胞葬障碍和 mtDNA-STING/NLRP3 的耦合轴

Vitiligo is a chronic depigmentary disorder initiated by oxidative stress, which activates inflammatory signaling and innate immunity, ultimately leading to melanocyte destruction. Although melanocyte defects have been widely studied, dermal fibroblasts-the predominant stromal regulators of cutaneous immunity-remain insufficiently characterized in vitiligo pathogenesis. Here, we demonstrate that subtoxic oxidative stress in normal human dermal fibroblasts (NHDFs) induces a VDAC1-dependent, non-apoptotic release of mitochondrial DNA (mtDNA), thereby linking redox imbalance to immune activation. Low-dose hydrogen peroxide preserved mitochondrial morphology while promoting VDAC1 oligomerization, forming pores that enabled selective mtDNA efflux from structurally intact mitochondria. The released mtDNA activated the cGAS-STING pathway and the NLRP3 inflammasome, driving the expression of IL-1β, IL-6, ICAM-1, and Occludin-a pattern consistent with a senescence-associated secretory phenotype. Pharmacological interventions using ethidium bromide, RU.521, VBIT-4, and exogenous mtDNA established mtDNA release as an upstream event in fibroblast innate immune activation. Notably, inhibiting VDAC1 oligomerization with VBIT-4 effectively prevented mtDNA leakage, attenuated fibroblast senescence and inflammatory signaling, and restored epidermal repigmentation in a vitiligo mouse model. These findings identify dermal fibroblasts as active sensors and amplifiers of oxidative stress via the VDAC1-mtDNA-cGAS-STING axis and highlight VDAC1 oligomerization as a promising therapeutic target.

Brown adipose tissue (BAT), a crucial heat-generating organ, regulates whole-body energy metabolism by mediating thermogenesis. BAT inflammation is implicated in the pathogenesis of mitochondrial dysfunction and impaired thermogenesis. However, the link between BAT inflammation and systematic metabolism remains unclear. Herein, we use mice with BAT deficiency of thioredoxin-2 (TRX2), a protein that scavenges mitochondrial reactive oxygen species (ROS), to evaluate the impact of BAT inflammation on metabolism and thermogenesis and its underlying mechanism. Our results show that BAT-specific TRX2 ablation improves systematic metabolic performance via enhancing lipid uptake, which protects mice from diet-induced obesity, hypertriglyceridemia, and insulin resistance. TRX2 deficiency impairs adaptive thermogenesis by suppressing fatty acid oxidation. Mechanistically, loss of TRX2 induces excessive mitochondrial ROS, mitochondrial integrity disruption, and cytosolic release of mitochondrial DNA, which in turn activate aberrant innate immune responses in BAT, including the cGAS/STING and the NLRP3 inflammasome pathways. We identify NLRP3 as a key converging point, as its inhibition reverses both the thermogenesis defect and the metabolic benefits seen under nutrient overload in BAT-specific Trx2-deficient mice. In conclusion, we identify TRX2 as a critical hub integrating oxidative stress, inflammation, and lipid metabolism in BAT, uncovering an adaptive mechanism underlying the link between BAT inflammation and systematic metabolism.

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Silica nanoparticles (SiNPs) are a nanometer powder widely used in various consumer products, engineering, the food industry, and medical applications. Environmental SiNPs have attracted attention owing to their exposure to various cardiovascular adverse events. Here, we exposed C57/BL6 mouse and HL-1 cells with different-sized SiNPs (50, 300 nm, and 1 μm) to investigate the underlying mechanism of its cardiovascular toxicity. Mice exposed to three-sized SiNPs showed significant weight loss after 21 days of treatment. Heart weight to tibia length ratio and histopathology staining indicated increased heart volume and cross-sectional area of myocardial fibers in mice exposed to SiNPs. In vivo and in vitro experiments results showed that exposure to SiNPs causes size-dependent mitochondrial damage and initiates mitophagy. Notably, compared to the damage caused by 300 nm and 1 μm SiNPs exposure, 50 nm SiNPs blocked autophagy flux, leading to excessive accumulation of mitochondrial DNA (mtDNA) in the cytoplasm, ultimately exacerbating downstream cGAS-STING pathway-mediated pyroptosis. This study revealed the potential health risks of SiNPs and helped to understand the differences in cytotoxicity caused by SiNPs of different sizes.

Microglial activation-induced neuroinflammation and impaired neuronal mitophagy are recognized as pivotal pathogeneses in Parkinson’s disease (PD). However, the role of microglial mitophagy in microglial activation during PD development remains unclear, and therapeutic interventions targeting this interaction are lacking. Rhapontigenin (Rhap), a stilbenoid enriched in Vitis vinifera, exhibits dual anti-neuroinflammatory and mitophagy-enhancing properties, but its therapeutic potential and mechanisms in PD are unexplored. This study aimed to investigate the therapeutic efficacy of Rhap on neurodegeneration in a PD model and explore its underlying mechanism. Here, we showed that Rhap administration significantly ameliorated motor deficits, dopaminergic neuron loss, and neuroinflammation in MPTP-induced PD mice. Mechanistically, Rhap suppressed neuroinflammation by inhibiting the cGAS-STING-NF-κB signaling axis in both PD model mice and MPP⁺-induced BV2 microglia. Crucially, its anti-inflammatory effects depend on the PINK1-mediated enhancement of microglial mitophagy to control cytosolic mtDNA leakage. Specifically, Rhap bound to PINK1 strengthened the PINK1-DRP1 interaction, promoted mitochondrial fission in damaged organelles, and enhanced mitophagy clearance. This mitophagy activation prevents cytosolic leakage of mitochondrial DNA (mtDNA), thereby attenuating mtDNA-cGAS-STING-NF-κB-derived neuroinflammation and subsequent neurodegeneration in PD. PINK1 deficiency in BV2 microglia abolished Rhap’s ability to suppress mtDNA-cGAS-STING-NF-κB activation and enhance mitophagy. Overall, our study reveals a previously unrecognized mechanism by which Rhap ameliorates PD-associated neurodegeneration through dual modulation of PINK1/DRP1-dependent microglial mitophagy and the mtDNA-cGAS-STING-NF-κB neuroinflammatory axis, suggesting a potential therapeutic strategy for PD and related neurodegenerative disorders.

No abstract available

No abstract available

Hepatocellular cell death and macrophage proinflammatory activation contribute to the pathology of various liver diseases, during which XBP1 plays an important role. However, the function and mechanism of XBP1 in thioacetamide (TAA)-induced acute liver injury (ALI) remains unknown. Here, we investigated the effects of XBP1 inhibition on promoting hepatocellular pyroptosis to activate macrophage STING signaling during ALI. While both TAA- and LPS-induced ALI triggered XBP1 activation in hepatocytes, hepatocyte-specific XBP1 knockout mice exhibited exacerbated ALI with increased hepatocellular pyroptosis and enhanced macrophage STING activation. Mechanistically, mtDNA released from TAA-stressed hepatocytes could be engulfed by macrophages, further inducing macrophage STING activation in a cGAS- and dose-dependent manner. XBP1 deficiency increased ROS production to promote hepatocellular pyroptosis by activating NLRP3/caspase-1/GSDMD signaling, which facilitated the extracellular release of mtDNA. Moreover, impaired mitophagy was found in XBP1 deficient hepatocytes, which was reversed by PINK1 overexpression. Mitophagy restoration also inhibited macrophage STING activation and ALI in XBP1 deficient mice. Activation of XBP1-mediated hepatocellular mitophagy and pyroptosis and macrophage STING signaling pathway were observed in human livers with ALI. Collectively, these findings demonstrate that XBP1 deficiency promotes hepatocyte pyroptosis by impairing mitophagy to activate mtDNA/cGAS/STING signaling of macrophages, providing potential therapeutic targets for ALI.

Silk fibroin (SF) has excellent biocompatibility and biodegradability as a biomaterial. The purity and molecular weight distribution of silk fibroin peptide (SFP) make it more suitable for medical application. In this study, SFP nanofibers (molecular weight ∼30kD) were prepared through CaCl2/H2O/C2H5OH solution decomposition and dialysis, and adsorbed naringenin (NGN) to obtain SFP/NGN NFs. In vitro results showed that SFP/NGN NFs increased the antioxidant activity of NGN and protected HK-2 cells from cisplatin-induced damage. In vivo results also showed that SFP/NGN NFs protected mice from cisplatin-induced acute kidney injury (AKI). The mechanism results showed that cisplatin induced mitochondrial damage, as well as increased mitophagy and mtDNA release, which activated the cGAS-STING pathway and induced the expression of inflammatory factors such as IL-6 and TNF-α. Interestingly, SFP/NGN NFs further activated mitophagy and inhibited mtDNA release and cGAS-STING pathway. Demonstrated that mitophagy-mtDNA-cGAS-STING signal axis was involved in the kidney protection mechanism of SFP/NGN NFs. In conclusion, our study confirmed that SFP/NGN NFs are candidates for protection of cisplatin-induced AKI, which is worthy of further study.

Metabolic cells exhibit low‐grade chronic inflsammation characterized by excessive production and secretion of proinflammatory cytokines and chemokines in response to overnutrition and energy excess. Mitochondrial dysfunction is closely associated with metabolic inflammation. PINK1 (phosphatase and tensin homology‐induced putative kinase 1) is a crucial pathway controlling mitochondrial autophagy, essential for maintaining mitochondrial quality control and metabolic homeostasis. The aim of this study was to investigate the role of PINK1 in metabolic inflammation. Our findings indicate that in adipocytes, palmitic acid (PA) activates the expression of PINK1. Additionally, knockdown of PINK1 exacerbates PA‐induced adipocyte inflammation. Mechanistically, PINK1 deficiency impairs mitochondrial function, leading to the release of mtDNA and further activation of the cGAS‐STING pathway. Therefore, targeting mitochondrial autophagy in adipocytes and the cGAS‐STING pathway may represent effective approaches to alleviate the chronic inflammation associated with obesity and related metabolic disorders.

Introduction Hypoxic-ischemic encephalopathy (HIE) involves neuroinflammation driven by microglial activation, yet regulatory mechanisms remain poorly defined. This study investigates how Retinoic Acid Receptor-Related Orphan Receptor Alpha (RORα) modulates mitophagy to suppress mtDNA-cGAS-STING-NLRP3 signaling in aging microglia, offering therapeutic potential for HIE. Methods A multi-omics approach combining single-cell RNA sequencing (scRNA-seq) of an HIE rat model, Weighted Gene Co-Expression Network Analysis (WGCNA), and LASSO regression identified RORα as a pivotal regulator. In vivo and in vitro HIE models with RORα overexpression were assessed via behavioral tests (morris water maze, tail suspension), reactive oxygen species (ROS) quantification, and molecular profiling (RT-qPCR, Western Blot, ELISA). Mitophagy inhibitor 3-MA was used to validate pathway dependence. Results Multi-omics integration revealed RORα as a hub gene linked to inflammatory and metabolic pathways. RORα activation enhanced mitophagy, reducing mtDNA leakage by 43% and cGAS-STING activity by 68%, which suppressed NLRP3 inflammasome activation (p < 0.01). This correlated with improved cognitive/motor function in HIE rats (p < 0.05) and attenuated ROS/IL-1β levels. Critically, 3-MA reversed RORα’s anti-inflammatory effects, confirming mitophagy dependence. Conclusion RORα alleviates HIE by resolving microglial neuroinflammation through mitophagic inhibition of mtDNA-cGAS-STING-NLRP3 signaling. These findings position RORα as a novel therapeutic target for HIE, bridging mitochondrial quality control and neuroimmunology.

ABSTRACT Acute lung injury (ALI) involves inflammatory cytokines and chemokines, resulting in lung and multiple organ injuries. This study explored the mechanism of mitophagy and cGAS/STING pathway in oleic acid (OA)‐induced ALI. Mice and pulmonary microvascular endothelial cells were divided into four groups: control group (Con), ALI group, FUNDC1 −/− control group (F‐Con), and FUNDC1 −/− ALI group (F‐ALI). After 24 h of modeling, proceed with tissue collection. Lung tissues were stained using hematoxylin eosin. Autophagosomes were observed by electron microscope and mtDNA was detected by qPCR. Western blot was used to analyze protein expression of pathways cGAS, STING, pTBK1, pIRF3, and pNF‐κB. Serum IFN‐β expression was detected by ELISA. Cellular morphological changes were observed using microscopy. LDH level, cGAS, and STING in endothelial cells were observed. Compared with control group, pathological changes in ALI group were significantly aggravated. Expressions of serum IFN‐β, cGAS, STING, pTBK1, pIRF3, and pNF‐κB in lung tissues of ALI mice were significantly higher than control group. After OA, the morphology of lung microvascular endothelial cells changed and LDH expression increased. After FUNDC1 gene was knocked out to inhibit mitophagy, autophagosomes were significantly reduced and mtDNA increased. Expressions of pathway proteins in lung tissues and cells of FUNDC1 −/− ALI group were higher than those of wild‐type ALI group. Serum IFN‐β expression also increased. Silencing FUNDC1 inhibits mitophagy. Subsequently, accumulated mtDNA activates cGAS/STING pathway, aggravating ALI pathological damage and inflammation, suggesting that mitophagy may provide protection in OA‐induced ALI through cGAS/STING pathway.

Pseudomonas aeruginosa (P. aeruginosa) infections pose a significant threat to public health, underscoring the need for deeper insights into host cellular defenses. This study explores the critical role of autophagy‐related protein 5 (ATG5) in lung epithelial cells during P. aeruginosa infection. Single‐cell RNA transcriptomics revealed a pronounced enrichment of autophagy pathways in type II alveolar epithelial cells (AEC2). Using a conditional Atg5 knockout murine model, we demonstrated that ATG5 deficiency in AEC2 compromises survival, hampers bacterial clearance, and increases pathogen dissemination. Additionally, the loss of ATG5 exacerbated inflammatory responses, notably through the activation of the AKT/PI3K/NF‐κB axis and pyroptosis, which culminated in severe lung injury and epithelial barrier disruption. Mechanistically, the absence of ATG5 disrupted mitophagy, leading to intensified mitochondrial damage. This exacerbated condition coupled with the activation of gasdermin D (GSDMD) by the noncanonical caspase‐11, enhancing the release of mitochondrial DNA (mtDNA), which in turn activated cGAS–STING–NLRP3 signaling in macrophages. These findings highlight the essential role of ATG5 in modulating immune responses and suggest potential therapeutic targets for managing P. aeruginosa‐induced pulmonary infections.

Manganese (Mn), the third most abundant transition metal in the earth’s crust, has widespread applications in the emerging field of organometallic catalysis and traditional industries. Excessive Mn exposure causes neurological syndrome resembling Parkinson’s disease (PD). The pathogenesis of PD is thought to involve microglia-mediated neuroinflammatory injury, with mitochondrial dysfunction playing a role in aberrant microglial activation. In the early stages of PD, PINK1/Parkin-mediated mitophagy contributes to the microglial inflammatory response via the cGAS/STING signaling pathway. Suppression of PINK1/Parkin-mediated mitophagy due to excessive Mn exposure exacerbates neuronal injury. Moreover, excessive Mn exposure leads to neuroinflammatory damage via the microglial cGAS-STING pathway. However, the precise role of microglial mitophagy in modulating neuroinflammation in Mn-induced parkinsonism and its underlying molecular mechanism remains unclear. Here, we observed that Mn-exposed mice exhibited neurobehavioral abnormalities and detrimental microglial activation, along with increased apoptosis of nerve cells, proinflammatory cytokines, and intracellular ROS. Furthermore, in vivo and in vitro experiments showed that excessive Mn exposure resulted in microglial mitochondrial dysfunction, manifested by increased mitochondrial ROS, decreased mitochondrial mass, and membrane potential. Additionally, with the escalating Mn dose, PINK1/Parkin-mediated mitophagy changed from activation to suppression. This was evidenced by decreased levels of LC3-II, PINK1, p-Parkin/Parkin, and increased levels of p62 protein expression level, as well as the colocalization between ATPB and LC3B due to excessive Mn exposure. Upregulation of mitophagy by urolithin A could mitigate Mn-induced mitochondrial dysfunction, as indicated by decreased mitochondrial ROS, increased mitochondrial mass, and membrane potential, along with improvements in neurobehavioral deficits and attenuated detrimental microglial activation. Using single-nucleus RNA-sequencing (snRNA-seq) analysis in the Mn-exposed mouse model, we identified the microglial cGAS-STING signaling pathway as a potential mechanism underlying Mn-induced neuroinflammation. This pathway is associated with an increase in cytosolic mtDNA levels, which activate STING signaling. These findings point to the induction of microglial mitophagy as a viable strategy to alleviate Mn-induced neuroinflammation through mtDNA-STING signaling.

Summary ZFAND6 is a zinc finger protein that interacts with TNF receptor-associated factor 2 (TRAF2) and polyubiquitin chains and has been linked to tumor necrosis factor (TNF) signaling. Here, we report a previously undescribed function of ZFAND6 in maintaining mitochondrial homeostasis by promoting mitophagy. Deletion of ZFAND6 in bone marrow-derived macrophages (BMDMs) upregulates reactive oxygen species (ROS) and the accumulation of damaged mitochondria due to impaired mitophagy. Consequently, mitochondrial DNA (mtDNA) is released into the cytoplasm, triggering the spontaneous expression of interferon-stimulated genes (ISGs) in a stimulator of interferon genes (STING) dependent manner, which leads to enhanced viral resistance. Mechanistically, ZFAND6 bridges a TRAF2-cIAP1 interaction and mediates the recruitment of TRAF2 to damaged mitochondria, which is required for the initiation of ubiquitin-dependent mitophagy. Our results suggest that ZFAND6 promotes the interactions of TRAF2 and cIAP1 and the clearance of damaged mitochondria by mitophagy to maintain mitochondrial homeostasis.

Aims: Type 2 diabetes mellitus (T2DM) significantly elevates the likelihood of atrial fibrillation (AF); However, the precise mechanisms remain incompletely elucidated. Mitochondrial dysfunction is a hallmark of diabetic cardiomyopathy, and recent evidence suggests that activation of the cGAS-STING signaling pathway may contribute to metabolic inflammation in the atria. This study aims to investigate the role of mitochondrial DNA (mtDNA)-mediated cGAS-STING activation in promoting diabetes-associated atrial fibrillation (AF) through cardiomyocyte-macrophage crosstalk. Methods and results: Using a high-fat diet combined with streptozotocin through intraperitoneal injection, we induced a diabetic mouse model. We observed increased AF inducibility, oxidative stress, and mitochondrial ultrastructural abnormalities, along with elevated expression of STING pathway components and pro-inflammatory cytokines in atrial tissue. RNA sequencing and histological analyses confirmed dysregulation of mitochondrial quality control (MQC), including impaired mitophagy, imbalance in fusion and fission, and reduced mitochondrial biogenesis. In vitro, HL-1 atrial cardiomyocytes exposed to high glucose and palmitic acid showed excessive production of mtROS and cytosolic release of mitochondrial DNA (mtDNA), which in turn triggered cGAS-STING activation. A transwell co-culture system revealed that cardiomyocyte-derived mtDNA was engulfed by RAW 264.7 macrophages, promoting M1 polarization of macrophages and further amplifying inflammatory signaling. Importantly, pharmacological intervention with the mitochondrial antioxidant mito-TEMPO or cardiomyocyte-specific STING knockdown suppressed inflammatory responses, reversed atrial remodeling, and reduced AF susceptibility. Notably, STING overexpression sustained inflammatory pathways independently of suppressing oxidative stress, highlighting cGAS-STING signaling as a downstream effector of mitochondrial damage. Conclusion: Impairment of mitochondrial quality control promotes atrial inflammation and remodeling in diabetes through mtDNA-induced cGAS-STING activation and cardiomyocyte-macrophage communication. Targeting this pathway may offer a novel strategy for AF management in metabolically compromised hearts.

The global prevalence of Neurological disorders has increased alarmingly in response to environmental and lifestyle changes. Atrazine (ATZ) is a difficult to degrade soil and water pollutant with well-known neurotoxicity. Melatonin (MT), an antioxidant with chemoprotective properties, has a potential therapeutic effect on cerebellar damage caused by ATZ exposure. The aim of this study was to explore the effects and underlying mechanisms of MT on the cerebellar inflammatory response and pyroptosis induced by ATZ exposure. In this study, C57BL/6J mice were treated with ATZ (170 mg/kg BW/day) and MT (5 mg/kg BW/day) for 28 days. Our results revealed that MT alleviated the histopathological changes, ultrastructural damage, oxidative stress and decrease of mitochondrial membrane potential (ΔΨm) in the cerebellum induced by ATZ exposure. ATZ exposure damaged the mitochondria leading to release of mitochondrial DNA (mtDNA) to the cytoplasm, MT activated the cyclic GMP-AMP synthetase interferon gene stimulator (cGAS-STING) axis to alleviate inflammation and pyroptosis caused by ATZ exposure. In general, our study provided new evidence that the cGAS-STING-NLRP3 axis plays an important role in the treatment of ATZ-induced cerebellar injury by MT.

Delayed healing of diabetic foot ulcers (DFUs) is driven by chronic inflammation and mitochondrial dysfunction. We identify the aryl hydrocarbon receptor (AhR) as a key regulator of immune and mitochondrial homeostasis in diabetic wounds. AhR expression was elevated in macrophages from human and murine DFUs. In AhR knockout mice, loss of AhR impaired M2 macrophage polarization and enhanced NLRP3 inflammasome activation via the cGAS–STING pathway. Mechanistically, AhR deficiency suppressed mitophagy, causing mitochondrial DNA leakage and sustained inflammatory signaling. To target this axis, we developed a FICZ-loaded GelMA hydrogel (GelMA–FICZ). Local application of GelMA–FICZ restored mitochondrial function, inhibited inflammasome activation, and significantly improved wound healing in diabetic mice. This study reveals a critical AhR–mitochondria–inflammasome pathway in DFUs and suggests a novel biomaterial-based immunomodulatory therapy for diabetic wound repair.

Tumor necrosis factor (TNF) is a key driver of several inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis, in which affected tissues show an interferon-stimulated gene signature. Here, we demonstrate that TNF triggers a type-I interferon response that is dependent on the cyclic guanosine monophosphate-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. We show that TNF inhibits PINK1-mediated mitophagy and leads to altered mitochondrial function and to an increase in cytosolic mtDNA levels. Using cGAS-chromatin immunoprecipitation (ChIP), we demonstrate that cytosolic mtDNA binds to cGAS after TNF treatment. Furthermore, TNF induces a cGAS-STING-dependent transcriptional response that mimics that of macrophages from rheumatoid arthritis patients. Finally, in an inflammatory arthritis mouse model, cGAS deficiency blocked interferon responses and reduced inflammatory cell infiltration and joint swelling. These findings elucidate a molecular mechanism linking TNF to type-I interferon signaling and suggest a potential benefit for therapeutic targeting of cGAS/STING in TNF-driven diseases.

Oncogenic KRAS causes the immunosuppressive tumor microenvironment (TME) to decrease the therapeutic response to radiotherapy (RT) and is associated with poor outcomes. In this study, we observed that constitutive dynamin-related protein 1 (DRP1) phosphorylation by oncogenic KRAS resulted in a decrease in mitochondrial DNA (mtDNA) content, leading to attenuated radiotherapy-induced cGAS/STING-driven type I IFN secretion. Targeting DRP1 phosphorylation enhanced cytosolic mtDNA release to promote cGAS/STING activation in response to radiotherapy. Disruption of cGAS-STING signaling limited the immune-enhancing effect of DRP1 targeting on type I IFN production and T-cell killing ability. Targeting DRP1 increased the therapeutic response and induced systemic antitumor immunity, accompanied by increased type I IFN signaling and intratumoral infiltration of immune cells in response to radiotherapy. Notably, the immune-enhancing effects of DRP1 inhibition were impaired by IFN receptor blockade. Moreover, dual targeting of DRP1 and MEK significantly enhanced the therapeutic response to RT in a KRASG12D-driven CRC model, providing its clinical relevance to target undruggable KRASG12D-driven CRC patients. Taken together, these findings reveal that DRP1 phosphorylation-mediated mtDNA decrease is a suppressor of antitumor immunity, suggesting that targeting DRP1 could be used to increase tumor immunogenicity and improve responsiveness to radiotherapy, especially in oncogenic KRAS-driven CRC patients.

Textiles and clothing are a primary source of microplastic pollution, releasing microfibers into the environment. In this study, lint microfibers in the top-loading washing machine lint filter (TWML) were provided by 10 volunteers and pooled into a single sample. The TWML consisted of irregularly shaped or fibrous particles, including heavy metals and microplastics. We dosed mice via oropharyngeal aspiration with TWML (10, 25, and 50 μg/mouse) for 90 days. The number of WBCs and the proportion of neutrophils in WBCs decreased in male and female mice exposed to the highest dose, respectively, and the proportion of RET in RBCs decreased in both sexes of mice. The total number of pulmonary immune cells increased with dose, accompanying an increase in the proportion of lymphocytes. Pulmonary immune cells aggregated around TWML, and among the inflammatory mediators measured in this study, only CXCL-1 and TGF-β levels increased significantly in the lungs of both sexes of mice. Infiltration of inflammatory cells and hyperplasia of mucous cells in the bronchial epithelium were found in the lung tissues of TWML-treated mice. When incubated at 40 μg/mL, alveolar macrophages were aggregated around the fibrous particles, as was observed in the lungs. The production of cell signaling-related secondary mediators increased significantly in TWML-treated cells. NGS analysis also indicated that the plasma membrane, cell periphery, and cell projections were the most affected cellular components, and that genes involved in protein synthesis and mitochondrial DNA replication were most downregulated in TWML-treated cells. Expression of mitochondrial dynamics- and cellular iron uptake-related proteins was inhibited following exposure to TWML, and those of anti-oxidant response-related proteins were clearly enhanced in the cells. Overall, we conclude that TWML-induced inflammatory lesions may be attributable to frustrated phagocytosis of alveolar macrophages. Additionally, TWML may disrupt cellular function through oxidative stress and damage to mitochondrial DNA replication.

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No abstract available

Introduction: Sterile inflammation has long been postulated to exacerbate heart failure (HF); however, a lack of viable biological targets and thorough understanding of the underlying mechanism posed formidable challenges in the development of novel therapeutic interventions. Hypothesis: Given proinflammatory mitochondrial DNA (mtDNA) are known to be elevated in HF plasma; and that cells release mitochondrial components in extracellular vesicles, we hypothesize that proinflammatory circulating Mitochondria-containing Extracellular Vesicle (MitoEV) is a mediator of HF sterile inflammation. Methods/Results: Using plasma samples of 12 Stage-D HFrEF and 12 healthy participants, plasma microparticles (MP) are co-stained with mitochondrial- and cytoplasmic-specific markers then quantified by volume-metric flow analysis. We found that there are significantly more mitochondrial-cytoplasmic double-positive MP (designated MitoEV, accounts for >70% of total MP) in HF relative to control. Next, using size-exclusion chromatography, we found MitoEV fractions are significantly more potent than soluble protein fractions to induce cytokine production in macrophage, implicating MitoEV as the source of HF plasma immunogenicity. Surface marker analysis by flow suggests that CD14+ monocytes is a major source of HF plasma MitoEV. Using THP-1 monocytes, we found that TLR9 activation by mtDNA results in injured mitochondria to be released as MitoEV (rather than removed by mitophagy), a pathway negatively regulated by beclin-1. Finally, boosting NAD by nicotinamide riboside preserves mitochondrial dysfunction and significantly reduces MitoEV release in primary monocytes. Conclusion: Our findings implicate circulating MitoEV released by monocytes upon mtDNA activation as a pivotal mediator of sterile inflammation in HF, demonstrating a promising avenue for potential therapeutic targeting. The study also provides a potential cellular mechanism to the anti-inflammatory effect of boosting NAD. Model: In HF circulation, mtDNA activates TLR9 of monocytes, injuring mitochondria, which are released as MitoEV (containing mtDNA) to amplify and propagate inflammation in an autocrine manner. Becn1, a key mediator of mitophagy, negatively regulates MitoEV release. Activated monocytes also release cytokines to further injure the heart, resulting in a vicious cycle. Boosting NAD protects against mtDNA-induced mitochondrial dysfunction, attenuates the MitoEV release, and ameliorates inflammation.

Introduction: Sterile inflammation is increasingly recognized as a key contributor to heart failure (HF) progression, yet the lack of clearly defined molecular mechanisms has limited the development of targeted therapies. Among proposed triggers, mitochondrial damage-associated molecular patterns (MitoDAMPs)—immunogenic molecules released from injured mitochondria—are elevated in the circulation of HF patients. Since mitochondria are frequently exported within extracellular vesicles (EVs), we hypothesized that mitochondria-containing extracellular vesicles (EV-Mito), a previously unrecognized subclass of circulating EVs, may act as immune-activating mediators in HF. Methods and Results: Plasma samples from patients with Stage D HFrEF (n=12) and healthy controls (n=12) were analyzed by flow cytometry using mitochondrial- and cytoplasmic-specific dyes. EV-Mito, defined as double-positive microparticles, were increased 3-fold in HF patients compared to controls (p = 0.0423) and accounted for over 70% of total plasma microparticles. EV-Mito isolated from HF plasma by size-exclusion chromatography followed by flow sorting induced robust IL1B and TNFα production in macrophages, confirming their immunogenicity. Surface marker profiling revealed CD14 enrichment, implicating monocytes as a primary source of EV-Mito in HF circulation. Mechanistically, mitochondrial DNA (mtDNA) activation of Toll-like receptor 9 (TLR9) in THP-1 monocytes induced mitochondrial dysfunction and impaired autophagy via disruption of the PI3K-III Complex II, resulting in EV-Mito release. Given the known role of NAD+ in preserving mitochondrial health, we tested nicotinamide riboside (an NAD+ precursor) supplementation in cultured monocytes and in HFrEF patients. While patient data are pending, NAD+ augmentation in primary monocytes preserved mitochondrial integrity and significantly reduced EV-Mito release by 30% (p = 0.0118). Conclusion: These findings identify EV-Mito as a mechanistically distinct and targetable mediator of inflammation in HF. By preventing their release, NAD+ augmentation offers a clinically feasible anti-inflammatory strategy with potential to improve outcomes in HF patients.

During the progression of severe sepsis, the oxidized mitochondrial DNA (mtDNA) in macrophages is cleaved by flap-structure-specific endonuclease 1 (FEN1) into small fragments, which are subsequently released into the cytosol and extracellular space to activate multiple pro-inflammatory signaling pathways such as NLRP3 inflammasome, cGAS-STING, and TLR9-NF-κB. Herein, biomimetic nanocomplexes (NCs) partially cloaked with macrophage membrane (MM) are developed to efficiently deliver FEN1 siRNA (siFEN1) into macrophages for sepsis management. To construct the NCs, membrane-penetrating, helical polypeptide (PG) first condenses siFEN1 and forms the cationic inner core, which is further coated with MM. By optimizing the membrane protein/siFEN1 weight ratios, partial membrane coating can be achieved, which enables the formation of NCs with both enhanced serum stability and efficient macrophage uptake efficiency. After systemic administration in cecal ligation and puncture-induced sepsis mice, the NCs exhibit prolonged blood circulation time and effective accumulation to the inflamed tissues, facilitated by MM-mediated charge neutralization of the cationic nanocore and inflammation homing. Subsequently, the NCs are efficiently internalized by macrophages through the interaction between the partially exposed polycationic core and the target cell membranes, provoking robust FEN1 silencing to suppress mtDNA fragmentation and leakage. Consequently, the NCs effectively restore immune homeostasis in sepsis mice, thereby mitigating cytokine storm and alleviating multiple organ failure.

Achilles tendinopathy represents a prototypical musculoskeletal disorder driven by a self-perpetuating “inflammaging” vicious cycle, where chronic inflammation and stem cell senescence mutually reinforce to precipitate tissue failure. Current therapeutics inadequately address the complex intercellular signaling fueling this loop. Herein, we present a reactive oxygen species (ROS)-responsive photothermal cascade nanoplatform (LT-NPs) that couples Licochalcone A delivery with mild near-infrared (NIR) hyperthermia (∼42 °C). Unlike conventional ablative therapies, this platform leverages mild thermal stress as a safe, generalized immunometabolic modulator. Mechanistically, we identify the mitochondrial DNA (mtDNA)-cGAS-STING axis as the pivotal “bridge” connecting mitochondrial dysfunction to immune dysregulation. The LT-NPs-NIR system dismantles this pathology via a synergistic “dual-lock” strategy: (1) mild photothermal heating induces heat shock protein 70 (HSP70) to seal mtDNA leakage; and (2) released Licochalcone A directly inhibits the downstream STING sensor. Crucially, this intervention re-engineers the dysregulated crosstalk between the immune niche and tendon stroma: by reprogramming M1 macrophages toward a reparative M2 phenotype and simultaneously rescuing tendon stem/progenitor cells (TSPCs) from senescence-associated secretory phenotype (SASP)-mediated senescence, the platform effectively uncouples the reciprocal feedback loop between inflammation and degeneration. In vivo, this orchestrated restoration of the microenvironment significantly suppresses heterotopic ossification and recovers biomechanical function. Consequently, the “mild photothermal cascade” concept establishes a versatile therapeutic paradigm, offering a scalable strategy to resolve the intricate inflammation-senescence crosstalk across a broad spectrum of age-related pathologies.

Abstract Background House dust mite (HDM) is the leading allergen for allergic rhinitis (AR). Although allergic sensitisation by inhaled allergens renders susceptible individuals prone to developing AR, the molecular mechanisms driving this process remain incompletely elucidated. Objective This study aimed to elucidate the molecular mechanisms underlying HDM‐induced AR. Methods We examined the expression of cytidine/uridine monophosphate kinase 2 (CMPK2), STING and the NLRP3 inflammasome in both AR patients and mice. Additionally, we investigated the role of CMPK2 and STING in the activation of the NLRP3 inflammasome in AR. Results The expression of CMPK2, STING and the NLRP3 inflammasome was significantly increased in the nasal mucosa of AR patients compared to non‐AR controls. A positive correlation was found between CMPK2 expression and the levels of STING, NLRP3, ASC, CASP1 and IL‐1β. HDM treatment up‐regulated the expression of CMPK2, and CMPK2 overexpression enhanced NLRP3 inflammasome activation in human nasal epithelial cells (HNEPCs). Additionally, mitochondrial reactive oxygen species (mtROS) production following HDM exposure contributed to mitochondrial dysfunction and the release of mitochondrial DNA (mtDNA), which activated the cyclic GMP‐AMP synthase (cGAS)‐STING pathway. Remarkably, depletion of mtDNA or inhibition of STING signalling reduced HDM‐induced NLRP3 inflammasome activation in HNEPCs. In vivo, genetic knockout of CMPK2 or STING alleviated NLRP3 inflammasome activation and ameliorated clinical symptoms of AR in mice. Conclusions Our results suggest that HDM promotes the activation of NLRP3 inflammasome through the up‐regulation of CMPK2 and ensuing mtDNA‐STING signalling pathway, hence revealing additional therapeutic target for AR. Key points Cytidine/uridine monophosphate kinase 2 (CMPK2) expression is up‐regulated in the nasal mucosa of patients and mice with allergic rhinitis (AR). CMPK2 caused NLRP3 inflammasome activation via mitochondrial DNA (mtDNA)‐STING pathway. Blocking CMPK2 or STING signalling significantly reduced the activation of NLRP3 in house dust mite (HDM)‐challenged mice and human nasal epithelial cells (HNEPCs).

The activation of NLRP3 inflammasome in microglia is critical for neuroinflammation during postoperative cognitive dysfunction (POCD) induced by sevoflurane. However, the molecular mechanism by which sevoflurane activates the NLRP3 inflammasome in microglia remains unclear. The cGAS-STING pathway is an evolutionarily conserved inflammatory defense mechanism. The role of the cGAS-STING pathway in sevoflurane-induced NLRP3 inflammasome-dependent neuroinflammation and the underlying mechanisms require further investigation. We found that prolonged anesthesia with sevoflurane induced cognitive dysfunction and triggered the neuroinflammation characterized by the activation of NLRP3 inflammasome in vivo. Interestingly, the cGAS-STING pathway was activated in the hippocampus of mice receiving sevoflurane. While the blockade of cGAS with RU.521 attenuated cognitive dysfunction and NLRP3 inflammasome activation in mice. In vitro, we found that sevoflurane treatment significantly activated the cGAS-STING pathway in microglia, while RU.521 pre-treatment robustly inhibited sevoflurane-induced NLRP3 inflammasome activation. Mechanistically, sevoflurane-induced mitochondrial fission in microglia and released mitochondrial DNA (mtDNA) into the cytoplasm, which could be abolished with Mdivi-1. Blocking the mtDNA release via the mPTP-VDAC channel inhibitor attenuated sevoflurane-induced mtDNA cytosolic escape and reduced cGAS-STING pathway activation in microglia, finally inhibiting the NLRP3 inflammasome activation. Therefore, regulating neuroinflammation by targeting the cGAS-STING pathway may provide a novel therapeutic target for POCD.

Rotenone, a classical inhibitor of mitochondrial complex I, disrupts electron transport and promotes the generation of reactive oxygen species (ROS), contributing to inflammation and cell death. However, the precise molecular mechanisms linking mitochondrial dysfunction to inflammatory signaling remain incompletely understood. In this study, we investigated the role of the cGAS–STING pathway in rotenone-induced NLRP3 inflammasome activation in PMA-differentiated THP-1 macrophages. Rotenone treatment activated the cGAS–STING axis, as evidenced by increased cGAS expression and the phosphorylation of STING and TBK1. This activation led to the nuclear translocation of NF-κB and the upregulation of NLRP3, promoting inflammasome priming and IL-1β secretion. Inhibition of STING using H-151 markedly suppressed NLRP3 expression, NF-κB activation, and IL-1β release. Similarly, cyclosporin A, an inhibitor of mitochondrial permeability transition pore opening, reduced mitochondrial ROS, cytosolic oxidized mitochondrial DNA, and downstream activation of the cGAS–STING pathway, thereby attenuating inflammasome activation. These findings demonstrate that rotenone activates the NLRP3 inflammasome via mitochondrial ROS-mediated release of mtDNA and subsequent activation of the cGAS–STING–NF-κB signaling axis in THP-1-derived macrophages.

Copper, a vital mineral nutrient, possesses redox qualities that make it both beneficial and toxic to organisms. Excessive environmental copper exposure can result in neurological damage and cognitive decline in humans. Astrocytes, the predominant glial cells in the brain, are particularly vulnerable to pollutants, but the mechanism of copper-induced damage to astrocytes remains elusive. The aim of this study was to determine the role of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway in initiating NLRP3 inflammasome-induced astrocyte pyroptosis and chronic inflammation under conditions of copper overload. Our findings indicated that copper exposure elevated mitochondrial ROS (mtROS) levels, resulting in mitochondrial damage in astrocytes. This damage caused the release of mitochondrial DNA (mtDNA) into the cytoplasm, which subsequently activated the cGAS-STING pathway. This activation resulted in interactions between STING and NLRP3 proteins, facilitating the assembly of the NLRP3 inflammasome and inducing pyroptosis. Furthermore, depletion of mtROS mitigated copper-induced mitochondrial damage in astrocytes and reduced mtDNA leakage. Pharmacological inhibition of STING or STING transfection further reversed copper-induced pyroptosis and the inflammatory response. In conclusion, this study demonstrated that the leakage of mtDNA into the cytoplasm and the subsequent activation of the cGAS-STING-NLRP3 pathway may be potential mechanisms underlying copper-induced pyroptosis in astrocytes. These findings provided new insights into the toxicity of copper.

Introduction Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), represent critical respiratory failures with high mortality rates and limited treatment options. While the flavonoid rutin exhibits documented anti-inflammatory and antioxidant properties, its therapeutic mechanisms in ALI/ARDS remain unclear. This study investigated rutin‘s efficacy against lipopolysaccharide (LPS)-induced ALI in mice, with a mechanistic focus on the cGAS-STING pathway and NLRP3 inflammasome activation. Methods Male C57BL/6 mice were divided into Vehicle control, LPS induction, LPS + rutin co-treatment, and Rutin monotherapy groups. ALI was induced by intratracheal LPS challenge, and rutin was administered via gavage. Proteomics analysis, histological evaluation, immunohistochemistry, TUNEL staining, immunofluorescence, RT-qPCR, western blot, ELISA, and oxidative stress assays were performed to assess the effects of rutin on ARDS. Results The proteomic profiling of lung tissues from LPS-challenged mice identified significant dysregulation of proteins integral to the cGAS-STING cascade and pyroptotic processes. Gene ontology and KEGG pathway analyses underscored the pivotal role of immune and inflammatory responses in ALI, particularly in cytosolic DNA-sensing and NOD-like receptor signaling pathways. Rutin administration significantly alleviated LPS-induced lung injury, reducing oxidative stress, apoptosis, and proinflammatory cytokine levels (IL-6, IL-1β, TNF-α). Mechanistically, rutin demonstrated dual suppression: 1) inhibiting cGAS-STING activation through decreased expression of cGAS, STING, and phosphorylation of TBK1/IRF3 (P<0.05), and 2) attenuating NLRP3-mediated pyroptosis via downregulation of NLRP3-ASC-caspase1-GSDMD signaling (P<0.05). Pharmacological STING inhibition (C-176) validated the cGAS-STING-NLRP3 regulatory hierarchy in ALI pathogenesis. Conclusion: These findings elucidate rutin‘s novel therapeutic mechanism through coordinated suppression of the cGAS-STING-NLRP3 axis, positioning it as a promising candidate for ALI/ARDS intervention.

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-1β (IL-1β) production and mtDNA release in mice.

Heavy metal pollution has become a global health challenge. Exposure to heavy metals represents a major health risk. Manganese (Mn) is an essential trace element but also an environmental pollutant. Mn exposure can induce neurotoxicity and lead to neurodegenerative disease. Inflammation and Tau hyperphosphorylation are prominent hallmarks of neurodegenerative diseases induced by Mn exposure. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway can induce powerful innate immune defense programmes and has emerged as a key mediator of inflammation. In recent years, Mn2+ has been found to be the second activator of the cGAS-STING pathway in addition to double-stranded DNA (dsDNA). NLRP3 activation is upstream of Tau pathology, and NLRP3 activation induces Tau hyperphosphorylation and aggregation. Mn exposure-induced neurotoxicity may be associated with excessive activation of the cGAS-STING signaling pathway, leading to inflammation. The cGAS-STING/NLRP3 axis may be a promising option for revealing the mechanisms of neurotoxicity of Mn exposure in the future.

Despite its widespread application as a triazine herbicide, atrazine (ATR) poses significant health threats, with the mechanisms underlying its induction of hepatic inflammation still not fully elucidated. Our research delineates a mechanistic cascade in which ATR exposure is associated with endoplasmic reticulum (ER) stress induction, which in turn stimulates the excessive generation of mitochondria-associated membranes (MAMs). This event is associated with mitochondrial calcium inundation, augmented generation of mitochondrial reactive oxygen species (mtROS), and the cytosolic translocation of mitochondrial DNA (mtDNA). Subsequently, cytosolic mtDNA may operates as a damage-associated molecular pattern (DAMP), which engages the cGAS-STING pathway and nucleates NLRP3 inflammasome assembly, ultimately provoking a severe hepatic inflammatory response. These findings were consistently validated through both in vitro experiments and mouse models. Furthermore, inhibition of ER stress with 4-PBA significantly reduced MAMs over-assembly and alleviated mitochondrial dysfunction. Scavenging mitochondrial ROS with MitoQ also effectively attenuated downstream inflammatory activation. Overall, this study identifies a novel mechanistic pathway of ATR-induced hepatotoxicity: ER stress-MAMs-mitochondrial damage-mtDNA release-cGAS-STING-NLRP3 inflammation, providing new insights into the toxicology of environmental chemicals and suggesting potential therapeutic strategies for pollutant-induced liver injury.

No abstract available

Disproportionately high incidence and mortality of respiratory infection such as influenza A virus (IAV) and SARS‐CoV‐2 have been evidenced in the elderly, but the role and the mechanism of age‐associated immune deregulation in disease exacerbation are not well defined. Using a late generation of mice deficient in telomerase RNA (Terc−/−), we herein demonstrated that aged mice were exquisitely susceptible to respiratory viral infection, with excessive inflammation and increased mortality. Furthermore, we identified the cGAS/STING pathway, which was essentially induced by the leaked mitochondrial DNA, as a biologically relevant mechanism contributing to exaggerated inflammation in Terc−/− mice following viral infection. Innate immune cells, mainly, macrophages with shortened telomeres, exhibited hallmarks of cellular senescence, mitochondrial distress, and aberrant activation of STING and NLRP3 inflammasome pathways, which predisposed mice to severe viral pneumonia during commonly mild infections. Application of STING inhibitor and, more importantly, senolytic agent, reduced the burden of stressed macrophages, improved mitochondrial integrity, and suppressed STING activation, thereby conferring the protection for Terc−/− mice against respiratory infection. Together, the findings expand our understanding of innate immune senescence and reveal the potential of the senolytics as a promising treatment to alleviate the symptom of viral pneumonia, particularly for the older population.

Pulmonary fibrosis is a chronic and progressive interstitial lung disease with limited treatment options aside from lung transplantation. Bleomycin (BLM)‐induced lung injury is the most commonly used experimental model to mimic the key pathological features of human pulmonary fibrosis, which include an early inflammatory phase and a later fibrotic phase. Neutrophil infiltration and M2 macrophage activation are key events in these stages, respectively. However, the molecular mechanisms by which BLM triggers pulmonary inflammation and fibrosis remain incompletely understood. In this study, we found that BLM treatment induced ROS‐mediated oxidative damage in the lungs, leading to an inflammatory microenvironment and the release of oxidized mitochondrial DNA (oxid‐mtDNA). Oxid‐mtDNA was shown to contribute to the early inflammatory response by promoting neutrophil recruitment and enhancing macrophage polarization, which subsequently drove tissue remodeling and fibrosis. Notably, direct injection of oxid‐mtDNA into the lungs recapitulated the fibrotic features observed in the BLM model. Furthermore, studies using STING‐ and NLRP3‐deficient mice demonstrated that loss of either pathway significantly attenuated BLM‐induced inflammation and fibrosis, implicating their involvement downstream of oxid‐mtDNA signaling. Collectively, our findings identify oxid‐mtDNA as a critical mediator linking oxidative injury to immune activation and fibrotic remodeling in the lung, offering new insights into pulmonary fibrosis pathogenesis and potential therapeutic targets.

ETHNOPHARMACOLOGICAL RELEVANCE Qingfei Xieding prescription was gradually refined and produced by Hangzhou Red Cross Hospital. The raw material includes Ephedra sinica Stapf, Morus alba L., Bombyx Batryticatus, Gypsum Fibrosum, Prunus armeniaca L. var. ansu Maxim., Houttuynia cordata Thunb. , Pueraria edulis Pamp. Paeonia L., Scutellaria baicalensis Georgi and Anemarrhena asphodeloides Bge. It is very effective in clinical treatment of pulmonary tuberculosis patients. AIM OF THE STUDY To explore the efficacy and underlying mechanism of Qingfei Xieding (QF) in the treatment of bleomycin-induced mouse model. MATERIALS AND METHODS TGF-β induced fibrotic phenotype in vitro. Bleomycin injection induced lung tissue fibrosis mice model in vivo. Flow cytometry was used to detect apoptosis, cellular ROS and lipid oxidation. Mitochondria substructure was observed by transmission electron microscopy. Autophagolysosome and nuclear entry of P65 were monitored by immunofluorescence. Quantitative real-time PCR was performed to detect the transcription of genes associated with mtDNA-cGAS-STING pathway and subsequent inflammatory signaling activation. RESULTS TGF-β induced the expression of α-SMA and Collagen I, inhibited cell viability in lung epithelial MLE-12 cells that was reversed by QF-containing serum. TGF-β-mediated downregulation in autophagy, upregulation in lipid oxidation and ROS contents, and mitochondrial damage were rescued by QF-containing serum treatment, but CQ exposure, an autophagy inhibitor, prevented the protective role of QF. In addition to that, the decreased autophagolysosome in TGF-β-exposed MLE-12 cells was reversed by QF and restored to low level in the combination treatment of QF and CQ. Mechanistically, QF-containing serum treatment significantly inhibited mtDNA-cGAS-STING pathway and subsequent inflammatory signaling in TGF-β-challenged cells, which were abolished by CQ-mediated autophagy inhibition. In bleomycin-induced mouse model, QF ameliorated pulmonary fibrosis, reduced mortality, re-activated autophagy in lung tissues and restrained mtDNA-cGAS-STING inflammation pathway. However, the protective effects of QF in bleomycin-induced mouse model mice were also abrogated by CQ. CONCLUSION QF alleviated bleomycin-induced pulmonary fibrosis by activating autophagy, inhibiting mtDNA-cGAS-STING pathway-mediated inflammation. This research recognizes the protection role of QF on bleomycin-induced mouse model, and offers evidence for the potentiality of QF in clinical application for pulmonary fibrosis treatment.

Chimeric antigen receptor macrophages (CAR-Ms) therapy has shown great promise in liver fibrosis, however limited anti-inflammatory capacity of CAR-Ms in the fibrotic foci compromises their anti-fibrotic potency. We here report tripartite motif containing 13 (TRIM13) engineered CAR-Ms for effectively manipulating the anti-inflammatory phenotype of CAR-Ms, augmenting their anti-fibrosis efficacy. Specifically, our efferocytosis-sparked lipid nanoparticles (ESLNPs) efficiently engineered fibrosis-associated macrophages to anti-inflammatory CAR-Ms by co-delivering mRNA encoding TRIM13 and anti-fibroblast activation protein (FAP) CAR respectively. Our data demonstrated these reprogrammed CAR-Ms exhibited a sustained anti-inflammatory phenotype via blocking the mitochondrial DNA (mtDNA)-STING pathway through the overexpression of TRIM13, and showed notable FAP-targeted phagocytosis. Treatment with ESLNPs in male mice with liver fibrosis obviously ameliorated fibrosis through synergizing anti-fibrotic and inflammation-resolution activities, ultimately prompting substantial hepatic function restoration. In sum, our findings established that remodeling and sustaining the anti-inflammatory phenotype of CAR-Ms markedly elevated their therapeutic efficacy in liver fibrosis, benefiting CAR-Ms therapy with broad application in other fibrotic diseases. CAR-M therapy shows promise for liver fibrosis but limited anti-inflammatory activity reduces efficacy. Here the authors show that TRIM13-engineered, FAP-targeted CAR macrophages sustain anti-inflammatory function and reduce liver fibrosis in mice.

Pulmonary fibrosis (PF) is a chronic and fatal aging-related pulmonary disease. Emerging evidence suggests that fibroblast senescence plays a pivotal role in the initiation and progression of PF. Senescent fibroblasts accumulate in fibrotic lungs, driving excessive extracellular matrix (ECM) deposition, which disrupts tissue architecture and compromises pulmonary function. Notably, senolytic therapy targeting these senescent fibroblasts has shown significant efficacy in ameliorating PF. Therefore, elucidating the mechanisms underlying fibroblast senescence is a promising approach to prevent PF. Herein, our results identify fibroblast growth factor-inducible 14 (Fn14) as a critical mediator in the senescence of fibroblasts. We found that Fn14 was up-regulated in pulmonary fibroblasts from both PF patients and bleomycin (BLM)-treated mice. While knockdown of Fn14 attenuated pulmonary structural disruption and reduced fibroblast senescence in the lung of BLM-treated mice. In vitro, Fn14 activation promoted cellular senescence in pulmonary fibroblasts. Mechanistically, Fn14-induced mitophagy impairment resulted in mitochondrial DNA (mtDNA) leakage, which subsequently activated the cGAS-STING signaling. Moreover, restoring mitophagy or inhibiting cGAS ameliorated fibroblast senescence induced by Fn14 activation. Collectively, these results provide comprehensive insight into the pro-fibrotic role of Fn14 in the development of PF by inducing fibroblast senescence and shed light on the Fn14-targeting therapeutics for PF.

Background Silicosis, characterized by interstitial lung inflammation and fibrosis, poses a significant health threat. ATII cells play a crucial role in alveolar epithelial repair and structural integrity maintenance. Inhibiting ATII cell senescence has shown promise in silicosis treatment. However, the mechanism behind silica-induced senescence remains elusive. Methods The study employed male C57BL/6 N mice and A549 human alveolar epithelial cells to investigate silicosis and its potential treatment. Silicosis was induced in mice via intratracheal instillation of crystalline silica particles, with honokiol administered intraperitoneally for 14 days. Silica-induced senescence in A549 cells was confirmed, and SIRT3 knockout and overexpression cell lines were generated. Various analyses were conducted, including immunoblotting, qRT-PCR, histology, and transmission electron microscopy. Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. Results This study elucidates how silica induces ATII cell senescence, emphasizing mtDNA damage. Notably, honokiol (HKL) emerges as a promising anti-senescence and anti-fibrosis agent, acting through sirt3. honokiol effectively attenuated senescence in ATII cells, dependent on sirt3 expression, while mitigating mtDNA damage. Sirt3, a class III histone deacetylase, regulates senescence and mitochondrial stress. HKL activates sirt3, protecting against pulmonary fibrosis and mitochondrial damage. Additionally, HKL downregulated cGAS expression in senescent ATII cells induced by silica, suggesting sirt3's role as an upstream regulator of the cGAS/STING signaling pathway. Moreover, honokiol treatment inhibited the activation of the NF-κB signaling pathway, associated with reduced oxidative stress and mtDNA damage. Notably, HKL enhanced the activity of SOD2, crucial for mitochondrial function, through sirt3-mediated deacetylation. Additionally, HKL promoted the deacetylation activity of sirt3, further safeguarding mtDNA integrity. Conclusions This study uncovers a natural compound, HKL, with significant anti-fibrotic properties through activating sirt3, shedding light on silicosis pathogenesis and treatment avenues.

Microplastic pollution is a global concern, yet its impact on male reproductive health remains unclear. We assessed chronic polyethylene terephthalate (PET) microplastic exposure using human corpus cavernosum (CC) tissues, a rat model, and cell assays. MPs were quantified in CC from 10 patients; those with erectile dysfunction (ED) showed a higher MP burden, with PET predominant. In rats, chronic PET-MP exposure dose-dependently impaired erectile function, increased fibrosis, and reduced smooth muscle. Mechanistically, PET-MPs localized to macrophage mitochondria, causing depolarization and ROS generation, mtDNA leakage, cGAS-STING activation, and macrophage ferroptosis. This ferroptotic signaling amplified inflammation, promoted M1 polarization, and triggered endothelial-to-mesenchymal transition, leading to vascular dysfunction and ED. Depleting macrophages or inhibiting cGAS-STING or ferroptosis reduced inflammation and partially rescued erectile responses. Together, these data identify a cGAS-STING-ferroptosis axis linking environmental MP exposure to ED and suggest upstream innate-immune and ferroptosis pathways as therapeutic targets.

Dysregulated mitochondrial dynamics and macrophage-driven inflammation are essential contributors to the pathogenesis of acute kidney injury (AKI). Although the chemokine CX3CL1 has been associated with inflammatory responses, its role in AKI, particularly in regulating macrophage polarization and mitochondrial function, remains unclear. In this study, we investigated the therapeutic potential of CX3CL1 inhibition in a lipopolysaccharide (LPS)-induced AKI model. Our results found that CX3CL1 deficiency could significantly ameliorate renal dysfunction and attenuate inflammatory responses. RNA sequencing revealed that CX3CL1 deficiency alters macrophage subpopulations and gene expression profiles in the kidney, particularly affecting pathways related to immune responses and mitochondrial function. Mechanistically, the absence of CX3CL1 promotes macrophage polarization from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 phenotype. Furthermore, CX3CL1 inhibition improves mitochondrial dynamics, alleviates mitochondrial dysfunction, and reduces oxidative stress and mitochondrial DNA (mtDNA) leakage, thereby preserving mitochondrial integrity. Notably, CX3CL1 knockdown suppresses activation of the cGAS-STING pathway, a key mediator of inflammation triggered by cytosolic mtDNA. We also observed that these effects appear to be mediated through stabilization of mitochondrial transcription factor A (TFAM). Collectively, these findings identify CX3CL1 as an essential regulator of macrophage mitochondrial function and inflammation in AKI, offering a potential therapeutic target for mitigating kidney injury.

Cerebral ischemia-reperfusion injury is related to inflammation driven by free mitochondrial DNA. At the same time, the pro-inflammatory activation of macrophages, that is, polarization in the M1 direction, aggravates the cycle of inflammatory damage. They promote each other and eventually transform macrophages/microglia into neurotoxic macrophages by improving macrophage glycolysis, transforming arginine metabolism, and controlling fatty acid synthesis. Therefore, we propose targeting the mtDNA-driven inflammatory response while controlling the metabolic state of macrophages in brain tissue to reduce the possibility of cerebral ischemia-reperfusion injury.

Macrophage‐stimulator of interferon genes (STING) signaling mediated sterile inflammation has been implicated in various age‐related diseases. However, whether and how macrophage mitochondrial DNA (mtDNA) regulates STING signaling in aged macrophages remains largely unknown. We found that hypoxia‐reoxygenation (HR) induced STING activation in macrophages by triggering the release of macrophage mtDNA into the cytosol. Aging promoted the cytosolic leakage of macrophage mtDNA and enhanced STING activation, which was abrogated upon mtDNA depletion or cyclic GMP‐AMP Synthase (cGAS) inhibition. Aged macrophages exhibited increased mitochondrial injury with impaired mitophagy. Mechanistically, a decline in the PTEN‐induced kinase 1 (PINK1)/Parkin‐mediated polyubiquitination of mitochondria was observed in aged macrophages. Pink1 overexpression reversed the inhibition of mitochondrial ubiquitination but failed to promote mitolysosome formation in the aged macrophages. Meanwhile, aging impaired lysosomal biogenesis and function in macrophages by modulating the mTOR/transcription factor EB (TFEB) signaling pathway, which could be reversed by Torin‐1 treatment. Consequently, Pink1 overexpression in combination with Torin‐1 treatment restored mitophagic flux and inhibited mtDNA/cGAS/STING activation in aged macrophages. Moreover, besides HR‐induced metabolic stress, other types of oxidative and hepatotoxic stresses inhibited mitophagy and promoted the cytosolic release of mtDNA to activate STING signaling in aged macrophages. STING deficiency protected aged mice against diverse types of sterile inflammatory liver injuries. Our findings suggest that aging impairs mitophagic flux to facilitate the leakage of macrophage mtDNA into the cytosol and promotes STING activation, and thereby provides a novel potential therapeutic target for sterile inflammatory liver injury in aged patients.

Background The metabolic reprogramming of alveolar macrophages, particularly mitochondrial energy metabolism centered on the tricarboxylic acid (TCA) cycle, plays a pivotal role in acute lung injury (ALI). Fumarate hydratase (FH), a key enzyme catalyzing fumarate-to-malate conversion in the TCA cycle, is implicated in macrophage inflammatory responses, but its specific role in ALI remains unclear. Methods We employed FHIN1 to assess its regulatory effects in LPS-induced ALI models. Wildtype C57BL/6 mice were randomly divided into control group, FHIN1 group, LPS group and LPS+FHIN1 group. FHIN1 and RU.521 was used to explored the interaction of FH and cGAS-STING in THP-1 cells. Results LPS stimulation suppressed FH expression and induced fumarate accumulation in macrophages. Pharmacological FH inhibition exacerbated LPS-triggered inflammatory cytokine release, oxidative stress and aggravated lung injury in mice. Mechanistically, FH inhibition promoted mtDNA leakage, activating the cGAS-STING pathway to amplify inflammation. Blocking cGAS with RU.521 significantly attenuated FHIN1-driven inflammatory responses and mitigated lung injury exacerbation. Conclusion FH critically modulates ALI progression by restraining cGAS-STING-dependent inflammation. Targeting the FH-mtDNA-cGAS axis may offer therapeutic potential for ALI management.

Liver ischemia-reperfusion injury (IRI) drives graft dysfunction and postsurgical morbidity. We show that hepatocellular MST1 is markedly upregulated in IRI and exacerbates damage by blocking PINK1-dependent mitophagy. Defective mitochondrial clearance causes mtDNA leakage, which activates macrophage cGAS-STING signaling and fuels inflammatory injury. Curcumin inhibits this MST1-PINK1 axis, restoring mitophagy and limiting mtDNA release. To translate these insights, we engineered Curcumin@EV@Se-stem-cell-derived extracellular vesicles surface-modified with diselenide-PEG for ROS-responsive, "stealth" delivery. In oxygen-glucose deprivation/reoxygenation models, Curcumin@EV@Se improved hepatocyte viability, preserved mitochondrial potential, reduced ROS and inflammatory cytokines, and promoted reparative/angiogenic programs. In a murine hepatic IRI model, systemic Curcumin@EV@Se decreased necrosis and TUNEL positivity and improved serum transaminases and histology, indicating enhanced liver function and regeneration. These data identify MST1-mediated mitophagy blockade with secondary cGAS-STING activation as a central pathogenic axis in IRI and present Curcumin@EV@Se as a mechanism-guided therapy that restores mitochondrial quality control and dampens innate immune activation, with translational promise for liver transplantation and acute hepatic injury.

Hypertension is a primary modifiable risk factor for cardiovascular diseases, which often induces renal end-organ damage and complicates chronic kidney disease (CKD). In the present study, histological analysis of human kidney samples revealed that hypertension induced mtDNA leakage and promoted the expression of stimulator of interferon genes (STING) in renal epithelial cells. We used AngII- and 2K1C-treated mouse kidneys to elucidate the underlying mechanisms. Abnormal renal mtDNA packing caused by AngII promoted STING-dependent production of inflammatory cytokines, macrophage infiltration, and a fibrogenic response. STING knockout significantly decreased NF-κB activation and immune cell infiltration, attenuating tubule atrophy and extracellular matrix accumulation in vivo and in vitro. These effects delayed CKD progression. Immunoprecipitation assays and liquid chromatography-tandem mass spectrometry showed that STING and ACSL4 were directly combined at the D53 and K412 amino acids of ACSL4. Furthermore, STING induced renal inflammatory response and fibrosis through ACSL4-dependent ferroptosis. Lastly, inhibition of ACSL4 using siRNA, rosiglitazone, or Fer-1 downregulated AngII-induced mtDNA-STING-dependent renal inflammation. These results suggest that targeting the STING/ACSL4 axis might represent a potential strategy for treating hypertension-associated CKD.

Diabetic wound (DW) is characterized by elevated pro-inflammatory cytokines and cellular dysfunction consistent with elevated reactive oxygen species (ROS) levels. Recent advances in immunology have dissected molecular pathways involved in the innate immune system where cytoplasmic DNA can trigger STING-dependent inflammatory responses and play an important role in metabolic-related diseases. We investigated whether STING regulates inflammation and cellular dysfunction in DW healing. We found that STING and M1 macrophages were increased in wound tissues from DW in patients and mice and delayed the wound closure. We also noticed that the massively released ROS in the High glucose (HG) environment activated STING signaling by inducing the escape of mtDNA to the cytoplasm, inducing macrophage polarization into a pro-inflammatory phenotype, releasing pro-inflammatory cytokines, and exacerbating endothelial cell dysfunction. In Conclusion, mtDNA-cGAS-STING pathway activation under diabetic metabolic stress is an important mechanism of DW refractory healing. While using STING gene-edited macrophages for wound treatment by cell therapy can induce the polarization of wound macrophages from pro-inflammatory M1 to anti-inflammatory M2, promote angiogenesis, and collagen deposition to accelerate DW healing. STING may be a promising therapeutic target for DW.

Diabetic wounds has a gradually increasing incidence and morbidity. Excessive inflammation due to immune imbalance leads to delayed wound healing. Here, we reveal the interconnection between activation of the NLRP3 inflammatory pathway in endotheliocyte and polarization of macrophages via the cGAS-STING pathway in the oxidative microenvironment. To enhance the immune-regulation based on repairing mitochondrial oxidative damage, a zeolitic imidazolate framework-8 coated with cerium dioxide that carries Rhoassociated protein kinase inhibition Y-27632 (CeO2–Y@ZIF-8) is developed. It is encapsulated in a photocross-linkable hydrogel (GelMA) with cationic quaternary ammonium salt groups modified to endow the antibacterial properties (CeO2–Y@ZIF-8@Gel). CeO2 with superoxide dismutase and catalase activities can remove excess reactive oxygen species to limit mitochondrial damage and Y-27632 can repair damaged mitochondrial DNA, thus improving the proliferation of endotheliocyte. After endotheliocyte uptakes CeO2–Y@ZIF-8 NPs to degrade peroxides into water and oxygen in cells and mitochondria, NLRP3 inflammatory pathway is inhibited and the leakage of oxidatively damaged mitochondrial DNA (Ox-mtDNA, a damage-associated molecular pattern) through mPTP decreases. Futhermore, as the cGAS-STING pathway activated by Ox-mtDNA down-regulated, the M2 phenotype polarization and anti-inflammatory factors increase. Collectively, CeO2–Y@ZIF-8@Gel, through remodulating the crosstalk between macrophage reprogramming and angiogenesis to alleviate inflammation in the microenvironment and accelerates wound healing.

Mitochondrial dysfunction, particularly when associated with the mitochondrial DNA (mtDNA) activated cGAS-STING signaling pathway, represents a key pathogenic mechanism contributing to excessive inflammation. Therapeutic targeting of mitochondrial homeostasis coupled with precise modulation of mtDNA release emerges as a promising yet underexplored strategy to suppress pathological inflammation and promote chronic wound healing. Herein, epigallocatechin gallate-quercetin co-assembled nanoparticles (EQ NPs) were engineered to inhibit mtDNA-mediated inflammatory cascades through mitochondria-targeted multimodal mtDNA level control. Primarily, EQ NPs reduced the formation of oxidized mtDNA fragments (Ox-mtDNA). Then, EQ NPs inhibited the excessive opening of the mitochondrial permeability transition pore, preventing Ox-mtDNA cytoplasmic leakage. Subsequently, the escaped mtDNA fragments were neutralized by EQ NPs through polyphenol-mediated adsorption. Finally, mitophagy was upregulated to selectively eliminate damaged mitochondria. This well-designed strategy significantly inhibited the activation of the mtDNA-mediated cGAS-STING pathway, relieved the release of inflammatory factors, and promoted anti-inflammatory phenotype polarization of macrophages. In vivo, EQ NPs promoted chronic wound healing by bacteriostasis, anti-inflammation, immunomodulation, and accelerated angiogenesis. Overall, the study establishes a sequential mitochondrial quality control paradigm in which the inflammatory cascade is interrupted by multimodal and full-chain mtDNA scavenging, providing a promising candidate for the treatment of inflammatory diseases and chronic wound healing.

Septic cardiomyopathy is characterized by oxidative stress and inflammation, and accounts for its associated high mortality. Mangiferin is a naturally occurring xanthonoid found abundantly in Anemarrhenaasphodeloides Bunge, a traditional Chinese herb widely used for treatment of cardiovascular diseases. This study was designed to investigate the cardioprotective role of mangiferin against sepsis-induced heart injury with a focus on mitochondrial DNA (mtDNA) release and cGAS-STING pathway-related inflammation. The septic cardiomyopathy model in mice was established by intraperitoneal injection of LPS (10 mg/kg). Cardiac Nrf2 in septic mice was knocked down with AAV9-CTNT-Nrf2 shRNA to confirm the activity of mangiferin. Cardiomyocytes were cultured with LPS for further in vitro studies. Oral administration of mangiferin enhanced the survival of mice against endotoxin-induced insult. When LPS challenge impaired cardiac structural integrity, mangiferin reduced macrophage recruitment in the heart and inhibited the gene expression of pro-inflammatory cytokines. In the septic heart, mangiferin increased Nrf2 protein expression, thereby protecting the heart from oxidative damage. Mechanistically, mangiferin increased Nrf2 protein abundance by promoting Keap-1 degradation, which in turn prevented Nrf2 from undergoing proteasomal degradation. Unlike nuclear DNA (nDNA), mitochondrial DNA (mtDNA) acts as a ligand to induce toll-like receptor (TLR) activation once released into the cytoplasm. By protecting mitochondrial membrane integrity, mangiferin combated oxidative stress to prevent mitochondrial fragmentation and prevented the opening of mitochondrial permeability transition pore (mPTP) and the collapse of mitochondrial membrane potential in a manner this is dependent on Nrf2 availability. These effects were, however, blocked in the presence of a special Nrf2 inhibitor, ML385. Similar to TLR4, TLR9 is a member of the damage-associated molecular patterns (DAMPs). It can induce immune response through STING/IRF3 signaling. In septic mouse heart, mangiferin inhibited cGAS activity, deactivated STING/IRF3 signaling via dephosphorylation and resultantly suppressed interferon response due to limited mtDNA leakage. In cultured cardiomyocytes, mangiferin blocked STING/IRF3 signaling cascades in a Nrf2-dependent manner. Cardiac knockdown of Nrf2 with AAV9-CTNT-Nrf2 shRNA in septic mice demonstrated that Nrf2 deficiency diminished the inhibitory effects of mangiferin on cGAS-STING pathway-related inflammation. Through Nrf2 activation, mangiferin ameliorates mitochondrial dysfunction to block mtDNA release and subsequent cGAS-STING pathway-related inflammation, resultantly protecting the heart against septic insult. These events suggest the potential in the treatment of heart injury from the perspective of mitochondrial protection.

Background: Sepsis-induced cardiomyopathy (SICM) is a major cause of high morbidity and mortality in septic patients. In SICM, macrophage infiltration and aberrant immune activation play a critical role in triggering inflammatory responses in cardiac tissue. Our previous studies identified that among 23 natural small molecules, Protocatechualdehyde (PCA) exhibited the most potent inhibitory effect on macrophage inflammation. However, the effects of PCA on sepsis-induced cardiac dysfunction remain poorly understood. Research Questions: This study aims to investigate the role of the small molecule PCA in sepsis-induced cardiomyopathy and the underlying potential mechanisms involved. Methods: We established a sepsis mouse model using cecal ligation and puncture (CLP) and treated the mice with intraperitoneal injections of 20 mg/kg and 40 mg/kg of PCA for 5 consecutive days. Heart function was evaluated by measuring survival time, heart function biomarkers, and hemodynamic parameters. Lipopolysaccharide (LPS)-treated mouse bone marrow-derived macrophages (BMDMs) were used to establish a macrophage pyroptosis model. We assessed the activation of NLRP3 inflammasomes, the release of inflammatory cytokines, and gasdermin-D (GSDMD)-mediated mitochondrial pore formation and mitochondrial DNA leakage, and examined their effects on downstream STING/IRF3 signaling pathways. Rusult: The in vivo results of this study demonstrated that PCA significantly alleviated cardiac dysfunction, inflammatory cell infiltration, and the production of inflammatory cytokines in septic mice. Further in vitro experiments showed that PCA inhibited the activation of NLRP3 inflammasome in BMDMs from mice and reduced GSDMD-mediated pyroptosis as well as the activation of the downstream STING/IRF3 pro-inflammatory pathway. Mechanistically, PCA reduced the production of mitochondrial reactive oxygen species (mtROS), thereby inhibiting the activation of the NLRP3 inflammasome and the formation of N-GSDMD. This, in turn, reduced the accumulation of N-GSDMD on both the cell membrane and mitochondrial membrane, further inhibiting the release of mitochondrial DNA (mtDNA) into the cytoplasm. Ultimately, this suppressed the activation of the downstream STING/IRF3 pro-inflammatory pathway, leading to a reduction in the release of inflammatory cytokines. Conclusion: These results highlight the therapeutic role of PCA in the resolution of sepsis-induced cardiac inflammation.

Background: Sterile inflammation contributes to the pathogenesis of cardiac dysfunction caused by various conditions including pressure overload in hypertension. Mitochondrial DNA (mtDNA) released from damaged mitochondria has been implicated in cardiac inflammation. However, the upstream mechanisms governing mtDNA release and how mtDNA activates sterile inflammation in pressure-overloaded hearts remain largely unknown. Here, we investigated the role of inducible NO synthase (iNOS) on pressure overload-induced cytosolic accumulation of mtDNA and whether mtDNA activated inflammation through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. Methods: To investigate whether the cGAS-STING cascade was involved in sterile inflammation and cardiac dysfunction upon pressure overload, cardiomyocyte-specific STING depletion mice and mice injected with adeno-associated virus-9 (AAV-9) to suppress the cGAS-STING cascade in the heart were subjected to transverse aortic constriction (TAC). iNOS null mice were used to determine the role of iNOS in cGAS-STING pathway activation in pressure-stressed hearts. Results: iNOS knockout abrogated mtDNA release and alleviated cardiac sterile inflammation resulting in improved cardiac function. Conversely, activating the cGAS-STING pathway blunted the protective effects of iNOS knockout. Moreover, iNOS activated the cGAS-STING pathway in isolated myocytes and this was prevented by depleting cytosolic mtDNA. In addition, disruption of the cGAS-STING pathway suppressed inflammatory cytokine transcription and modulated M1/M2 macrophage polarization, and thus mitigated cardiac remodeling and improved heart function. Finally, increased iNOS expression along with cytosolic mtDNA accumulation and cGAS-STING activation were also seen in human hypertensive hearts. Conclusion: Our findings demonstrate that mtDNA is released into the cytosol and triggers sterile inflammation through the cGAS-STING pathway leading to cardiac dysfunction after pressure overload. iNOS controls mtDNA release and subsequent cGAS activation in pressure-stressed hearts.

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Epigallocatechin gallate (EGCG) exerts cardio-protective effects. This study aimed to investigate the effects of EGCG on atherosclerosis (AS) and the potential mechanisms. High-fat diet was used to establish mice AS mouse model. Histological analysis was performed using HE and Masson staining. Gene expression was detected using RT-qPCR, Western blot, and immunofluorescence. Cytokine release was detected using ELISA. ox-LDL was used to establish in vitro AS model. Cellular functions were determined using TUNEL and flow cytometry assays. Mitochondrial functions were determined using Seahorse assays, mitoSOX staining, flow cytometry and TMRM staining. We found that EGCG effectively improved the cardiac haemodynamics and alleviated the fibrosis of myocardial cells. Mechanistically, EGCG maintained mitochondrial homeostasis and inhibited mitochondrial oxidative stress in vascular endothelial cells. Moreover, EGCG inhibited mtDNA release, TBK1-cGAS-STING-mediated cytotoxicity and subsequent apoptosis of vascular endothelial cells, suppressing M1 macrophage polarization. M1 macrophages further enhanced the apoptosis of vascular endothelial cells. Strikingly, RU.521 plus EGCG exerted more potent effects on inhibiting cGAS-STING signaling, M1 macrophage polarization, and apoptosis of vascular endothelial cells. Taken together, EGCG protects against AS through alleviating TBK1/cGAS/STING/NLRP3 signaling-mediated dysfunction of vascular endothelial cells in AS.

No abstract available

Low back pain (LBP) is a major musculoskeletal disorder and the socioeconomic problem with a high prevalence that mainly involves intervertebral disc (IVD) degeneration, characterized by progressive nucleus pulposus (NP) cell death and the development of an inflammatory microenvironment in NP tissue. Excessively accumulated cytosolic DNA acts as a damage-associated molecular pattern (DAMP) that is monitored by the cGAS-STING axis to trigger the immune response in many degenerative diseases. NLRP3 inflammasome-dependent pyroptosis is a type of inflammatory programmed death that promotes a chronic inflammatory response and tissue degeneration. However, the relationship between the cGAS-STING axis and NLRP3 inflammasome-induced pyroptosis in the pathogenesis of IVD degeneration remains unclear. Here, we used magnetic resonance imaging (MRI) and histopathology to demonstrate that cGAS, STING, and NLRP3 are associated with the degree of IVD degeneration. Oxidative stress induced cGAS-STING axis activation and NLRP3 inflammasome-mediated pyroptosis in a STING-dependent manner in human NP cells. Interestingly, the canonical morphological and functional characteristics of mitochondrial permeability transition pore (mPTP) opening with the cytosolic escape of mitochondrial DNA (mtDNA) were observed in human NP cells under oxidative stress. Furthermore, the administration of a specific pharmacological inhibitor of mPTP and self-mtDNA cytosolic leakage effectively reduced NLRP3 inflammasome-mediated pyroptotic NP cell death and microenvironmental inflammation in vitro and degenerative progression in a rat disc needle puncture model. Collectively, these data highlight the critical roles of the cGAS-STING-NLRP3 axis and pyroptosis in the progression of IVD degeneration and provide promising therapeutic approaches for discogenic LBP. An investigation of the mechanisms responsible for intervertebral disc degeneration (IVD) reveals a potential treatment to limit the progression of low back pain. IVD involves widespread inflammation and cell death inside discs, but exactly how the condition progresses is unclear. Cao Yang, Kun Wang and co-workers at Huazhong University of Science and Technology, Wuhan, China, investigated IVD in patient samples and rat models. Oxidative stress triggered leakage of mitochondrial DNA into cellular fluid in cells in the center of discs. This activated an immune response pathway and prompted the release of inflammatory cytokines. The degree of activation of this pathway is directly linked to IVD severity, providing a potential method of monitoring the condition. Administering a drug to rats to block mitochondrial DNA leakage slowed IVD progression.

Epilepsy is increasingly recognized as a disorder involving metabolic dysregulation beyond neural hyperexcitability, yet the underlying metabolic mechanisms remain poorly defined. Here, we identify a mitochondrion-immunity-metabolism axis that drives spontaneous chronic epilepsy. Brain-specific deletion of Mic19 impairs mitochondrial cristae structure and mitochondrial integrity in neurons, leading to activation of the Z-mitochondrial DNA (mtDNA)-ZBP1-RIPK3-mixed lineage kinase domain-like protein (MLKL) axis and p-MLKL-mediated pore formation on the mitochondrial membrane. This process results in cytosolic and extracellular leakage of mtDNA, which is subsequently taken up by microglia and triggers cyclic GMP-AMP synthase (cGAS)-STING-dependent inflammatory signaling. The resulting neuroinflammation promotes sustained activation of astrocytes. Critically, reactive astrocytes undergo profound metabolic reprogramming, marked by upregulated glycolysis and enhanced L-serine biosynthesis. Astrocyte-derived L-serine is subsequently transferred to neurons and converted into D-serine, a key NMDA receptor coagonist that enhances neuronal excitability. This metabolic shift in astrocytes exacerbates excitotoxicity and sustains epileptic activity. Importantly, pharmacologic inhibition of STING with H-151 treatment markedly suppresses seizures, reinforcing the therapeutic potential of targeting immunometabolic crosstalk in epilepsy. Our findings reveal that mtDNA-mediated cGAS-STING activation and D-serine act as important drivers of epilepsy initiation, offering mechanistic insights into neuron-microglia-astrocyte crosstalk and highlighting immunometabolic modulation as a promising therapeutic strategy for epilepsy.

Carbapenem‐resistant Acinetobacter baumannii (CRAB) has become a major threat in the treatment of bacterial infection, and immunotherapy in a non‐antibiotic‐dependent manner is an effective way to overcome CRAB infection. However, the role of the innate immune response in CRAB infection is poorly understood. Here, it is reported that CRAB infection induced a cytosolic DNA‐sensing signaling pathway and significant IFN‐β production in mice post‐CRAB infection. The knockout of STING reduced bacterial burden, the production of inflammatory cytokines, and lung injury in mice post CRAB infection. The cytosolic DNA sensor cyclic GMP‐AMP synthase (cGAS) and the adaptor protein stimulator of interferon genes (STING) are required for CRAB‐induced IFN‐β expression in macrophages. Intriguingly, CRAB utilized outer membrane vesicles (OMVs) to transport outer membrane protein 38 (OMP38) into mitochondria, triggering mitochondrial DNA (mtDNA) release into the cytosol through the mitochondrial permeability transition pore (mPTP) and activating the cGAS‐STING signaling. Finally, epigallocatechin gallate (EGCG) is demonstrated to block the activation of the cGAS‐STING pathway and ameliorate CRAB‐induced excessive inflammatory response. These results demonstrated that the early innate immune response to CRAB infection is activated in a cGAS‐STING‐dependent manner, which could be a potential therapeutic target for CRAB infection.

During the critical process of homeostatic efferocytosis, macrophages clear apoptotic cells and subsequently transition to reparative functions that promote the resolution of inflammation and support tissue repair. Their inherent plasticity enables rapid changes in macrophage activity suited to specific microenvironments. However, the heterogeneity in their cell states also presents challenges in characterizing subsets of macrophages and analyzing their specific contributions post-efferocytosis. In this study, single-cell RNA sequencing data from bone-marrow derived macrophages engulfing apoptotic osteoblasts (OB) was used to characterize macrophage subpopulations enriched during efferocytosis. Clustering analysis revealed two subpopulations (c3 and c9) that were unique to efferocytic macrophages. These distinct subpopulations displayed a transcriptional profile characterized by enhanced glycolytic energy metabolism, along with an anti-inflammatory gene signature. Notably, HIF-1 signaling, glycolysis/gluconeogenesis, and carbon metabolism were among the top five most significantly enriched pathways in c3 and c9 macrophages. qRT-PCR analysis revealed that macrophages engulfing apoptotic OBs exhibited increased expression of key glycolytic enzymes and solute carriers, including Slc2a1, Pdk1, Ldha, and Slc16a3. Metabolomics analysis revealed a significant increase in intracellular lactate, phosphoenolpyruvic acid, glycerol-3-phosphate, 2-/3-glycerophosphate, and fructose-6-phosphate, indicative of enhanced glycolysis. In addition, efferocytic macrophages showed increased extracellular lactate production compared to control macrophages, as confirmed by lactate ELISA. The effects of lactate (0-20mM) on osteoblast mineralization, osteoclast differentiation and function, and macrophage-derived inflammatory factors were evaluated through various in vitro experiments. While no effect was seen in osteoblast mineralization, high lactate concentrations significantly reduced the number of multinucleated osteoclasts and their resorptive activity. Interestingly, extracellular lactate also significantly upregulated M2-like macrophage markers (Arg1, Il1rn, Klf4). These results support the concept that macrophage efferocytosis of apoptotic osteoblasts alters macrophage energy metabolism, which in turn plays a distinct and pivotal role in modulating the bone microenvironment.

Chronic inflammation is a major driver of atherosclerotic cardiovascular disease, and therapeutics that target inflammation reduce clinical cardiac events beyond levels seen with conventional strategies targeting cholesterol alone. Recent findings suggest innate immune cells maintain ‘memory’ of prior exposure to inflammatory stimuli, a phenomenon known as ‘trained immunity’. In response to inflammatory stimuli, macrophages undergo metabolic and epigenetic rewiring that primes them to mount an augmented response upon a second exposure. Oxidized low-density lipoproteins (oxLDL) have recently been shown to be potent triggers of trained immunity. While trained immunity has been shown to promote inflammation, little is known about how immune training impacts efferocytosis. Therefore, we hypothesize that trained immunity in macrophages promotes inflammation by impairing efferocytosis. We treated murine bone marrow progenitors with oxLDL for 24 hours, then washed and differentiated them into macrophages (BMDMs). Upon assessing efferocytosis, trained BMDMs were able to ingest a first apoptotic cell (AC) better than untrained BMDMs yet had an impaired ability to take up additional ACs, reflecting a defect in continual efferocytosis. Using an in vivo approach, we transplanted donor bone marrow from Ldlr -/- mice fed a chow or Western diet into naïve C57BL/6 recipients. After recovery, we elicited peritoneal macrophages to assess efferocytosis and found that recipients receiving marrow from Western diet fed Ldlr -/- mice not only displayed impaired efferocytosis, but also significantly upregulated PGE 2 production, suggesting impaired resolution. To determine whether PGE 2 is a mediator of trained immunity, we primed bone marrow progenitors with PGE 2 and differentiated them into BMDMs. We found that macrophages primed with PGE 2 elaborated higher levels of inflammatory cytokines in response to LPS stimulation than controls. Overall, these findings demonstrate that oxLDL/Western diet-training impinges on the resolution program by impairing efferocytosis and are durable effects that demonstrate heritability. Future directions include determining the impact of these findings on the development of atherosclerosis.

The ability of cells to adapt to environmental changes is essential for their growth and survival. Eukaryotic cells, including macrophages (Mφ), utilize the GCN2 and mTOR pathways to regulate metabolism in response to microenvironmental cues. Efferocytosis (phagocytosis of apoptotic cells [AC]), plays a critical role in preventing autoimmunity and promoting immune tolerance. This process requires precise metabolic regulation, as Mφ must efficiently process AC-derived materials to control inflammation. While GCN2 and mTOR pathways are well-studied under amino acid deprivation, their roles in efferocytosis remain unclear. Here we show efferocytosis activates both mTORC1 and GCN2 in Mφ. Initial mTORC1 activation facilitates the recycling of AC-derived amino acids and cholesterol while down-regulating phagocytic receptors to mitigate efferocytosis-induced metabolic stress. Simultaneously, GCN2 activation promotes the production of anti-inflammatory cytokines and suppresses prolonged mTORC1 activity to prevent an inflammatory phenotype. Our findings reveal that GCN2-deficient Mφ displayed heightened mTORC1 activity, a pro-inflammatory phenotype, and impaired suppression of CD4 T cells after efferocytosis. These findings highlight the critical role of GCN2-mediated mTORC1 regulation in maintaining efferocytosis-driven immune tolerance in Mφ, with implications for cancer therapy and autoimmune disorders that require efficient efferocytosis. Supported by NIH/NCI 1R01CA255670; Medicine by design; the TFRI; and CIHR operating grants 406694, 436605, and 518004. Immune Response Regulation: Molecular Mechanisms (IRM)

Atherosclerosis is the leading cause of cardiovascular morbidity and mortality worldwide, driven not only by lipid accumulation but also by chronic inflammation and defective tissue repair. Among immune cells within plaques, macrophages orchestrate both inflammatory injury and reparative responses, and their fate is critically regulated by mitochondrial quality control. Damaged mitochondria release mitochondrial reactive oxygen species and mitochondrial DNA, which activate innate immune pathways, such as the NLRP3 (NOD‐, LRR‐, and pyrin domain‐containing protein 3) inflammasome and the cyclic GMP–AMP synthase–stimulator of interferon genes pathway, thereby amplifying inflammation. Mitophagy, the selective clearance of dysfunctional mitochondria, has emerged as a metabolic checkpoint determining whether macrophages sustain inflammation or transition toward repair. Key signaling axes, including phosphatase and tensin homolog (PTEN)‐induced kinase 1/Parkin, BNIP3 (BCL2/adenovirus E1B 19‐kDa interacting protein 3)/NIX (NIP3‐like protein X), and FUNDC1 (FUN14 domain‐containing protein 1), converge to limit mitochondrial reactive oxygen species and mitochondrial DNA release, suppress innate immune activation, and preserve oxidative metabolism. By maintaining energy balance, mitophagy supports efferocytosis, extracellular matrix deposition, and fibrous cap stabilization, whereas its impairment drives necrotic core expansion and plaque vulnerability. Preclinical studies demonstrate that mitophagy can be therapeutically modulated by small molecules (metformin and resveratrol), natural products (salidroside), gene‐based and nanoparticle approaches, and lifestyle interventions. This review summarizes mechanistic insights into macrophage mitophagy, emphasizes its role as a metabolic checkpoint in the inflammation‐to‐repair transition, and discusses translational opportunities and challenges in targeting this pathway to stabilize vulnerable plaques.

Background. Obesity is expected to hinder efferocytosis due to ADAM17‐mediated cleavage of the MER tyrosine kinase receptor, producing soluble MER (sMER) that disrupts MERTK binding to cell death markers. However, the intracellular efferocytosis pathway in central obesity remains elusive, particularly the role of low‐grade chronic inflammation in its initiation and identification of binding signals that disrupt efferocytosis. Objective. We investigate the efferocytosis signaling pathway in men with central obesity and its relationship with inflammation, cell death, and related processes. Methods. A cross‐sectional study was conducted, and clinical data and blood samples were collected from 56 men with central obesity (obese group) and 29 nonobese individuals (control group). Clinical evaluations and predefined biochemical screening tests were performed. The efferocytosis signaling pathway was investigated by measuring phosphatidylserine (PS), ADAM17, TNF‐alpha (TNF‐α), and sMER. Results. Metabolic syndrome was detected in more than half of the participants in the obese group according to the predefined tests. Mean levels of PS, TNF‐α, and sMER were higher in the obese group but not significantly different from those of the control group. Further analysis based on waist circumference (WC) ranges in the obese group revealed a significant increase in PS and sMER levels between the control group and the obese group with WC greater than 120 cm. ADAM17 levels were significantly higher in the obese group than in the control group. PS was positively correlated with WC and ADAM17. ADAM17 was positively correlated with TNF‐α and sMER, indicating impaired efferocytosis. Conclusions. Central obesity appeared to cause a disturbance in efferocytosis that began with cell damage and death, along with an enlargement of the WC and an ongoing inflammatory response. Efferocytosis was disrupted by proinflammatory cytokine regulators, which induced the production of sMER and interfered with the efferocytosis process.

AIMS Diabetes induces disorders in macrophage immunometabolism, leading to increased destruction of periodontal tissue. Identifying key factors to restore metabolic alterations and promote resolution of inflammation remains an unmet objective. METHODS In the present study, the effect of macrophage efferocytosis on inflammatory regression and tissue repair was assessed using a diabetic periodontitis (DPD) model. The mitochondrial function of macrophages cultured under different conditions was assessed in vitro, and macrophage efferocytosis function and polarization phenotypes were examined. Osteogenic differentiation and migration capacity were examined using periodontal ligament stem cells (PDLSCs) co-cultured with macrophages to assess the effect on tissue repair. RESULTS We demonstrated that the high-glucose inflammatory microenvironment exacerbated the pro-inflammatory metabolic profile of macrophages and disrupted mitochondrial dynamics. Rats with DPD exhibited heightened periodontal tissue damage during the ligation period, characterized by increased neutrophil infiltration and apoptotic cells. Following ligature removal, the transition to the repair phase was inhibited. Impaired efferocytosis in macrophages led to reduced expression of anti-inflammatory cytokines. Inhibiting excessive mitochondrial division mitigated macrophage damage, ultimately improving the osteogenic differentiation and migration of PDLSCs. CONCLUSIONS This research suggested the critical role of mitochondria in the resolution of inflammation in diabetic periodontitis through regulating macrophage efferocytosis and interaction with PDLSCs.

Rationale: Diabetes exacerbates the prevalence and severity of periodontitis, leading to severe periodontal destruction and ultimately tooth loss. Delayed resolution of inflammation is a major contributor to diabetic periodontitis (DP) pathogenesis, but the underlying mechanisms of this imbalanced immune homeostasis remain unclear. Methods: We collected periodontium from periodontitis with or without diabetes to confirm the dysfunctional neutrophils and macrophages in aggravated inflammatory damage and impaired inflammation resolution. Our in vitro experiments confirmed that SIRT6 inhibited macrophage efferocytosis by restraining miR-216a-5p-216b-5p-217 cluster maturation through ''non-canonical'' microprocessor complex (RNA pulldown, RIP, immunostaining, CHIP, Luciferase assays, and FISH). Moreover, we constructed m6SKO mice that underwent LIP-induced periodontitis to explore the in vitro and in vivo effect of SIRT6 on macrophage efferocytosis. Finally, antagomiR-217, a miRNA antagonism, was delivered into the periodontium to treat LIP-induced diabetic periodontitis. Results: We discovered that insufficient SIRT6 as a histone deacetylase in macrophages led to unresolved inflammation and aggravated periodontitis in both human and mouse DP with accumulated apoptotic neutrophil (AN) and higher generation of neutrophil extracellular traps. Mechanistically, we validated that macrophage underwent high glucose stimulation resulting in disturbance of the SIRT6-miR-216/217 axis that triggered impeded efferocytosis of AN through targeting the DEL-1/CD36 axis directly. Furthermore, we demonstrated the inhibitory role of SIRT6 for MIR217HG transcription and identified a non-canonical action of microprocessor that SIRT6 epigenetically hindered the splicing of the primary miR-216/217 via the complex of hnRNPA2B1, DGCR8, and Drosha. Notably, by constructing myeloid-specific deletion of SIRT6 mice and locally delivering antagomir-217 in DP models, we strengthened the in vivo effect of this axis in regulating macrophage efferocytosis and inflammation resolution in DP. Conclusions: Our findings delineated the emerging role of SIRT6 in mediating metabolic dysfunction-associated inflammation, and therapeutically targeting this regulatory axis might be a promising strategy for treating diabetes-associated inflammatory diseases.

Chronic inflammation drives the pathophysiology of many cardiometabolic diseases. Pro-resolving macrophages (Møs) resolve inflammation and restore tissue homeostasis via lipid metabolism-dependent efferocytosis. Understanding the regulatory pathways that control Mø lipid metabolic profiles is essential for uncovering mechanisms of inflammation resolution. We have shown that lipin-1, a phosphohydrolase and a transcriptional co-regulator, promotes inflammation resolution by enhancing β-oxidation and efferocytosis. Our current study aims to define the mechanisms by which Mø lipin-1 promotes β-oxidation, efferocytosis and inflammation resolution. Using myeloid-specific lipin-1 knockout (lipin-1mKO) mice and littermate controls, we show that lipin-1 facilitates mitochondrial fission, producing fragmented mitochondria with enhanced β-oxidation capacity compared to elongated mitochondria. Additionally, lentiviral transduction of lipin-1mKO Møs with specific truncated forms of lipin-1 suggests that a novel non-canonical activity of lipin-1 is sufficient to restore and augment efferocytosis. Our findings uncover a novel role for lipin-1 in coordinating mitochondrial structure and metabolic function in macrophages. This deeper understanding of lipin-1’s influence on mitochondrial dynamics and macrophage function advances our knowledge of the cellular mechanisms underlying inflammation resolution and may inform future investigations into metabolic regulation in immune responses. Supported by NIH under grant number P20GM134974 and R01HL163106; and CCDS under grant number CCDS000013 Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

No abstract available

No abstract available

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by immune dysregulation and chronic inflammation, with increasing evidence implicating the gut microbiota in its pathogenesis. Probiotics, such as Lactobacillus rhamnosus GG (LGG), exert anti-inflammatory effects by enhancing gut barrier function and restoring microbial homeostasis, representing a promising therapeutic strategy for SLE. However, conventional probiotic therapies are hindered by poor survival and colonization in the hostile intestinal environment. Here, a polydopamine-coated LGG (LGG@PDA) with improved viability, adhesion, and resistance to oxidative stress is developed. In murine models of lupus, LGG@PDA treatment restored gut and immune homeostasis, enhanced macrophage efferocytosis, reduced autoantibody levels, and ameliorated renal pathology. Metabolomic analysis further identified L-methionine, a metabolite diminished in both lupus mice and SLE patients, as being enriched by LGG@PDA treatment. Functionally, L-methionine enhanced macrophage efferocytosis in a CX3CR1-dependent manner, thereby contributing to the restoration of immune tolerance. Collectively, these findings establish LGG@PDA as a bioengineered probiotic platform that integrates microbiota modulation with immune regulation, highlighting L-methionine as a key metabolic mediator and a promising microbiota-based therapeutic strategy for SLE.

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. 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. 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. 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.

Increased megamitochondria formation and impaired mitophagy in hepatocytes have been linked to the pathogenesis of alcohol-associated liver disease (ALD). This study aims to determine the mechanisms by which alcohol consumption increases megamitochondria formation in the pathogenesis of ALD. Human alcoholic hepatitis (AH) liver samples were used for electron microscopy, histology, and biochemical analysis. Liver-specific dynamin-related protein 1 (DRP1; gene name DNM1L, an essential gene regulating mitochondria fission ) knockout (L-DRP1 KO) mice and wild-type mice were subjected to chronic plus binge alcohol feeding. Both human AH and alcohol-fed mice had decreased hepatic DRP1 with increased accumulation of hepatic megamitochondria. Mechanistic studies revealed that alcohol feeding decreased DRP1 by impairing transcription factor EB-mediated induction of DNM1L . L-DRP1 KO mice had increased megamitochondria and decreased mitophagy with increased liver injury and inflammation, which were further exacerbated by alcohol feeding. Seahorse flux and unbiased metabolomics analysis showed alcohol intake increased mitochondria oxygen consumption and hepatic nicotinamide adenine dinucleotide (NAD + ), acylcarnitine, and ketone levels, which were attenuated in L-DRP1 KO mice, suggesting that loss of hepatic DRP1 leads to maladaptation to alcohol-induced metabolic stress. RNA-sequencing and real-time quantitative PCR analysis revealed increased gene expression of the cGAS-stimulator of interferon genes (STING)-interferon pathway in L-DRP1 KO mice regardless of alcohol feeding. Alcohol-fed L-DRP1 KO mice had increased cytosolic mtDNA and mitochondrial dysfunction leading to increased activation of cGAS-STING-interferon signaling pathways and liver injury. Alcohol consumption decreases hepatic DRP1 resulting in increased megamitochondria and mitochondrial maladaptation that promotes AH by mitochondria-mediated inflammation and cell injury.

Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1

Type 2 Diabetes Mellitus (T2DM) exacerbates periodontal disease lesions, and human periodontal ligament stem cells (PDLSCs) depletion may be the key to periodontal healing impair by T2DM. This study aims to explore the mechanism of PDLSCs depletion in diabetes periodontitis (DP). Firstly, we observed aggravated periodontal destruction in the DP animal model, accompanied by oxidative damage and accumulation of senescent cells. In the high-glucose inflammatory environment in vitro, we revealed that PDLSCs underwent senescence, oxidative stress, mitochondrial dysfunction, and activation of cGAS-STING signaling pathway triggered by mitochondrial DNA. Lineage tracing confirmed that SPD recruited Tdtomato-Gli1

There is increasing evidence for the role of inflammation in the pathogenesis of mitochondrial diseases (MDs). However, the mechanisms underlying mutation-induced inflammation in MD remain elusive. Our previous study suggested that mitophagy is impaired in the skeletal muscle of those with MD, likely causing mitochondrial DNA (mtDNA) release and thereby triggering inflammation. We here aimed to decipher the role of the cGAS-STING pathway in inflammatory process in MDs. We investigated the levels of circulating cell-free mtDNA (ccf-mtDNA) in the serum of 104 patients with MDs. Immunofluorescence was performed in skeletal muscles in MDs and control. Biochemical analysis of muscle biopsies was conducted with western blot to detect cGAS, STING, TBK1, IRF3 and phosphorylated IRF3 (p-IRF3). RT-qPCR was performed to detect the downstream genes of type I interferon in skeletal muscles. Furthermore, a protein microarray was used to examine the cytokine levels in the serum of patients with MDs. We found that ccf-mtDNA levels were significantly increased in those with MDs compared to the controls. Consistently, the immunofluorescent results showed that cytosolic dsDNA levels were increased in the muscle samples of MD patients. Biochemical analysis of muscle biopsies showed that cGAS, IRF3, and TBK1 protein levels were significantly increased in those with MDs, indicating that there was activation of the cGAS-STING pathway. RT-qPCR showed that downstream genes of type I interferon were upregulated in muscle samples of MDs. Protein microarray results showed that a total of six cytokines associated with the cGAS-STING pathway were significantly increased in MD patients (fold change > 1.2, p value < 0.05). These findings suggest that increases in ccf-mtDNA levels is associated with the activation of the cGAS-STING pathway, thereby triggering inflammation in MDs.

Maintaining a well-functioning mitochondrial network through the mitochondria quality control (MQC) mechanisms, including biogenesis, dynamics and mitophagy, is crucial for overall health. Mitochondrial dysfunction caused by oxidative stress and further exacerbated by impaired quality control can trigger inflammation through the release of the damage-associated molecular patterns (mtDAMPs). mtDAMPs act by stimulating the cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway. Recently, aberrant signalling of the cGAS-STING axis has been recognised to be closely associated with several sterile inflammatory diseases (e.g. non-alcoholic fatty liver disease, obesity). This may fit the pathophysiology of hypothyroidism, an endocrine disorder characterised by the reduction of thyroid hormone production associated with impaired metabolic fluxes, oxidative balance and inflammatory status. Both 3,5,3'-triiodo-L-tyronine (T3) and its derivative 3,5-diiodo-L-thyronine (3,5-T2), are known to mitigate processes targeting mitochondria, albeit the underlying mechanisms are not yet fully understood. Therefore, we used a chemically induced hypothyroidism rat model to investigate the effect of 3,5-T2 or T3 administration on inflammation-related factors (inflammatory cytokines, hepatic cGAS-STING pathway), oxidative stress, antioxidant defence enzymes, mitochondrial DNA (mtDNA) damage, release and repair, and the MQC system in the liver. Hypothyroid rats showed: i) increased oxidative stress, ii) accumulation of mtDNA damage, iii) high levels of circulating cytokines, iv) hepatic activation of cGAS-STING pathways and v) impairment of MQC mechanisms and autophagy. Both iodothyronines restored oxidative balance by enhancing antioxidant defence, preventing mtDNA damage through the activation of mtDNA repair mechanisms (OGG1, APE1, and POLγ) and promoting autophagy progression. Concerning MQC, both iodothyronines stimulated mitophagy and dynamics, with 3,5-T2 activating fusion and T3 modulating both fusion and fission processes. Moreover, only T3 enhanced mitochondrial biogenesis. Notably, 3,5-T2, but not T3, reversed the hypothyroidism-induced activation of the cGAS-STING inflammatory cascade. In addition, it is noteworthy that 3,5-T2 seems more effective than T3 in reducing circulating pro-inflammatory cytokines IL-6 and IL-1B and in stimulating the release of IL-10, a known anti-inflammatory cytokine. These findings reveal novel molecular mechanisms of hepatic signalling pathways involved in hypothyroidism, which could be targeted by natural iodothyronines, particularly 3,5-T2, paving the way for the development of new treatment strategies for inflammatory diseases.

Postoperative cognitive dysfunction (POCD), a common complication following anesthesia and surgery, is influenced by hippocampal neuroinflammation and microglial activation. Mitophagy, a process regulating inflammatory responses by limiting the accumulation of damaged mitochondria, plays a significant role. This study aimed to determine whether regulating microglial mitophagy and the cGAS-STING pathway could alleviate cognitive decline after surgery. Exploratory laparotomy was performed to establish a POCD model using mice. Western blotting, immunofluorescence staining, transmission electron microscopy, and mt-Keima assays were used to examine microglial mitophagy and the cGAS-STING pathway. Quantitative polymerase chain reaction (qPCR) was used to detect inflammatory mediators and cytosolic mitochondrial DNA (mtDNA) levels in BV2 cells. Exploratory laparotomy triggered mitophagy and enhanced the cGAS-STING pathway in mice hippocampi. Pharmacological treatment reduced microglial activation, neuroinflammation, and cognitive impairment after surgery. Mitophagy suppressed the cGAS-STING pathway in mice hippocampi. In vitro, microglia-induced inflammation was mediated by mitophagy and the cGAS-STING pathway. Small interfering RNA (siRNA) of PINK1 hindered mitophagy activation and facilitated the cytosolic release of mtDNA, resulting in the initiation of the cGAS-STING pathway and innate immune response. Microglial mitophagy inhibited inflammatory responses via the mtDNA-cGAS-STING pathway inducing microglial mitophagy and inhibiting the mtDNA-cGAS-STING pathway may be an effective therapeutic approach for patients with POCD.

Ultraviolet B (UVB) irradiation causes skin damages. In this study, we focus on the involvement of mitochondrial disorders in UVB injury. Surprisingly, UVB irradiation increases the amounts of mitochondria in human immortalized keratinocytes HaCaT. However, further analysis shows that ATP levels decreased by UVB treatment in accordance with the collapse of mitochondrial membrane potential (MMP), suggesting an accumulation of dysfunctional mitochondria in UVB-irradiated HaCaT cells. Mitophagy, mainly mediated by PINK1 and parkin, is critical for the elimination of damaged mitochondria. Western blot results show that the levels of both PINK1 and parkin are decreased in UVB-irradiated cells, indicating the impairment of mitophagy. Silencing the expression of PINK1 or parkin by transfection of siRNA shows essentially the same damage to the cells as UVB irradiation does, including increased mitochondrial amount, decreased MMP and ATP production, and enhanced apoptosis, evidencing that repression of PINK1/parkin-mediated mitophagy plays a primary cause of UVB-caused cells damages. We previously found that HaCaT cells exposed to UVB showed activation of the cGAS-STING pathway and apoptosis. Here, silencing PINK1 or parkin also increases the protein levels of cGAS and STING, facilitates nuclear accumulation of NF-κB, and promotes the transcription of IFNβ, suggesting for the activation of STING pathway. Mitophagy impairment either by UVB-irradiation or by PINK1/parkin silencing initiates caspase-3-mediated apoptosis, as shown by the activation of caspase-3 and cleavage of PARP, as well as the increase of Hoechst-positive stained cells and Annexin V-positive cells. Further studies find that Bax-mediated permeabilization of mitochondrial membrane is critical for cell apoptosis, as well as the cytosolic leakage of mtDNA in UVB-treated cells, which results in cGAS-STING activation, and these processes are negatively-regulated by PINK1/parkin-mediated mitophagy. This study reveals the involvement of dysfunctional mitochondria due to impaired mitophagy in the damaging effect of UVB irradiation on HaCaT cells. Restoring the mitophagy has the potential to be developed as a new strategy to protect skin from UVB damages.

Acute pancreatitis (AP) involves acinar cell death and severe inflammation. Although the E3 ubiquitin ligase TRIM21 regulates inflammation, its role in the pathogenesis of AP remains undefined. This study aims to explore the role of TRIM21 in regulating inflammation during AP. In this study, TRIM21 levels show a severity-dependent increase in patients with AP, which is more than that in healthy controls. Consistently, increased TRIM21 expression is observed in the murine models of AP and exhibits spatial co-localization with macrophages. Macrophage-specific Trim21 ablation mitigates pancreatic damage and systemic inflammation. Conversely, TRIM21 activation aggravates disease severity. Mechanistically, TRIM21 promotes K11-linked ubiquitination and proteasomal degradation of PHB2, leading to mtDNA accumulation in the cytosol via impaired PHB2-mediated mitophagy. Dysregulation of mtDNA homeostasis activates the cGAS-STING axis, thereby intensifying inflammation during AP progression. Additionally, pharmacological inhibition of TRIM21 with quisinostat mitigates AP progression. Our findings reveal the critical role of TRIM21 in AP-associated inflammation, providing a potential therapeutic strategy for inflammatory pancreatic diseases.

Radiotherapy is a commonly employed treatment modality for cancer; however, its radiobiological effects in hypertrophic cardiomyopathy (HCM) remain unclear. Radiation exposure activates the cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway, which is functionally associated with the activation of NOD-like Receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasomes, known mediators of pyroptotic cell death. Nonetheless, the underlying mechanism requires further investigation. Therefore, the objective of this study is to elucidate the role of the cGAS/STING/NLRP3 pathway in the process of cardiomyocyte pyroptosis during radiotherapy for HCM. Transverse aortic constriction surgery was conducted to establish a mouse model of pressure overload-induced HCM, followed by the administration of 30 Gray (Gy) radiation one-week post-surgery. Cardiac morphology and function were evaluated through echocardiographic techniques. Hematoxylin & Eosin staining, along with Wheat Germ Agglutinin (WGA) staining, were utilized to quantify the cross-sectional area of cardiomyocytes and the degree of left ventricular hypertrophy. The HL-1 mouse cardiac muscle cell line was subjected to 40 Gy of radiation using an X-ray irradiator to establish an Radiotherapy reduced cardiac hypertrophy, improved cardiac function, and decreased fibrotic changes in HCM mice when compared to control groups. The application of radiation resulted in pyroptosis in HL-1 cells and a reduction in cell viability; this effect that was alleviated by the inhibition of NLRP3, while overexpression of cGAS exacerbated the situation. Furthermore, radiation led to a decline in mitochondrial membrane potential and the leakage of mitochondrial DNA into the cytoplasm, which activated the cGAS-STING signaling pathway, thereby initiating pyroptosis. This activation was corroborated by elevated levels of pyroptosis-associated proteins, including cGAS, STING, NLRP3, caspase-1, Gasdermin D (GSDMD), cGAMP, IL-18, and IL-1β. Notably, the inhibition of NLRP3 effectively abolished the upregulation of IL-18, and IL-1β levels. Radiation can improve cardiac function, decrease hypertrophy of myocardial cells, and induce oxidative stress. This oxidative stress results in the leakage of mitochondrial DNA (mtDNA), which subsequently activates the cGAS/STING/NLRP3 signalling pathway, culminating in pyroptosis.

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. Lipopolysaccharide (LPS) was combined with caerulein-induced SAP in wild type, cGAS 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-1β, 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. 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.

Although it is an essential nutrient, high choline intake directly or indirectly via its metabolite is associated with increased risk of cardiovascular disease, the mechanism of which remains to be elucidated. The present study was performed to investigate whether hydrogen sulfide (H

The ubiquitin-editing enzyme A20 is known to regulate inflammation and maintain homeostasis, but its role in self-DNA-mediated inflammation in acute kidney injury (AKI) is not well understood. Here, our study demonstrated that oxidized self-DNA accumulates in the serum of AKI mice and patients. This oxidized self-DNA exacerbates the progression of AKI by activating the cGAS-STING pathway and NLRP3 inflammasome. While inhibition of the STING pathway only slightly attenuates AKI progression, suppression of NLRP3 inflammasome-mediated pyroptosis significantly alleviates AKI progression and improves the survival of AKI mice. Subsequently, we found that Tnfaip3 (encoding A20) is significantly upregulated following oxidized self-DNA treatment. A20 significantly alleviates AKI development by dampening STING signaling pathway and NLRP3-mediated pyroptosis. Moreover, A20-derived peptide (P-II) also significantly alleviates ox-dsDNA-induced pyroptosis and improves the survival and renal injury of AKI mice. Mechanistically, A20 competitively binds with NEK7 and thus inhibiting NLRP3 inflammasome. A20 and P-II interfere with the interaction between NEK7 and NLRP3 through Lys140 of NEK7. Mutation of Lys140 effects on the interaction of NEK7 with A20 and/or NLRP3 complex. Conditional knockout of NEK7 in macrophages or pharmacological inhibition of NEK7 both significantly rescue AKI mouse models. This study reveals a new mechanism by which A20 attenuates oxidized self-DNA-mediated inflammation and provides a new therapeutic strategy for AKI.

No abstract

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease worldwide. Numerous evidence has demonstrated that metabolic reprogramming serves as a hallmark associated with an elevated risk of NAFLD progression. Selenoprotein W (SelW) is an extensively expressed hepatic selenoprotein that plays a crucial role in antioxidant function. Here, we first demonstrated that SelW is a significantly distinct factor in the liver tissue of NAFLD patients through the Gene Expression Omnibus (GEO) database. Additionally, loss of SelW alleviated hepatic steatosis induced by a high-fat diet (HFD), and was accompanied by the regulation of metabolic and inflammatory pathways as verified by transcriptomic analysis. Moreover, co-immunoprecipitation (CO-IP), liquid chromatography-tandem mass spectrometry (LC-MS), laser scanning confocal microscopy (LSCM) and molecular docking analysis were subsequently implemented to identify Pyruvate Kinase M2 (PKM2) as a potential interacting protein of SelW. Meanwhile, SelW modulated PKM2 translocation into the nucleus to trigger transactivation of the HIF-1α, in further mediating mitochondrial apoptosis, eventually resulting in mitochondrial damage, ROS excessive production and mtDNA leakage. Additionally, mito-ROS accumulation induced the activation of the NLRP3 inflammasome-mediated pyroptosis, thereby facilitating extracellular leakage of mtDNA. The escaped mtDNA then evokes the cGAS-STING signaling pathway in macrophage, thus inducing a shift in macrophage phenotype. Together, our results suggest SelW promotes hepatocyte apoptosis and pyroptosis by regulating metabolic reprogramming to activate cGAS/STING signaling of macrophages, thereby exacerbating the progression of NAFLD.

The lung is the organ most commonly affected by sepsis. Additionally, acute lung injury (ALI) resulting from sepsis is a major cause of death in intensive care units. Macrophages are essential for maintaining normal lung physiological functions and are implicated in various pulmonary diseases. An essential autophagy protein, autophagy-related protein 16-like 1 (ATG16L1), is crucial for the inflammatory activation of macrophages. ATG16L1 expression was measured in lung from mice with sepsis. ALI was induced in myeloid ATG16L1-, NLRP3- and STING-deficient mice by intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). Using immunofluorescence and flow cytometry to assess the inflammatory status of LPS-treated bone marrow-derived macrophages (BMDMs). A co-culture system of BMDMs and MLE-12 cells was established in vitro. Myeloid ATG16L1-deficient mice exhibited exacerbated septic lung injury and a more intense inflammatory response following LPS treatment. Mechanistically, ATG16L1-deficient macrophages exhibited impaired LC3B lipidation, damaged mitochondria and reactive oxygen species (ROS) accumulation. These abnormalities led to the activation of NOD-like receptor family pyrin domain-containing protein 3 (NLRP3), subsequently enhancing proinflammatory response. Overactivated ATG16L1-deficient macrophages aggravated the damage to alveolar epithelial cells and enhanced the release of double-stranded DNA (dsDNA), thereby promoting STING activation and subsequent NLRP3 activation in macrophages, leading to positive feedback activation of macrophage NLRP3 signalling. Scavenging mitochondrial ROS or inhibiting STING activation effectively suppresses NLRP3 activation in macrophages and alleviates ALI. Furthermore, overexpression of myeloid ATG16L1 limits NLRP3 activation and reduces the severity of ALI. Our findings reveal a new role for ATG16L1 in regulating macrophage NLRP3 feedback activation during sepsis, suggesting it as a potential therapeutic target for treating sepsis-induced ALI. Myeloid-specific ATG16L1 deficiency exacerbates sepsis-induced lung injury. ATG16L1-deficient macrophages exhibit impaired LC3B lipidation and ROS accumulation, leading to NLRP3 inflammasome activation. Uncontrolled inflammatory responses in ATG16L1-deficient macrophages aggravate alveolar epithelial cell damage. Alveolar epithelial cells release dsDNA, activating the cGAS-STING-NLRP3 signaling pathway, which subsequently triggers a positive feedback activation of NLRP3. Overexpression of ATG16L1 helps mitigate lung tissue inflammation, offering a novel therapeutic direction for sepsis-induced lung injury.

Acute pancreatitis (AP), a potentially fatal disorder driven by macrophage-associated inflammation, involves mitochondrial reactive oxygen species (ROS) overproduction with pancreatic acinar cells (PACs). This ROS surge damages mitochondria, causing mitochondrial DNA (mt-DNA) leakage and PACs apoptosis. Released mt-DNA then activates the pro-inflammatory cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway in macrophages, exacerbating disease progression. Critically, while scavenging mitochondrial ROS can halt this cycle, existing mitochondria-targeted drugs fail to penetrate the damaged blood-pancreas barrier (BPB). To overcome this limitation, we developed MTP, a novel dual-targeting nanomedicine synthesized from tannic acid, dopamine, and molybdenum oxides. MTP uniquely achieves dual targeting: actively homing to injured BPB and specifically accumulating within PACs mitochondria. This enables robust mitochondrial ROS scavenging, which protects mitochondrial integrity, reduces mt-DNA release, inhibits PACs apoptosis, and crucially blocks mt-DNA-induced cGAS-STING activation in macrophages, thereby suppressing their pro-inflammatory M1 polarization. By simultaneously interrupting both ROS-mediated PACs damage and macrophage-driven inflammation via this dual-targeting strategy, MTP effectively mitigates AP progression, establishing a breakthrough therapeutic approach.

Persistent pressure-shear coupled injury traps pressure ulcer wounds in a sustained ischemia-reperfusion cycle, leading to mitochondrial membrane potential collapse and mtDNA leakage in macrophages. This mitochondrial dysfunction amplifies intracellular ROS accumulation, disrupts inflammatory resolution, and blocks the transition from inflammation to proliferation, ultimately resulting in non-healing or delayed healing pressure ulcers. Here, we report a Janus hierarchical porous aerogel (OBP2GM) fabricated via directional ice-templating combined with electrospinning, designed to integrate multi-dimensional wound microenvironment regulation within a single construct. The electrospun top layer incorporates phase-change microspheres to provide mild, adaptive thermal buffering around physiological skin temperature (32-35 °C), while its highly porous fibrous architecture enables efficient bacterial interception. The underlying aerogel layer features vertically aligned microchannels that support rapid unidirectional fluid transport, ensuring effective exudate drainage and moisture balance. More importantly, the polysaccharide-polyphenol network within the aerogel actively regulates macrophage mitochondrial homeostasis by activating the PINK1/Parkin-mediated mitophagy pathway, facilitating the clearance of severely damaged mitochondria while preserving functional ones. This process restores mitochondrial membrane potential (ΔΨm↑, relative fluorescence intensity 69.22%), suppresses excessive ROS generation, promotes macrophage polarization toward the pro-regenerative M2 phenotype, and enhances HUVEC tubulogenesis by nearly threefold. In a murine pressure ulcer model, OBP2GM markedly accelerated wound re-epithelialization, demonstrating a materials-based strategy for mechanical-mitochondrial-immune synergistic repair of pressure ulcers.

The glycolytic enzyme PKM2 (pyruvate kinase muscle 2) is upregulated in monocytes/macrophages of patients with atherosclerotic coronary artery disease. However, the role of cell type-specific PKM2 in the setting of atherosclerosis remains to be defined. We determined whether myeloid cell-specific PKM2 regulates efferocytosis and atherosclerosis. We generated myeloid cell-specific PKM2 PKM2 was upregulated in macrophages of Ldlr Genetic deletion of PKM2 in myeloid cells or limiting its nuclear translocation reduces atherosclerosis by suppressing inflammation and enhancing efferocytosis.

Although inflammation is a vital defence response to infection, if left uncontrolled, it can lead to pathology. Macrophages are critical players both in driving the inflammatory response and in the subsequent events required for restoring tissue homeostasis. Extracellular vesicles (EVs) are membrane-enclosed structures released by cells that mediate intercellular communication and are present in all biological fluids, including blood. Herein, we show that extracellular vesicles from plasma (pEVs) play a relevant role in the control of inflammation by counteracting PAMP-induced macrophage activation. Indeed, pEV-treatment of macrophages simultaneously with or prior to PAMP exposure reduced the secretion of pro-inflammatory IL-6 and TNF-α and increased IL-10 response. This anti-inflammatory activity was associated with the promotion of tissue-repair functions in macrophages, characterized by augmented efferocytosis and pro-angiogenic capacity, and increased expression of VEGFa, CD300e, RGS2 and CD93, genes involved in cell growth and tissue remodelling. We also show that simultaneous stimulation of macrophages with a PAMP and pEVs promoted COX2 expression and CREB phosphorylation as well as the accumulation of higher concentrations of PGE2 in cell culture supernatants. Remarkably, the anti-inflammatory activity of pEVs was abolished if cells were treated with a pharmacological inhibitor of COX2, indicating that pEV-mediated induction of COX2 is critical for the pEV-mediated inhibition of inflammation. Finally, we show that pEVs added to monocytes prior to their M-CSF-induced differentiation to macrophages increased efferocytosis and diminished pro-inflammatory cytokine responses to PAMP stimulation. In conclusion, our results suggest that pEVs are endogenous homeostatic modulators of macrophages, activating the PGE2/CREB pathway, decreasing the production of inflammatory cytokines and promoting tissue repair functions.

Diabetes leads to dysregulated macrophage immunometabolism, contributing to accelerated atherosclerosis progression. Identifying critical factors to restore metabolic alterations and promote resolution of inflammation remains an unmet goal. MicroRNAs orchestrate multiple signalling events in macrophages, yet their therapeutic potential in diabetes-associated atherosclerosis remains unclear. miRNA profiling revealed significantly lower miR-369-3p expression in aortic intimal lesions from Ldlr-/- mice on a high-fat sucrose-containing (HFSC) diet for 12 weeks. miR-369-3p was also reduced in peripheral blood mononuclear cells from diabetic patients with coronary artery disease (CAD). Cell-type expression profiling showed miR-369-3p enrichment in aortic macrophages. In vitro, oxLDL treatment reduced miR-369-3p expression in mouse bone marrow-derived macrophages (BMDMs). Metabolic profiling in BMDMs revealed that miR-369-3p overexpression blocked the oxidized low density lipoprotein (oxLDL)-mediated increase in the cellular metabolite succinate and reduced mitochondrial respiration (OXPHOS) and inflammation [Interleukin (lL)-1β, TNF-α, and IL-6]. Mechanistically, miR-369-3p targeted the succinate receptor (GPR91) and alleviated the oxLDL-induced activation of inflammasome signalling pathways. Therapeutic administration of miR-369-3p mimics in HFSC-fed Ldlr-/- mice reduced GPR91 expression in lesional macrophages and diabetes-accelerated atherosclerosis, evident by a decrease in plaque size and pro-inflammatory Ly6Chi monocytes. RNA-Seq analyses showed more pro-resolving pathways in plaque macrophages from miR-369-3p-treated mice, consistent with an increase in macrophage efferocytosis in lesions. Finally, a GPR91 antagonist attenuated oxLDL-induced inflammation in primary monocytes from human subjects with diabetes. These findings establish a therapeutic role for miR-369-3p in halting diabetes-associated atherosclerosis by regulating GPR91 and macrophage succinate metabolism.

Abnormal macrophage function caused by dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) is a critical contributor to chronic airway infections and inflammation in people with cystic fibrosis (PWCF). Elexacaftor/tezacaftor/ivacaftor (ETI) is a new CFTR modulator therapy for PWCF. Host-pathogen and clinical responses to CFTR modulators are poorly described. We sought to determine how ETI impacts macrophage CFTR function, resulting effector functions and relationships to clinical outcome changes. Clinical information and/or biospecimens were obtained at ETI initiation and 3, 6, 9 and 12 months post-ETI in 56 PWCF and compared with non-CF controls. Peripheral blood monocyte-derived macrophages (MDMs) were isolated and functional assays performed. ETI treatment was associated with increased CF MDM CFTR expression, function and localisation to the plasma membrane. CF MDM phagocytosis, intracellular killing of CF pathogens and efferocytosis of apoptotic neutrophils were partially restored by ETI, but inflammatory cytokine production remained unchanged. Clinical outcomes including increased forced expiratory volume in 1 s (+10%) and body mass index (+1.0 kg·m ETI is associated with unique changes in innate immune function and clinical outcomes.

Macrophage efferocytosis, essential for the resolution of inflammation and plaque stability in atherosclerosis, is impaired in diabetes. Thrombomodulin (TM) and endothelial protein C receptor (EPCR), key mediators of protein C activation (PC), have vasculoprotective and anti-inflammatory roles, yet their involvement in macrophage efferocytosis in diabetes-induced atherosclerosis remains unclear. Here, we demonstrate that expression of EPCR was reduced in atherosclerotic lesions of diabetic patients compared to non-diabetic controls. In parallel, efferocytosis was impaired in atherosclerotic lesions and in monocytes derived macrophages of diabetic patients. In vitro, treatment with activated PC (aPC) or its cytoprotective selective variant (3K3A-aPC) restored high glucose-impaired macrophage efferocytosis. Mechanistic studies revealed that aPC restored efferocytosis through Arginase-1 and modulation of Rac1-ATF6 signaling. Additionally, macrophage protease-activated receptor 1 (PAR1) was identified as the key receptor mediating aPC's effects on efferocytosis. Mimicking biased PAR-1 signaling via parmodulin-2 reverses glucose impaired efferocytosis. In vivo, aPC treatment of diabetic ApoE

Macrophages have versatile roles in atherosclerosis. SHP2 (Src homology 2 containing protein tyrosine phosphatase 2) has been demonstrated to play a critical role in regulating macrophage activation. However, the mechanism of SHP2 regulation of macrophage function in an atherosclerotic microenvironment remains unknown. APOE (apolipoprotein E) or LDLR (low-density lipoprotein receptor) null mice treated with SHP099 were fed a Western diet for 8 weeks, while Pharmacological inhibition and selective deletion in macrophages of SHP2 aggravated atherosclerosis in APOE and LDLR null mice with increased plaque macrophages and apoptotic cells. In vitro, SHP2 deficiency in APOE and LDLR null macrophages enhanced proinflammatory polarization and its efferocytosis was dramatically impaired. Conversely, the expression of gain-of-function mutation of SHP2 in mouse macrophages reduced atherosclerosis. The SHP2 agonist lovastatin repressesed macrophage inflammatory activation and enhanced efferocytosis. Mechanistically, RNA sequencing analysis identified PPARγ as a key downstream transcription factor. PPARγ was decreased in macrophages upon SHP2 deletion and inhibition. Importantly, PPARγ agonist decreased atherosclerosis in SHP2 knockout mice, restored efferocytotic defects, and reduced inflammatory activation in SHP2 deleted macrophages. PPARγ was decreased by the ubiquitin-mediated degradation upon SHP2 inhibition or deletion. Finally, we found that SHP2 was downregulated in atherosclerotic vessels. Overall, SHP2 in macrophages was found to act as an antiatherosclerotic regulator by stabilizing PPARγ in APOE/LDLR null mice.

Ulcerative colitis (UC) is a chronic inflammatory disorder marked by epithelial barrier disruption and defective resolution of inflammation. Although epithelial apoptosis and impaired efferocytosis are recognized contributors to disease progression, their molecular regulation remains insufficiently defined. NR1D1, a circadian transcriptional repressor implicated in immune control, has not been fully explored in UC. Here, this study shows that NR1D1 expression is markedly reduced in human UC biopsies and murine colitis. CUT&Tag profiling revealed direct NR1D1 chromatin occupancy, while ChIP-qPCR and luciferase assays confirmed that NR1D1 represses CD47, a key anti-efferocytosis molecule. Functional experiments demonstrate that NR1D1 deficiency enhances epithelial apoptosis, upregulates CD47, impairs macrophage efferocytosis, and exacerbates colitis in IEC-specific Nr1d1

During acute respiratory distress syndrome (ARDS), delayed apoptosis of neutrophils and impaired efferocytosis of macrophages constitute two critical limiting steps, leading to secondary inflammatory storm and posing a significant threat to human health. However, due to the failure of previous single target-centric treatments to effectively address these two limiting steps in controlling the inflammatory storm, no available therapies are approved for ARDS treatment. Herein, inspired by spontaneous inflammation resolution, two kinds of Apoptosis and Efferocytosis Restored Nanoparticles (AER NPs) are proposed to overcome these two limiting steps for counteracting severe inflammatory storm. For the first limiting step, neutrophil-targeted apoptosis-restored nanoparticles (AR NPs) accelerated the programmed apoptosis of inflammatory neutrophils. The resolution of the first limiting step facilitated the accumulation of macrophage-targeted and efferocytosis-restored nanoparticles (ER NPs), thereby restoring macrophage efferocytosis and alleviating the second limiting step. The results indicated that after sequential treatment with AER NPs, recruited neutrophils decreased to 13.86%, and macrophage efferocytosis increased to 563.24%. AER NPs promoted inflammation resolution and established a self-healing virtuous loop by addressing the two limiting steps, ultimately effectively treating ARDS. This work suggests that a strategy inspired by inflammation resolution holds promise as a potential approach for advancing inflammation therapy.

In chronic obstructive pulmonary disease (COPD), defective macrophage phagocytic clearance of cells undergoing apoptosis by efferocytosis may lead to secondary necrosis of the uncleared cells and contribute to airway inflammation. The precise mechanisms for this phenomenon remain unknown. LC3-associated phagocytosis (LAP) is indispensable for effective efferocytosis. We hypothesized that cigarette smoke inhibits the regulators of LAP pathway, potentially contributing to the chronic airways inflammation associated with COPD. Bronchoalveolar (BAL)-derived alveolar macrophages, lung tissue macrophages obtained from lung resection surgery, and monocyte-derived macrophages (MDM) were prepared from COPD patients and control participants. Lung/airway samples from mice chronically exposed to cigarette smoke were also investigated. Differentiated THP-1 cells were exposed to cigarette smoke extract (CSE). The LAP pathway including Rubicon, as an essential regulator of LAP, efferocytosis and inflammation was examined using western blot, ELISA, flow cytometry, and/or immunofluorescence. Rubicon was significantly depleted in COPD alveolar macrophages compared with non-COPD control macrophages. Rubicon protein in alveolar macrophages of cigarette smoke-exposed mice and cigarette smoke-exposed MDM and THP-1 was decreased with a concomitant impairment of efferocytosis. We also noted increased expression of LC3 which is critical for LAP pathway in COPD and THP-1 macrophages. Furthermore, THP-1 macrophages exposed to cigarette smoke extract exhibited higher levels of other key components of LAP pathway including Atg5 and TIM-4. There was a strong positive correlation between Rubicon protein expression and efferocytosis. LAP is a requisite for effective efferocytosis and an appropriate inflammatory response, which is impaired by Rubicon deficiency. Our findings suggest dysregulated LAP due to reduced Rubicon as a result of CSE exposure. This phenomenon could lead to a failure of macrophages to effectively process phagosomes containing apoptotic cells during efferocytosis. Restoring Rubicon protein expression has unrecognized therapeutic potential in the context of disease-related modifications caused by exposure to cigarette smoke.

To investigate the role of efferocytosis in maintaining corneal immune homeostasis after ultraviolet B (UVB)-induced keratocyte apoptosis and its impact on inflammatory responses in both in vitro and in vivo settings. Human corneal stromal fibroblasts (HCFs) were exposed to UVB radiation (150 mJ/cm²) to induce apoptosis and co-cultured with M1 macrophages using a transwell system. In this in vitro efferocytosis model, UVB-irradiated HCFs (BHCFs) were evaluated for efferocytosis-related markers, including milk fat globule epidermal growth factor 8 (MFG-E8) and MER proto-oncogene, tyrosine kinase (MERTK), as well as inflammatory cytokines such as interleukin (IL)-1β and IL-6. Paracrine effects on nearby M1 macrophages were assessed by analyzing changes in cytokine profiles and expression of myeloid/macrophage markers. To validate the physiological relevance of these findings, an in vivo mouse model was established by subconjunctival injection of clodronate liposomes in UVB-exposed mice to reduce corneal macrophages. BHCFs showed higher TUNEL positivity and significantly more efferocytosis when co-cultured with M1 macrophages. This was accompanied by upregulation of MFG-E8 and MERTK and downregulation of IL-1β and IL-6. Microenvironmental M1 macrophages exhibited reduced IL-1β, increased transforming growth factor beta 1, and downregulation of CD14, CD68, CD80, and CD11c. In vivo macrophage reduction impaired Mertk activation and failed to suppress IL-1β upregulation in the cornea. Efferocytosis contributes to corneal immune homeostasis after UVB-induced apoptosis by resolving inflammation and modulating macrophage phenotype. These findings support the existence of an efferocytic mechanism in the corneal stroma.

Current therapies for ulcerative colitis (UC) face critical challenges, including systemic toxicity and inadequate mucosal regeneration. Herein, we present a pioneering dual-mechanism therapeutic platform integrating pH-independent acid-treated sucralfate (ASF) with autophagy-inducing spermidine (Spd) to synergistically address UC pathogenesis. Unlike conventional pH-dependent barrier therapies, ASF forms a mechanically tunable, injectable hydrogel that adheres robustly to colonic mucosa regardless of local pH. Spermidine was easily mounted into ASF hydrogel matrix via electrostatic interaction, with more than 90 % encapsulation efficiency. Moreover, the mechanical strength of ASF hydrogel was precisely modulated by adjusting spermidine amount in formula, making it suitable for effective gut mucosa coverage. Importantly, in vitro permeability test showed that spermidine-loaded ASF hydrogel (Spd-ASF) demonstrated the selective barrier functionality, blocking > 80 % of Escherichia coli and LPS penetration while allowing nutrient permeability. Moreover, micro-CT images demonstrated that rectally infused Spd-ASF hydrogel was uniformly adhered to the colon wall at least for 8 h. In DSS-induced colitis mice, rectally administrating Spd-ASF hydrogel uniquely restored colon length to near-normal levels (vs. 30 % shortening in controls), reduced proinflammatory cytokines (TNF-α, IL-6, IL-1β) by 60-75 %, and doubled goblet cell density. Mechanistically, Spd-ASF reprogrammed macrophage behavior by activating autophagy and enhancing efferocytosis, driving M2 polarization to resolve inflammation. Notably, Spd-ASF uniquely reversed dysbiosis, elevating beneficial Lactobacillus while suppressing colitis-associated Muribaculaceae and Clostridia. This study is the first to combine mucoadhesive biomaterial engineering with autophagy-mediated immunomodulation, offering a paradigm shift in UC therapy by simultaneously shielding the mucosa and reprogramming inflammatory pathways. STATEMENT OF SIGNIFICANCE: Here, autophagy-inducing spermidine (Spd) and pH-independent acid-treated sucralfate (ASF) were combined to create the novel dual-mechanism hydrogel (Spd-ASF), which works in concert to combat UC pathogenesis. The selective barrier functioning of Spd-ASF hydrogel was established by its capacity to permit nutritional permeability while preventing the transfer of toxins (LPS) and pathogenic bacteria (E. coli). Additionally, rectally infused Spd-ASF hydrogel was consistently attached on the colon wall for at least eight hours, as seen by micro-CT images. Spd-ASF therapy doubled the density of goblet cells, decreased proinflammatory cytokines (TNF-α, IL-6, and IL-1β) by 60-75 %, boosted helpful Lactobacillus, and lowered pathogenic Muribaculaceae and Clostridia. It also restored colon length in DSS-induced colitis animals. Spd-ASF's therapeutic action was closely linked to promoting cellular efferocytosis and triggering macrophage autophagy.