Neuropeptide substance P attenuates colitis by suppressing inflammation and ferroptosis via the cGAS-STING signaling pathway.

Neuropeptide substance P (SP) belongs to a family of bioactive peptides and regulates many human diseases. This study aims to investigate the role and underlying mechanisms of SP in colitis. Here, activated SP-positive neurons and increased SP expression were observed in dextran sodium sulfate (DSS)-induced colitis lesions in mice. Administration of exogenous SP efficiently ameliorated the clinical symptoms, impaired intestinal barrier function, and inflammatory response. Mechanistically, SP protected mitochondria from damage caused by DSS or TNF-α exposure, preventing mitochondrial DNA (mtDNA) leakage into the cytoplasm, thereby inhibiting the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. SP can also directly prevent STING phosphorylation through the neurokinin-1 receptor (NK1R), thereby inhibiting the activation of the TBK1-IRF3 signaling pathway. Further studies revealed that SP alleviated the DSS or TNF-α-induced ferroptosis process, which was associated with repressing the cGAS-STING signaling pathway. Notably, we identified that the NK1R inhibition reversed the effects of SP on inflammation and ferroptosis via the cGAS-STING pathway. Collectively, we unveil that SP attenuates inflammation and ferroptosis via suppressing the mtDNA-cGAS-STING or directly acting on the STING pathway, contributing to improving colitis in an NK1R-dependent manner. These findings provide a novel mechanism of SP regulating ulcerative colitis (UC) disease.

FGF21 attenuates neuroinflammation following subarachnoid hemorrhage through promoting mitophagy and inhibiting the cGAS-STING pathway.

BACKGROUND: Subarachnoid hemorrhage (SAH) represents a form of cerebrovascular event characterized by a notable mortality and morbidity rate. Fibroblast growth factor 21 (FGF21), a versatile hormone predominantly synthesized by the hepatic tissue, has emerged as a promising neuroprotective agent. Nevertheless, the precise impacts and underlying mechanisms of FGF21 in the context of SAH remain enigmatic. METHODS: To elucidate the role of FGF21 in inhibiting the microglial cGAS-STING pathway and providing protection against SAH-induced cerebral injury, a series of cellular and molecular techniques, including western blot analysis, real-time polymerase chain reaction, immunohistochemistry, RNA sequencing, and behavioral assays, were employed. RESULTS: Administration of recombinant fibroblast growth factor 21 (rFGF21) effectively mitigated neural apoptosis, improved cerebral edema, and attenuated neurological impairments post-SAH. Transcriptomic analysis revealed that SAH triggered the upregulation of numerous genes linked to innate immunity, particularly those involved in the type I interferon (IFN-I) pathway and microglial function, which were notably suppressed upon adjunctive rFGF21 treatment. Mechanistically, rFGF21 intervention facilitated mitophagy in an AMP-activated protein kinase (AMPK)-dependent manner, thereby preventing mitochondrial DNA (mtDNA) release into the cytoplasm and dampening the activation of the DNA-sensing cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. Conditional knockout of STING in microglia markedly ameliorated the inflammatory response and mitigated secondary brain injuries post-SAH. CONCLUSION: Our results present the initial evidence that FGF21 confers a protective effect against neuroinflammation-associated brain damage subsequent to SAH. Mechanistically, we have elucidated a novel pathway by which FGF21 exerts this neuroprotection through inhibition of the cGAS-STING signaling cascade.

Mitochondrial DNA and Electron Transport Chain Protein Levels Are Altered in Peripheral Nerve Tissues from Donors with HIV Sensory Neuropathy: A Pilot Study.

Distal sensory polyneuropathy (DSP) and distal neuropathic pain (DNP) remain significant challenges for older people with HIV (PWH), necessitating enhanced clinical attention. HIV and certain antiretroviral therapies (ARTs) can compromise mitochondrial function and impact mitochondrial DNA (mtDNA) replication, which is linked to DSP in ART-treated PWH. This study investigated mtDNA, mitochondrial fission and fusion proteins, and mitochondrial electron transport chain protein changes in the dorsal root ganglions (DRGs) and sural nerves (SuNs) of 11 autopsied PWH. In antemortem standardized assessments, six had no or one sign of DSP, while five exhibited two or more DSP signs. Digital droplet polymerase chain reaction was used to measure mtDNA quantity and the common deletions in isolated DNA. We found lower mtDNA copy numbers in DSP+ donors. SuNs exhibited a higher proportion of mtDNA common deletion than DRGs in both groups. Mitochondrial electron transport chain (ETC) proteins were altered in the DRGs of DSP+ compared to DSP- donors, particularly Complex I. These findings suggest that reduced mtDNA quantity and increased common deletion abundance may contribute to DSP in PWH, indicating diminished mitochondrial activity in the sensory neurons. Accumulated ETC proteins in the DRG imply impaired mitochondrial transport to the sensory neuron's distal portion. Identifying molecules to safeguard mitochondrial integrity could aid in treating or preventing HIV-associated peripheral neuropathy.

Heat-Stress Impacts on Developing Bovine Oocytes: Unraveling Epigenetic Changes, Oxidative Stress, and Developmental Resilience.

Extreme temperature during summer may lead to heat stress in cattle and compromise their productivity. It also poses detrimental impacts on the developmental capacity of bovine budding oocytes, which halt their fertility. To mitigate the adverse effects of heat stress, it is necessary to investigate the mechanisms through which it affects the developmental capacity of oocytes. The primary goal of this study was to investigate the impact of heat stress on the epigenetic modifications in bovine oocytes and embryos, as well as on oocyte developmental capacity, reactive oxygen species, mitochondrial membrane potential, apoptosis, transzonal projections, and gene expression levels. Our results showed that heat stress significantly reduced the expression levels of the epigenetic modifications from histone H1, histone H2A, histone H2B, histone H4, DNA methylation, and DNA hydroxymethylation at all stages of the oocyte and embryo. Similarly, heat stress significantly reduced cleavage rate, blastocyst rate, oocyte mitochondrial-membrane potential level, adenosine-triphosphate (ATP) level, mitochondrial DNA copy number, and transzonal projection level. It was also found that heat stress affected mitochondrial distribution in oocytes and significantly increased reactive oxygen species, apoptosis levels and mitochondrial autophagy levels. Our findings suggest that heat stress significantly impacts the expression levels of genes related to oocyte developmental ability, the cytoskeleton, mitochondrial function, and epigenetic modification, lowering their competence during the summer season.

[Knockdown mitochondrial transcription factor A (TFAM) inhibits autophagy, proliferation, invasion and migration in human cervical cancer and osteosarcoma cells].

Objective To investigate the effects of mitochondrial transcription factor A (TFAM) on mitochondrial function, autophagy, proliferation, invasion, and migration in cervical cancer HeLa cells and osteosarcoma U2OS cells. Methods TFAM small-interfering RNA (si-TFAM) was transfected to HeLa and U2OS cells for downregulating TFAM expression. Mito-Tracker Red CMXRos staining combined with laser confocal microscopy was used to detect mitochondrial membrane potential (MMP). MitoSOXTM Red labeling was used to test mitochondrial reactive oxygen species (mtROS) levels. The expression of mitochondrial DNA (mtDNA) was detected by real-time quantitative PCR. Changes in the number of autophagosomes were detected by immunofluorescence cytochemistry. Western blot analysis was used to detect the expressions of TFAM, autophagy microtubule associated protein 1 light chain 3A/B (LC3A/B), autophagy associated protein 2A (ATG2A), ATG2B, ATG9A, zinc finger transcription factor Snail, matrix metalloproteinase 2 (MMP2) and MMP9. CCK-8 assay and plate clony formation assay were used to detect cell proliferation, while TranswellTM assay and scratch healing assay were used to detect changes in cell invasion and migration. Results The downregulation of TFAM expression resulted in a decrease in MMP and mtDNA copy number, but an increase in mtROS production. The protein content of LC3A/B decreased significantly compared to the control group and the number of autophagosomes in the cytoplasm decreased significantly. The expressions of ATG2B and ATG9A in the early stage of autophagy were significantly reduced. The expressions of Snail, MMP2 and MMP9 proteins in HeLa and U2OS cells were also decreased. The proliferation, invasion and migration ability of HeLa and U2OS cells were inhibited after being interfered with TFAM expression. Conclusion Downregulation of TFAM expression inhibits mitochondrial function, delays autophagy process and reduces the proliferation, invasion and migration ability of cervical cancer cells and osteosarcoma cells.

Mechanisms of mitochondrial dysfunction in ovarian aging and potential interventions.

Mitochondria plays an essential role in regulating cellular metabolic homeostasis, proliferation/differentiation, and cell death. Mitochondrial dysfunction is implicated in many age-related pathologies. Evidence supports that the dysfunction of mitochondria and the decline of mitochondrial DNA copy number negatively affect ovarian aging. However, the mechanism of ovarian aging is still unclear. Treatment methods, including antioxidant applications, mitochondrial transplantation, emerging biomaterials, and advanced technologies, are being used to improve mitochondrial function and restore oocyte quality. This article reviews key evidence and research updates on mitochondrial damage in the pathogenesis of ovarian aging, emphasizing that mitochondrial damage may accelerate and lead to cellular senescence and ovarian aging, as well as exploring potential methods for using mitochondrial mechanisms to slow down aging and improve oocyte quality.

Lysosomal dysfunction and overload of nucleosides in thymidine phosphorylase deficiency of MNGIE.

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.

Therapeutic landscape in systemic lupus erythematosus: mtDNA activation of the cGAS-STING pathway.

Mitochondrial DNA (mtDNA) serves as a pivotal immune stimulus in the immune response. During stress, mitochondria release mtDNA into the cytoplasm, where it is recognized by the cytoplasmic DNA receptor cGAS. This activation initiates the cGAS-STING-IRF3 pathway, culminating in an inflammatory response. The cGAS-STING pathway has emerged as a critical mediator of inflammatory responses in microbial infections, stress, autoimmune diseases, chronic illnesses, and tissue injuries. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by connective tissue involvement across various bodily systems. Its hallmark is the production of numerous autoantibodies, which prompt the immune system to target and damage the body's own tissues, resulting in organ and tissue damage. Increasing evidence implicates the cGAS-STING pathway as a significant contributor to SLE pathogenesis. This article aims to explore the role of the mtDNA-triggered cGAS-STING pathway and its mechanisms in SLE, with the goal of providing novel insights for clinical interventions.

Oscillatory shear stress promotes endothelial senescence and atherosclerosis via STING activation.

Endothelial dysfunction is an initiating factor in atherosclerosis. Endothelial cells (ECs) are constantly subject to blood flow shear stress, and atherosclerotic plaques tend to occur in aortic bends or bifurcations impaired by low oscillatory shear stress (OSS). However, the mechanism that how OSS affects the initiation and progression of atherosclerosis remains to be explored. Here, we first reported that OSS can promote endothelial dysfunction and atherogenesis in vivo and in vitro by activating STING pathway. Mechanistically, at atherosclerosis-prone areas, OSS caused mitochondria damage in ECs, leading to the leakage of mitochondrial DNA (mtDNA) into the cytoplasm. The cytoplasmic mtDNA was recognized by cGAS to produce cGAMP, activating the STING pathway and leading to endothelial senescence, which resulted in endothelial dysfunction and atherosclerosis. We found that STING was activated in plaques of atherosclerotic patients and in aortic arch ECs of high-fat diet (HFD)-fed ApoeKO mice, as well as in ECs exposed to OSS. STING-specific deficiency in ECs attenuates endothelial senescence and resulted in a significant reduction in aortic arch plaque area in HFD-fed ApoeKO mice. Consistently, specific deficiency or pharmacological inhibition of STING attenuated OSS-induced senescence and endothelial dysfunction. Pharmacological depletion of mtDNA ameliorated OSS-induced senescence and endothelial dysfunction. Taken together, our study linked hemodynamics and endothelial senescence, and revealed a novel mechanism by which OSS leads to endothelial dysfunction. Our study provided new insights into the development of therapeutic strategies for endothelial senescence and atherosclerosis.

Development of Cu(II) 4-hydroxybenzoylhydrazone complexes that induce mitochondrial DNA damage and mitochondria-mediated apoptosis in liver cancer.

Cisplatin remains the most widely used chemotherapeutic agent in cancer treatment; however, its inherent drawbacks have fueled the development of novel metalloanticancer drugs. In this study, two novel Cu(II) complexes (Cu1 and Cu2) were designed and synthesized. Notably, these Cu(II) complexes showed higher cytotoxicity against HL-7402 cells than cisplatin. Moreover, Cu(II) complexes significantly inhibited liver cancer growth in a xenograft model. A mechanism study revealed that the Cu(II) complexes reduced the mitochondrial membrane potential of cancer cells, produced excessive reactive oxygen species (ROS), induced mitochondrial DNA (mtDNA) damage, and ultimately facilitated cancer cell apoptosis.

Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

The Arabidopsis Mitochondrial Nucleoid-Associated Protein WHIRLY2 Is Required for a Proper Response to Salt Stress.

In the last years, plant organelles have emerged as central coordinators of responses to internal and external stimuli, which can induce stress. Mitochondria play a fundamental role as stress sensors being part of a complex communication network between the organelles and the nucleus. Among the different environmental stresses, salt stress poses a significant challenge and requires efficient signaling and protective mechanisms. By using the why2 T-DNA insertion mutant and a novel knock-out mutant prepared by CRISPR/Cas9-mediated genome editing, this study revealed that WHIRLY2 is crucial for protecting mitochondrial DNA (mtDNA) integrity during salt stress. Loss-of-function mutants show an enhanced sensitivity to salt stress. The disruption of WHIRLY2 causes the impairment of mtDNA repair that results in the accumulation of aberrant recombination products, coinciding with severe alterations in nucleoid integrity and overall mitochondria morphology besides a compromised redox-dependent response and misregulation of antioxidant enzymes. The results of this study revealed that WHIRLY2-mediated structural features in mitochondria (nucleoid compactness and cristae) are important for an effective response to salt stress.

High glucose-induced p66Shc mitochondrial translocation regulates autophagy initiation and autophagosome formation in syncytiotrophoblast and extravillous trophoblast.

BACKGROUND: p66Shc, as a redox enzyme, regulates reactive oxygen species (ROS) production in mitochondria and autophagy. However, the mechanisms by which p66Shc affects autophagosome formation are not fully understood. METHODS: p66Shc expression and its location in the trophoblast cells were detected in vivo and in vitro. Small hairpin RNAs or CRISPR/Cas9, RNA sequencing, and confocal laser scanning microscope were used to clarify p66Shc's role in regulating autophagic flux and STING activation. In addition, p66Shc affects mitochondrial-associated endoplasmic reticulum membranes (MAMs) formation were observed by transmission electron microscopy (TEM). Mitochondrial function was evaluated by detected cytoplastic mitochondrial DNA (mtDNA) and mitochondrial membrane potential (MMP). RESULTS: High glucose induces the expression and mitochondrial translocation of p66Shc, which promotes MAMs formation and stimulates PINK1-PRKN-mediated mitophagy. Moreover, mitochondrial localized p66Shc reduces MMP and triggers cytosolic mtDNA release, thus activates cGAS/STING signaling and ultimately leads to enhanced autophagy and cellular senescence. Specially, we found p66Shc is required for the interaction between STING and LC3II, as well as between STING and ATG5, thereby regulates cGAS/STING-mediated autophagy. We also identified hundreds of genes associated several biological processes including aging are co-regulated by p66Shc and ATG5, deletion either of which results in diminished cellular senescence. CONCLUSION: p66Shc is not only implicated in the initiation of autophagy by promoting MAMs formation, but also helps stabilizing active autophagic flux by activating cGAS/STING pathway in trophoblast.

Lack of detectable sex differences in the mitochondrial function of Caenorhabditis elegans.

BACKGROUND: Sex differences in mitochondrial function have been reported in multiple tissue and cell types. Additionally, sex-variable responses to stressors including environmental pollutants and drugs that cause mitochondrial toxicity have been observed. The mechanisms that establish these differences are thought to include hormonal modulation, epigenetic regulation, double dosing of X-linked genes, and the maternal inheritance of mtDNA. Understanding the drivers of sex differences in mitochondrial function and being able to model them in vitro is important for identifying toxic compounds with sex-variable effects. Additionally, understanding how sex differences in mitochondrial function compare across species may permit insight into the drivers of these differences, which is important for basic biology research. This study explored whether Caenorhabditis elegans, a model organism commonly used to study stress biology and toxicology, exhibits sex differences in mitochondrial function and toxicant susceptibility. To assess sex differences in mitochondrial function, we utilized four male enriched populations (N2 wild-type male enriched, fog-2(q71), him-5(e1490), and him-8(e1498)). We performed whole worm respirometry and determined whole worm ATP levels and mtDNA copy number. To probe whether sex differences manifest only after stress and inform the growing use of C. elegans as a mitochondrial health and toxicologic model, we also assessed susceptibility to a classic mitochondrial toxicant, rotenone. RESULTS: We detected few to no large differences in mitochondrial function between C. elegans sexes. Though we saw no sex differences in vulnerability to rotenone, we did observe sex differences in the uptake of this lipophilic compound, which may be of interest to those utilizing C. elegans as a model organism for toxicologic studies. Additionally, we observed altered non-mitochondrial respiration in two him strains, which may be of interest to other researchers utilizing these strains. CONCLUSIONS: Basal mitochondrial parameters in male and hermaphrodite C. elegans are similar, at least at the whole-organism level, as is toxicity associated with a mitochondrial Complex I inhibitor, rotenone. Our data highlights the limitation of using C. elegans as a model to study sex-variable mitochondrial function and toxicological responses.

Mitochondria-Selective Dicationic Small-Molecule Ligand Targeting G-Quadruplex Structures for Human Colorectal Cancer Therapy.

Mitochondria are important drug targets for anticancer and other disease therapies. Certain human mitochondrial DNA sequences capable of forming G-quadruplex structures (G4s) are emerging drug targets of small molecules. Despite some mitochondria-selective ligands being reported for drug delivery against cancers, the ligand design is mostly limited to the triphenylphosphonium scaffold. The ligand designed with lipophilic small-sized scaffolds bearing multipositive charges targeting the unique feature of high mitochondrial membrane potential (MMP) is lacking and most mitochondria-selective ligands are not G4-targeting. Herein, we report a new small-sized dicationic lipophilic ligand to target MMP and mitochondrial DNA G4s to enhance drug delivery for anticancer. The ligand showed marked alteration of mitochondrial gene expression and substantial induction of ROS production, mitochondrial dysfunction, DNA damage, cellular senescence, and apoptosis. The ligand also exhibited high anticancer activity against HCT116 cancer cells (IC50, 3.4 µM) and high antitumor efficacy in the HCT116 tumor xenograft mouse model (∼70% tumor weight reduction).

Leveraging new methods for comprehensive characterization of mitochondrial DNA in esophageal squamous cell carcinoma.

BACKGROUND: Mitochondria play essential roles in tumorigenesis; however, little is known about the contribution of mitochondrial DNA (mtDNA) to esophageal squamous cell carcinoma (ESCC). Whole-genome sequencing (WGS) is by far the most efficient technology to fully characterize the molecular features of mtDNA; however, due to the high redundancy and heterogeneity of mtDNA in regular WGS data, methods for mtDNA analysis are far from satisfactory. METHODS: Here, we developed a likelihood-based method dMTLV to identify low-heteroplasmic mtDNA variants. In addition, we described fNUMT, which can simultaneously detect non-reference nuclear sequences of mitochondrial origin (non-ref NUMTs) and their derived artifacts. Using these new methods, we explored the contribution of mtDNA to ESCC utilizing the multi-omics data of 663 paired tumor-normal samples. RESULTS: dMTLV outperformed the existing methods in sensitivity without sacrificing specificity. The verification using Nanopore long-read sequencing data showed that fNUMT has superior specificity and more accurate breakpoint identification than the current methods. Leveraging the new method, we identified a significant association between the ESCC overall survival and the ratio of mtDNA copy number of paired tumor-normal samples, which could be potentially explained by the differential expression of genes enriched in pathways related to metabolism, DNA damage repair, and cell cycle checkpoint. Additionally, we observed that the expression of CBWD1 was downregulated by the non-ref NUMTs inserted into its intron region, which might provide precursor conditions for the tumor cells to adapt to a hypoxic environment. Moreover, we identified a strong positive relationship between the number of mtDNA truncating mutations and the contribution of signatures linked to tumorigenesis and treatment response. CONCLUSIONS: Our new frameworks promote the characterization of mtDNA features, which enables the elucidation of the landscapes and roles of mtDNA in ESCC essential for extending the current understanding of ESCC etiology. dMTLV and fNUMT are freely available from https://github.com/sunnyzxh/dMTLV and https://github.com/sunnyzxh/fNUMT , respectively.

Mitochondrial (mt)DNA-cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling promotes pyroptosis of macrophages via interferon regulatory factor (IRF)7/IRF3 activation to aggravate lung injury during severe acute pancreatitis.

BACKGROUND: Macrophage proinflammatory activation contributes to the pathology of severe acute pancreatitis (SAP) and, simultaneously, macrophage functional changes, and increased pyroptosis/necrosis can further exacerbate the cellular immune suppression during the process of SAP, where cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) plays an important role. However, the function and mechanism of cGAS-STING in SAP-induced lung injury (LI) remains unknown. METHODS: Lipopolysaccharide (LPS) was combined with caerulein-induced SAP in wild type, cGAS -/- and sting -/- mice. Primary macrophages were extracted via bronchoalveolar lavage and peritoneal lavage. Ana-1 cells were pretreated with LPS and stimulated with nigericin sodium salt to induce pyroptosis in vitro. RESULTS: SAP triggered NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation-mediated pyroptosis of alveolar and peritoneal macrophages in mouse model. Knockout of cGAS/STING could ameliorate NLRP3 activation and macrophage pyroptosis. In addition, mitochondrial (mt)DNA released from damaged mitochondria further induced macrophage STING activation in a cGAS- and dose-dependent manner. Upregulated STING signal can promote NLRP3 inflammasome-mediated macrophage pyroptosis and increase serum interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α levels and, thus, exacerbate SAP-associated LI (SAP-ALI). Downstream molecules of STING, IRF7, and IRF3 connect the mtDNA-cGAS-STING axis and the NLRP3-pyroptosis axis. CONCLUSIONS: Negative regulation of any molecule in the mtDNA-cGAS-STING-IRF7/IRF3 pathway can affect the activation of NLRP3 inflammasomes, thereby reducing macrophage pyroptosis and improving SAP-ALI in mouse model.

Membrane damage by MBP-1 is mediated by pore formation and amplified by mtDNA.

Eosinophils play a crucial role in host defense while also contributing to immunopathology through the release of inflammatory mediators. Characterized by distinctive cytoplasmic granules, eosinophils securely store and rapidly release various proteins exhibiting high toxicity upon extracellular release. Among these, major basic protein 1 (MBP-1) emerges as an important mediator in eosinophil function against pathogens and in eosinophil-associated diseases. While MBP-1 targets both microorganisms and host cells, its precise mechanism remains elusive. We demonstrate that formation of small pores by MBP-1 in lipid bilayers induces membrane permeabilization and disrupts potassium balance. Additionally, we reveal that mitochondrial DNA (mtDNA) present in eosinophil extracellular traps (EETs) amplifies MBP-1 toxic effects, underscoring the pivotal role of mtDNA in EETs. Furthermore, we present evidence indicating that absence of CpG methylation in mtDNA contributes to the regulation of MBP-1-mediated toxicity. Taken together, our data suggest that the mtDNA scaffold within extracellular traps promotes MBP-1 toxicity.

Mitochondrial DNA-Activated cGAS-STING Signaling in Environmental Dry Eye.

Purpose: The cGAS-STING pathway has been shown to be an important mediator of inflammation. There is emerging evidence of the importance of this signaling cascade in a variety of inflammatory diseases settings. Here, we present evidence that the mitochondrial DNA (mtDNA) damage-mediated cGAS-STING pathway plays an important role in the induction of inflammation in environmental dry eye (DE). Methods: RT-qPCR and Western blot were used to assess the induction of the cGAS-STING pathway and inflammatory cytokines in environmental DE mouse model, primary human corneal epithelial cells (pHCECs), and patients with DE. RNA sequencing was used to determine mRNA expression patterns of high osmotic pressure (HOP)-stimulated pHCECs. mtDNA was detected with electron microscopy, flow cytometry, and immunofluorescent staining. mtDNA was isolated and transfected into pHCECs for evaluating the activation of the cGAS-STING pathway. Results: The expression levels of cGAS, STING, TBK1, IRF3, and IFNß were significantly increased in an environmental DE model and HOP-stimulated pHCECs. The STING inhibitor decreased the expression of inflammatory factors in DE. An upregulation of STING-mediated immune responses and IRF3 expression mediated by TBK1 were observed in the HOP group. HOP stimulation induced mitochondrial oxidative damage and the leakage of mtDNA into the cytoplasm. Then, mtDNA activated the cGAS-STING pathway and induced intracytoplasmic STING translocated to the Golgi apparatus. Finally, we also found activated cGAS-STING signaling in the human conjunctival blot cell of patients with DE. Conclusions: Our findings suggest that the cGAS-STING pathway is activated by recognizing cytoplasmic mtDNA leading to STING translocation, further exacerbating the development of inflammation in environmental DE.

Mitochondrial genome transfer drives metabolic reprogramming in adjacent colonic epithelial cells promoting TGFß1-mediated tumor progression.

Although nontumor components play an essential role in colon cancer (CC) progression, the intercellular communication between CC cells and adjacent colonic epithelial cells (CECs) remains poorly understood. Here, we show that intact mitochondrial genome (mitochondrial DNA, mtDNA) is enriched in serum extracellular vesicles (EVs) from CC patients and positively correlated with tumor stage. Intriguingly, circular mtDNA transferred via tumor cell-derived EVs (EV-mtDNA) enhances mitochondrial respiration and reactive oxygen species (ROS) production in CECs. Moreover, the EV-mtDNA increases TGFß1 expression in CECs, which in turn promotes tumor progression. Mechanistically, the intercellular mtDNA transfer activates the mitochondrial respiratory chain to induce the ROS-driven RelA nuclear translocation in CECs, thereby transcriptionally regulating TGFß1 expression and promoting tumor progression via the TGFß/Smad pathway. Hence, this study highlights EV-mtDNA as a major driver of paracrine metabolic crosstalk between CC cells and adjacent CECs, possibly identifying it as a potential biomarker and therapeutic target for CC.