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Mitochondrial DNA stress primes the antiviral innate immune response

LETTER

doi:10.1038/nature14156

A.Phillip West 1,William Khoury-Hanold 2,Matthew Staron 2,Michal C.Tal 2{,Cristiana M.Pineda 1,Sabine https://www.doczj.com/doc/8f2816603.html,ng 1,Megan Bestwick 1{,Brett A.Duguay 3,Nuno Raimundo 1{,Donna A.MacDuff 4,Susan M.Kaech 2,5,James R.Smiley 3,Robert E.Means 1,Akiko Iwasaki 2,5&Gerald S.Shadel 1,6

Mitochondrial DNA (mtDNA)is normally present at thousands of copies per cell and is packaged into several hundred higher-order structures termed nucleoids 1.The abundant mtDNA-binding pro-tein TFAM (transcription factor A,mitochondrial)regulates nucleoid architecture,abundance and segregation https://www.doczj.com/doc/8f2816603.html,plete mtDNA depletion profoundly impairs oxidative phosphorylation,triggering calcium-dependent stress signalling and adaptive metabolic responses 3.How-ever,the cellular responses to mtDNA instability,a physiologically relevant stress observed in many human diseases and ageing,remain poorly defined 4.Here we show that moderate mtDNA stress elicited by TFAM deficiency engages cytosolic antiviral signalling to enhance the expression of a subset of interferon-stimulated genes.Mechanis-tically,we find that aberrant mtDNA packaging promotes escape of mtDNA into the cytosol,where it engages the DNA sensor cGAS (also known as MB21D1)and promotes STING (also known as TMEM173)–IRF3-dependent signalling to elevate interferon-stimulated gene expression,potentiate type I interferon responses and confer broad viral resistance.Furthermore,we demonstrate that herpesviruses induce mtDNA stress,which enhances antiviral signalling and type I interferon responses during infection.Our results further demon-strate that mitochondria are central participants in innate immunity,identify mtDNA stress as a cell-intrinsic trigger of antiviral signal-ling and suggest that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully engage antiviral innate immunity.

To explore the cellular responses to mtDNA stress in the absence of oxidative phosphorylation deficiency,we employed a TFAM hetero-zygous knockout (Tfam 1/2)mouse model.Cells and tissues from these animals exhibit modest or no significant differences in mtDNA-encoded transcripts and oxygen consumption rates,despite an approximately 50%depletion of mtDNA (Extended Data Fig.1a–c)5,6.In addition to mtDNA depletion,Tfam 1/2mouse embryonic fibroblasts (MEFs)have reduced oxidative mtDNA damage repair capacity and markedly altered mtDNA packaging,organization and distribution (Fig.1a)6.Nucleoids in Tfam 1/2MEFs were less numerous and exhibited a larger size distri-bution (Fig.1a and Extended Data Fig.1d).Thus,Tfam 1/2cells provide a robust model to characterize cellular responses triggered by moderate mtDNA stress.

Gene expression profiling of Tfam 1/2MEFs revealed an unexpec-ted enrichment of interferon-stimulated genes (ISGs)and antiviral sig-nalling factors (Fig.1b).Of the 45most overexpressed genes,39were ISGs,including many with direct antiviral activity (Ifi44,Ifit1,Ifit3,Oasl2,Rtp4)7,8.We also observed increased expression of cytoplasmic RNA and DNA sensors,such as Ddx58and Ifih1and p200family pro-teins Ifi203,Ifi204and Ifi205,as well as transcription factors Irf7,Stat1and Stat2,ISGs that function to positively reinforce the antiviral res-ponse.Direct measurement of basal ISG mRNA and protein expression

in Tfam 1/2MEFs validated the microarray results (Fig.1c,d).Finally,Tfam 1/2MEFs expressed three-to fourfold more Ifnb and Ifna4upon transfection with the IFIH1agonist poly(I:C)(Fig.1e),consistent with enhanced type I interferon responses.

Department of Pathology,Yale School of Medicine,New Haven,Connecticut 06520,USA.2Department of Immunobiology,Yale School of Medicine,New Haven,Connecticut 06520,USA.3Li Ka Shing Institute of Virology,Department of Medical Microbiology and Immunology,University of Alberta,Edmonton,Alberta T6G 2S2,Canada.4Department of Pathology and Immunology,Washington University School of Medicine,St Louis,Missouri 63110,USA.5Howard Hughes Medical Institute,Chevy Chase,Maryland 20815-6789,USA.6Department of Genetics,Yale School of Medicine,New Haven,

Connecticut 06520,USA.{Present addresses:Institute for Stem Cell Biology and Regenerative Medicine,Stanford University School of Medicine,Stanford,California 94305,USA (M.C.T.);Department of

Chemistry,Linfield College,McMinnville,Oregon 97128,USA (M.B.);Institute for Cellular Biochemistry,Universita

¨tsmedizin Go ¨ttingen,37073Go ¨ttingen,Germany (N.R.).

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W T 1W T 2Figure 1|Tfam 1/2cells exhibit mtDNA stress,elevated ISG expression and augmented type I interferon responses.a ,Confocal microscopy images of MEFs stained with anti-DNA (DNA)and anti-HSP60(Mito.)antibodies.b ,Heat maps of microarray analyses.Genes in Tfam 1/2MEFs exhibiting statistically significant (P ,0.05),twofold or greater increases over wild type (WT)are shown.c ,d ,Quantitative real-time-PCR (qRT–PCR)(c )and western blots (d )of basal ISG expression in two littermate wild-type and Tfam 1/2MEF lines.e ,qRT–PCR analysis of type I interferon expression in MEFs 9h after cytosolic delivery of poly(I:C).Error bars indicate 6s.e.m.of triplicate technical replicates and are representative of three independent experiments.***P ,0.001.

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To ensure that the mtDNA stress and ISG expression phenotypes were not unique to Tfam 1/2MEFs,we employed inducible TFAM de-pletion models (TF D ).Analogous to Tfam 1/2cells,TF D MEFs and bone-marrow-derived macrophages (BMDMs)displayed mtDNA stress phenotypes,augmented ISG expression,and heightened type I inter-feron responses to poly(I:C)(Extended Data Fig.1d–i).Collectively,these data indicate that TFAM depletion induces mtDNA nucleoid stress that triggers antiviral ‘priming’,characterized by basally elevated ISG expression and potentiated type I interferon production.

Since mitochondrial stress can trigger the release of mtDNA into the cytosol to engage the NLRP3inflammasome,we assayed for extra-mitochondrial mtDNA in TF D cells 9,10.Analysis of pure cytosolic ex-tracts revealed a three-to fourfold increase of specific mtDNA fragments from the D-loop regulatory region,indicating liberation of immunosti-mulatory mtDNA into the cytosol (Extended Data Fig.2)11.Confocal and electron microscopy of TF D cells revealed significantly elongated,interconnected mitochondrial networks consistent with a hyperfused phenotype (Fig.1a and Extended Data Figs 1e,g and 3a,b).Since mi-tochondrial fission facilitates proper nucleoid distribution and removal of damaged mtDNA,we examined whether mitochondrial hyperfusion in TF D cells governed mtDNA stress-induced ISG expression 12,13.Knock-down of mitofusin 1(Mfn1)induced fission and largely abrogated ISG expression in TF D MEFs (Extended Data Fig.3c–e).Moreover,deple-tion of the mtDNA quality-control enzyme endo/exonuclease (59–39),endonuclease G-like (EXOG)exacerbated ISG expression in Tfam 1/2MEFs (Extended Data Fig.3f)14.Collectively,these data indicate that TFAM depletion promotes accumulation of aberrant mtDNA,which accesses the cytosol to engage innate immune signalling.

We next examined the involvement of the cytosolic DNA sensor cGAS in mtDNA stress signalling,as it mediates ISG expression in response

to exogenous and endogenous immunostimulatory DNA species 15–17.Knockdown of cGAS in Tfam 1/2MEFs or TFAM depletion in cGas 2/2MEFs largely abrogated ISG expression (Fig.2a).Furthermore,ISG mRNAs in TF D cells were reduced 70–90%in the absence of STING,indicating cGAS–STING signalling is the predominant driver of mtDNA stress-induced ISG expression (Fig.2b).STING signals via the TBK1–IRF3/7axis to trigger antiviral gene expression,and knockdown of ei-ther TBK1or IRF3robustly blocked ISG expression in Tfam 1/2MEFs (Fig.2c,d)18,19.Consistent with IRF3activating ISG transcription,we observed enhanced nuclear accumulation of IRF3in TF D cells (Fig.2e).Finally,using cGas 2/2MEFs reconstituted with hemagglutinin (HA)-tagged,murine cGAS,we observed prominent re-localization of cGAS from nuclear and/or cytoplasmic pools to the vicinity of aberrant mtDNA nucleoids in TF D MEFs (Fig.2f,g).Taken together,these results indi-cate that mtDNA stress facilitates cGAS-dependent sensing of cyto-plasmic mtDNA,resulting in STING–TBK1–IRF3signalling to trigger ISG expression.

To establish functional significance of mtDNA stress-induced anti-viral priming,we challenged MEFs with herpes simplex virus 1(HSV-1)or vesicular stomatitis virus (VSV)that express green fluorescent pro-tein (GFP)for easy detection.In contrast to wild-type cells,which dis-played robust viral GFP expression post-infection,Tfam 1/2MEFs were markedly resistant to HSV-1and VSV (Fig.3a).In addition,Tfam 1/2MEFs exhibited heightened type I interferon and ISG expression upon viral challenge,consistent with potentiated type I interferon responses in these cells (Extended Data Fig.4a).Similar results were obtained upon challenge with the rodent gammaherpesvirus MHV-68(Fig.3b and Ex-tended Data Fig.4b).Furthermore,TF D BMDMs displayed augmented antiviral gene expression and markedly lower HSV-1-and VSV-encoded mRNA and protein 6–24h post-infection (Extended Data Fig.4c–f).

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Figure 2|mtDNA stress triggers ISG expression in a cGAS-and STING-dependent fashion.

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b ,ISG expression in Tfam 1/2MEFs transfected with the indicated short interfering RNAs (siRNAs;top panels),or wild-type (WT),cGas 2/2(a ),and Sting 2/2(b )MEFs transfected with TFAM siRNAs (bottom panels).Ctrl,control.

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d ,ISG expression in Tfam 1/2MEFs transfected with th

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g ,cGAS 2/2MEFs reconstituted wit

h cGAS–HA were transfected with the indicated siRNAs for 96h,then stained with anti-DNA (DNA),anti-HSP60(Mito.)and anti-HA (cGAS–HA)antibodies and imaged.cGAS co-localization scoring was performed as described in the

Methods.Error bars indicate 6s.e.m.of triplicate technical (a –d )or biological (g )replicates and are representative of three independent experiments.*P ,0.05;**P ,0.01;***P ,0.001.

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