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Bacterial Invasion: Linking Autophagy and Innate Immunity Crohn’s disease is a chronic inflammatory bowel disorder that has been associated with polymorphisms in the genes encoding the pattern-recognition receptor NOD2 and the autophagic regulator ATG16L1. A new study demonstrates that NOD2 recruits ATG16L1 at bacterial entry sites, thereby bridging innate immunity and autophagy. Lorenzo Galluzzi1,2,3, Oliver Kepp1,2,3, Laurence Zitvogel2,3,4, and Guido Kroemer1,2,3 The etiology of chronic inflammatory disorders of the bowel, such as Crohn’s disease, is largely unknown; however, recent epidemiological studies have suggested the existence of a strong genetic predisposition interacting with hitherto undetermined environmental triggers to render susceptible individuals at risk. More than 30 distinct loci have indeed been involved in the genetic susceptibility to Crohn’s disease, including genes implicated in autophagy, maintenance of epithelial barrier integrity, innate immunity, and secondary immune responses [1]. In particular, Crohn’s disease has been associated with polymorphisms affecting the essential autophagic modulator ATG16L1 and the pattern-recognition receptor (PRR) NOD2 [2]. Although both ATG16L1 and NOD2 had previously been shown to be required for the proper recognition and disposal of intracellular pathogens (including enteric bacteria that may contribute to the etiology of Crohn’s disease) [3,4], until recently a mechanistic link between these proteins was missing. Now, Travassos et al. [5] have demonstrated that NOD2 recruits ATG16L1 at the plasma membrane of infected cells to activate the generation of autophagosomes around invading bacteria, thereby pointing to a functional link between autophagy and innate immunity that is critical for the etiology of Crohn’s disease. Autophagy is a finely regulated, evolutionarily conserved multi-step process by which damaged, supernumerary or ectopic intracellular entities are sent to lysosomes for degradation [6]. While in a few experimental settings autophagy may contribute to cell death [7,8], in most circumstances autophagy constitutes a cytoprotective mechanism that is
activated in response to a wide range of stressful conditions [9]. Moreover, baseline levels of autophagy have been shown to be required for the maintenance of intracellular homeostasis — for instance, by preventing the accumulation of aggregate-prone proteins or uncoupled (and hence potentially dangerous) mitochondria — and to exert oncosuppressive functions [10]. Thus, the autophagic pathway has been implicated in human disorders as different as neurodegeneration, cancer, aging and infectious diseases [2]. In particular, autophagy has emerged as a critical mechanism of host defense against viral, bacterial and parasitic infections (in this context, it has been dubbed ‘xenophagy’), and defective autophagy has been linked to increased susceptibility to infectious diseases, both in vitro and in vivo [11]. During the initial phase of autophagy, an isolation membrane that presumably derives from the endoplasmic reticulum begins to surround the cytosolic material to be degraded. This process can occur in a relatively non-specific fashion, for instance when the autophagic pathway is triggered by nutrient deprivation (and hence is aimed at generating novel substrates to meet the cell’s energetic/anabolic demands), or it can be highly specific, for instance in response to pathogen invasion [12]. Multiple AuTophaGy-related (ATG) proteins, including the well-known ATG6/Beclin-1 and ATG8/LC3, are required for the correct execution of the autophagic program [12]. ATG16L1 also represents an essential autophagy modulator, as demonstrated by the fact that ATG16L1-deficient mice die in the first day of life (similar to atg52/2 and atg72/2 animals), presumably due to their inability to adapt to early postnatal starvation by activating autophagy [13]. ATG16L1-deficient cells are characterized by ineffective recruitment of the ATG5–ATG12
complex to the isolation membrane, and hence by an overall impairment of the autophagic machinery [13]. The Crohn’s disease-associated atg16l1 risk allele encodes a protein with a threonine-to-alanine substitution (T300A) in its carboxy-terminal domain, which contains tryptophan-aspartate (WD) repeats [4]. Although this domain of ATG16L1 is not conserved and is actually dispensable for the ATG16L1-dependent recruitment of ATG5–ATG12 complexes, as well as for starvation-induced autophagy, the ATG16L1 T300A mutant may exhibit a reduced stability and therefore fail to localize the autophagic machinery to invading bacteria [4]. This hypothesis is supported by the observation that ATG16L1-hypomorphic mice (generated by the intronic insertion of a gene-trap vector within atg16l1) normally develop into adulthood, yet progressively accumulate histological abnormalities in the bowel that closely resemble changes in the ileum from those patients with Crohn’s disease who are homozygous for the allele encoding the T300A ATG16L1 mutant [14]. Notably, in both settings, Paneth cells, which normally provide a barrier to bacterial invasion by secreting antimicrobial products, exhibit morphological and ultrastructural alterations, and, in ATG16L1-hypomorphic animals, these cells lose the capacity to secrete the antimicrobial peptide lysozyme into the intestinal lumen [14]. This suggests that defects in the molecular machinery for autophagy may contribute to Crohn’s disease by affecting distinct cell types of the bowel, including local macrophages and Paneth cells. In macrophages, the absence of ATG16L1 has been associated with enhanced cytokine production in response to ligation of Toll-like receptors (TLRs) [13], which are known to act as PRRs and therefore play a prominent role in innate immunity. PRRs, which include (but are not limited to) NODs and TLRs, are characterized by the ability to recognize invading pathogens as well as other ‘danger signals’ and directly activate specific signal transduction pathways that alert host defenses [15]. Such biochemical cascades may vary depending on both cell-extrinsic and cell-intrinsic variables, including cell type and initiating stimulus. Thus, in
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Figure 1. Schematic outline of the alterations that affect the NOD2–ATG16L1 signaling axis and xenophagy in Crohn’s disease. (A) Normal intestinal epithelium. Paneth cells react to pathogens by efficiently secreting antimicrobial products into the intestinal lumen. In addition, local macrophages mount a proficient xenophagic response mediated by the interaction between NOD2 and ATG16L1 at bacterial entry sites, resulting in the recruitment and activation of ATG5–ATG12 complexes. (B) Epithelium at risk for Crohn’s disease. ATG16L1-deficient Paneth cells exhibit ultrastructural alterations and (at least partially) lose the capacity to secrete antimicrobial peptides into the intestinal lumen in response to infection. Although the ATG16L1 T300A mutant retains the ability to recruit ATG5–ATG12 complexes, it displays a reduced stability and therefore fails to actively localize the autophagic machinery to invading bacteria. Similarly, the xenophagic response to intracellular pathogens is highly inefficient in macrophages that express the NOD2FS variant, as NOD2FS prevents ATG16L1 from localizing at bacterial entry sites by retaining it in the cytoplasm.
non-myeloid cells infected by Shigella flexneri, NOD1 can activate the pro-inflammatory transcription factor NF-kB (via the receptor-interacting kinase RIP2) or can elicit a regulated form of necrosis (necroptosis, which also has inflammatory outcomes) [16]. Similarly, NOD2 has been shown to respond to bacterial products by triggering a RIP2-dependent signal transduction cascade that eventually results in NF-kB activation [17]. Consistent with the critical role of NODs in innate immunity, both nod12/2 and nod22/2 mice exhibit an increased susceptibility to infection by intracellular bacteria [18,19]. The most prevalent nod2 polymorphism in Crohn’s disease is L1007insC, resulting in a truncated NOD2 protein (NOD2FS) that fails to activate NF-kB in response to peptidoglycan [20]. Altogether, these observations suggested that both autophagy and innate immunity are involved in the
pathogenesis of Crohn’s disease, and strongly pointed to some kind of cooperation between these processes, yet the molecular nature of this crosstalk was elusive. This gap has now been filled by Travassos and colleagues [5], who have demonstrated that peptidoglycan sensing is functionally connected to the initiation of autophagy thanks to the interaction between NODs and ATG16L1 (Figure 1). In a variety of experimental settings (including ‘classical’ human cancer cell lines, nod12/2 and nod22/2 mice, as well as immortalized cells derived from Crohn’s disease patients), these authors have demonstrated that NOD1 and NOD2 are critical for the autophagic response to invasive bacteria because they recruit ATG16L1 to bacterial entry sites at the plasma membrane. The interaction between NODs and ATGL16L1 (and the consequent induction of autophagy) persisted in rip22/2 mouse embryonic
fibroblasts challenged with S. flexneri, suggesting that NODs mediate autophagy independent of NF-kB [5]. Most importantly, the authors found that the Crohn’s disease-associated truncated variant of NOD2 (NOD2FS, which unlike its wild-type counterpart is localized exclusively to the cytoplasm) fails to recruit ATG16L1 to the plasma membrane upon infection with intracellular bacteria. Co-immunoprecipitation assays revealed that, similar to wild-type NOD2, NOD2FS retains the ability to bind ATG16L1. However, in NOD2FS-expressing cells, ATG16L1 failed to relocalize to the sites of bacterial invasion, suggesting that NOD2FS might suppress the autophagy-modulatory functions of ATG16L1 by retaining it in the cytoplasm and hence preventing it from localizing at the plasma membrane. Accordingly, macrophages derived from the bone marrow of
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mice homozygous for the murine NOD2 mutation corresponding to the human Crohn’s disease-associated L1007insC mutation displayed a striking xenophagy defect in response to S. flexneri infection [5]. Taken together, the results by Travassos et al. [5] establish a mechanistic link between modulators of innate immunity (i.e., NODs) and the cellular machinery for autophagy (and ATG16L1 in particular), which cooperate in the control of bacterial invasion. Thus, the NOD2–ATG16L1 axis appears for the first time as a unique pathway, the deregulation of which plays a central role in the etiology of Crohn’s disease, with obvious therapeutic implications. Future investigations will have to elucidate whether the products of other loci that have been associated with Crohn’s disease also interact with the molecular machinery for xenophagy. References 1. Van Limbergen, J., Wilson, D.C., and Satsangi, J. (2009). The genetics of Crohn’s disease. Annu. Rev. Genomics Hum. Genet. 10, 89–116. 2. Levine, B., and Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell 132, 27–42. 3. Inohara, N., Ogura, Y., Fontalba, A., Gutierrez, O., Pons, F., Crespo, J., Fukase, K., Inamura, S., Kusumoto, S., Hashimoto, M., et al. (2003). Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J. Biol. Chem. 278, 5509–5512.
4. Kuballa, P., Huett, A., Rioux, J.D., Daly, M.J., and Xavier, R.J. (2008). Impaired autophagy of an intracellular pathogen induced by a Crohn’s disease associated ATG16L1 variant. PLoS ONE 3, e3391. 5. Travassos, L.H., Carneiro, L.A., Ramjeet, M., Hussey, S., Kim, Y.G., Magalhaes, J.G., Yuan, L., Soares, F., Chea, E., Le Bourhis, L., et al. (2009). Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry. Nat. Immunol. 11, 55–62. 6. Galluzzi, L., Vicencio, J.M., Kepp, O., Tasdemir, E., Maiuri, M.C., and Kroemer, G. (2008). To die or not to die: that is the autophagic question. Curr. Mol. Med. 8, 78–91. 7. Kroemer, G., Galluzzi, L., and Brenner, C. (2007). Mitochondrial membrane permeabilization in cell death. Physiol. Rev. 87, 99–163. 8. Kroemer, G., Galluzzi, L., Vandenabeele, P., Abrams, J., Alnemri, E.S., Baehrecke, E.H., Blagosklonny, M.V., El-Deiry, W.S., Golstein, P., Green, D.R., et al. (2009). Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ. 16, 3–11. 9. Kroemer, G., and Levine, B. (2008). Autophagic cell death: the story of a misnomer. Nat. Rev. Mol. Cell Biol. 9, 1004–1010. 10. Morselli, E., Galluzzi, L., Kepp, O., Vicencio, J.M., Criollo, A., Maiuri, M.C., and Kroemer, G. (2009). Anti- and pro-tumor functions of autophagy. Biochim. Biophys. Acta 1793, 1524–1532. 11. Virgin, H.W., and Levine, B. (2009). Autophagy genes in immunity. Nat. Immunol. 10, 461–470. 12. Mizushima, N., Levine, B., Cuervo, A.M., and Klionsky, D.J. (2008). Autophagy fights disease through cellular self-digestion. Nature 451, 1069–1075. 13. Saitoh, T., Fujita, N., Jang, M.H., Uematsu, S., Yang, B.G., Satoh, T., Omori, H., Noda, T., Yamamoto, N., Komatsu, M., et al. (2008). Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature 456, 264–268.
siRNAs: The Hidden Face of the Small RNA World MicroRNAs are believed to control many physiological processes in animals. Now, two studies show that some of their presumptive functions are actually fulfilled by another class of RNAs — siRNAs. Herve´ Seitz Three classes of small regulatory RNAs are known in animals: microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). The first two classes share common features: they are loaded on the same effector proteins (the Ago subfamily of the ‘Argonaute’ protein family) and they are generated by the cleavage of double-stranded RNA by nucleases of the RNase III family. But miRNAs and siRNAs differ in their biogenesis (Figure 1): while both
classes are processed by a nuclease called ‘Dicer’, the biogenesis of most miRNAs also involves an enzyme called ‘Drosha’ (and its partner protein, Dgcr8). miRNAs were discovered earlier than siRNAs (in mammals, endogenous siRNAs were uncovered two years ago [1–3], seven years later than miRNAs [4]). Therefore, microRNAs have been more extensively studied: before their siRNA cousins were even discovered, miRNAs had been implicated in a broad range of biological processes, notably in the control of development (reviewed
14. Cadwell, 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. (2008). A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature 456, 259–263. 15. Carneiro, L.A., Magalhaes, J.G., Tattoli, I., Philpott, D.J., and Travassos, L.H. (2008). Nod-like proteins in inflammation and disease. J. Pathol. 214, 136–148. 16. Galluzzi, L., and Kroemer, G. (2009). Shigella targets the mitochondrial checkpoint of programmed necrosis. Cell Host Microbe 5, 107–109. 17. Hasegawa, M., Fujimoto, Y., Lucas, P.C., Nakano, H., Fukase, K., Nunez, G., and Inohara, N. (2008). A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB activation. EMBO J. 27, 373–383. 18. Viala, J., Chaput, C., Boneca, I.G., Cardona, A., Girardin, S.E., Moran, A.P., Athman, R., Memet, S., Huerre, M.R., Coyle, A.J., et al. (2004). Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat. Immunol. 5, 1166–1174. 19. Kobayashi, K.S., Chamaillard, M., Ogura, Y., Henegariu, O., Inohara, N., Nunez, G., and Flavell, R.A. (2005). Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 307, 731–734. 20. Girardin, S.E., Boneca, I.G., Viala, J., Chamaillard, M., Labigne, A., Thomas, G., Philpott, D.J., and Sansonetti, P.J. (2003). Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278, 8869–8872. 1INSERM,
U848, F-94805 Villejuif, France. Gustave Roussy, F-94805 Villejuif, France. 3Universite´ Paris-Sud/Paris XI, F-94270 Le Kremlin Biceˆtre, France. 4INSERM, U805, F-94805 Villejuif, France. E-mail:
[email protected] 2Institut
DOI: 10.1016/j.cub.2009.12.016
in [5]). Mammalian siRNAs have been shown to repress transposable elements and a few non-transposable genes (far less than miRNA-regulated genes) [1,2]. In this light, the strong phenotypic defects of Dicer-defective mice [6] were usually interpreted as a consequence of their lack of miRNAs. The recent discovery of endogenous siRNAs could challenge this belief: as Dicer participates in the biogenesis of both miRNAs and siRNAs (Figure 1), these defects could actually be due to a lack of siRNAs. Two papers in this issue of Current Biology indeed show that the observed defects in mouse oocyte development must be due to siRNAs [7,8]. In order to sort out the contribution of miRNAs and siRNAs to the spectacular phenotypes of Dicer-deficient oocytes, Suh and collaborators [7] prepared a conditional knock-out of Dgcr8. While
