Oxidized mitochondrial DNA: a protective signal gone awry

Abstract

Despite the emergence of mitochondria as key regulators of innate immunity, the mechanisms underlying the generation and release of immunostimulatory alarmins by stressed mitochondria remains nebulous. We propose that the major mitochondrial alarmin in myeloid cells is oxidized mitochondrial DNA (Ox-mtDNA). Fragmented Ox-mtDNA enters the cytosol where it activates the NLRP3 inflammasome and generates IL-1β, IL-18, and cGAS-STING to induce type I interferons and interferon-stimulated genes. Inflammasome activation further enables the circulatory release of Ox-mtDNA by opening gasdermin D pores. We summarize new data showing that, in addition to being an autoimmune disease biomarker, Ox-mtDNA converts beneficial transient inflammation into long-lasting immunopathology. We discuss how Ox-mtDNA induces short- and long-term immune activation, and highlight its homeostatic and immunopathogenic functions.

Keywords: NLRP3 inflammasome; Ox-mtDNA; autoimmunity; cGAS–STING; cell-free DNA; immunopathology; inflammation; stressed mitochondria.

Figure 2.

(Left) Cytosolic Ox-mtDNA escaping from stressed mitochondria stimulates proinflammatory signaling pathways through multiple modalities. (i) Cytosolic Ox-mtDNA binds to cyclic GMP-AMP (cGAMP) synthase (cGAS) to induce cGAMP production, which triggers stimulator of interferon genes (STING) phosphorylation, resulting in the nuclear translocation of TBK1-phosphorylated (P) interferon regulatory factor 3 (IRF3) to induce type I interferon (IFN) production [7,9,22,55,60]. (ii) Endosomal Toll-like receptor 9 (TLR9) recognizes extramitochondrial DNA to trigger NF-κB-dependent inflammatory cytokine induction [72]. (iii) Cytosolic Ox-mtDNA fragments bind to NLRP3 and activate the inflammasome, leading to caspase-1 activation, proteolytic processing of pro-IL-1β and pro-IL-18, and the formation of pyroptotic pores assembled by the N-terminal region of gasdermin D (GSDMD-N), enabling secretion of IL-1β, IL-18, and Ox-mtDNA into the extracellular space [6,17,22]. However, whether the released Ox-mtDNA is naked or bound to accessory proteins remains to be defined. The horizontal transfer of mtDNA via extracellular vesicles (EVs) serves as an alternative strategy for bystander cells to acquire Ox-mtDNA [117]. Nevertheless, how Ox-mtDNA is incorporated into EVs remains unknown. Upon pathogen encounter, neutrophils extrude Ox-mtDNA coated by transcription factor A, mitochondrial (TFAM) via neutrophil extracellular traps (NETs) [65,77]. (Right) Neighboring cells take up circulating Ox-mtDNA, presumably by endocytosis [22,119]. In the recipient cell, Ox-mtDNA binds to TLR9, triggering the induction of NF-κB-dependent proinflammatory cytokines and chemokines, and induces type I IFNs via the cGAS–STING pathway [6,9,65,77]. Type I IFNs in turn induce the expression of IFN-stimulated genes (ISGs) through IRF9 [7].

Figure 1.. Ox-mtDNA fragments cleaved by FEN1 escape to the cytosol in murine myeloid cells.

NLRP3 inflammasome activators (e.g., ATP, nigericin, alum, or MSU) stimulate [Ca²⁺]_m uptake via the MCU [118] to trigger mPTP opening in the IMM, resulting in VDAC oligomerization in the OMM [22]. Simultaneously, by upregulating the expression of rate-limiting CMPK2, LPS-induced priming initiates new mtDNA synthesis [17] which relies on ATP-dependent POLG [17,52] and MGME1 [22,29,30]. Newly synthesized mtDNA is oxidized by mtROS, whose production is stimulated in response to mitochondrial stress and ETC uncoupling triggered by NLRP3 activators, yielding Ox-mtDNA [17,52]. Ox-mtDNA is either repaired by OGG1, or cleaved by FEN1 into short (500–650 bp) fragments that escape to the cytosol via IMM pores, whose opening is mPTP-dependent, and OMM VDAC channels [22,25,32]. Activation of BAX and BAK [9,38] or gasdermin D cleavage [33] can also lead to OMM permeabilization and mtDNA release. However, it remains to be determined whether DNA released via these pores is oxidized and cleaved by FEN1. Abbreviations: CMPK2, cytidine/uridine monophosphate kinase 2; dNTP, deoxynucleoside triphosphate; FEN1, flap structure-specific endonuclease 1; GSDMD, gasdermin D; IMM, inner mitochondrial membrane; IMS, mitochondrial intermembrane space; LPS, lipopolysaccharide; MCU, mitochondrial calcium uniporter; MGME1, mitochondrial genome maintenance exonuclease 1; mPTP, mitochondrial permeability transition pore; MSU, monosodium urate; mtDNA, mitochondrial DNA; OGG1, 8-oxoguanine DNA glycosylase 1; OMM, outer mitochondrial membrane; Ox-mtDNA, oxidized mtDNA; POLG, DNA polymerase γ; ROS, reactive oxygen species; TLR4, Toll-like receptor 4; VDAC, voltage-dependent anion channel 1.