Mitochondrial DNA in inflammation and immunity

Review23 March 2020Open Access Mitochondrial DNA in inflammation and immunity Joel S Riley Corresponding Author Joel S Riley [email protected] orcid.org/0000-0001-9170-5716 Cancer Research UK Beatson Institute, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Stephen WG Tait Corresponding Author Stephen WG Tait [email protected] orcid.org/0000-0001-7697-132X Cancer Research UK Beatson Institute, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Joel S Riley Corresponding Author Joel S Riley [email protected] orcid.org/0000-0001-9170-5716 Cancer Research UK Beatson Institute, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Stephen WG Tait Corresponding Author Stephen WG Tait [email protected] orcid.org/0000-0001-7697-132X Cancer Research UK Beatson Institute, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Author Information Joel S Riley *,1,2 and Stephen WG Tait *,1,2 1Cancer Research UK Beatson Institute, Glasgow, UK 2Institute of Cancer Sciences, University of Glasgow, Glasgow, UK *Corresponding author. Tel: +44 141 330 6283; E-mail: [email protected] *Corresponding author. Tel: +44 141 330 8703; E-mail: [email protected] EMBO Reports (2020)21:e49799https://doi.org/10.15252/embr.201949799 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitochondria are cellular organelles that orchestrate a vast range of biological processes, from energy production and metabolism to cell death and inflammation. Despite this seemingly symbiotic relationship, mitochondria harbour within them a potent agonist of innate immunity: their own genome. Release of mitochondrial DNA into the cytoplasm and out into the extracellular milieu activates a plethora of different pattern recognition receptors and innate immune responses, including cGAS-STING, TLR9 and inflammasome formation leading to, among others, robust type I interferon responses. In this Review, we discuss how mtDNA can be released from the mitochondria, the various inflammatory pathways triggered by mtDNA release and its myriad biological consequences for health and disease. Glossary 5hmC 5-Hydroxymethylcytosine 5mC 5-Methylcytosine AGS Aicardi–Goutieres syndrome AIM2 Absent in melanoma 2 APC Antigen-presenting cell ASC Apoptosis-associated speck-like protein containing a CARD ATP Adenosine triphosphate BAK Bcl-2 homologous antagonist/killer BAX Bcl-2-associated X protein BID BH3 interacting-domain death agonist CARD Caspase activation and recruitment domain CD47 Cluster of differentiation 47 CDN Cyclic dinucleotide cGAMP Cyclic guanosine monophosphate–adenosine monophosphate cGAS Cyclic GMP-AMP synthase CLR C-type lectin receptor CMPK2 Cytidine/Uridine monophosphate kinase 2 DAMP Damage-associated molecular pattern DC Dendritic cell DNase Deoxyribonuclease dsDNA Double-stranded DNA ER Endoplasmic reticulum EV Extracellular vesicle GTP Guanosine-5′-triphosphate HMGB1 High-mobility group protein 1 HSV-1 Herpes simplex virus-1 IAP Inhibitor of apoptosis protein IFNAR Interferon-α/β receptor IFN-β Interferon-β IFN-γ Interferon-γ IL-18 Interleukin-18 IL-1R Interleukin-1 receptor IL-1β Interleukin-1β IL-6 Interleukin-6 IRF3 Interferon regulatory factor 3 ISG Interferon-stimulated gene K+ Potassium LPS Lipopolysaccharide LRR Leucine-rich repeat MAPK Mitogen-activated protein kinase MAVS Mitochondrial anti-viral signalling protein MDA5 Melanoma differentiation-associated protein 5 MEF Mouse embryonic fibroblast MiDAS Mitochondrial dysfunction-associated senescence MI Myocardial infarction MOMP Mitochondrial outer membrane permeabilisation mPTP Mitochondrial permeability transition pore mtDNA Mitochondrial DNA NASH Non-alcoholic fatty liver disease NET Neutrophil extracellular trap NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NLRC4 NLR Family CARD Domain Containing 4 NLR Nucleotide oligomerisation domain-like receptor NLRP1 NLR Family Pyrin Domain Containing 1 NLRP3 NACHT, LRR and PYD domain-containing protein 3 NOD Nucleotide oligomerisation domain ODN Oligodeoxynucleotide OPA1 Optic Atrophy 1 Mitochondrial Dynamin Like GTPase PAMP Pathogen-associated molecular pattern pDC Plasmacytoid dendritic cell PD-L1 Programmed death-ligand 1 PINK1 Phosphatase and tensin homolog-induced kinase 1 PMA Phorbol 12-myristate 13-acetate PNPase Polynucleotide phosphorylase PRR Pattern recognition receptor PYD Pyrin domain RAGE Receptor for advanced glycation endproducts RIG-I Retinoic acid-inducible gene I RIP1 Receptor-interacting serine/threonine-protein kinase 1 RLR Retinoic acid-inducible gene-I-like receptors RNP IC Ribonucleotide immune complex ROS Reactive oxygen species SAMDH1 Sterile alpha motif domain and HD domain-containing protein 1 SIRS Systemic inflammatory response syndrome SLE Systemic lupus erythematosus ssDNA Single-stranded DNA STING Stimulator of interferon genes SUV3 Suppressor of Var1 TBK1 TANK-binding kinase 1 TFAM Transcription factor A, mitochondrial TLR9 Toll-like receptor 9 TLR Toll-like receptor TNF Tumour necrosis factor TREX1 Three Prime Repair Exonuclease 1 tRNA Transfer RNA VDAC Voltage-dependent anion channel Introduction Serving as a first line of defence, the innate immune system guards us against a plethora of insults and invading microorganisms. Infection by pathogenic agents is detected in cells by pattern recognition receptors (PRRs) which recognise specific pathogen-associated molecular patterns (PAMPs). PRRs can be broadly classified into four distinct groups: NOD-like receptors (NLRs), Toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and C-type lectin receptors (CLRs) 1. Upon detection of a PAMP, PRRs initiate a multitude of different signalling pathways, which culminate in the up-regulation of various type I interferons, pro-inflammatory chemokines and cytokines. These prime the adaptive immune system and create a hostile environment for the microorganism in which to survive. Additionally, damage-associated molecular patterns (DAMPs) are immune triggers that arise from the cell itself, such as proteins or DNA, and can activate innate immune pathways 2. Mitochondria first appeared in eukaryotic cells about two billion years ago as α-proteobacterium, in what is thought to be an endosymbiotic relationship 3, 4. Over time, these bacteria evolved to become the much-studied organelle that we know today, playing crucial roles in metabolism, calcium homeostasis and cell death. Nevertheless, they have maintained an independent genome, which encodes 37 genes, comprised of 13 mRNAs forming key components of the oxidative phosphorylation system, in addition to 2 ribosomal RNA components and 22 tRNAs 3, 4. An estimated 1,000 proteins are located in the mitochondria, all of which, except those encoded by mtDNA, are translated in the cytosol and imported into the mitochondria 5. Mitochondrial DNA itself is a circular molecule of double-stranded (ds)DNA. Transcription of both the heavy and light strand results in long, full-length transcripts which are processed by RNase enzymes to produce mature mRNA, tRNA and ribosomal RNA. In mammals, the polymerase responsible for mtDNA replication is DNA polymerase γ, but as POLγ cannot replicate dsDNA, the DNA helicase Twinkle is required to act directly before to unwind the DNA structure. Newly synthesised single-stranded (ss)DNA is bound by mitochondrial single-stranded DNA-binding protein to prevent secondary structure formation and attack by nucleases. Mitochondrial DNA replication has recently been reviewed extensively elsewhere 6; here, we focus on the unique aspects of mtDNA which make it immunostimulatory. We will then discuss how mtDNA which is ejected from the mitochondria under specific circumstances can activate different innate immune pathways, including cGAS-STING signalling, inflammasomes and Toll-like receptors. We will also focus on the role of mtDNA in the formation of neutrophil extracellular traps (NETs) and the transfer of mtDNA between cells. Mitochondrial DNA as a stimulator of the immune system Potentially stemming from its bacterial origin, mitochondrial DNA is sensed as "foreign", suggesting that it is seen differently to "self" DNA in cells. One example of this can be seen in its methylation status, where many studies have reported mtDNA to be hypomethylated compared to nuclear DNA 7, 8, despite the presence of DNA methyltransferases in the mitochondria 9, 10. Some groups have reported aberrant methylation patterns of mtDNA, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at CpG motifs 9-14, although others have proposed technical limitations to this work and using more sensitive techniques report that mtDNA is devoid of CpG methylation 15. Clearly, more effort is required in determining the precise degree of methylation in mtDNA, but if studies showing an absence of CpG methylation are correct, then mtDNA would harbour unmethylated CpG motifs similar to bacterial DNA, which could potentially activate pattern recognition receptors such as TLR9, absent in melanoma 2 (AIM2) and cGAS 15-18. Mitochondrial DNA replication and transcription itself may represent a rich source of potential activators of DNA pattern recognition receptors; for example, RNA:DNA hybrids form during transcription, in addition to long stretches of ssDNA and R-loops composed of RNA:DNA hybrids with a non-template ssDNA which can be recognised by cGAS 16. Mitochondrial DNA exists in the mitochondrial matrix in close proximity to the electron transport chain, a major source of reactive oxygen species. Due to this, it is particularly vulnerable to oxidation, resulting in mtDNA mutations which can contribute to the pathogenesis of cancer 17, diabetes 18 and ageing 19. It was thought the cell had limited capacity to repair mtDNA; however, multiple repair pathways are now well characterised 20. Mitochondrial DNA is often schematically represented as a plasmid structure; however, this is an over-simplification. Rather, super-resolution imaging has revealed that it is densely compacted into nucleoids consisting of one copy of mtDNA and a number of different proteins 21, the most notable of which is mitochondrial transcription factor A (mtTFA, commonly referred to as TFAM). It might be assumed that the compaction of mtDNA into protein structures shields DNA from recognition, but this is not the case as we shall discuss further in this Review, and in fact, a number of studies have shown that TFAM itself might be immunostimulatory 13, 14. In a landmark study in 2004, Collins et al 22 found that injecting mtDNA into the joints of mice resulted in localised inflammation and arthritis. Further investigation revealed that the inflammation was dependent on the presence of oxidatively damaged bases in the mtDNA, as injection of an oligodeoxynucleotide (ODN) with the same sequence but without the oxidised residue had no effect. The observation that mtDNA can elicit potent immune responses opened a whole new field of research, and it is now appreciated that mtDNA can stimulate many PRRs, including cGAS, TLR9 and inflammasomes (Fig 1). Release of mtDNA from mitochondria and subsequent recognition by PRRs occurs during many cellular processes, including infection, cell death and neurodegeneration, and this will be the focus of the rest of this Review. Figure 1. Overview of pro-inflammatory signalling pathways engaged by mitochondrial DNAMitochondrial DNA (mtDNA) can trigger various pro-inflammatory signalling pathways by endosomal localised TLR9 or via cytosolic cGAS-STING or via cytosolic inflammasome (AIM2 or NLRP3). Top: TLR9 binds mtDNA in the endosome eliciting an NF-κB-dependent pro-inflammatory signalling program. Middle: cGAS recognises mtDNA in the cytosol and activates endoplasmic reticulum (ER)-localised STING triggering an interferon response. Bottom: mtDNA-dependent inflammasome activity leads to caspase-1-dependent maturation or pro-inflammatory IL-1 and IL-8. Download figure Download PowerPoint mtDNA-dependent activation of cGAS-STING signalling mtDNA release in infection Through necessity, cells have evolved elegant systems to detect the presence of invading pathogenic DNA. Cyclic GMP-AMP synthase (cGAS) is one such direct detector, which binds dsDNA to form a dimer 23, 24. cGAS then undergoes a conformational change which facilitates the conversion of ATP and GTP into 2′3′-cyclic GMP-AMP (cGAMP) 25-31. cGAMP is a second messenger, which binds the endoplasmic reticulum (ER)-resident protein stimulator of interferon genes (STING) inducing a conformational change in its C-terminal tail. TANK-binding kinase 1 (TBK1) is recruited to STING which phosphorylates it and the transcription factor interferon regulatory factor 3 (IRF3), eliciting the transcription of hundreds of interferon stimulatory genes (ISGs) that are potently anti-viral 32 (Fig 2). cGAS was assumed to be primarily cytosolic to avoid persistent activation by self-DNA in the nucleus, but recent work has shown it to be present in the nucleus 33, 34 and at the plasma membrane 35. A recent attempt to resolve these discrepancies by Volkmann et al 36 reveals a more complex model than the cytosolic DNA sensing paradigm. The authors show that the majority of cGAS protein is nuclear, and they propose a model where cGAS must be "desequestered" prior to its full activation. However, it remains unclear how cytosolic DNA can be detected by cGAS, if cGAS is tethered in the nuclear compartment. Three independent studies were the first to show that mtDNA released from mitochondria is able to activate cGAS-STING signalling 37-39. White et al and Rongvaux et al explored mtDNA release in the context of cell death (discussed later in this Review), whereas West et al provided evidence that TFAM deficiency promotes mitochondrial stress and mis-packaged mtDNA, resulting in their ejection into the cytoplasm where they bind and activate cGAS initiating a type I interferon response 39 (Fig 2). Of pathophysiological relevance, infection with Herpes simplex virus-1 (HSV-1) or vesicular stomatitis virus (VSV) results in mtDNA stress, TFAM depletion and mtDNA entrance into the cytoplasm. The cytoplasmic mtDNA is then sensed by cGAS, triggering cGAS-STING signalling leading to the up-regulation of a plethora of interferon genes, conferring an anti-viral state on the cell. Importantly, Tfam+/− cells, which exhibit mtDNA stress, are more resistant to infection with HSV-1 or VSV than wild-type cells, as they have heightened ISG expression owing to mtDNA release. Mechanistically, the HSV-1 virus encodes a nuclease, UL12.5, which localises to the mitochondria and degrades mtDNA, resulting in complete loss of mtDNA in infected cells 40, 41. Removal of mtDNA in infected cells does not appear to impact HSV replication 42. Furthermore, exonuclease activity is required for effective viral DNA production to maintain cell-to-cell infectivity, though whether this is related to UL12.5's mtDNA-targeted nuclease activity is unknown 43. Figure 2. mtDNA-dependent activation of cGAS-STING signallingVarious mitochondrial stresses including bacterial or viral infection can lead to mtDNA release. Alternatively, activation of BAX and BAK leads to outer mitochondrial membrane permeabilisation (MOMP) and mtDNA release. Once cytoplasmic, mtDNA can bind the DNA sensing protein cGAS that catalyses the production of the secondary messenger 2′3′ cyclic GMP–AMP (2′3′cGAMP) from ATP and GTP. cGAMP binds the adaptor molecule STING on the ER leading to activation of TBK1 kinase. Active TBK1 phosphorylates the transcription factor IRF3 initiating a type I interferon response. Download figure Download PowerPoint Curiously, infection with RNA viruses, such as dengue virus, also elicits a cGAS-STING response, despite cGAS being a DNA-specific PRR 44. Several studies have now shown that dengue virus causes the release of predominantly oxidised mtDNA into the cytosol, where it can activate both cGAS 45, 46 and TLR9 47. Dengue virus has evolved strategies to circumvent cytosolic mtDNA-induced cGAS signalling during infection by encoding proteases which target cGAS and STING for degradation, thus ensuring persistence of the virus 46, 48, 49. Infection with the bacterial pathogen Mycobacterium tuberculosis triggers cGAS activation and subsequent IRF3-dependent type I interferon response 50-52. This was assumed to be solely due to detection of mycobacterium DNA, but other studies have identified a role for mitochondrial stress and ensuing release of mtDNA into the cytoplasm 53. This observation is strain-dependent but does propose a role for mitochondrial stress and dynamics on the M. tuberculosis-induced release of mtDNA. Previous work has observed cytochrome c release from mitochondria in cells infected with M. tuberculosis, indicating that there may be a possible role for BAX/BAK-dependent mitochondrial permeabilisation (discussed in detail later) in infection-related mtDNA release 54 (Fig 2). Pathogen-infected cells often secrete IL-1β due to inflammasome activation. A recent report by Aarreberg et al discovers a link between IL-1β secretion in infected cells, which can then activate a cGAS-STING-dependent type I interferon response in surrounding bystander cells. Interestingly, IL-1β stimulation of bystander cells increases mitochondrial mass, decreases mitochondrial membrane potential and induces mtDNA release 55. However, mtDNA release is observed in the absence of detectable cytochrome c release and cell death, suggesting that this is not the mechanism of mtDNA release, although it does not rule out limited mitochondrial permeabilisation seen by us and others in the context of infection (see below). This is not the first time IL-1R signalling has been implicated in cell-intrinsic defence 56-58, but it is the first to suggest that mtDNA release plays a key role in the initiation of cGAS-STING signalling in the bystander cells. mtDNA activation of cGAS-STING during cell death During programmed cell death, the pro-apoptotic proteins BAX and BAK permeabilise the mitochondrial outer membrane to allow the passage of pro-apoptotic molecules to move from the inner membrane space into the cytosol, where they can initiate a caspase cascade, resulting in a rapid cell death 59. White et al and Rongvaux et al showed that in the absence of apoptotic caspase activation, mtDNA activates cGAS in a promiscuous manner, which in vivo leads to mildly elevated IFN-β protein levels in blood, though a level sufficient to induce the expression of interferon-stimulated genes 37, 38 (Fig 3). This suggests that apoptotic caspases play a crucial role in dampening type I interferon responses in dying cells, maintaining the "immune-silent" nature of apoptosis (Fig 3). Further work has shown that apoptotic caspases directly cleave cGAS, IRF3 and mitochondrial anti-viral signalling protein (MAVS), key proteins required for the production of type I interferon 60, supporting the notion that caspases dampen the immune response during cell death. High-resolution imaging studies have further expanded our understanding of how mtDNA is released from the mitochondria during cell death. We and others recently showed that BAX and BAK can permeabilise the mitochondrial outer membrane, but in the context of caspase inhibition these pores grow dramatically, sufficient to allow inner membrane herniation and extrusion of mtDNA 61-63 (Fig 3). We found that under caspase-inhibited conditions, mitochondrial permeabilisation leads to down-regulation of inhibitor of apoptosis proteins (IAPs), NF-κB-inducing kinase (NIK) activation and an NF-κB transcriptional program, in addition to mtDNA release-induced cGAS-STING activation 64. The cytokines and chemokines up-regulated via NF-κB after mitochondrial permeabilisation can serve to promote macrophage activation 64, 65. This leads to robust anti-tumour effects, highlighting a potential therapeutic role for caspase inhibition in cancer treatment 64. Collectively, these results help to reconcile how predominantly cytosolic cGAS can be activated by mtDNA during cell death. Nevertheless, a number of unresolved questions remain. Firstly, is inner membrane permeabilisation a regulated process, and if so, how? A rapid inner membrane permeabilisation of sufficient size to allow the passage of small ions is observed minutes after outer membrane permeabilisation 61, but is insufficient to allow mtDNA nucleoid extrusion and is probably only transient, as inner membrane potential can be maintained after outer membrane permeabilisation 66-69. Secondly, there are cell type differences in the degree of inner membrane permeabilisation, as different studies report varying degrees of mtDNA release during cell death 61, 62, implying that specific cell-intrinsic factors play a role in inner membrane permeabilisation. Finally, the physiological relevance of cell death-related mtDNA release is unknown. Most cell types undergo rapid and complete caspase-dependent apoptosis in vivo, presumably limiting any potential for mtDNA-driven inflammation during cell death. However, some cell types, for instance cardiomyocytes, display deficient caspase activity downstream of mitochondrial permeabilisation 70. Such cells might generate a greater type I anti-viral interferon response after mitochondrial permeabilisation. Alternatively, cGAMP might transfer from apoptotic to healthy cells, serving as an "early warning" defence system, instructing healthy cells to transcribe genes important for their survival (Fig 4) 71, 72. Figure 3. BAX/BAK-dependent initiation of inflammationFollowing a pro-apoptotic stress, BAX and BAK are activated leading to mitochondrial outer membrane permeabilisation. This enables the release of caspase-activating proteins from the mitochondrial intermembrane space. Following this, macropores form on the mitochondrial outer membrane causing extrusion and permeabilisation of the inner membrane. This enables release of mtDNA. Mitochondrial double-stranded RNA (dsRNA) can also be released. Collective release of these molecules triggers inflammation via MAVS, cGAS-STING and NF-κB. Caspase activity is anti-inflammatory, in part, through direct cleavage and inactivation of inflammatory signalling molecules. Download figure Download PowerPoint Figure 4. Non-cell autonomous effects of mtDNA(A) Upon pathogen encounter, neutrophils can extrude DNA (both nuclear and mitochondrial) that forms an extracellular trap for extracellular microbes. Due to pro-inflammatory properties, these DNA neutrophil extracellular traps (NETs) can also have pathological effects in diseases such as lupus. (B) mtDNA can transfer via exosomes or in intact mitochondria to neighbouring cells, impacting on the metabolism and survival of the recipient cell. Inflammatory responses to mtDNA can also have non-cell autonomous effects. The cGAS-induced secondary messenger cGAMP has been shown to transfer via gap junctions eliciting anti-viral interferon responses in neighbouring cells. Download figure Download PowerPoint In addition to DNA, mitochondria also possess dsRNA which is known to be potently immunogenic 73. Mitochondrial dsRNA arises from transcription of both the heavy and lights strands of mtDNA; however, although the light strand is rapidly degraded the heavy strand is not, and nearly all the dsRNA detected in the cytoplasm are of mitochondrial origin. The mitochondrial helicase SUV3 and polynucleotide phosphorylase PNPase dampen the accumulation of dsRNA, but when these are depleted, dsRNA accumulates in the cytoplasm where it activates a type I interferon response driven by the dsRNA receptor MDA5 74. Silencing of BAX and BAK suppresses the type I interferon response, strongly suggesting that BAX/BAK-dependent mitochondrial outer membrane permeabilisation is responsible for mitochondrial dsRNA escape into the cytoplasm 74 Furthermore, patients with mutations leading to a decrease in PNPT1, the gene that encodes PNPase protein, exhibit greater accumulation of dsRNA and elevated interferon levels in their serum 74. Mitochondrial outer membrane permeabilisation is a rapid and complete event, spreading to all mitochondria in a cell. Following formation of BAX/BAK pores, pro-apoptotic proteins such as cytochrome c are released from the intermembrane space where they initiate the caspase cascade, culminating in cell death. However, we have found that under conditions of sub-lethal stress, a limited number of mitochondria in a cell can undergo permeabilisation, called minority MOMP, leading to genomic instability and transformation 75. A recent report by Brokatsky et al reveals a link between pathogen invasion and activation of mitochondrial cell death machinery 76. In this study, it was found that various pathogens can induce limited mitochondrial permeabilisation. It remains unclear how pathogens can trigger minority MOMP, but nevertheless they can, resulting in mtDNA release (presumably through BAX/BAK pores), stimulating cGAS-STING activation and cytokine secretion 76. How else might mtDNA be released from mitochondria? Another potential mechanism for mtDNA release from mitochondria is through the mitochondrial permeability transition pore (mPTP) 77, 78. The exact composition of the pore is unclear, although there seems to be consensus that cyclophilin D is present 79. The mPTP spans the mitochondrial inner membrane and forms in response to high mitochondrial calcium concentration and various other cellular stresses. However, the mPTP is predicted to only allow the efflux of molecules smaller than 1.5 kDa, much smaller than a mtDNA nucleoid 80, 81. In line with this, studies have shown that only fragments of mtDNA can pass through the mPTP 77, 82, 83. It remains possible that sustained opening of the pore can lead to swelling of the mitochondria and subsequent rupture of the inner membrane, which would permit the efflux of mtDNA into the cytoplasm. The involvement of mPTP in mtDNA release during cell death has been ruled out 61, but chitosan, a vaccine adjuvant, appears to induce a cGAS-STING- and mPTP-dependent type I interferon response. This is possibly due to mtDNA release, though a direct role for mtDNA has not been rigorously assessed 84. An intriguing recent report suggests that cells experiencing mitochondrial stress caused by the lack of mitochondrial endonuclease G release mtDNA through pores formed by oligomers of the voltage-dependent anion channel (VDAC) 85. As mitochondrial DNA release is thought to play a role in the pathogenesis of lupus 86, 87, a role for VDAC pore formation was tested in an in vivo model of lupus-like disease. Using the VDAC1 oligomerisation inhibitor VBIT-4, the authors were able to reduce lupus-like symptoms in lupus-prone mice, providing a rationale to target VDAC-mediated mtDNA release in this disease 85. Therapeutic targeting of mtDNA-dependent cGAS-STING activity There is currently intense interest in the development of inhibitors and activators of the cGAS-STING pathway, depending on the disease. In humans, the systemic inflammatory disease Aicardi–Goutières syndrome (AGS) is characterised by mutations in a number of different genes involved in DNA sensing 88. For example, TREX1, a DNA exonuclease, is frequently mutated in human patients with AGS and systemic lupus erythematosus (SLE) 89-91, and co-deletion of cGAS, STING, Interferon-α/β receptor (IFNAR) or IRF3 rescues this phenotype 92-98. Accumulation of cytosolic DNA appears to be a defining characteristic of AGS and SLE, as deletions in DNA- and RNA-related genes including SAMDH1, a DNA exonuclease and RnaseH2 are frequent 99-102. Gain-of-function mutations in STING itself lead to an up-regulation of type I interferon responses and lupus-like symptoms in patients 103, 104. DNase II deficiency in humans leads to autoinflammation with increased type I IFN 105 and in mice causes arthritis 106. This is thought to be due to the lack of self-DNA degradation in dead cells engulfed by macrophages resulting in sustained cGAS-STING stimulation 98, 106, 107, and AIM2 inflammasome formation 108, 109 with a possible contribution of endosomal TLRs 108. Myocardial infarction (MI) is another condition known to involve a strong inflammatory component. King et al 110 showed that ischaemic cell death and engulfment by macrophages drives an IRF3-dependent type I IFN response. Genetic or pharmacological disruption of cGAS-STING signalling in mice improved their outcomes post-MI, proposing this signalling axis as suitable for therapeutic intervention in patients 110, 111. While it is not clear if this is due to mtDNA release per se, increased mtDNA in plasma from patients with heart disease has been frequently observed 112-114. Clearly, inhibiting the cGAS-STING pathway in these disease settings might be beneficial to patients. Small molecules targeting both cGAS 115, 116 and STING 117 have been developed, with STING antag