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• W2006714910 abstract "•Invasive bacteria induce autophagy in the intestinal epithelium•Pathobionts and overt pathogens induce epithelial cell autophagy•Intestinal epithelial autophagy requires MyD88•Intestinal epithelial autophagy limits bacterial dissemination The mammalian intestine is colonized with a diverse community of bacteria that perform many beneficial functions but can threaten host health upon tissue invasion. Epithelial cell-intrinsic innate immune responses are essential to limit the invasion of both commensal and pathogenic bacteria and maintain beneficial host-bacterial relationships; however, little is known about the role of various cellular processes, notably autophagy, in controlling bacterial interactions with the intestinal epithelium in vivo. We demonstrate that intestinal epithelial cell autophagy protects against tissue invasion by both opportunistically invasive commensals and the invasive intestinal pathogen Salmonella Typhimurium. Autophagy is activated following bacterial invasion of epithelial cells through a process requiring epithelial cell-intrinsic signaling via the innate immune adaptor protein MyD88. Additionally, mice deficient in intestinal epithelial cell autophagy exhibit increased dissemination of invasive bacteria to extraintestinal sites. Thus, autophagy is an important epithelial cell-autonomous mechanism of antibacterial defense that protects against dissemination of intestinal bacteria. The mammalian intestine is colonized with a diverse community of bacteria that perform many beneficial functions but can threaten host health upon tissue invasion. Epithelial cell-intrinsic innate immune responses are essential to limit the invasion of both commensal and pathogenic bacteria and maintain beneficial host-bacterial relationships; however, little is known about the role of various cellular processes, notably autophagy, in controlling bacterial interactions with the intestinal epithelium in vivo. We demonstrate that intestinal epithelial cell autophagy protects against tissue invasion by both opportunistically invasive commensals and the invasive intestinal pathogen Salmonella Typhimurium. Autophagy is activated following bacterial invasion of epithelial cells through a process requiring epithelial cell-intrinsic signaling via the innate immune adaptor protein MyD88. Additionally, mice deficient in intestinal epithelial cell autophagy exhibit increased dissemination of invasive bacteria to extraintestinal sites. Thus, autophagy is an important epithelial cell-autonomous mechanism of antibacterial defense that protects against dissemination of intestinal bacteria. The mammalian intestine is home to a complex and diverse population of bacteria. The members of this microbial community are essential for host metabolism and digestion, thereby creating a mutualistic relationship. Although the overall host-microbial relationship is symbiotic, the bacterial components of this community span a wide spectrum of lifestyles, encompassing benign commensals, opportunistically pathogenic pathobionts, and overt pathogens. The intestinal epithelium interfaces directly with this diverse community of bacteria and is the first line of defense against bacterial invasion of host tissues. The epithelium must therefore be equipped with a diverse array of defenses against bacterial attachment and invasion. One way in which the epithelium defends against bacterial penetration is by secreting antimicrobial proteins that limit bacterial contact with the epithelial surface (Vaishnava et al., 2011Vaishnava S. Yamamoto M. Severson K.M. Ruhn K.A. Yu X. Koren O. Ley R. Wakeland E.K. Hooper L.V. The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine.Science. 2011; 334: 255-258Crossref PubMed Scopus (963) Google Scholar). However, certain intestinal pathogens, such as Salmonella enterica Serovar Typhimurium (Salmonella Typhimurium), or opportunistically invasive commensal bacteria, such as Enterococcus faecalis, can evade this first line of innate defense and enter epithelial cells (Eichelberg and Galán, 1999Eichelberg K. Galán J.E. Differential regulation of Salmonella typhimurium type III secreted proteins by pathogenicity island 1 (SPI-1)-encoded transcriptional activators InvF and hilA.Infect. Immun. 1999; 67: 4099-4105PubMed Google Scholar; Klare et al., 2001Klare I. Werner G. Witte W. Enterococci. Habitats, infections, virulence factors, resistances to antibiotics, transfer of resistance determinants.Contrib. Microbiol. 2001; 8: 108-122Crossref PubMed Google Scholar; Müller et al., 2012Müller A.J. Kaiser P. Dittmar K.E.J. Weber T.C. Haueter S. Endt K. Songhet P. Zellweger C. Kremer M. Fehling H.J. Hardt W.D. Salmonella gut invasion involves TTSS-2-dependent epithelial traversal, basolateral exit, and uptake by epithelium-sampling lamina propria phagocytes.Cell Host Microbe. 2012; 11: 19-32Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This raises the question of whether there are epithelial cell-intrinsic immune mechanisms that detect invading bacteria and limit their further dissemination. Autophagy is an evolutionarily ancient process in which cytoplasmic materials are targeted to the lysosome for degradation. Portions of the cytoplasm are sequestered into double-membrane structures, called autophagosomes, that fuse with lysosomes, delivering their contents for degradation by lysosomal enzymes (Deretic and Levine, 2009Deretic V. Levine B. Autophagy, immunity, and microbial adaptations.Cell Host Microbe. 2009; 5: 527-549Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). The process involves the concerted action of several cytoplasmic proteins. These include LC3, which becomes lipid-conjugated and associates with the autophagosome membrane, and ATG5, which is conjugated to ATG12 and associates with the elongating isolation membrane (Mizushima et al., 2002Mizushima N. Ohsumi Y. Yoshimori T. Autophagosome formation in mammalian cells.Cell Struct. Funct. 2002; 27: 421-429Crossref PubMed Scopus (755) Google Scholar). A primary function of autophagy is to maintain cellular homeostasis by degrading cytoplasmic contents during cellular starvation and by recycling damaged organelles and proteins (Rabinowitz and White, 2010Rabinowitz J.D. White E. Autophagy and metabolism.Science. 2010; 330: 1344-1348Crossref PubMed Scopus (1474) Google Scholar). More recently, autophagy has been shown to be critical for the recognition and degradation of intracellular pathogens, thus functioning as an innate barrier to infection (Deretic and Levine, 2009Deretic V. Levine B. Autophagy, immunity, and microbial adaptations.Cell Host Microbe. 2009; 5: 527-549Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar; Levine et al., 2011Levine B. Mizushima N. Virgin H.W. Autophagy in immunity and inflammation.Nature. 2011; 469: 323-335Crossref PubMed Scopus (2415) Google Scholar). Bacterial targets of autophagy include the intestinal pathogens S. Typhimurium (Jia et al., 2009Jia K. Thomas C. Akbar M. Sun Q. Adams-Huet B. Gilpin C. Levine B. Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance.Proc. Natl. Acad. Sci. USA. 2009; 106: 14564-14569Crossref PubMed Scopus (87) Google Scholar; Kuballa et al., 2008Kuballa P. Huett A. Rioux J.D. Daly M.J. Xavier R.J. Impaired autophagy of an intracellular pathogen induced by a Crohn’s disease associated ATG16L1 variant.PLoS ONE. 2008; 3: e3391Crossref PubMed Scopus (284) Google Scholar; Rioux et al., 2007Rioux J.D. Xavier R.J. Taylor K.D. Silverberg M.S. Goyette P. Huett A. Green T. Kuballa P. Barmada M.M. Datta L.W. et al.Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis.Nat. Genet. 2007; 39: 596-604Crossref PubMed Scopus (1470) Google Scholar; Wild et al., 2011Wild P. Farhan H. McEwan D.G. Wagner S. Rogov V.V. Brady N.R. Richter B. Korac J. Waidmann O. Choudhary C. et al.Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth complexes in bacteria.Science. 2011; 333: 228-233Crossref PubMed Scopus (956) Google Scholar) and Listeria monocytogenes (Py et al., 2007Py B.F. Lipinski M.M. Yuan J. Autophagy limits Listeria monocytogenes intracellular growth in the early phase of primary infection.Autophagy. 2007; 3: 117-125PubMed Google Scholar). Autophagy limits the replication of both of these bacterial species in cell culture models (Py et al., 2007Py B.F. Lipinski M.M. Yuan J. Autophagy limits Listeria monocytogenes intracellular growth in the early phase of primary infection.Autophagy. 2007; 3: 117-125PubMed Google Scholar; Wild et al., 2011Wild P. Farhan H. McEwan D.G. Wagner S. Rogov V.V. Brady N.R. Richter B. Korac J. Waidmann O. Choudhary C. et al.Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth complexes in bacteria.Science. 2011; 333: 228-233Crossref PubMed Scopus (956) Google Scholar). Intestinal epithelial cell autophagy is also critical for resistance against S. Typhimurium infection in the nematode Caenorhabditis elegans (Jia et al., 2009Jia K. Thomas C. Akbar M. Sun Q. Adams-Huet B. Gilpin C. Levine B. Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance.Proc. Natl. Acad. Sci. USA. 2009; 106: 14564-14569Crossref PubMed Scopus (87) Google Scholar). However, the importance of autophagy for mammalian intestinal immunity remains underexplored. Such functions are likely to be especially important in the intestinal epithelium, which interfaces with a dense microbial community that harbors invasive bacteria. Genetic studies of inflammatory bowel disease (IBD) have revealed important roles for autophagy pathway proteins in intestinal immune homeostasis. IBD is a chronic inflammatory disease of the intestine that arises from dysregulated interactions with the resident microbiota (Xavier and Podolsky, 2007Xavier R.J. Podolsky D.K. Unravelling the pathogenesis of inflammatory bowel disease.Nature. 2007; 448: 427-434Crossref PubMed Scopus (3234) Google Scholar). Recent studies have identified polymorphisms in genes of the autophagy pathway that are linked to Crohn’s disease, a type of IBD in which the inflammation is localized to the distal small intestine and variable regions in the colon. Mutations in the critical autophagy gene ATG16L1 are associated with a predisposition to Crohn’s disease in humans (Hampe et al., 2007Hampe J. Franke A. Rosenstiel P. Till A. Teuber M. Huse K. Albrecht M. Mayr G. De La Vega F.M. Briggs J. et al.A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1.Nat. Genet. 2007; 39: 207-211Crossref PubMed Scopus (1544) Google Scholar; Rioux et al., 2007Rioux J.D. Xavier R.J. Taylor K.D. Silverberg M.S. Goyette P. Huett A. Green T. Kuballa P. Barmada M.M. Datta L.W. et al.Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis.Nat. Genet. 2007; 39: 596-604Crossref PubMed Scopus (1470) Google Scholar; Wellcome Trust Case Control Consortium, 2007Wellcome Trust Case Control ConsortiumGenome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls.Nature. 2007; 447: 661-678Crossref PubMed Scopus (7775) Google Scholar). However, the ATG16L1 polymorphisms associated with Crohn’s disease do not confer autophagy defects in mice, suggesting that the inflammatory phenotypes arise from autophagy-independent functions of ATG16L1 (Cadwell et al., 2008Cadwell K. Liu J.Y. Brown S.L. Miyoshi H. Loh J. Lennerz J.K. Kishi C. Kc W. Carrero J.A. Hunt S. et al.A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells.Nature. 2008; 456: 259-263Crossref PubMed Scopus (1161) Google Scholar). Rather, the mutations cause defects in granule formation in Paneth cells, a specialized epithelial cell lineage that secretes abundant antimicrobial proteins (Cadwell et al., 2008Cadwell K. Liu J.Y. Brown S.L. Miyoshi H. Loh J. Lennerz J.K. Kishi C. Kc W. Carrero J.A. Hunt S. et al.A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells.Nature. 2008; 456: 259-263Crossref PubMed Scopus (1161) Google Scholar; Cadwell et al., 2010Cadwell K. Patel K.K. Maloney N.S. Liu T.-C. Ng A.C.Y. Storer C.E. Head R.D. Xavier R. Stappenbeck T.S. Virgin H.W. Virus-plus-susceptibility gene interaction determines Crohn’s disease gene Atg16L1 phenotypes in intestine.Cell. 2010; 141: 1135-1145Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar). Thus, it is not yet clear whether bona fide autophagy plays a role in maintaining intestinal homeostasis in vivo. Here we report that intestinal epithelial cell autophagy is essential for mammalian intestinal defense against invasive bacteria. We show that epithelial autophagy is activated in the mouse intestinal epithelium by a pathogen, S. Typhimurium, as well as by E. faecalis, an opportunistically invasive commensal. Further, we find that epithelial autophagy is specifically triggered by bacterial invasion of epithelial cells and requires epithelial cell-intrinsic MyD88 signaling. Finally, we use mice with an epithelial cell-specific deletion of a critical autophagy factor to show that epithelial cell autophagy is critical for limiting extraintestinal spread of S. Typhimurium. Our observations thus reveal that autophagy is a key epithelial cell-autonomous mechanism of antibacterial defense that protects against dissemination of intestinal bacteria. Our findings provide insight into how the mammalian intestinal epithelium maintains homeostasis with a diverse intestinal microbiota and establish a key role for autophagy in innate immune defense of the intestine. LC3 is an essential autophagy protein that is recruited from the cytoplasm to the autophagosome membrane (Mizushima et al., 2002Mizushima N. Ohsumi Y. Yoshimori T. Autophagosome formation in mammalian cells.Cell Struct. Funct. 2002; 27: 421-429Crossref PubMed Scopus (755) Google Scholar). A classical assay for autophagy activation uses immunofluorescence to visually assess the recruitment of cytoplasmic LC3 to autophagosomes, which are detected as morphologically distinct punctate structures (Mizushima et al., 2010Mizushima N. Yoshimori T. Levine B. Methods in mammalian autophagy research.Cell. 2010; 140: 313-326Abstract Full Text Full Text PDF PubMed Scopus (3531) Google Scholar). To determine whether bacteria activate autophagy in the intestinal epithelium in vivo, we assessed LC3 distribution in the intestinal epithelial cells of mice of differing bacterial colonization status. Germfree mice are microbiologically sterile, thus allowing us to compare LC3 localization in the presence and absence of intestinal bacteria. Both germfree and conventionally raised (conventional) mice exhibited a diffuse, cytoplasmic distribution of LC3, indicating that a specific pathogen-free (SPF) microbiota was not sufficient to activate epithelial autophagosome formation, as indicated by LC3 staining (Figure 1A). To test whether an invasive bacterial pathogen could elicit autophagosome formation, we orally infected mice with S. Typhimurium. At 24 hr after S. Typhimurium inoculation into both germfree and conventional mice, we observed punctate LC3+ structures in small intestinal epithelial cells, indicating autophagosome formation (Figure 1A). Following S. Typhimurium infection of germfree mice, the LC3+ puncta were positioned apically relative to the nucleus. In contrast, puncta were positioned both apically and basolaterally relative to the epithelial cell nuclei in S. Typhimurium-infected conventional mice (Figure 1A). Although the cause of this difference is not clear, it may be due to different routes of S. Typhimurium entry (e.g., apical versus basolateral) in the two different host settings (Chieppa et al., 2006Chieppa M. Rescigno M. Huang A.Y.C. Germain R.N. Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement.J. Exp. Med. 2006; 203: 2841-2852Crossref PubMed Scopus (577) Google Scholar; Müller et al., 2012Müller A.J. Kaiser P. Dittmar K.E.J. Weber T.C. Haueter S. Endt K. Songhet P. Zellweger C. Kremer M. Fehling H.J. Hardt W.D. Salmonella gut invasion involves TTSS-2-dependent epithelial traversal, basolateral exit, and uptake by epithelium-sampling lamina propria phagocytes.Cell Host Microbe. 2012; 11: 19-32Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar; Niess et al., 2005Niess J.H. Brand S. Gu X. Landsman L. Jung S. McCormick B.A. Vyas J.M. Boes M. Ploegh H.L. Fox J.G. et al.CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance.Science. 2005; 307: 254-258Crossref PubMed Scopus (1287) Google Scholar). We also performed z stack reconstructions of the fluorescent images in multiple focal planes to verify that the LC3+ structures were located within epithelial cells (Movie S1). We next characterized the location and timing of epithelial LC3+ autophagosome formation following S. Typhimurium infection. Numbers of LC3+ puncta were highest in the distal small intestine (ileum) and diminished in the middle and proximal regions (jejunum and duodenum, respectively) (Figures 1B and S1A available online). LC3+ autophagosomes were more abundant in epithelial cells inhabiting the ileal villus tips compared to the cells located closer to the crypts (Figure S1A). LC3+ puncta were also observed in colonic epithelial cells following S. Typhimurium infection but were rare relative to the numbers in the terminal ileum (Figure S1B). Numbers of LC3+ autophagosomes were highest in the terminal ileum at ∼24 hr following S. Typhimurium infection and diminished at 48 and 72 hr postinfection (Figure 1C). Thus, autophagosome formation is a rapid and transient response of the intestinal epithelium to oral S. Typhimurium infection. Drawing on these initial observations, all subsequent analysis was performed on ileal tissues, with epithelial cells visualized at the midpoint between the crypt base and villus tip. During recruitment to the autophagosome, LC3-I is lipidated to yield LC3-II, which becomes associated with the autophagosome membrane (Pankiv et al., 2007Pankiv S. Clausen T.H. Lamark T. Brech A. Bruun J.-A. Outzen H. Øvervatn A. Bjørkøy G. Johansen T. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy.J. Biol. Chem. 2007; 282: 24131-24145Crossref PubMed Scopus (3306) Google Scholar). Western blot analysis of isolated ileal epithelial cells showed increased conversion of LC3-I to LC3-II at 24 hr after S. Typhimurium infection, consistent with autophagy activation (Figures 1D and 1E). This conversion was diminished after 72 hr (Figures 1D and 1E), which is in accord with the reduced numbers of LC3+ autophagosomes observed by immunofluorescence (Figure 1C). To assess whether autophagosomes colocalized with intracellular bacteria, we orally challenged germfree mice with S. Typhimurium constitutively expressing GFP (S. Typhimurium-GFP). At 24 hr after challenge, we could visualize S. Typhimurium within enterocytes (Figure 1F). Analysis of serially cut sections with a no-primary-antibody control verified that the GFP signal was not due to nonspecific autofluorescence (Figure S1C), which is frequently observed in fixed small intestinal tissues (Salzman et al., 2010Salzman N.H. Hung K. Haribhai D. Chu H. Karlsson-Sjöberg J. Amir E. Teggatz P. Barman M. Hayward M. Eastwood D. et al.Enteric defensins are essential regulators of intestinal microbial ecology.Nat. Immunol. 2010; 11: 76-83Crossref PubMed Scopus (869) Google Scholar; Vaishnava et al., 2011Vaishnava S. Yamamoto M. Severson K.M. Ruhn K.A. Yu X. Koren O. Ley R. Wakeland E.K. Hooper L.V. The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine.Science. 2011; 334: 255-258Crossref PubMed Scopus (963) Google Scholar). The GFP signal was specific to S. Typhimurium, as no signal above background was detected in uninfected germfree tissues (Figure S1D). Upon merging the LC3 and GFP channels, we found that some S. Typhimurium-GFP colocalized with LC3 (Figure 1F), consistent with the targeting of S. Typhimurium to autophagosomes. The colocalization of LC3 and GFP was determined to have a Pearson’s coefficient of 0.89, indicating a strong, positive relationship between the two signals (Figure 1G). Approximately 40% of intracellular S. Typhimurium colocalized with LC3 (Figure 1H), while ∼70% of the LC3+ puncta colocalized with S. Typhimurium-GFP (Figure 1I). These results demonstrate that autophagosome formation occurs in epithelial cells in response to S. Typhimurium infection and that the majority of autophagosomes colocalize with bacteria. To determine whether the epithelial autophagic response to S. Typhimurium was representative of a general response to invading bacteria, we asked whether epithelial autophagy could also be activated by opportunistically invasive members of the intestinal microbiota. To test this idea, we orally challenged germfree mice with Enterococcus faecalis, a Gram-positive, opportunistically invasive member of the human intestinal microbiota (Klare et al., 2001Klare I. Werner G. Witte W. Enterococci. Habitats, infections, virulence factors, resistances to antibiotics, transfer of resistance determinants.Contrib. Microbiol. 2001; 8: 108-122Crossref PubMed Google Scholar). At 24 hr postinfection, we detected E. faecalis (harboring an episomal GFP-expressing plasmid) in epithelial cells of the small intestine (Figures 2A and 2D ). Coincident with the ability of E. faecalis to invade epithelial cells, we observed numerous LC3+ autophagosomes (Figures 2B and 2E). In contrast to the apical localization of the LC3+ puncta observed after S. Typhimurium colonization of germfree mice (Figure 1A), the majority of the E. faecalis-induced autophagosomes were located on the basolateral side of the nucleus. As discussed above, we suggest that this may be due to differing routes of epithelial cell entry for S. Typhimurium and E. faecalis. Western blot analysis of isolated ileal epithelial cells revealed increased conversion of LC3-I to LC3-II at 24 hr following E. faecalis colonization, consistent with autophagy activation (Figures 2F and 2G). In contrast, a noninvasive member of the microbiota, Lactobacillus salivarius, colonized the small intestine to approximately the same extent as E. faecalis (Figure 2C) but was not detected within epithelial cells (Figures 2A and 2D) and did not trigger formation of LC3+ autophagosomes (Figures 2B and 2E). Together, these results suggest that epithelial cell autophagy can be activated by opportunistically invasive members of the microbiota. We further characterized the autophagic response of intestinal epithelial cells using transmission electron microscopy (TEM). At 24 hr after introduction of S. Typhimurium into germfree mice, we observed double-membraned autophagosomes within small intestinal epithelial cells (Figures 3B and 3C ) (Mizushima et al., 2010Mizushima N. Yoshimori T. Levine B. Methods in mammalian autophagy research.Cell. 2010; 140: 313-326Abstract Full Text Full Text PDF PubMed Scopus (3531) Google Scholar). The double membranes enclosed bacteria and were absent from the intestinal epithelial cells of germfree mice (Figure 3A). We also observed double-membrane structures surrounding bacteria in the epithelial cells of mice colonized for 24 hr with E. faecalis (Figures 3D and 3E). These results support the idea that invading bacteria activate autophagy within intestinal epithelial cells and are targeted to the autophagosomes. Our findings above suggested that bacterial invasion of epithelial cells is required to activate intestinal epithelial autophagy. To further test this idea, we used genetically altered S. Typhimurium. The SPI-1 pathogenicity island encompasses genes essential for S. Typhimurium entry into epithelial cells, and an S. Typhimurium mutant engineered to lack this island (ΔSPI-1) is defective in its ability to invade gut epithelia (Eichelberg and Galán, 1999Eichelberg K. Galán J.E. Differential regulation of Salmonella typhimurium type III secreted proteins by pathogenicity island 1 (SPI-1)-encoded transcriptional activators InvF and hilA.Infect. Immun. 1999; 67: 4099-4105PubMed Google Scholar). The wild-type and ΔSPI-1 strains colonized germfree mice to equivalent levels after 24 hr (Figure S2), yet the numbers of LC3+ autophagosomes formed in response to the ΔSPI-1 mutant were dramatically reduced relative to the wild-type strain (Figures 4A and 4B ). This suggests that cellular invasion is required for S. Typhimurium to activate epithelial autophagy. To corroborate this finding, we tested a second isogenic S. Typhimurium mutant strain lacking a single component of the type III secretion apparatus, InvA. Deletion of invA also inhibits epithelial cell entry by S. Typhimurium (Everest et al., 1999Everest P. Ketley J. Hardy S. Douce G. Khan S. Shea J. Holden D. Maskell D. Dougan G. Evaluation of Salmonella typhimurium mutants in a model of experimental gastroenteritis.Infect. Immun. 1999; 67: 2815-2821PubMed Google Scholar; Galán et al., 1992Galán J.E. Ginocchio C. Costeas P. Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family.J. Bacteriol. 1992; 174: 4338-4349Crossref PubMed Scopus (443) Google Scholar), and, like the ΔSPI-1 strain, the ΔinvA mutant elicited reduced numbers of LC3+ autophagosomes in the small intestinal epithelium (Figures 4A and 4B). These results support the idea that bacterial entry into epithelial cells is a prerequisite for autophagy activation. Prior studies have shown that bacterial induction of autophagy in macrophages requires activation of innate immune signaling pathways (Shi and Kehrl, 2008Shi C.-S. Kehrl J.H. MyD88 and Trif target Beclin 1 to trigger autophagy in macrophages.J. Biol. Chem. 2008; 283: 33175-33182Crossref PubMed Scopus (321) Google Scholar; Travassos et al., 2010Travassos L.H. Carneiro L.A.M. Ramjeet M. Hussey S. Kim Y.-G. Magalhães J.G. Yuan L. Soares F. Chea E. Le Bourhis L. et al.Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry.Nat. Immunol. 2010; 11: 55-62Crossref PubMed Scopus (1025) Google Scholar). At the same time, other intestinal epithelial cell-intrinsic innate immune responses, such as expression of the antimicrobial protein RegIIIγ, are dependent on MyD88 (Brandl et al., 2007Brandl K. Plitas G. Schnabl B. DeMatteo R.P. Pamer E.G. MyD88-mediated signals induce the bactericidal lectin RegIII γ and protect mice against intestinal Listeria monocytogenes infection.J. Exp. Med. 2007; 204: 1891-1900Crossref PubMed Scopus (310) Google Scholar; Vaishnava et al., 2011Vaishnava S. Yamamoto M. Severson K.M. Ruhn K.A. Yu X. Koren O. Ley R. Wakeland E.K. Hooper L.V. The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine.Science. 2011; 334: 255-258Crossref PubMed Scopus (963) Google Scholar), which signals downstream of Toll-like receptors (TLRs), the interleukin-1 receptor (IL-1R), and the IL-18 receptor (IL-18R) (Akira et al., 2006Akira S. Uematsu S. Takeuchi O. Pathogen recognition and innate immunity.Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8720) Google Scholar). We therefore tested whether bacterial induction of epithelial autophagy was similarly dependent on MyD88. As in conventional wild-type mice, epithelial LC3 showed a cytoplasmic distribution in conventional Myd88−/− mice (Figures 5A and 5B ). However, unlike wild-type mice, Myd88−/− mice did not show detectable LC3+ autophagosome formation after oral challenge with S. Typhimurium (Figures 5A and 5B), indicating that MyD88 is essential for bacterial activation of autophagy in small intestinal epithelial cells. We next investigated the cellular origin of the MyD88 signals required to elicit epithelial autophagy. We generated mice with an epithelial cell-specific deletion of Myd88 (Myd88ΔIEC) (Ismail et al., 2011Ismail A.S. Severson K.M. Vaishnava S. Behrendt C.L. Yu X. Benjamin J.L. Ruhn K.A. Hou B. DeFranco A.L. Yarovinsky F. Hooper L.V. Gammadelta intraepithelial lymphocytes are essential mediators of host-microbial homeostasis at the intestinal mucosal surface.Proc. Natl. Acad. Sci. USA. 2011; 108: 8743-8748Crossref PubMed Scopus (210) Google Scholar) by crossing mice carrying a loxP-flanked (floxed, fl) Myd88 allele (Myd88fl/fl) (Hou et al., 2008Hou B. Reizis B. DeFranco A.L. Toll-like receptors activate innate and adaptive immunity by using dendritic cell-intrinsic and -extrinsic mechanisms.Immunity. 2008; 29: 272-282Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) with mice expressing Cre recombinase under the control of the intestinal epithelial cell (IEC)-specific villin promoter (Madison et al., 2002Madison B.B. Dunbar L. Qiao X.T. Braunstein K. Braunstein E. Gumucio D.L. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine.J. Biol. Chem. 2002; 277: 33275-33283Crossref PubMed Scopus (555) Google Scholar). At 24 hr after oral challenge with S. Typhimurium, the Myd88ΔIEC mice retained a cytoplasmic distribution of epithelial LC3, whereas their Myd88fl/fl littermates showed LC3+ autophagosome formation (Figures 5C and 5D). Western blot analysis of isolated ileal epithelial cells revealed increased conversion of LC3-I to L" @default.
• W2006714910 created "2016-06-24" @default.
• W2006714910 creator A5015807309 @default.
• W2006714910 creator A5040267549 @default.
• W2006714910 creator A5042396622 @default.
• W2006714910 creator A5049357046 @default.
• W2006714910 date "2013-06-01" @default.
• W2006714910 modified "2023-10-16" @default.
• W2006714910 title "Intestinal Epithelial Autophagy Is Essential for Host Defense against Invasive Bacteria" @default.
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