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• W2065609114 abstract "Listeria monocytogenes is a facultative intracellular pathogen that invades both phagocytic and non-phagocytic cells. Recent studies have shown that L. monocytogenes infection activates the autophagy pathway. However, the innate immune receptors involved and the downstream signaling pathways remain unknown. Here, we show that macrophages deficient in the TLR2 and NOD/RIP2 pathway display defective autophagy induction in response to L. monocytogenes. Inefficient autophagy in Tlr2−/− and Nod2−/− macrophages led to a defect in bacteria colocalization with the autophagosomal marker GFP-LC3. Consequently, macrophages lacking TLR2 and NOD2 were found to be more susceptible to L. monocytogenes infection, as were the Rip2−/− mice. Tlr2−/− and Nod2−/− cells showed perturbed NF-κB and ERK signaling. However, autophagy against L. monocytogenes was dependent selectively on the ERK pathway. In agreement, wild-type cells treated with a pharmacological inhibitor of ERK or ERK-deficient cells displayed inefficient autophagy activation in response to L. monocytogenes. Accordingly, fewer bacteria were targeted to the autophagosomes and, consequently, higher bacterial growth was observed in cells deficient in the ERK signaling pathway. These findings thus demonstrate that TLR2 and NOD proteins, acting via the downstream ERK pathway, are crucial to autophagy activation and provide a mechanistic link between innate immune receptors and induction of autophagy against cytoplasm-invading microbes, such as L. monocytogenes. Listeria monocytogenes is a facultative intracellular pathogen that invades both phagocytic and non-phagocytic cells. Recent studies have shown that L. monocytogenes infection activates the autophagy pathway. However, the innate immune receptors involved and the downstream signaling pathways remain unknown. Here, we show that macrophages deficient in the TLR2 and NOD/RIP2 pathway display defective autophagy induction in response to L. monocytogenes. Inefficient autophagy in Tlr2−/− and Nod2−/− macrophages led to a defect in bacteria colocalization with the autophagosomal marker GFP-LC3. Consequently, macrophages lacking TLR2 and NOD2 were found to be more susceptible to L. monocytogenes infection, as were the Rip2−/− mice. Tlr2−/− and Nod2−/− cells showed perturbed NF-κB and ERK signaling. However, autophagy against L. monocytogenes was dependent selectively on the ERK pathway. In agreement, wild-type cells treated with a pharmacological inhibitor of ERK or ERK-deficient cells displayed inefficient autophagy activation in response to L. monocytogenes. Accordingly, fewer bacteria were targeted to the autophagosomes and, consequently, higher bacterial growth was observed in cells deficient in the ERK signaling pathway. These findings thus demonstrate that TLR2 and NOD proteins, acting via the downstream ERK pathway, are crucial to autophagy activation and provide a mechanistic link between innate immune receptors and induction of autophagy against cytoplasm-invading microbes, such as L. monocytogenes. Listeria monocytogenes is a Gram-positive food-borne pathogen that causes listeriosis, a severe form of gastroenteritis with a possible nervous system infection, particularly in immunocompromised individuals, pregnant women, and neonates (1Vázquez-Boland J.A. Kuhn M. Berche P. Chakraborty T. Domínguez-Bernal G. Goebel W. González-Zorn B. Wehland J. Kreft J. Clin. Microbiol. Rev. 2001; 14: 584-640Crossref PubMed Scopus (1694) Google Scholar, 2Corr S.C. O'Neill L.A. Cell Microbiol. 2009; 11: 703-709Crossref PubMed Scopus (65) Google Scholar). L. monocytogenes mediates its own uptake into phagocytic cells and non-phagocytic cells (e.g. enterocytes, hepatocytes, fibroblasts, and endothelial cells) via bacterial invasion factors called internalins (3Ireton K. Cell Microbiol. 2007; 9: 1365-1375Crossref PubMed Scopus (71) Google Scholar, 4Mengaud J. Ohayon H. Gounon P. Mege R.M. Cossart P. Cell. 1996; 84: 923-932Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Once inside the phagosome, a decrease in pH activates Listeria cytolysin listeriolysin O. Listeriolysin O then blocks phagolysosomal fusion and degrades the vacuolar membrane, leading to the escape of L. monocytogenes into the cytosol (5Hamon M. Bierne H. Cossart P. Nat. Rev. Microbiol. 2006; 4: 423-434Crossref PubMed Scopus (459) Google Scholar, 6Portnoy D.A. Auerbuch V. Glomski I.J. J. Cell Biol. 2002; 158: 409-414Crossref PubMed Scopus (335) Google Scholar). 90 min after infection, approximately 80% of the L. monocytogenes are observed in the cytosol (7Henry R. Shaughnessy L. Loessner M.J. Alberti-Segui C. Higgins D.E. Swanson J.A. Cell Microbiol. 2006; 8: 107-119Crossref PubMed Scopus (97) Google Scholar). Entry into the cytoplasm is also assisted by phosphatidylinositol phospholipase C and phosphatidylcholine phospholipase C two bacterial phospholipases that hydrolyze host lipids to produce diacylglycerol and inositol phosphate, and ceramide, respectively, additionally playing a major role in subverting host cellular responses (8Gründling A. Gonzalez M.D. Higgins D.E. J. Bacteriol. 2003; 185: 6295-6307Crossref PubMed Scopus (113) Google Scholar, 9Poussin M.A. Goldfine H. Infect. Immun. 2005; 73: 4410-4413Crossref PubMed Scopus (30) Google Scholar). Three to five hours after infection, L. monocytogenes in the cytosol utilizes its ActA protein to polymerize host actin forming comet-like tail that propels bacterial movement and spread from cell to cell (10Monack D.M. Theriot J.A. Cell Microbiol. 2001; 3: 633-647Crossref PubMed Scopus (83) Google Scholar). The innate immune response depends on pathogen recognition receptors for detection of pathogen-associated molecular patterns. These receptors include Toll-like receptors (TLRs), 2The abbreviations used are: TLRToll-like receptorNLRNod-like receptorDCdendritic cellCQchloroquineWBWestern blotting. RIG-I-like receptors, and Nod-like receptors (NLRs) family of proteins (11Creagh E.M. O'Neill L.A. Trends Immunol. 2006; 27: 352-357Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). TLRs are transmembrane proteins for sensing extracellular pathogens whereas NLRs sense pathogen-associated molecular patterns in the cytosolic compartment. NLRs consist of more than 20 family members, including Nucleotide Oligomerization domain 1 (NOD1), NOD2, NLRP3, and NLRC4 (12Kanneganti T.D. Lamkanfi M. Núñez G. Immunity. 2007; 27: 549-559Abstract Full Text Full Text PDF PubMed Scopus (794) Google Scholar, 13Kawai T. Akira S. Int. Immunol. 2009; 21: 317-337Crossref PubMed Scopus (1180) Google Scholar, 14Anand P.K. Malireddi R.K.S. Kanneganti T.D. Front. Microbiol. 2011; 2: 12Crossref PubMed Scopus (70) Google Scholar). NOD1 is expressed ubiquitously, whereas NOD2 is expressed mainly in the myeloid cells such as macrophages and dendritic cells (DCs) (12Kanneganti T.D. Lamkanfi M. Núñez G. Immunity. 2007; 27: 549-559Abstract Full Text Full Text PDF PubMed Scopus (794) Google Scholar). NOD1/NLRC1 and NOD2/NLRC2 recognize peptidoglycan components γ-d-glutamyl-meso-diaminopimelic acid and muramyl dipeptide, respectively (15Girardin S.E. Boneca I.G. Viala J. Chamaillard M. Labigne A. Thomas G. Philpott D.J. Sansonetti P.J. J. Biol. Chem. 2003; 278: 8869-8872Abstract Full Text Full Text PDF PubMed Scopus (1906) Google Scholar, 16Girardin S.E. Boneca I.G. Carneiro L.A. Antignac A. Jéhanno M. Viala J. Tedin K. Taha M.K. Labigne A. Zähringer U. Coyle A.J. DiStefano P.S. Bertin J. Sansonetti P.J. Philpott D.J. Science. 2003; 300: 1584-1587Crossref PubMed Scopus (1248) Google Scholar). L. monocytogenes activates a cytosolic surveillance system that results in the expression of interferon β-regulated genes. Furthermore, host defense against L. monocytogenes is mediated by the secretion of IFN- γ, TNFα, IL-1β, IL-6, IL-12, IL-18, CCL2, MIP2, CXCL1, and the coexpression of costimulatory molecules CD40, CD80, and CD86 on antigen-presenting cells (17O'Riordan M. Yi C.H. Gonzales R. Lee K.D. Portnoy D.A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 13861-13866Crossref PubMed Scopus (242) Google Scholar, 18McCaffrey R.L. Fawcett P. O'Riordan M. Lee K.D. Havell E.A. Brown P.O. Portnoy D.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 11386-11391Crossref PubMed Scopus (156) Google Scholar). Toll-like receptor Nod-like receptor dendritic cell chloroquine Western blotting. Autophagy is a highly conserved cellular catabolic process that removes damaged organelles and degrades long-lived proteins during periods of starvation, thereby playing a crucial role during cell survival and death (19Yang Z. Klionsky D.J. Curr. Top Microbiol. Immunol. 2009; 335: 1-32Crossref PubMed Scopus (582) Google Scholar, 20Glick D. Barth S. Macleod K.F. J. Pathol. 2010; 221: 3-12Crossref PubMed Scopus (2066) Google Scholar, 21Pattingre S. Espert L. Biard-Piechaczyk M. Codogno P. Biochimie. 2008; 90: 313-323Crossref PubMed Scopus (436) Google Scholar). Autophagy also has an essential role in the innate defense mechanism, i.e. it eliminates cytoplasm-invading microbes by forming a double-layered membrane that wraps around the cytosolic bacteria so that it can be degraded via fusion with lysosomes (22Deretic V. Trends Immunol. 2005; 26: 523-528Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 23Deretic V. Curr. Opin. Cell Biol. 2009; 22: 1-11Google Scholar, 24Deretic V. Levine B. Cell Host Microbe. 2009; 5: 527-549Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar, 25Huang J. Brumell J.H. Curr. Top Microbiol. Immunol. 2009; 335: 189-215PubMed Google Scholar, 26Campoy E. Colombo M.I. Biochim. Biophys. Acta. 2009; 1793: 1465-1477Crossref PubMed Scopus (88) Google Scholar). Autophagy was recently shown to be protective in elimination of bacterial pathogens (27Py B.F. Lipinski M.M. Yuan J. Autophagy. 2007; 3: 117-125Crossref PubMed Scopus (186) Google Scholar, 28Nakagawa I. Amano A. Mizushima N. Yamamoto A. Yamaguchi H. Kamimoto T. Nara A. Funao J. Nakata M. Tsuda K. Hamada S. Yoshimori T. Science. 2004; 306: 1037-1040Crossref PubMed Scopus (937) Google Scholar, 29Birmingham C.L. Smith A.C. Bakowski M.A. Yoshimori T. Brumell J.H. J. Biol. Chem. 2006; 281: 11374-11383Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar, 30Gutierrez M.G. Master S.S. Singh S.B. Taylor G.A. Colombo M.I. Deretic V. Cell. 2004; 119: 753-766Abstract Full Text Full Text PDF PubMed Scopus (1757) Google Scholar). In the context of L. monocytogenes, earlier reports have shown that TLR2 and NOD proteins play a protective role during L. monocytogenes infection. TLR2 is required for macrophage activation (31Flo T.H. Halaas O. Lien E. Ryan L. Teti G. Golenbock D.T. Sundan A. Espevik T. J. Immunol. 2000; 164: 2064-2069Crossref PubMed Scopus (254) Google Scholar, 32Machata S. Tchatalbachev S. Mohamed W. Jänsch L. Hain T. Chakraborty T. J. Immunol. 2008; 181: 2028-2035Crossref PubMed Scopus (74) Google Scholar, 33Seki E. Tsutsui H. Tsuji N.M. Hayashi N. Adachi K. Nakano H. Futatsugi-Yumikura S. Takeuchi O. Hoshino K. Akira S. Fujimoto J. Nakanishi K. J. Immunol. 2002; 169: 3863-3868Crossref PubMed Scopus (241) Google Scholar). Similarly, the NOD1-NOD2/RIP2 pathway has been shown to be critical for host defense against L. monocytogenes in vitro and in vivo (34Kim Y.G. Park J.H. Shaw M.H. Franchi L. Inohara N. Núñez G. Immunity. 2008; 28: 246-257Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 35Park J.H. Kim Y.G. McDonald C. Kanneganti T.D. Hasegawa M. Body-Malapel M. Inohara N. Núñez G. J. Immunol. 2007; 178: 2380-2386Crossref PubMed Scopus (391) Google Scholar). However, the role of extracellular TLRs and the cytosolic NOD proteins in autophagy of L. monocytogenes remain unknown. Here we show that the innate immune receptors TLR2 and NOD/RIP2 pathways activate autophagy via ERK activation, leading to degradation of L. monocytogenes within autophagosomes. All reagents were obtained from Sigma unless otherwise stated. The following antibodies were used: anti-LC3 from Novus Biologicals, anti-ERK, anti-phospho-pERK, anti-IκB, anti-pIκB (Cell Signaling Technology, Inc.), anti-actin, and anti-tubulin (Sigma). HRP-labeled anti-rabbit and anti-mouse antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. All fluorescently labeled secondary antibodies were obtained from Molecular Probes (Invitrogen). Rapamycin was obtained from LC Laboratories. NF-κB inhibitor SN50 (catalog no. 481480) and MEK inhibitor PD98059 (catalog no. 513000) were obtained from Calbiochem. Nod1−/−, Nod2−/−, Rip2−/−, Tlr2−/−, Tlr4−/− mice backcrossed to the C57BL/6 background for at least 10 generations have been described before (57Lamkanfi M. Malireddi R.K. Kanneganti T.D. J. Biol. Chem. 2009; 284: 20574-20581Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 58Kanneganti T.D. Lamkanfi M. Kim Y.G. Chen G. Park J.H. Franchi L. Vandenabeele P. Núñez G. Immunity. 2007; 26: 433-443Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). All mice were housed in a pathogen-free facility. Bone marrow-derived macrophages were prepared from the femurs of 6- to 10-week-old male mice by using supernatant from L cells as differentiation medium. Wild-type L. monocytogenes and isogenic mutants were grown in brain heart infusion medium at 37 °C overnight to mid-log phase for macrophage infections. Briefly, L. monocytogenes were washed twice with PBS and macrophages were infected for 30 min with a multiplicity of infection of 1:1 unless stated otherwise, and the medium was replaced with fresh medium. After 45 min of infection, gentamicin (10 μg/ml) was added to limit the growth of extracellular bacteria. Where mentioned, CQ (50 μm) was added 30 min after infection. Where mentioned, NF-κB inhibitor SN50 (15 μM) or MEK inhibitor PD98059 (50 μM) were preincubated with macrophages 1 h before infection and maintained during the course of the experiment. For in vivo experiments, adult mice (6–10 weeks old) were used. For infection, wild-type and Rip2−/− mice were infected intraperitoneally with 3 × 105 L. monocytogenes. Bacterial loads in the liver and spleen were determined on day 3 after infection by serial dilution of the lysates on brain heart infusion medium. At different times after infection, cells were lysed in radioimmune precipitation assay lysis buffer supplemented with complete protease inhibitor mixture (Roche) and PhosSTOP (Roche). Lysates were resolved on SDS-PAGE and transferred to PVDF membranes by electroblotting. Membranes were blocked in 5% nonfat milk and incubated overnight with primary antibody at 4 °C and for 1 h with secondary HRP-tagged antibody at room temperature. The membranes were developed with ECL (Pierce). Cells were resuspended in nucleofector buffer (Amaxa) at a density of 2 × 106 with 2 μg of GFP-LC3 plasmid. Cells were nucleoporated according to the manufacturer's instructions (Amaxa) and seeded onto glass coverslips. After 4 h, cells were infected with L. monocytogenes at a multiplicity of infection of 1:1. The cells were fixed at the desired time points with 4% paraformaldehyde and processed for immunoflourescence as described previously (59Anand P.K. Anand E. Bleck C.K. Anes E. Griffiths G. PLoS ONE. 2010; 5e10136Crossref PubMed Scopus (91) Google Scholar). Cells on coverslips were mounted on slides with ProLong® anti-gold antifade reagent (Invitrogen) and analyzed with a Nikon C1 confocal microscope with a ×40 objective lens using the manufacturer's original software. The images were processed later with the ImageJ program. For transmission electron microscopy, cells were fixed in a solution of 2.5% glutaraldehyde in 0.1 m cacodylate buffer (pH 7.4). Cells were then embedded and sectioned for transmission electron microscopy by the Cell and Tissue Imaging Core Facility of St. Jude Children's Research Hospital. Cells were grown on coverslips and infected with L. monocytogenes at a multiplicity of infection of 1:1 as described above. Intracellular growth curves of L. monocytogenes were generated as described previously (60Portnoy D.A. Jacks P.S. Hinrichs D.J. J. Exp. Med. 1988; 167: 1459-1471Crossref PubMed Scopus (652) Google Scholar). Quantifications of the number of autophagosomes per cell and LC3 - positive L. monocytogenes were performed by direct visualization on a Nikon C1 confocal microscope. For intracellular growth curves, at least three independent experiments were done in triplicate, and the means ± S.E. are reported. p values were calculated using the two-tailed Student's t test. Densitometry analysis of the immunoblots was performed by the ImageJ program. Bacterial counts of infected mice were analyzed by Mann-Whitney U test. L. monocytogenes is an intracellular pathogen that escapes the phagosome and replicates in the host cytosol. Recent studies have suggested that cytoplasm-invading bacteria are targets for degradation by autophagy (36Rich K.A. Burkett C. Webster P. Cell Microbiol. 2003; 5: 455-468Crossref PubMed Scopus (198) Google Scholar). L. monocytogenes was observed to induce autophagy and colocalized with autophagosomes in mouse embryonic fibroblasts and RAW 264.7 macrophages (27Py B.F. Lipinski M.M. Yuan J. Autophagy. 2007; 3: 117-125Crossref PubMed Scopus (186) Google Scholar, 37Birmingham C.L. Canadien V. Gouin E. Troy E.B. Yoshimori T. Cossart P. Higgins D.E. Brumell J.H. Autophagy. 2007; 3: 442-451Crossref PubMed Scopus (192) Google Scholar). We first evaluated whether L. monocytogenes induces autophagy in bone marrow-derived macrophages by employing autophagy marker, LC3, as a readout (38Klionsky D.J. Abeliovich H. Agostinis P. Agrawal D.K. Aliev G. Askew D.S. Baba M. Baehrecke E.H. Bahr B.A. Ballabio A. Bamber B.A. Bassham D.C. Bergamini E. Bi X. Biard-Piechaczyk M. Blum J.S. Bredesen D.E. Brodsky J.L. Brumell J.H. Brunk U.T. Bursch W. Camougrand N. Cebollero E. Cecconi F. Chen Y. Chin L.S. Choi A. Chu C.T. Chung J. Clarke P.G. Clark R.S. Clarke S.G. Clavé C. Cleveland J.L. Codogno P. Colombo M.I. Coto-Montes A. Cregg J.M. Cuervo A.M. Debnath J. Demarchi F. Dennis P.B. Dennis P.A. Deretic V. Devenish R.J. Di Sano F. Dice J.F. Difiglia M. Dinesh-Kumar S. Distelhorst C.W. Djavaheri-Mergny M. Dorsey F.C. Dröge W. Dron M. Dunn Jr., W.A. Duszenko M. Eissa N.T. Elazar Z. Esclatine A. Eskelinen E.L. Fésüs L. Finley K.D. Fuentes J.M. Fueyo J. Fujisaki K. Galliot B. Gao F.B. Gewirtz D.A. Gibson S.B. Gohla A. Goldberg A.L. Gonzalez R. González-Estévez C. Gorski S. Gottlieb R.A. Häussinger D. He Y.W. Heidenreich K. Hill J.A. Høyer-Hansen M. Hu X. Huang W.P. Iwasaki A. Jäättelä M. Jackson W.T. Jiang X. Jin S. Johansen T. Jung J.U. Kadowaki M. Kang C. Kelekar A. Kessel D.H. Kiel J.A. Kim H.P. Kimchi A. Kinsella T.J. Kiselyov K. Kitamoto K. Knecht E. Komatsu M. Kominami E. Kondo S. Kovács A.L. Kroemer G. Kuan C.Y. Kumar R. Kundu M. Landry J. Laporte M. Le W. Lei H.Y. Lenardo M.J. Levine B. Lieberman A. Lim K.L. Lin F.C. Liou W. Liu L.F. Lopez-Berestein G. López-Otín C. Lu B. Macleod K.F. Malorni W. Martinet W. Matsuoka K. Mautner J. Meijer A.J. Meléndez A. Michels P. Miotto G. Mistiaen W.P. Mizushima N. Mograbi B. Monastyrska I. Moore M.N. Moreira P.I. Moriyasu Y. Motyl T. Münz C. Murphy L.O. Naqvi N.I. Neufeld T.P. Nishino I. Nixon R.A. Noda T. Nürnberg B. Ogawa M. Oleinick N.L. Olsen L.J. Ozpolat B. Paglin S. Palmer G.E. Papassideri I. Parkes M. Perlmutter D.H. Perry G. Piacentini M. Pinkas-Kramarski R. Prescott M. Proikas-Cezanne T. Raben N. Rami A. Reggiori F Rohrer B Rubinsztein DC Ryan KM Sadoshima J Sakagami H Sakai Y Sandri M Sasakawa C Sass M Schneider C Seglen PO Seleverstov O Settleman J Shacka JJ Shapiro IM Sibirny A Silva-Zacarin EC Simon HU Simone C Simonsen A Smith M.A. Spanel-Borowski K. Srinivas V. Steeves M. Stenmark H. Stromhaug P.E. Subauste C.S. Sugimoto S. Sulzer D. Suzuki T. Swanson M.S. Tabas I. Takeshita F. Talbot N.J. Tallóczy Z. Tanaka K. Tanaka K. Tanida I. Taylor G.S. Taylor J.P. Terman A. Tettamanti G. Thompson C.B. Thumm M. Tolkovsky A.M. Tooze S.A. Truant R. Tumanovska L.V. Uchiyama Y. Ueno T. Uzcátegui N.L. van der Klei I. Vaquero E.C. Vellai T. Vogel M.W. Wang H.G. Webster P. Wiley J.W. Xi Z. Xiao G. Yahalom J. Yang J.M. Yap G. Yin X.M. Yoshimori T. Yu L. Yue Z. Yuzaki M. Zabirnyk O. Zheng X. Zhu X. Deter R.L. Autophagy. 2008; 4: 151-175Crossref PubMed Scopus (1970) Google Scholar). LC3 is present in mammalian cells as a cytoplasmic form (LC3-I) or in a membrane-associated form (LC3-II). LC3-I is lipidated by phosphatidylethanolamine (LC3-PE) to the LC3-II form that associates to the autophagosome membrane and migrates on SDS-PAGE with a downward shift in molecular weight. First, we infected macrophages with L. monocytogenes for 0.5 h at 37 °C. At different times post-infection, protein lysates were collected. As shown in Fig. 1A, control cells showed only basal levels of autophagy. However, there was a time-dependent increase in LC3-II expression after infection with the pathogen. Using this antibody, LC3-I was barely detectable because of the lower sensitivity of the presently available anti-LC3 antibodies for LC3-I (39Mizushima N. Yoshimori T. Autophagy. 2007; 3: 542-545Crossref PubMed Scopus (1954) Google Scholar). Further, this response was not dependent on the bacterial dose because LC3-II expression increased equally well at a higher multiplicity of infection (Fig. 1A, right panel). As the processing of LC3-I to LC3-II increases, there is a corresponding increase in autophagosome formation (40Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5433) Google Scholar). We next employed GFP-LC3 to observe this phenomenon. Uninfected macrophages transfected with GFP-LC3 showed diffuse cytoplasmic distribution of the protein (Fig. 1B, left panel). However, after infection with L. monocytogenes, an increase in autophagosomes in the form of GFP-LC3 puncta were observed (Fig. 1B, right panel) that was approximately 4 times more than in control cells (Fig. 1C). We also observed autophagy at the ultrastructure level by electron microscopy. As shown in Fig. 1D, autophagosomes showing double-membraned structures were observed after infection with L. monocytogenes. LC3-II associates with both the outer and the inner membrane of autophagosomes. As autophagosomes mature by fusion with lysosomes, LC3-II present on the inner membrane of this organelle is also degraded (38Klionsky D.J. Abeliovich H. Agostinis P. Agrawal D.K. Aliev G. Askew D.S. Baba M. Baehrecke E.H. Bahr B.A. Ballabio A. Bamber B.A. Bassham D.C. Bergamini E. Bi X. Biard-Piechaczyk M. Blum J.S. Bredesen D.E. Brodsky J.L. Brumell J.H. Brunk U.T. Bursch W. Camougrand N. Cebollero E. Cecconi F. Chen Y. Chin L.S. Choi A. Chu C.T. Chung J. Clarke P.G. Clark R.S. Clarke S.G. Clavé C. Cleveland J.L. Codogno P. Colombo M.I. Coto-Montes A. Cregg J.M. Cuervo A.M. Debnath J. Demarchi F. Dennis P.B. Dennis P.A. Deretic V. Devenish R.J. Di Sano F. Dice J.F. Difiglia M. Dinesh-Kumar S. Distelhorst C.W. Djavaheri-Mergny M. Dorsey F.C. Dröge W. Dron M. Dunn Jr., W.A. Duszenko M. Eissa N.T. Elazar Z. Esclatine A. Eskelinen E.L. Fésüs L. Finley K.D. Fuentes J.M. Fueyo J. Fujisaki K. Galliot B. Gao F.B. Gewirtz D.A. Gibson S.B. Gohla A. Goldberg A.L. Gonzalez R. González-Estévez C. Gorski S. Gottlieb R.A. Häussinger D. He Y.W. Heidenreich K. Hill J.A. Høyer-Hansen M. Hu X. Huang W.P. Iwasaki A. Jäättelä M. Jackson W.T. Jiang X. Jin S. Johansen T. Jung J.U. Kadowaki M. Kang C. Kelekar A. Kessel D.H. Kiel J.A. Kim H.P. Kimchi A. Kinsella T.J. Kiselyov K. Kitamoto K. Knecht E. Komatsu M. Kominami E. Kondo S. Kovács A.L. Kroemer G. Kuan C.Y. Kumar R. Kundu M. Landry J. Laporte M. Le W. Lei H.Y. Lenardo M.J. Levine B. Lieberman A. Lim K.L. Lin F.C. Liou W. Liu L.F. Lopez-Berestein G. López-Otín C. Lu B. Macleod K.F. Malorni W. Martinet W. Matsuoka K. Mautner J. Meijer A.J. Meléndez A. Michels P. Miotto G. Mistiaen W.P. Mizushima N. Mograbi B. Monastyrska I. Moore M.N. Moreira P.I. Moriyasu Y. Motyl T. Münz C. Murphy L.O. Naqvi N.I. Neufeld T.P. Nishino I. Nixon R.A. Noda T. Nürnberg B. Ogawa M. Oleinick N.L. Olsen L.J. Ozpolat B. Paglin S. Palmer G.E. Papassideri I. Parkes M. Perlmutter D.H. Perry G. Piacentini M. Pinkas-Kramarski R. Prescott M. Proikas-Cezanne T. Raben N. Rami A. Reggiori F Rohrer B Rubinsztein DC Ryan KM Sadoshima J Sakagami H Sakai Y Sandri M Sasakawa C Sass M Schneider C Seglen PO Seleverstov O Settleman J Shacka JJ Shapiro IM Sibirny A Silva-Zacarin EC Simon HU Simone C Simonsen A Smith M.A. Spanel-Borowski K. Srinivas V. Steeves M. Stenmark H. Stromhaug P.E. Subauste C.S. Sugimoto S. Sulzer D. Suzuki T. Swanson M.S. Tabas I. Takeshita F. Talbot N.J. Tallóczy Z. Tanaka K. Tanaka K. Tanida I. Taylor G.S. Taylor J.P. Terman A. Tettamanti G. Thompson C.B. Thumm M. Tolkovsky A.M. Tooze S.A. Truant R. Tumanovska L.V. Uchiyama Y. Ueno T. Uzcátegui N.L. van der Klei I. Vaquero E.C. Vellai T. Vogel M.W. Wang H.G. Webster P. Wiley J.W. Xi Z. Xiao G. Yahalom J. Yang J.M. Yap G. Yin X.M. Yoshimori T. Yu L. Yue Z. Yuzaki M. Zabirnyk O. Zheng X. Zhu X. Deter R.L. Autophagy. 2008; 4: 151-175Crossref PubMed Scopus (1970) Google Scholar, 39Mizushima N. Yoshimori T. Autophagy. 2007; 3: 542-545Crossref PubMed Scopus (1954) Google Scholar). An increase in LC3-II levels indicates either enhanced autophagosome formation or decreased turnover of LC3-II (or autophagic flux) caused by delayed fusion with lysosomes. To better interpret the increase in LC3-II levels after L. monocytogenes infection, we treated macrophages with chloroquine (CQ), a lysosomotropic agent that inhibits autophagosome fusion with lysosomes and therefore autophagic degradation. As expected, control cells treated with CQ showed an increase in the expression of the lipidated LC3-II form that showed a similar higher trend when infected macrophages were treated with CQ (Fig. 1E), suggesting the presence of an efficient autophagic flux. Infection with L. monocytogenes led to autophagy induction. Thus, we sought to decipher the molecular sensor for host cells that triggers autophagy after L. monocytogenes infection. We first focused on the role of TLR2 in autophagy activation upon L. monocytogenes infection. Macrophages, either wild-type (WT) or Tlr2−/−, were infected with L. monocytogenes, and LC3 lipidation was evaluated by Western blotting (WB) 2 h and 4 h after infection. Cells deficient in TLR2 showed inefficient autophagy induction after infection with L. monocytogenes (Fig. 2A and B). However, a normal autophagic response was induced with rapamycin, a classical inducer of autophagy (Fig. 2A), thus indicating that autophagy induction only in response to L. monocytogenes is compromised in Tlr2−/− cells. TLR4 has no role in L. monocytogenes recognition. In agreement, Tlr4−/− macrophages showed LC3-II induction comparable with that of WT macrophages (Fig. 2, A and B). Further, autophagic flux in control or infected cells, measured after exposure to CQ, was found to be equally efficient (Fig. 2A). We next used GFP-LC3 to determine the formation of autophagosomes in WT and Tlr2−/− cells. Transfection of WT macrophages showed distinct GFP-LC3 punctate structures after infection with L. monocytogenes (Fig. 2C). Consistent with our WB data, the number of GFP-LC3 puncta were markedly reduced in TLR2-deficient cells infected with L. monocytogenes (Fig. 2, C and D). This was not because of reduced uptake, as it was found to be similar in wild-type and Tlr2−/− macrophages (supplemental Fig. S1A). The induction of autophagosome formation was normal when these cells were treated with rapamycin (Fig. 2, C and D). These data demonstrate that autophagy induction upon L. monocytogenes infection is dependent on TLR2 signaling. Soon after its entry into a phagosome within a host cell, L. monocytogenes escapes into the cytosol, where it is recognized by intracellular receptors NOD1 and NOD2 (35Park J.H. Kim Y.G. McDonald C. Kanneganti T.D. Hasegawa M. Body-Malapel M. Inohara N. Núñez G. J. Immunol. 2007; 178: 2380-2386Crossref PubMed Scopus (391) Google Scholar, 41Mosa A. Trumstedt C. Eriksson E. Soehnlein O. Heuts F. Janik K. Klos A. Dittrich-Breiholz O. Kracht M. Hidmark A. Wigzell H. Rottenberg M.E. Infect. Immun. 2009; 77: 2908-2918Crossref PubMed Scopus (26) Google Scholar). To resolve whether any of these cytosolic receptors are required for activation of autophagy, we next infected Nod1−/− or Nod2−/− macrophages with L. monocytogenes and evaluated LC3 lipidation by WB at various times post-infection. Cells that were deficient in either NOD1 or NOD2 showed a marked reduction in autophagy induction. However, these cells showed a normal autophagic response to rapamycin (Fig. 3, A and B). Further, autophagic flux in control or infected cells, measured after exposure to CQ, was found to be equally efficient (Fig. 3A). We next infected GFP-LC3 transfected macrophages with L. monocytogenes. Consistent with our WB data, fewer GFP-LC3 puncta were seen in NOD1- or NOD2- deficient cells infected with L. monocytogenes as compared with WT cells (Fig. 3, C and D). This was not because of reduced uptake, as it was found to be similar in WT and Nod1−/− or Nod2−/− macrophages (supplemental Fig. S1A). The induction of autophagosome formation was normal when Nod1−/− or Nod2−/− cells were treated with rapamycin, indicating that the response is selective for L. monocytogenes infection (Fig. 3, C and D). These results suggest that both NOD1 and NOD2 are essential for induction of autophagy after infection of macrophages with L. monocytogenes. NOD1 and NOD2 signaling pathway involves the downstream adaptor RIP2 kinase. Earlier reports have suggested contradictory results in regard to the role for RIP2 in autophagy induction by stimulation with NOD ligands (42Travassos L.H. Carneiro L.A. Ramjeet M." @default.
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• W2065609114 title "TLR2 and RIP2 Pathways Mediate Autophagy of Listeria monocytogenes via Extracellular Signal-regulated Kinase (ERK) Activation" @default.
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