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NEWS AND COMMENTARY mtDNA stress triggers interferon production
Stressed mitochondria sound the alarm Michael J White and Benjamin T Kile Immunology and Cell Biology advance online publication, 10 March 2015; doi:10.1038/icb.2015.31
M
itochondria are a central hub for antiviral immune defense. For example, the elimination of infected cells is, at least in part, driven by mitochondria-dependent (intrinsic) apoptosis. Mitochondria also act as a platform for the assembly of protein signaling complexes that drive the production of type I interferons (IFNs), potent antiviral cytokines which limit viral replication. In a recent issue of Nature, West et al.1 describe an additional role for mitochondria and their unique genome in mediating antiviral immunity. West and colleagues examined the cellular response to mitochondrial (mt) DNA stress, a phenomenon observed in virally infected cells,2 autoimmune diseases such as systemic lupus erythematosus, and age-related pathologies including cancer.3,4 They began with mice heterozygous for a knockout allele of the mtDNA-binding protein transcription factor A, mitochondrial (TFAM). TFAM regulates mtDNA architecture, abundance and segregation. The authors found that Tfam+/ − mouse embryonic fibroblasts (MEFs) exhibited mtDNA depletion, reduced oxidative mtDNA damage repair capacity, and markedly altered mtDNA packaging, organization and distribution. Collectively, they term this ‘mitochondrial DNA stress’. Expression profiling revealed enrichment of interferon-stimulated genes (ISGs) in Tfam+/ − MEFs, and these cells exhibited an enhanced type I interferon response upon transfection with poly(I:C). Similar results were obtained in MEFs or bone marrow-derived macrophages (BMDMs) where TFAM was inducibly depleted. This suggested that mtDNA stress leads to antiviral priming. MJ White, BT Kile are at ACRF Chemical Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia and Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia E-mail: [email protected]
Antiviral signaling pathways that initiate IFN production rely partly on receptors that monitor the cytosol for the presence of nucleic acids. Our lab and that of Richard Flavell recently demonstrated that, in the absence of a functional apoptotic caspase cascade, mtDNA released during Bcl-2 family-mediated mitochondrial apoptosis triggers the stimulator of interferon genes (STING) pathway.5,6 Cytosolic mtDNA binds to cyclic GMP–AMP synthase (cGAS), thereby catalyzing production of cyclic GMP–AMP dinucleotide (cGAMP), which binds to and activates STING. STING induces type I IFN transcription via the Tbk1–Irf3 signaling axis. Given prior evidence that mitochondrial stress can trigger the release of mtDNA into the cytosol,7 West and colleagues assayed for extra-mitochondrial mtDNA in TFAM-depleted cells and found a three- to four-fold increase in mtDNA fragments present in cytosolic extracts. Induction of mitochondrial fission, which facilitates removal of damaged mtDNA, significantly reduced ISG expression. West et al. therefore probed the role of the cGAS/STING pathway by short interfering RNA-mediated depletion in Tfam+/ − MEFs. Knockdown of cGAS, STING, Tbk1 or Irf3 ablated the ISG signature observed in TFAM-deficient cells. Thus, mtDNA stress leads to mtDNA leakage into the cytosol and subsequent activation of IFN production via the cGAS/STING pathway. As might be expected, given their augmented levels of ISG expression, Tfam+/ − MEFs and Tfam-depleted BMDMs were largely resistant to infection with herpes simplex virus 1 (HSV-1), vesicular stomatitis virus or rodent gammaherpesvirus MHV-68. Tfam+/ − mice were also resistant to lymphocytic choriomeningitis virus Armstrong infection. The authors therefore sought to establish whether mtDNA stress mediates antiviral responses in normal cells. Consistent with a prior report,2 they observed significant
mtDNA stress and DNA depletion in wildtype MEFs following challenge with HSV-1, MHV-68 and HSV-2. No such effect was seen in cells infected with vesicular stomatitis virus, influenza, lymphocytic choriomeningitis virus or vaccinia, suggesting virallyinduced mtDNA stress may be a specific byproduct of herpesvirus infection. HSV is known to induce the depletion of host mtDNA via the action of a virally encoded nuclease called UL12.5, which localizes to the mitochondria.2 West and colleagues infected MEFs and BMDMs with a replicationincompetent retrovirus encoding only UL12 M185 (a truncated but functionally equivalent form of UL12.5) and found that it caused many of the hallmarks of mtDNA stress, along with TFAM depletion and elevated ISG expression. When they infected cells with a UL12-deficient HSV-1 strain, a significant reduction in mtDNA stress, Tbk1 phosphorylation, IFNβ and ISG expression was observed relative to cells infected with the UL12-sufficient control. In mice inoculated intravaginally with HSV, the UL12-deficient strain elicited a lower type I IFN response and spread more quickly to the dorsal root ganglia. Collectively, these data suggest that herpesvirus-induced mtDNA stress is a critical danger signal to the infected cell that allows it to engage the innate immune system (Figure 1). This fascinating study provides further support to the notion that mtDNA is a damage-associated molecular pattern capable of triggering an antiviral response. It also raises some intriguing questions. Why would a virus deliberately trigger mtDNA stress and risk mtDNA reaching the cytosol? Logic would suggest that mtDNA depletion must assist in subverting some aspect of host defense, but whether that is the case, and what that aspect might be, remains to be established. By what mechanism does mtDNA stress leads to mtDNA escaping to
News and Commentary 2 Herpes virus
Mitochondrial stress
Antiviral gene expression
release in the setting of virally-induced mtDNA stress is distinct from apoptotic mtDNA release. Much remains to be understood about how the mitochondria and its genome contribute to the regulation of immune responses.
Nucleus
IRF3
TBK1
mtDNA release
cGAS
STING
Figure 1 Herpes virus infection triggers mitochondrial stress that results in mtDNA release into the cytosol. This subsequently activates cGAS and the downstream STING–TBK1–IRF3 pathway, leading to induction of antiviral gene expression and inhibition of viral replication.
the cytosol? Bcl-2 family-mediated apoptosis has been shown to trigger mtDNA release, but it is unclear whether mtDNA is released through the pores formed by Bak and Bax (the essential pro-death executioners of the intrinsic apoptosis pathway) in the mitochondrial outer membrane, or via some other
Immunology and Cell Biology
route.5,6 Does herpesvirus-induced mtDNA stress lead to the release of other mitochondrial proteins such as cytochrome c. The latter triggers activation of the apoptotic caspase cascade, which would be expected to cause cell death, not IFN production. On balance then, this would suggest that mtDNA
1 West AP, Khoury-Hanold W, Staron M, Tal MC, Pineda CM, Lang SM et al. Mitochondrial DNA stress primes the antiviral innate immune response. Nature e-pub ahead of print 2 February 2015; doi:10.1038/ nature14156. 2 Saffran HA, Pare JM, Corcoran JA, Weller SK, Smiley JR. Herpes simplex virus eliminates host mitochondrial DNA. EMBO Rep 2007; 8: 188–193. 3 Lee HT, Lin CS, Chen WS, Liao HT, Tsai CY, Wei YH. Leukocyte mitochondrial DNA alteration in systemic lupus erythematosus and its relevance to the susceptibility to lupus nephritis. Int J Mol Sci 2012; 13: 8853–8868. 4 Wallace DC. Mitochondria and cancer. Cancer 2012; 12: 685–698. 5 Rongvaux A, Jackson R, Harman CC, Li T, West AP, de Zoete MR et al. Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA. Cell 2014; 159: 1563–1577. 6 White MJ, McArthur K, Metcalf D, Lane RM, Cambier JC, Herold MJ et al. Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production. Cell 2014; 159: 1549–1562. 7 Shimada K, Crother TR, Karlin J, Dagvadorj J, Chiba N, Chen S et al. Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity 2012; 36: 401–414.
NEWS AND COMMENTARY mtDNA stress triggers interferon production
Stressed mitochondria sound the alarm Michael J White and Benjamin T Kile Immunology and Cell Biology advance online publication, 10 March 2015; doi:10.1038/icb.2015.31
M
itochondria are a central hub for antiviral immune defense. For example, the elimination of infected cells is, at least in part, driven by mitochondria-dependent (intrinsic) apoptosis. Mitochondria also act as a platform for the assembly of protein signaling complexes that drive the production of type I interferons (IFNs), potent antiviral cytokines which limit viral replication. In a recent issue of Nature, West et al.1 describe an additional role for mitochondria and their unique genome in mediating antiviral immunity. West and colleagues examined the cellular response to mitochondrial (mt) DNA stress, a phenomenon observed in virally infected cells,2 autoimmune diseases such as systemic lupus erythematosus, and age-related pathologies including cancer.3,4 They began with mice heterozygous for a knockout allele of the mtDNA-binding protein transcription factor A, mitochondrial (TFAM). TFAM regulates mtDNA architecture, abundance and segregation. The authors found that Tfam+/ − mouse embryonic fibroblasts (MEFs) exhibited mtDNA depletion, reduced oxidative mtDNA damage repair capacity, and markedly altered mtDNA packaging, organization and distribution. Collectively, they term this ‘mitochondrial DNA stress’. Expression profiling revealed enrichment of interferon-stimulated genes (ISGs) in Tfam+/ − MEFs, and these cells exhibited an enhanced type I interferon response upon transfection with poly(I:C). Similar results were obtained in MEFs or bone marrow-derived macrophages (BMDMs) where TFAM was inducibly depleted. This suggested that mtDNA stress leads to antiviral priming. MJ White, BT Kile are at ACRF Chemical Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia and Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia E-mail: [email protected]
Antiviral signaling pathways that initiate IFN production rely partly on receptors that monitor the cytosol for the presence of nucleic acids. Our lab and that of Richard Flavell recently demonstrated that, in the absence of a functional apoptotic caspase cascade, mtDNA released during Bcl-2 family-mediated mitochondrial apoptosis triggers the stimulator of interferon genes (STING) pathway.5,6 Cytosolic mtDNA binds to cyclic GMP–AMP synthase (cGAS), thereby catalyzing production of cyclic GMP–AMP dinucleotide (cGAMP), which binds to and activates STING. STING induces type I IFN transcription via the Tbk1–Irf3 signaling axis. Given prior evidence that mitochondrial stress can trigger the release of mtDNA into the cytosol,7 West and colleagues assayed for extra-mitochondrial mtDNA in TFAM-depleted cells and found a three- to four-fold increase in mtDNA fragments present in cytosolic extracts. Induction of mitochondrial fission, which facilitates removal of damaged mtDNA, significantly reduced ISG expression. West et al. therefore probed the role of the cGAS/STING pathway by short interfering RNA-mediated depletion in Tfam+/ − MEFs. Knockdown of cGAS, STING, Tbk1 or Irf3 ablated the ISG signature observed in TFAM-deficient cells. Thus, mtDNA stress leads to mtDNA leakage into the cytosol and subsequent activation of IFN production via the cGAS/STING pathway. As might be expected, given their augmented levels of ISG expression, Tfam+/ − MEFs and Tfam-depleted BMDMs were largely resistant to infection with herpes simplex virus 1 (HSV-1), vesicular stomatitis virus or rodent gammaherpesvirus MHV-68. Tfam+/ − mice were also resistant to lymphocytic choriomeningitis virus Armstrong infection. The authors therefore sought to establish whether mtDNA stress mediates antiviral responses in normal cells. Consistent with a prior report,2 they observed significant
mtDNA stress and DNA depletion in wildtype MEFs following challenge with HSV-1, MHV-68 and HSV-2. No such effect was seen in cells infected with vesicular stomatitis virus, influenza, lymphocytic choriomeningitis virus or vaccinia, suggesting virallyinduced mtDNA stress may be a specific byproduct of herpesvirus infection. HSV is known to induce the depletion of host mtDNA via the action of a virally encoded nuclease called UL12.5, which localizes to the mitochondria.2 West and colleagues infected MEFs and BMDMs with a replicationincompetent retrovirus encoding only UL12 M185 (a truncated but functionally equivalent form of UL12.5) and found that it caused many of the hallmarks of mtDNA stress, along with TFAM depletion and elevated ISG expression. When they infected cells with a UL12-deficient HSV-1 strain, a significant reduction in mtDNA stress, Tbk1 phosphorylation, IFNβ and ISG expression was observed relative to cells infected with the UL12-sufficient control. In mice inoculated intravaginally with HSV, the UL12-deficient strain elicited a lower type I IFN response and spread more quickly to the dorsal root ganglia. Collectively, these data suggest that herpesvirus-induced mtDNA stress is a critical danger signal to the infected cell that allows it to engage the innate immune system (Figure 1). This fascinating study provides further support to the notion that mtDNA is a damage-associated molecular pattern capable of triggering an antiviral response. It also raises some intriguing questions. Why would a virus deliberately trigger mtDNA stress and risk mtDNA reaching the cytosol? Logic would suggest that mtDNA depletion must assist in subverting some aspect of host defense, but whether that is the case, and what that aspect might be, remains to be established. By what mechanism does mtDNA stress leads to mtDNA escaping to
News and Commentary 2 Herpes virus
Mitochondrial stress
Antiviral gene expression
release in the setting of virally-induced mtDNA stress is distinct from apoptotic mtDNA release. Much remains to be understood about how the mitochondria and its genome contribute to the regulation of immune responses.
Nucleus
IRF3
TBK1
mtDNA release
cGAS
STING
Figure 1 Herpes virus infection triggers mitochondrial stress that results in mtDNA release into the cytosol. This subsequently activates cGAS and the downstream STING–TBK1–IRF3 pathway, leading to induction of antiviral gene expression and inhibition of viral replication.
the cytosol? Bcl-2 family-mediated apoptosis has been shown to trigger mtDNA release, but it is unclear whether mtDNA is released through the pores formed by Bak and Bax (the essential pro-death executioners of the intrinsic apoptosis pathway) in the mitochondrial outer membrane, or via some other
Immunology and Cell Biology
route.5,6 Does herpesvirus-induced mtDNA stress lead to the release of other mitochondrial proteins such as cytochrome c. The latter triggers activation of the apoptotic caspase cascade, which would be expected to cause cell death, not IFN production. On balance then, this would suggest that mtDNA
1 West AP, Khoury-Hanold W, Staron M, Tal MC, Pineda CM, Lang SM et al. Mitochondrial DNA stress primes the antiviral innate immune response. Nature e-pub ahead of print 2 February 2015; doi:10.1038/ nature14156. 2 Saffran HA, Pare JM, Corcoran JA, Weller SK, Smiley JR. Herpes simplex virus eliminates host mitochondrial DNA. EMBO Rep 2007; 8: 188–193. 3 Lee HT, Lin CS, Chen WS, Liao HT, Tsai CY, Wei YH. Leukocyte mitochondrial DNA alteration in systemic lupus erythematosus and its relevance to the susceptibility to lupus nephritis. Int J Mol Sci 2012; 13: 8853–8868. 4 Wallace DC. Mitochondria and cancer. Cancer 2012; 12: 685–698. 5 Rongvaux A, Jackson R, Harman CC, Li T, West AP, de Zoete MR et al. Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA. Cell 2014; 159: 1563–1577. 6 White MJ, McArthur K, Metcalf D, Lane RM, Cambier JC, Herold MJ et al. Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production. Cell 2014; 159: 1549–1562. 7 Shimada K, Crother TR, Karlin J, Dagvadorj J, Chiba N, Chen S et al. Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity 2012; 36: 401–414.
