Mitochondrial DNA release and sensing in innate immune responses

Abstract

Mitochondria are pleiotropic organelles central to an array of cellular pathways including metabolism, signal transduction, and programmed cell death. Mitochondria are also key drivers of mammalian immune responses, functioning as scaffolds for innate immune signaling, governing metabolic switches required for immune cell activation, and releasing agonists that promote inflammation. Mitochondrial DNA (mtDNA) is a potent immunostimulatory agonist, triggering pro-inflammatory and type I interferon responses in a host of mammalian cell types. Here we review recent advances in how mtDNA is detected by nucleic acid sensors of the innate immune system upon release into the cytoplasm and extracellular space. We also discuss how the interplay between mtDNA release and sensing impacts cellular innate immune endpoints relevant to health and disease.

Keywords: NLRP3; TLR9; cGAS-STING; inflammation; innate immunity; mitochondria; mitochondrial DNA.

Figure 1

Pathways of mitochondrial DNA (mtDNA) release. (1) Formation of BAX/BAK pores drives herniation of the inner mitochondrial membrane (IMM) and mtDNA release for sensing by cGAS. It is not currently understood how mtDNA leaves these herniated structures. (2) Damaged mtDNA and impaired mtDNA replication intermediates are sequestered into outer mitochondrial membrane (OMM)-localized endosomes through a process that requires ATAD3, SAMM50, and VPS35, and may involve BAX. It is proposed that when this pathway is overwhelmed, mtDNA ends up in the cytosol to engage cGAS. (3) Reactive oxygen species (ROS), which are generated from the electron transport chain, may oxidize mtDNA in the mitochondrial matrix. Oxidized mtDNA (ox-mtDNA) may be released through cooperation between the mitochondrial permeability transition pore (mPTP) and voltage dependent anion channel (VDAC). There is some evidence that mtDNA crosses the IMM without the mPTP, yet how this happens is not clear. Ox-mtDNA is a ligand for the NLRP3 inflammasome, although it may also activate cGAS or AIM2. (4) Mitochondria-derived vesicles (MDVs) may mediate mtDNA release, with some subtypes merging with endolysosomes or possibly breaking apart to allow for sensing by TLR9 and cGAS sensing. Gasdermin D (GSDMD) may also facilitate release of mtDNA from endolysosomes. (5) Gasdermin D can localize to mitochondria when cardiolipin is exposed to form pores that allow for mtDNA release into the cytosol. Figure created with BioRender.com.

Figure 2

mtDNA sensing pathways. Mitochondrial nucleic acids are sensed by an array of pattern recognition receptors (PRRs). These PRRs engage signaling leading to the activation of transcription factors nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 and 7 (IRF3/7), which induce inflammatory cytokines, type I interferons (IFN-I), and interferon-stimulated genes (ISGs). (1) Mitochondrial double-stranded RNA (mtRNA) may be endocytosed for sensing by toll-like receptor 7 (TLR7), whereas mtDNA entering the endosomal pathway can be sensed by TLR9. Both TLRs signal through adaptor myeloid differentiation primary response 88 (MyD88) to activate NF-κB and IRFs. Vesicles derived from mitochondria may also carry mtRNA or mtDNA to the endolysosomal compartment for TLR sensing. (2) Cytosolic mtRNA can be sensed by retinoic acid-inducible gene I-like receptors (RLRs), which signal through mitochondrial antiviral signaling protein (MAVS), a protein that scaffolds on the mitochondrial outer membrane and drives signaling to NF-κB and IRFs. (3) Cyclic GMP-AMP synthase (cGAS) senses mtDNA and produces cGAMP, which binds and activates stimulator of interferon genes (STING) on the endoplasmic reticulum to promote IRF3/7 activity. The nucleic acid sensor Z-DNA binding protein 1 (ZBP1) can specifically bind Z-form mtDNA (Z-mtDNA) and complex with cGAS to augment IFN-I signaling through receptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1 and RIPK3) mediated phosphorylation of signal transducer and activator of transcription 1 (STAT1), which boosts ISG factor 3 (ISGF3) complex activation and ISG expression. Interferon gamma inducible protein 16 (IFI16) can also sense DNA and enhance cGAS activity, although whether this is through direct binding with cGAS is not known. (4) Absent in melanoma (AIM2) and NLR family pyrin domain containing 3 (NLRP3) sense mtDNA and oxidized mtDNA (ox-mtDNA), respectively. Oligomeric inflammasomes containing apoptosis-associated speck-like protein containing a caspase recruit domain (ASC) trigger Caspase-1 activation, leading to interleukin-18 (IL-18), interleukin-1β (IL-1β), and Gasdermin D (GSDMD) cleavage. GSDMD-NT then forms pores in the plasma membrane leading to cytokine and DAMP release and pyroptotic cell death. Figure created with BioRender.com.