Oxidized DNA fragments exit mitochondria via mPTP- and VDAC-dependent channels to activate NLRP3 inflammasome and interferon signaling.

Mitochondrial DNA (mtDNA) escaping stressed mitochondria provokes inflammation via cGAS-STING pathway activation and, when oxidized (Ox-mtDNA), it binds cytosolic NLRP3, thereby triggering inflammasome activation. However, it is unknown how and in which form Ox-mtDNA exits stressed mitochondria in non-apoptotic macrophages. We found that diverse NLRP3 inflammasome activators rapidly stimulated uniporter-mediated calcium uptake to open mitochondrial permeability transition pores (mPTP) and trigger VDAC oligomerization. This occurred independently of mtDNA or reactive oxygen species, which induce Ox-mtDNA generation. Within mitochondria, Ox-mtDNA was either repaired by DNA glycosylase OGG1 or cleaved by the endonuclease FEN1 to 500-650 bp fragments that exited mitochondria via mPTP- and VDAC-dependent channels to initiate cytosolic NLRP3 inflammasome activation. Ox-mtDNA fragments also activated cGAS-STING signaling and gave rise to pro-inflammatory extracellular DNA. Understanding this process will advance the development of potential treatments for chronic inflammatory diseases, exemplified by FEN1 inhibitors that suppressed interleukin-1ß (IL-1ß) production and mtDNA release in mice.

Oxidized mitochondrial DNA: a protective signal gone awry.

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-1ß, 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.

Inflammatory signaling in NASH driven by hepatocyte mitochondrial dysfunctions.

Liver steatosis, inflammation, and variable degrees of fibrosis are the pathological manifestations of nonalcoholic steatohepatitis (NASH), an aggressive presentation of the most prevalent chronic liver disease in the Western world known as nonalcoholic fatty liver (NAFL). Mitochondrial hepatocyte dysfunction is a primary event that triggers inflammation, affecting Kupffer and hepatic stellate cell behaviour. Here, we consider the role of impaired mitochondrial function caused by lipotoxicity during oxidative stress in hepatocytes. Dysfunction in oxidative phosphorylation and mitochondrial ROS production cause the release of damage-associated molecular patterns from dying hepatocytes, leading to activation of innate immunity and trans-differentiation of hepatic stellate cells, thereby driving fibrosis in NASH.

1'-Acetoxychavicol acetate inhibits NLRP3-dependent inflammasome activation via mitochondrial ROS suppression.

The nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing (NLRP) 3 inflammasome is a multiprotein complex that triggers Caspase-1-mediated IL-1ß production and pyroptosis, and its dysregulation is associated with the pathogenesis of inflammatory diseases. 1'-Acetoxychavicol acetate (ACA) is a natural compound in the rhizome of tropical ginger Alpinia species with anti-microbial, anti-allergic and anti-cancer properties. In this study, we found that ACA suppressed NLRP3 inflammasome activation in mouse bone marrow-derived macrophages and human THP-1 monocytes. ACA inhibited Caspase-1 activation and IL-1ß production by NLRP3 agonists such as nigericin, monosodium urate (MSU) crystals, and ATP. Moreover, it suppressed oligomerization of the adapter molecule, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1-mediated cleavage of pyroptosis executor Gasdermin D. Mechanistically, ACA inhibited generation of mitochondrial reactive oxygen species (ROS) and prevented release of oxidized mitochondrial DNA, which trigger NLRP3 inflammasome activation. ACA also prevented NLRP3 inflammasome activation in vivo, as evidenced in the MSU crystal-induced peritonitis and dextran sodium sulfate-induced colitis mouse models accompanied by decreased Caspase-1 activation. Thus, ACA is a potent inhibitor of the NLRP3 inflammasome for prevention of NLRP3-associated inflammatory diseases.

Oxidized mitochondrial DNA activates the cGAS-STING pathway in the neuronal intrinsic immune system after brain ischemia-reperfusion injury.

In the context of stroke and revascularization therapy, brain ischemia-reperfusion injury is a significant challenge that leads to oxidative stress and inflammation. Central to the cell's intrinsic immunity is the cGAS-STING pathway, which is typically activated by unusual DNA structures. The involvement of oxidized mitochondrial DNA (ox-mtDNA)-an oxidative stress byproduct-in this type of neurological damage has not been fully explored. This study is among the first to examine the effect of ox-mtDNA on the innate immunity of neurons following ischemia-reperfusion injury. Using a rat model of transient middle cerebral artery occlusion and a cellular model of oxygen-glucose deprivation/reoxygenation, we have discovered that ox-mtDNA activates the cGAS-STING pathway in neurons. Importantly, pharmacologically limiting the release of ox-mtDNA into the cytoplasm reduces inflammation and improves neurological functions. Our findings suggest that targeting ox-mtDNA release may be a valuable strategy to attenuate brain ischemia-reperfusion injury following revascularization therapy for acute ischemic stroke.

Microglial CMPK2 promotes neuroinflammation and brain injury after ischemic stroke.

Neuroinflammation plays a significant role in ischemic injury, which can be promoted by oxidized mitochondrial DNA (Ox-mtDNA). Cytidine/uridine monophosphate kinase 2 (CMPK2) regulates mtDNA replication, but its role in neuroinflammation and ischemic injury remains unknown. Here, we report that CMPK2 expression is upregulated in monocytes/macrophages and microglia post-stroke in humans and mice, respectively. Microglia/macrophage CMPK2 knockdown using the Cre recombination-dependent adeno-associated virus suppresses the inflammatory responses in the brain, reduces infarcts, and improves neurological outcomes in ischemic CX3CR1Cre/ERT2 mice. Mechanistically, CMPK2 knockdown limits newly synthesized mtDNA and Ox-mtDNA formation and subsequently blocks NLRP3 inflammasome activation in microglia/macrophages. Nordihydroguaiaretic acid (NDGA), as a CMPK2 inhibitor, is discovered to reduce neuroinflammation and ischemic injury in mice and prevent the inflammatory responses in primary human monocytes from ischemic patients. Thus, these findings identify CMPK2 as a promising therapeutic target for ischemic stroke and other brain disorders associated with neuroinflammation.

Dioscin Alleviates Periodontitis by Inhibiting NLRP3 Inflammasome Activation via Regulation of K+ Homeostasis and Mitochondrial Function.

Gingival inflammation and alveolar bone loss are characteristic manifestations of periodontitis. Interleukin (IL)-1ß, the maturation of which is mainly regulated by NOD-like receptor protein (NLRP) 3 inflammasome, not only amplifies the inflammatory response but also triggers osteoclastogenesis, thereby accelerating the progression of periodontitis. Dioscin, a natural steroid saponin, has been shown to inhibit NLRP3 inflammasome. Nevertheless, research on the effectiveness of Dioscin for the management of periodontitis remains scarce. In this study, Dioscin was found to dramatically reduce the integral components of NLRP3 inflammasome, ultimately limiting IL-1ß secretion. Notably, the inhibitory impact of Dioscin on NLRP3 inflammasome might be exerted by curbing the generation of mitochondrial (mt) reactive oxygen species (ROS) and oxidized (ox) mtDNA, which were mediated by inhibition of K+ efflux. Furthermore, Dioscin effectively alleviated periodontitis in mice. Overall, the results established that Dioscin could alleviate periodontitis by inhibiting NLRP3 inflammasome via modulation of the K+ efflux-mtROS-ox-mtDNA pathway, holding the potential to treat periodontitis and other NLRP3-driven inflammatory diseases.

Holistic Assessment Based On Hepatocyte Mitochondria: Lycopene Repairs Oxidized mtDNA to Alleviate Mitochondrial Stress Induced by Atrazine.

Atrazine (ATZ) is a highly persistent herbicide that harms organism health. Lycopene (LYC) is an antioxidant found in plants and fruits. The aim of this study is to investigate the mechanisms of atrazine-induced mitochondrial damage and lycopene antagonism in the liver. The mice were divided into seven groups by randomization: blank control (Con group), vehicle control (Vcon group), 5 mg/kg lycopene (LYC group), 50 mg/kg atrazine (ATZ1 group), ATZ1+LYC group, 200 mg/kg atrazine (ATZ2 group), and ATZ2+LYC group. The present study performed a holistic assessment based on mitochondria to show that ATZ causes the excessive fission of mitochondria and disrupts mitochondrial biogenesis. However, the LYC supplementation reverses these changes. ATZ causes increased mitophagy and exacerbates the production of oxidized mitochondrial DNA (Ox-mtDNA) and mitochondrial stress. This study reveals that LYC could act as an antioxidant to repair Ox-mtDNA and restore the disordered mitochondrial function caused by ATZ.

Nanocatalytic Innate Immunity Activation by Mitochondrial DNA Oxidative Damage for Tumor-Specific Therapy.

The innate immune system plays a key role in protecting the human body from tumors, which, unfortunately, is largely counteracted by their immune-suppression function. Such an immune suppression has been reported to be induced by the immunosuppressive microenvironment, including the exhausted cytotoxic T lymphocytes (CTLs) and tumor-promoting M2-polarized macrophages. Here, a novel tumor-immunotherapeutic modality based on the nanocatalytic innate immunity activation by tumor-specific mitochondrial DNA (mtDNA) oxidative damage is proposed. In detail, a nanocatalytic medicine, Fe2+ -Ru2+ -loaded mesoporous silica nanoparticle named as MSN-Ru2+ /Fe2+ (MRF), is constructed to induce oxidative damage in the mtDNA of tumor cells. Such an oxidative mtDNA is able to escape from the tumor cells and acts as an immunogenic damage-associated molecular pattern to M1-polarize tumor-associated macrophages (TAMs), resulting in the reactivated immunoresponse of macrophages against cancer cells, and the subsequent inflammatory response of innate immunity. Most importantly, the treatment strategy based on regulating the innate immune response of TAMs not only stops the primary tumor progression, but also almost completely inhibits the growth of distant tumors in the periods of treatments.