Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome Kiichi Nakahira 1 , Jeffrey Adam Haspel 1,2 , Vijay A K Rathinam 3 , Seon-Jin Lee 1 , Tamas Dolinay 1 , Hilaire C Lam 1 , Joshua A Englert 1 , Marlene Rabinovitch 4 , Manuela Cernadas 1 , Hong Pyo Kim 1,5 , Katherine A Fitzgerald 3 , Stefan W Ryter 1 & Augustine M K Choi 1 Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. Here we demonstrate that depletion of the autophagic proteins LC3B and beclin 1 enhanced the activation of caspase-1 and secretion of interleukin 1β (IL-1β) and IL-18. Depletion of autophagic proteins promoted the accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial reactive oxygen species (ROS). Cytosolic mtDNA contributed to the secretion of IL-1β and IL-18 in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity. Autophagy, an evolutionarily conserved cellular process, facilitates the turnover of damaged proteins and organelles such as mitochondria 1 . During autophagy, cytosolic constituents are engulfed into double- membrane-bound vesicles called ‘autophagosomes’, which are subse- quently delivered to the lysosomes for degradation 1 . Accumulating evidence suggests that autophagic dysfunction is associated with aging and human diseases, including cancer, and neurodegenerative disorders 2 . Autophagy also participates in the clearance of intracel- lular bacteria, viruses and protozoa from host cells 2,3 . In addition, autophagy affects diverse immune system functions such as anti- gen presentation, lymphocyte development and cytokine secretion by cells of the immune response 3 . The involvement of autophagy in the secretion of proinflammatory cytokines has been demonstrated in mice with deletion of the gene encoding the autophagy protein ATG16L1, which produce excessive amounts of interleukin 1β (IL-1β) and IL-18 in response to lipopolysaccharide (LPS) and other patho- gen-associated molecular patterns 4 . That observation suggested that mutations in genes encoding autophagy proteins may directly or indirectly deregulate the secretion of IL-1β and IL-18. However, the mechanism by which autophagy regulates cytokine secretion is poorly understood. In macrophages, the secretion of IL-1β and IL-18 is controlled by the inflammatory signaling platform called the ‘inflammasome’ 5–7 . This is a multiprotein complex that mediates the cleavage and activation of caspase-1, leading to the maturation and secretion of IL-1β and IL-18. Cytoplasmic receptors of the NALP (also called NLR) fam- ily are critical components of the inflammasome and interact with apoptosis-associated adaptor protein ASC, which recruits the precur- sor form of caspase-1. Mice lacking NALP3, ASC or caspase-1 show a considerable defect in the production of mature IL-1β and IL-18 after challenge with LPS and ATP and are resistant to LPS-induced lethality 8,9 . Given the pivotal role of caspase-1 activation in the secre- tion of IL-1β and IL-18 and in LPS-induced inflammation in vivo, we hypothesized that autophagy-related proteins might regulate the secretion of proinflammatory cytokines via modulation of caspase-1 activation. In this study, we describe a pathway by which autophagic proteins regulate caspase-1-mediated innate immune responses through their role in preserving mitochondrial homeostasis. RESULTS Deficiency in autophagic proteins activates caspase-1 To examine the role of autophagic proteins during inflammasome acti- vation, we isolated macrophages from mice with deletion of autophagy genes, pretreated the cells with LPS and then stimulated them with ATP. ATP-driven activation of caspase-1 in LPS-primed macrophages is an established model for NALP3 inflammsome–mediated activa- tion of caspase-1 in vitro, which acts through the signaling pathways mediated by the purinergic receptor P2X7 (P2X7 receptor) and Toll- 1 Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA. 2 Pulmonary and Critical Care Medicine Section, VA Boston Healthcare System, Boston, Massachusetts, USA. 3 Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA. 4 The Wall Center for Pulmonary Vascular Diseases, School of Medicine, Stanford University, Stanford, California, USA. 5 School of Biological Sciences, College of Natural Sciences, University of Ulsan, Ulsan, Korea. Correspondence should be addressed to A.M.K.C. (amchoi@rics.bwh.harvard.edu). Received 18 November; accepted 6 December; published online 12 December 2010; doi:10.1038/ni.1980 222 VOLUME 8 NUMBER 3 MARCH 2011 NATURE IMMUNOLOGY ARTICLES © 2011 Nature America, Inc. All rights reserved.