2 appears to be connected to the TNF-α-dependent and early activation of Listeria macrophages through internal signals linking the regulation of late trafficking events with the onset of the innate Listeria immune response. IntroductionInfection with a sublethal dose of Listeria monocytogenes (LM) 4The abbreviations used are: LM, L. monocytogenes; dpi, days post-infection; MØ, macrophages; LMP, L. monocytogenes phagosomes-like vesicles; TLR, Toll-like receptor; MIIC, MHC class II antigen-processing compartment; HKLM, heat-killed LM; CFU, colony-forming unit(s); PNS, post-nuclear supernatant(s); BM-DM, bone marrow-derived MØ; ASMase, acid sphingomyelinase; RI, replication index; TRITC, tetramethylrhodamine isothiocyanate; Ctsd, cathepsin-D. triggers an innate immune response in which MØs play a central role. In fact, LM replicates intracellularly, and the number of bacteria is limited by activated MØs, which prevent the dissemination of LM into the bloodstream. Recent studies suggest that the onset of LM innate immune response in MØs involves at least three intracellular stages (1Kobayashi K.S. Chamaillard M. Ogura Y. Henegariu O. Inohara N. Nuñez G. Flavell R.A. Science. 2005; 307: 731-734Crossref PubMed Scopus (1461) Google Scholar, 2Leber J.H. Crimmins G.T. Raghavan S. Meyer-Morse N.P. Cox J.S. Portnoy D.A. PLoS Pathog. 2008; 4: e6Crossref PubMed Scopus (178) Google Scholar, 3Bauler L.D. Duckett C.S. O'Riordan M.X. PLoS Pathog. 2008; 4: e1000142Crossref PubMed Scopus (84) Google Scholar). The first stage corresponds with LM internalization by the Toll-like receptor (TLR) recognition system and several scavenger receptors (4Crombie R. Silverstein R. J. Biol. Chem. 1998; 273: 4855-4863Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 5Agaisse H. Burrack L.S. Philips J.A. Rubin E.J. Perrimon N. Higgins D.E. Science. 2005; 309: 1248-1251Crossref PubMed Scopus (251) Google Scholar, 6Cheng L.W. Viala J.P. Stuurman N. Wiedemann U. Vale R.D. Portnoy D.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 13646-13651Crossref PubMed Scopus (106) Google Scholar, 7Philips J.A. Rubin E.J. Perrimon N. Science. 2005; 309: 1251-1253Crossref PubMed Scopus (301) Google Scholar, 8Vishnyakova T.G. Kurlander R. Bocharov A.V. Baranova I.N. Chen Z. Abu-Asab M.S. Tsokos M. Malide D. Basso F. Remaley A. Csako G. Eggerman T.L. Patterson A.P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16888-16893Crossref PubMed Scopus (52) Google Scholar, 9Rennemeier C. Hammerschmidt S. Niemann S. Inamura S. Zähringer U. Kehrel B.E. FASEB J. 2007; 21: 3118-3132Crossref PubMed Scopus (76) Google Scholar). Next, the phagosomal stage promotes the degradation of LM via the action of oxidative pathways, such as phox and inducible NOS enzymatic complexes (10Unanue E.R. Curr. Opin. Immunol. 1997; 9: 35-43Crossref PubMed Scopus (172) Google Scholar, 11Prada-Delgado A. Carrasco-Marin E. Bokoch G.M. Alvarez-Dominguez C. J. Biol. Chem. 2001; 276: 19059-19065Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Pamer E.G. Nat. Rev. Immunol. 2004; 4: 812-823Crossref PubMed Scopus (644) Google Scholar, 13Zenewicz L.A. Shen H. Microbes Infect. 2007; 9: 1208-1215Crossref PubMed Scopus (138) Google Scholar), and nonoxidative microbicidal components, such as endosomal-lysosomal proteins (11Prada-Delgado A. Carrasco-Marin E. Bokoch G.M. Alvarez-Dominguez C. J. Biol. Chem. 2001; 276: 19059-19065Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Prada-Delgado A. Carrasco-Marín E. Peña-Macarro C. Del Cerro-Vadillo E. Fresno-Escudero M. Leyva-Cobián F. Alvarez-Dominguez C. Traffic. 2005; 6: 252-265Crossref PubMed Scopus (42) Google Scholar, 15del Cerro-Vadillo E. Madrazo-Toca F. Carrasco-Marín E. Fernandez-Prieto L. Beck C. Leyva-Cobián F. Saftig P. Alvarez-Dominguez C. J. Immunol. 2006; 176: 1321-1325Crossref PubMed Scopus (44) Google Scholar). Lastly, a newly described cytosolic surveillance system mediated by NOD-like receptors senses degraded phagosomal bacteria (2Leber J.H. Crimmins G.T. Raghavan S. Meyer-Morse N.P. Cox J.S. Portnoy D.A. PLoS Pathog. 2008; 4: e6Crossref PubMed Scopus (178) Google Scholar, 3Bauler L.D. Duckett C.S. O'Riordan M.X. PLoS Pathog. 2008; 4: e1000142Crossref PubMed Scopus (84) Google Scholar) and induces the production of MØ-derived pro-inflammatory cytokines/chemokines MCP-1, TNF-α, IL-6, and IL-12. The culmination of these intracellular responses is the transformation of LM-primed MØs into powerful microbicidal cells that promote the clearance of LM and contribute to adaptive immunity as antigen-presenting cells. LM-primed MØs respond to different signals to acquire features of microbicidal cells and antigen-presenting cells. TNF-α is produced by LM-primed MØs and controls the early signals that grant these cells the ability to kill intracellular bacteria. IFN-γ is produced by NK and T cells and regulates the late signals that transform MØs into listericidal cells and activated antigen-presenting cells with high MHC class II expression (10Unanue E.R. Curr. Opin. Immunol. 1997; 9: 35-43Crossref PubMed Scopus (172) Google Scholar, 12Pamer E.G. Nat. Rev. Immunol. 2004; 4: 812-823Crossref PubMed Scopus (644) Google Scholar, 13Zenewicz L.A. Shen H. Microbes Infect. 2007; 9: 1208-1215Crossref PubMed Scopus (138) Google Scholar). Although the overall picture of LM-driven innate immunity is well described (1Kobayashi K.S. Chamaillard M. Ogura Y. Henegariu O. Inohara N. Nuñez G. Flavell R.A. Science. 2005; 307: 731-734Crossref PubMed Scopus (1461) Google Scholar, 10Unanue E.R. Curr. Opin. Immunol. 1997; 9: 35-43Crossref PubMed Scopus (172) Google Scholar, 12Pamer E.G. Nat. Rev. Immunol. 2004; 4: 812-823Crossref PubMed Scopus (644) Google Scholar, 13Zenewicz L.A. Shen H. Microbes Infect. 2007; 9: 1208-1215Crossref PubMed Scopus (138) Google Scholar), the intracellular molecules connecting the phagosomal and cytosolic stages are currently unknown. Moreover, the trafficking components involved in the innate immune response that promotes the transformation of the less bactericidal phagosomes into fully competent microbicidal and MHC class II antigen-processing compartments (MIICs) remain elusive.Our group has been analyzing the role of MØs in LM-driven innate immunity and in deciphering the phagosomal, nonoxidative components for several years. We have previously reported the direct participation of the soluble lysosomal protease cathepsin-D (Ctsd) in LM degradation in the phagosome (14Prada-Delgado A. Carrasco-Marín E. Peña-Macarro C. Del Cerro-Vadillo E. Fresno-Escudero M. Leyva-Cobián F. Alvarez-Dominguez C. Traffic. 2005; 6: 252-265Crossref PubMed Scopus (42) Google Scholar, 15del Cerro-Vadillo E. Madrazo-Toca F. Carrasco-Marín E. Fernandez-Prieto L. Beck C. Leyva-Cobián F. Saftig P. Alvarez-Dominguez C. J. Immunol. 2006; 176: 1321-1325Crossref PubMed Scopus (44) Google Scholar, 16Carrasco-Marín E. 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We based our hypothesis on two observations: (i) different MIIC and phagocytic vesicles contain LAMP-1 and LIMP-2 as characteristic markers (11Prada-Delgado A. Carrasco-Marin E. Bokoch G.M. Alvarez-Dominguez C. J. Biol. Chem. 2001; 276: 19059-19065Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 17Schaible U.E. Sturgill-Koszycki S. Schlesinger P.H. Russell D.G. J. Immunol. 1998; 160: 1290-1296PubMed Google Scholar, 19Huynh K.K. Eskelinen E.L. Scott C.C. Malevanets A. Saftig P. Grinstein S. EMBO J. 2007; 26: 313-324Crossref PubMed Scopus (458) Google Scholar, 20Binker M.G. Cosen-Binker L.I. Terebiznik M.R. Mallo G.V. McCaw S.E. Eskelinen E.L. Willenborg M. Brumell J.H. Saftig P. Grinstein S. Gray-Owen S.D. Cell Microbiol. 2007; 9: 2153-2166Crossref PubMed Scopus (62) Google Scholar, 23Harding C.V. Geuze H.J. J. Cell Biol. 1992; 119: 531-542Crossref PubMed Scopus (137) Google Scholar, 24von Delwig A. Musson J.A. Shim H.K. Lee J.J. Walter N. Harding C.V. Williamson E.D. Robinson J.H. Scand. J. Immunol. 2005; 62: 243-250Crossref PubMed Scopus (9) Google Scholar, 25Schramm M. Herz J. Haas A. Krönke M. Utermöhlen O. Cell Microbiol. 2008; 10: 1839-1853Crossref PubMed Scopus (52) Google Scholar, 26Jutras I. Houde M. Currier N. Boulais J. Duclos S. LaBoissière S. Bonneil E. Kearney P. Thibault P. Paramithiotis E. Hugo P. Desjardins M. Mol. Cell. Proteomics. 2008; 7: 697-715Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and (ii) both proteins appear as key regulators of late trafficking events (19Huynh K.K. Eskelinen E.L. Scott C.C. Malevanets A. Saftig P. Grinstein S. EMBO J. 2007; 26: 313-324Crossref PubMed Scopus (458) Google Scholar, 27Sandoval I.V. Martinez-Arca S. Valdueza J. Palacios S. Holman G.D. J. Biol. Chem. 2000; 275: 39874-39885Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 28Kuronita T. Eskelinen E.L. Fujita H. Saftig P. Himeno M. Tanaka Y. J. Cell Sci. 2002; 115: 4117-4131Crossref PubMed Scopus (121) Google Scholar, 29Reczek D. Schwake M. Schröder J. Hughes H. Blanz J. Jin X. Brondyk W. Van Patten S. Edmunds T. Saftig P. Cell. 2007; 131: 770-783Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Therefore, they were good candidates for the molecules that connect the late trafficking processes with innate immunity to LM.We infected LIMP-2- and LAMP-1-deficient mice (LIMP-2−/− and LAMP-1−/−, respectively) with LM and found that only the LIMP-2−/− mice were highly susceptible to LM infection and displayed defective MØ activation. This MØ defect affected the amount of LM able to escape to the cytosol, the production of early acute phase pro-inflammatory cytokines, and the ability of LM phagosomes to interact with MIIC vesicles. To confirm a role for LIMP-2 in phagocytic late trafficking events such as phagosome-lysosome fusion or bacterial proteolysis, we used CHO cells that overexpressed LIMP-2. Our results suggest that in concert with active Rab5a, LIMP-2 regulated the phagosomal fusion machinery of the late endosomes-lysosomes and the cytosolic levels of LM.DISCUSSIONIn this study, we have investigated the role of LAMP-1 and LIMP-2 in bacterial immunity. We present evidence for the specific role of LIMP-2/SCARB2 in the innate immune response to L. monocytogenes and in phagocytosis, whereas LAMP-1 seems to play no important role. Studies with LM-infected LIMP-2−/− BM-DM and LIMP-2−/− mice indicated that LIMP-2 participates in two processes in the activation of LM-primed MØ. First, LIMP-2 tightly controls the number of cytosolic LM and the induction of acute phase pro-inflammatory cytokines such as MCP-1, TNF-α, and IL-6. Therefore, it appears that low cytosolic LM numbers modulate the normal production of these cytokines in BM-DM. However, the production of late pro-inflammatory cytokines, such as IFN-γ and IL-10, was not regulated by LIMP-2. LIMP-2 also participates in late trafficking events transforming LM phagosomes into microbicidal and antigen processing compartments without affecting early intracellular processes. In fact, LIMP-2−/− BM-DM display impaired listericidal function and low expression of cell surface markers characteristic of activated MØ (i.e. F4/80 and IAb), albeit a normal oxidative burst capacity. They showed impaired interactions of LM phagosomes with late endosomes and lysosomes. Moreover, we observed decreased recruitment of peptide-loaded MHC II molecules in LIMP-2−/− BM-DM and hardly any co-localization of LM with MHC-II molecules compared with the WT cells, indicating impaired interactions with late MIIC vesicles. However, we also observed normal levels of several other endosomal proteins in the LM phagosomes from LIMP-2−/− BM-DM, such as Rab5a, mCtsd, and ASMase, suggesting normal interactions with endosomes. LIMP-2 appears to require active Rab5a to regulate all of these late trafficking processes. Nevertheless, Rab5a seems to precede the role of LIMP-2 in phagocytosis as previously suggested in MØ with inhibited Rab5a expression, which presented reduced LIMP-2 translocation to LM phagosomes (11Prada-Delgado A. Carrasco-Marin E. Bokoch G.M. Alvarez-Dominguez C. J. Biol. Chem. 2001; 276: 19059-19065Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In fact, experiments in LIMP-2-transfected CHO cells and co-transfection with Rab5a-inactive forms abrogated LIMP-2 effects in different LM phagocytic parameters such as LM killing, phagosome-lysosome fusion, and late phagocytic rates.The proposed linkage of LAMP-1 and Rab7 in different phagocytic systems (19Huynh K.K. Eskelinen E.L. Scott C.C. Malevanets A. Saftig P. Grinstein S. EMBO J. 2007; 26: 313-324Crossref PubMed Scopus (458) Google Scholar, 20Binker M.G. Cosen-Binker L.I. Terebiznik M.R. Mallo G.V. McCaw S.E. Eskelinen E.L. Willenborg M. Brumell J.H. Saftig P. Grinstein S. Gray-Owen S.D. Cell Microbiol. 2007; 9: 2153-2166Crossref PubMed Scopus (62) Google Scholar) and the recent demonstration that ASMase functions in the interaction between LM phagosomes and endosomes (25Schramm M. Herz J. Haas A. Krönke M. Utermöhlen O. Cell Microbiol. 2008; 10: 1839-1853Crossref PubMed Scopus (52) Google Scholar) suggest the possibility that late endocytic vesicles are subdivided into different compartments that are targeted by different trafficking regulators (18Harrison R.E. Brumell J.H. Khandani A. Bucci C. Scott C.C. Jiang X. Finlay B.B. Grinstein S. Mol. Biol. Cell. 2004; 15: 3146-3154Crossref PubMed Scopus (131) Google Scholar, 19Huynh K.K. Eskelinen E.L. Scott C.C. Malevanets A. Saftig P. Grinstein S. EMBO J. 2007; 26: 313-324Crossref PubMed Scopus (458) Google Scholar, 21Sun J. Deghmane A.E. Soualhine H. Hong T. Bucci C. Solodkin A. Hmama Z. J. Leukocyte Biol. 2007; 82: 1437-1445Crossref PubMed Scopus (70) Google Scholar).The specific intracellular action of LIMP-2/SCARB2 in LM phagocytosis and the lack of involvement in bacterial cell surface adherence or TLR2/TLR4 signaling correlate with the role assigned to LIMP-2 in endocytosis participating in late trafficking events (27Sandoval I.V. Martinez-Arca S. Valdueza J. Palacios S. Holman G.D. J. Biol. Chem. 2000; 275: 39874-39885Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 28Kuronita T. Eskelinen E.L. Fujita H. Saftig P. Himeno M. Tanaka Y. J. Cell Sci. 2002; 115: 4117-4131Crossref PubMed Scopus (121) Google Scholar, 29Reczek D. Schwake M. Schröder J. Hughes H. Blanz J. Jin X. Brondyk W. Van Patten S. Edmunds T. Saftig P. Cell. 2007; 131: 770-783Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 38Kuronita T. Hatano T. Furuyama A. Hirota Y. Masuyama N. Saftig P. Himeno M. Fujita H. Tanaka Y. Traffic. 2005; 6: 895-906Crossref PubMed Scopus (20) Google Scholar) and also correlate with TLR2/TLR4-independent killing of LM by activated MØ (39Edelson B.T. Unanue E.R. J. Immunol. 2002; 169: 3869-3875Crossref PubMed Scopus (195) Google Scholar, 40O'Connell R.M. Vaidya S.A. Perry A.K. Saha S.K. Dempsey P.W. Cheng G. J. Immunol. 2005; 174: 1602-1607Crossref PubMed Scopus (74) Google Scholar, 41Herskovits A.A. Auerbuch V. Portnoy D.A. PLoS Pathog. 2007; 3: e51Crossref PubMed Scopus (125) Google Scholar). In fact, LIMP-2−/− BM-DM showed a failure in listericidal abilities and acute phase cytokines production, whereas they displayed normal LPS responses and oxidative burst capacity. This intracellular specificity of LIMP-2/SCARB2 for late endocytic vesicles appears dependent on the presence of a coiled-coil domain in the luminal region and a preceding N-terminal transmembrane segment (36Blanz J. Groth J. Zachos C. Wehling C. Saftig P. Schwake M. Human Mol. Genet. 2010; 19: 563-572Crossref PubMed Scopus (74) Google Scholar, 38Kuronita T. Hatano T. Furuyama A. Hirota Y. Masuyama N. Saftig P. Himeno M. Fujita H. Tanaka Y. Traffic. 2005; 6: 895-906Crossref PubMed Scopus (20) Google Scholar). These LIMP-2/SCARB2 domains are absent in other CD36 family members such as CLA-1/SCARB1 or CD36, which have been shown to participate in bacterial recognition and uptake (7Philips J.A. Rubin E.J. Perrimon N. Science. 2005; 309: 1251-1253Crossref PubMed Scopus (301) Google Scholar, 8Vishnyakova T.G. Kurlander R. Bocharov A.V. Baranova I.N. Chen Z. Abu-Asab M.S. Tsokos M. Malide D. Basso F. Remaley A. Csako G. Eggerman T.L. Patterson A.P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16888-16893Crossref PubMed Scopus (52) Google Scholar, 9Rennemeier C. Hammerschmidt S. Niemann S. Inamura S. Zähringer U. Kehrel B.E. FASEB J. 2007; 21: 3118-3132Crossref PubMed Scopus (76) Google Scholar). The fine division of tasks among the CD36 family of scavenger-like molecules seems highlighted by the differential roles of CLA-1/SCARB1 and LIMP-2/SCARB2 in LM phagocytosis. In this regard, CLA-1/SCARB1 participates in the recognition and uptake of LM (5Agaisse H. Burrack L.S. Philips J.A. Rubin E.J. Perrimon N. Higgins D.E. Science. 2005; 309: 1248-1251Crossref PubMed Scopus (251) Google Scholar). However, LIMP-2/SCARB2 functions in the late phagosomal trafficking events involved in the onset of the innate immune response to LM but not in bacterial adherence (this study).Listeriosis is characterized by the effect of two cytokines involved in MØ activation: TNF-α and IFN-γ. Although TNF-α acts as an early signal in innate immunity, IFN-γ is a late signal. In fact, IFN-γ constitutes a late pro-inflammatory cytokine, bridging the innate and adaptive immune response and facilitating the full clearance of LM from the spleen. Another difference between TNF-α and IFN-γ signaling is the endogenous and exogenous role of TNF-α to activate MØs. LM-primed MØs produce TNF-α that serves as a feedback mechanism to activate MØs exogenously. MØs show several activating states before turning into powerful bactericidal cells with antigen-presenting abilities. It has been claimed that the exogenous action of TNF-α promotes an early activating state in MØs that triggers the cytosolic microbicidal mechanisms (12Pamer E.G. Nat. Rev. Immunol. 2004; 4: 812-823Crossref PubMed Scopus (644) Google Scholar, 13Zenewicz L.A. Shen H. Microbes Infect. 2007; 9: 1208-1215Crossref PubMed Scopus (138) Google Scholar, 39Edelson B.T. Unanue E.R. J. Immunol. 2002; 169: 3869-3875Crossref PubMed Scopus (195) Google Scholar). The exogenous effect of IFN-γ acts in MØ as a late activation signal that induces a strong microbicidal cascade affecting different subcellular compartments, phagosomes, endosomes, and cytosol (10Unanue E.R. Curr. Opin. Immunol. 1997; 9: 35-43Crossref PubMed Scopus (172) Google Scholar, 12Pamer E.G. Nat. Rev. Immunol. 2004; 4: 812-823Crossref PubMed Scopus (644) Google Scholar, 13Zenewicz L.A. Shen H. Microbes Infect. 2007; 9: 1208-1215Crossref PubMed Scopus (138) Google Scholar).LIMP-2 participates in a second process involved in exogenous MØ activation, the early signals modulated by TNF-α. In fact, WT BM-DM responds to listericidal signals induced by TNF-α and IFN-γ. LIMP-2−/− BM-DM responds normally to IFN-γ listericidal signals but failed to respond to TNF-α listericidal signals, showing high rates of LM growth. We have compiled all of these data in a proposed model (Fig. 4) for LIMP-2 role in early activation of LM-primed MØ, connecting late trafficking events with the acute phase pro-inflammatory cytokines signaling in listeriosis.The phagocytosis of LM appears to be regulated by the function of Rab5a (steps 1–3 in Fig. 4) (11Prada-Delgado A. Carrasco-Marin E. Bokoch G.M. Alvarez-Dominguez C. J. Biol. Chem. 2001; 276: 19059-19065Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 15del Cerro-Vadillo E. Madrazo-Toca F. Carrasco-Marín E. Fernandez-Prieto L. Beck C. Leyva-Cobián F. Saftig P. Alvarez-Dominguez C. J. Immunol. 2006; 176: 1321-1325Crossref PubMed Scopus (44) Google Scholar). Rab5a activation (step 2.1 in Fig. 4) promotes early interactions with the endosomal compartment and transport to LM phagosomes containing Ctsd-endosomal hydrolytic enzymes that, upon activation, have listericidal potential (15del Cerro-Vadillo E. Madrazo-Toca F. Carrasco-Marín E. Fernandez-Prieto L. Beck C. Leyva-Cobián F. Saftig P. Alvarez-Dominguez C. J. Immunol. 2006; 176: 1321-1325Crossref PubMed Scopus (44) Google Scholar) (step 2 in Fig. 4). In fact, Ctsd enzymatic action inactivates listeriolysin O, which is the LM virulence factor responsible for phagosomal membrane lysis, and decreases the bacterial viability within the phagosomal lumen (16Carrasco-Marín E. Madrazo-Toca F. de los Toyos J.R. Cacho-Alonso E. Tobes R. Pareja E. Paradela A. Albar J.P. Chen W. Gomez-Lopez M.T. Alvarez-Dominguez C. Mol. Microbiol. 2009; 72: 668-682Crossref PubMed Scopus (23) Google Scholar). Next, specific late endocytic vesicles transform the LM phagosomes into MIIC (steps 2.2–2.3 in Fig. 4). The action of LIMP-2 on the membrane permeability of LM phagosomes, which confines the nondegraded LM to the phagosomal compartment, contributes to bacterial proteolysis by other listericidal oxidative or nonoxidative mechanisms. LIMP-2 regulation of the transformation of phagosomes into MIIC (MIIC-Phg in Fig. 4) would also limit the number of cytosolic LM bacteria. Notably, only low numbers of cytosolic LM appear to be sensed by the cytosolic surveillance system of NOD2 receptors (Fig. 4, step 2.4), activating the production of acute phase pro-inflammatory cytokines/chemokines such as MCP-1, TNF-α, and IL-6 (Fig. 4, step 3) (2Leber J.H. Crimmins G.T. Raghavan S. Meyer-Morse N.P. Cox J.S. Portnoy D.A. PLoS Pathog. 2008; 4: e6Crossref PubMed Scopus (178) Google Scholar, 22Via L.E. Deretic D. Ulmer R.J. Hibler N.S. Huber L.A. Deretic V. J. Biol. Chem. 1997; 272: 13326-13331Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). The secretion of these cytokines/chemokines such as TNF-α may act as a feedback loop that activates MØ to transform LM phagosomes into proteolytic and MIIC compartments that control the number of bacteria accessing the cytosolic compartment (Fig. 4, step 4). In conclusion, LIMP-2-mediated regulation of the late trafficking events of MØ may be due to TNF-α signaling pathways induced during the innate response to L. monocytogenes. IntroductionInfection with a sublethal dose of Listeria monocytogenes (LM) 4The abbreviations used are: LM, L. monocytogenes; dpi, days post-infection; MØ, macrophages; LMP, L. monocytogenes phagosomes-like vesicles; TLR, Toll-like receptor; MIIC, MHC class II antigen-processing compartment; HKLM, heat-killed LM; CFU, colony-forming unit(s); PNS, post-nuclear supernatant(s); BM-DM, bone marrow-derived MØ; ASMase, acid sphingomyelinase; RI, replication index; TRITC, tetramethylrhodamine isothiocyanate; Ctsd, cathepsin-D. triggers an innate immune response in which MØs play a central role. In fact, LM replicates intracellularly, and the number of bacteria is limited by activated MØs, which prevent the dissemination of LM into the bloodstream. Recent studies suggest that the onset of LM innate immune response in MØs involves at least three intracellular stages (1Kobayashi K.S. Chamaillard M. Ogura Y. Henegariu O. Inohara N. Nuñez G. Flavell R.A. Science. 2005; 307: 731-734Crossref PubMed Scopus (1461) Google Scholar, 2Leber J.H. Crimmins G.T. Raghavan S. Meyer-Morse N.P. Cox J.S. Portnoy D.A. PLoS Pathog. 2008; 4: e6Crossref PubMed Scopus (178) Google Scholar, 3Bauler L.D. Duckett C.S. O'Riordan M.X. PLoS Pathog. 2008; 4: e1000142Crossref PubMed Scopus (84) Google Scholar). The first stage corresponds with LM internalization by the Toll-like receptor (TLR) recognition system and several scavenger receptors (4Crombie R. Silverstein R. J. Biol. Chem. 1998; 273: 4855-4863Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 5Agaisse H. Burrack L.S. Philips J.A. Rubin E.J. Perrimon N. Higgins D.E. Science. 2005; 309: 1248-1251Crossref PubMed Scopus (251) Google Scholar, 6Cheng L.W. Viala J.P. Stuurman N. Wiedemann U. Vale R.D. Portnoy D.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 13646-13651Crossref PubMed Scopus (106) Google Scholar, 7Philips J.A. Rubin E.J. Perrimon N. 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Immunol. 2004; 4: 812-823Crossref PubMed Scopus (644) Google Scholar, 13Zenewicz L.A. Shen H. Microbes Infect. 2007; 9: 1208-1215Crossref PubMed Scopus (138) Google Scholar), and nonoxidative microbicidal components, such as endosomal-lysosomal proteins (11Prada-Delgado A. Carrasco-Marin E. Bokoch G.M. Alvarez-Dominguez C. J. Biol. Chem. 2001; 276: 19059-19065Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Prada-Delgado A. Carrasco-Marín E. Peña-Macarro C. Del Cerro-Vadillo E. Fresno-Escudero M. Leyva-Cobián F. Alvarez-Dominguez C. Traffic. 2005; 6: 252-265Crossref PubMed Scopus (42) Google Scholar, 15del Cerro-Vadillo E. Madrazo-Toca F. Carrasco-Marín E. Fernandez-Prieto L. Beck C. Leyva-Cobián F. Saftig P. Alvarez-Dominguez C. J. Immunol. 2006; 176: 1321-1325Crossref PubMed Scopus (44) Google Scholar). Lastly, a newly described cytosolic surveillance system mediated by NOD-like receptors senses degraded phagosomal bacteria (2Leber J.H. Crimmins G.T. Raghavan S. Meyer-Morse N.P. Cox J.S. Portnoy D.A. PLoS Pathog. 2008; 4: e6Crossref PubMed Scopus (178) Google Scholar, 3Bauler L.D. Duckett C.S. O'Riordan M.X. PLoS Pathog. 2008; 4: e1000142Crossref PubMed Scopus (84) Google Scholar) and induces the production of MØ-derived pro-inflammatory cytokines/chemokines MCP-1, TNF-α, IL-6, and IL-12. The culmination of these intracellular responses is the transformation of LM-primed MØs into powerful microbicidal cells that promote the clearance of LM and contribute to adaptive immunity as antigen-presenting cells. LM-primed MØs respond to different signals to acquire features of microbicidal cells and antigen-presenting cells. TNF-α is produced by LM-primed MØs and controls the early signals that grant these cells the ability to kill intracellular bacteria. IFN-γ is produced by NK and T cells and regulates the late signals that transform MØs into listericidal cells and activated antigen-presenting cells with high MHC class II expression (10Unanue E.R. Curr. Opin. Immunol. 1997; 9: 35-43Crossref PubMed Scopus (172) Google Scholar, 12Pamer E.G. Nat. Rev. Immunol. 2004; 4: 812-823Crossref PubMed Scopus (644) Google Scholar, 13Zenewicz L.A. Shen H. Microbes Infect. 2007; 9: 1208-1215Crossref PubMed Scopus (138) Google Scholar). Although the overall picture of LM-driven innate immunity is well described (1Kobayashi K.S. Chamaillard M. Ogura Y. Henegariu O. Inohara N. Nuñez G. Flavell R.A. Science. 2005; 307: 731-734Crossref PubMed Scopus (1461) Google Scholar, 10Unanue E.R. Curr. Opin. Immunol. 1997; 9: 35-43Crossref PubMed Scopus (172) Google Scholar, 12Pamer E.G. Nat. Rev. Immunol. 2004; 4: 812-823Crossref PubMed Scopus (644) Google Scholar, 13Zenewicz L.A. Shen H. Microbes Infect. 2007; 9: 1208-1215Crossref PubMed Scopus (138) Google Scholar), the intracellular molecules connecting the phagosomal and cytosolic stages are currently unknown. Moreover, the trafficking components involved in the innate immune response that promotes the transformation of the less bactericidal phagosomes into fully competent microbicidal and MHC class II antigen-processing compartments (MIICs) remain elusive.Our group has been analyzing the role of MØs in LM-driven innate immunity and in deciphering the phagosomal, nonoxidative components for several years. We have previously reported the direct participation of the soluble lysosomal protease cathepsin-D (Ctsd) in LM degradation in the phagosome (14Prada-Delgado A. Carrasco-Marín E. Peña-Macarro C. Del Cerro-Vadillo E. Fresno-Escudero M. Leyva-Cobián F. Alvarez-Dominguez C. Traffic. 2005; 6: 252-265Crossref PubMed Scopus (42) Google Scholar, 15del Cerro-Vadillo E. Madrazo-Toca F. Carrasco-Marín E. Fernandez-Prieto L. Beck C. Leyva-Cobián F. Saftig P. Alvarez-Dominguez C. J. Immunol. 2006; 176: 1321-1325Crossref PubMed Scopus (44) Google Scholar, 16Carrasco-Marín E. 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Our results suggest that in concert with active Rab5a, LIMP-2 regulated the phagosomal fusion machinery of the late endosomes-lysosomes and the cytosolic levels of LM.
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