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Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome.

ABSTRACT: 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 1? (IL-1?) 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-1? 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.

SUBMITTER: Nakahira K

PROVIDER: S-EPMC3079381 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome.

Nakahira Kiichi K Haspel Jeffrey Adam JA Rathinam Vijay A K VA Lee Seon-Jin SJ Dolinay Tamas T Lam Hilaire C HC Englert Joshua A JA Rabinovitch Marlene M Cernadas Manuela M Kim Hong Pyo HP Fitzgerald Katherine A KA Ryter Stefan W SW Choi Augustine M K AM

Nature immunology 20101212 3

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 ...[more]

PMID: 21151103

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Project description:Genotoxic insult causes nuclear and mitochondrial DNA damages with macroautophagy/autophagy induction. The role of mitochondrial DNA (mtDNA) damage in the requirement of autophagy for nuclear DNA (nDNA) stability is unclear. Using site-specific DNA damage approaches, we show that specific nDNA damage alone does not require autophagy for repair unless in the presence of mtDNA damage. We provide evidence that after IR exposure-induced mtDNA and nDNA damages, autophagy suppression causes non-apoptotic mitochondrial permeability, by which mitochondrial ENDOG (endonuclease G) is released and translocated to nuclei to sustain nDNA damage in a TET (tet methylcytosine dioxygenase)-dependent manner. Furthermore, blocking lysosome function is sufficient to increase the amount of mtDNA leakage to the cytosol, accompanied by ENDOG-free mitochondrial puncta formation with concurrent ENDOG nuclear accumulation. We proposed that autophagy eliminates the mitochondria specified by mtDNA damage-driven mitochondrial permeability to prevent ENDOG-mediated genome instability. Finally, we showed that HBx, a hepatitis B viral protein capable of suppressing autophagy, also causes mitochondrial permeability-dependent ENDOG mis-localization in nuclei and is linked to hepatitis B virus (HBV)-mediated hepatocellular carcinoma development.Abbreviations: 3-MA: 3-methyladenine; 5-hmC: 5-hydroxymethylcytosine; ACTB: actin beta; ATG5: autophagy related 5; ATM: ATM serine/threonine kinase; DFFB/CAD: DNA fragmentation factor subunit beta; cmtDNA: cytosolic mitochondrial DNA; ConA: concanamycin A; CQ: chloroquine; CsA: cyclosporin A; Dox: doxycycline; DSB: double-strand break; ENDOG: endonuclease G; GFP: green fluorescent protein; Gy: gray; H2AX: H2A.X variant histone; HBV: hepatitis B virus; HBx: hepatitis B virus X protein; HCC: hepatocellular carcinoma; I-PpoI: intron-encoded endonuclease; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOMP: mitochondrial outer membrane permeability; mPTP: mitochondrial permeability transition pore; mtDNA: mitochondrial DNA; nDNA: nuclear DNA; 4-OHT: 4-hydroxytamoxifen; rDNA: ribosomal DNA; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TET: tet methylcytosine dioxygenase; TFAM: transcription factor A, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Project description:Modified vaccinia virus Ankara (MVA) is an attenuated double-stranded DNA poxvirus currently developed as a vaccine vector against HIV/AIDS. Profiling of the innate immune responses induced by MVA is essential for the design of vaccine vectors and for anticipating potential adverse interactions between naturally acquired and vaccine-induced immune responses. Here we report on innate immune sensing of MVA and cytokine responses in human THP-1 cells, primary human macrophages and mouse bone marrow-derived macrophages (BMDMs). The innate immune responses elicited by MVA in human macrophages were characterized by a robust chemokine production and a fairly weak pro-inflammatory cytokine response. Analyses of the cytokine production profile of macrophages isolated from knockout mice deficient in Toll-like receptors (TLRs) or in the adapter molecules MyD88 and TRIF revealed a critical role for TLR2, TLR6 and MyD88 in the production of IFNbeta-independent chemokines. MVA induced a marked up-regulation of the expression of RIG-I like receptors (RLR) and the IPS-1 adapter (also known as Cardif, MAVS or VISA). Reduced expression of RIG-I, MDA-5 and IPS-1 by shRNAs indicated that sensing of MVA by RLR and production of IFNbeta and IFNbeta-dependent chemokines was controlled by the MDA-5 and IPS-1 pathway in the macrophage. Crosstalk between TLR2-MyD88 and the NALP3 inflammasome was essential for expression and processing of IL-1beta. Transcription of the Il1b gene was markedly impaired in TLR2(-/-) and MyD88(-/-) BMDM, whereas mature and secreted IL-1beta was massively reduced in NALP3(-/-) BMDMs or in human THP-1 macrophages with reduced expression of NALP3, ASC or caspase-1 by shRNAs. Innate immune sensing of MVA and production of chemokines, IFNbeta and IL-1beta by macrophages is mediated by the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways. Delineation of the host response induced by MVA is critical for improving our understanding of poxvirus antiviral escape mechanisms and for designing new MVA vaccine vectors with improved immunogenicity.

Project description:Backgrounds and Aims: Na+ is an important nutrient and its intake, mainly from salt (NaCl), is essential for normal physiological function. However, high salt intake may lead to vascular injury, independent of a rise in blood pressure (BP). Canonical NALP3 inflammasome activation is a caspase-1 medicated process, resulting in the secretion of IL-18 and IL-1β which lead to endothelial dysfunction. However, some researches uncovered a direct and inflammasome-independent role of NALP3 in renal injury. Thus, this study was designed to investigate the possible mechanisms of NALP3 in high salt induced endothelial dysfunction. Methods and Results: Changes in endothelial function were measured by investigating mice (C57BL/6J, NALP3-/- and wild-type, WT) fed with normal salt diet (NSD) or high salt diet (HSD) for 12W, and thoracic aortic rings from C57BL/6J mice cultured in high-salt medium. Changes of tube formation ability, intracellular reactive oxygen species (ROS), and NALP3 inflammasome expression were detected using mouse aortic endothelial cells (MAECs) cultured in high-salt medium. Consumption of HSD for 12W did not affect BP or body weight in C57BL/6J mice. Endothelium-dependent relaxation (EDR) decreased significantly in C57BL/6J mice fed with HSD for 12W, and in isolated thoracic aortic rings cultured in high-salt medium for 24 h. Results from the aortic ring assay also revealed that the angiogenic function of thoracic aortas was impaired by either consumption of HSD or exposure to high-salt medium. NALP3-/- mice fed with HSD showed a relatively mild decrease in EDR function when compared with WT mice. Tube length of thoracic aortic rings from NALP3-/- mice was longer than those from WT mice after receiving high-salt treatment. Inhibiting NALP3 with a NALP3 antagonist, small interfering (si) RNA experiments using si-NALP3, and decomposing ROS significantly improved tube formation ability in MAECs under high salt medium. NALP3 expression was increased in MAECs cultured with high salt treatment and inhibiting NALP3 reversed the down-regulation of p-eNOS induced by high salt in MAECs. Conclusion: High salt intake impairs endothelial function, which is at least in part mediated by increasing NALP3 expression.

Dataset Information

Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome.

Publications

Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome.

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