Release Of Mitochondrial DNA Research Articles

Airway Epithelial cGAS Is Critical for Induction of Experimental Allergic Airway Inflammation.

DNA damage could lead to the accumulation of cytosolic DNA, and the cytosolic DNA-sensing pathway has been implicated in multiple inflammatory diseases. However, the role of cytosolic DNA-sensing pathway in asthma pathogenesis is still unclear. This article explored the role of airway epithelial cyclic GMP-AMP synthase (cGAS), the major sensor of cytosolic dsDNA, in asthma pathogenesis. Cytosolic dsDNA accumulation in airway epithelial cells (ECs) was detected in the setting of allergic inflammation both in vitro and in vivo. Mice with cGAS deletion in airway ECs were used for OVA- or house dust mite (HDM)-induced allergic airway inflammation. Additionally, the effects of cGAS knockdown on IL-33-induced GM-CSF production and the mechanisms by which IL-33 induced cytosolic dsDNA accumulation in human bronchial epithelial (HBE) cells were explored. Increased accumulation of cytosolic dsDNA was observed in airway epithelium of OVA- or HDM-challenged mice and in HBE cells treated with IL-33. Deletion of cGAS in the airway ECs of mice significantly attenuated the allergic airway inflammation induced by OVA or HDM. Mechanistically, cGAS participates in promoting TH2 immunity likely via regulating the production of airway epithelial GM-CSF. Furthermore, Mito-TEMPO could reduce IL-33-induced cytoplasmic dsDNA accumulation in HBE cells possibly through suppressing the release of mitochondrial DNA into the cytosol. In conclusion, airway epithelial cGAS plays an important role via sensing the cytosolic dsDNA in asthma pathogenesis and could serve as a promising therapeutic target against allergic airway inflammation.

MtDNA Activates cGAS Signaling and Suppresses the YAP-Mediated Endothelial Cell Proliferation Program to Promote Inflammatory Injury

Cytosolic DNA acts as a universal danger-associated molecular pattern (DAMP) signal; however, the mechanisms of self-DNA release into the cytosol and its role in inflammatory tissue injury are not well understood. We found that the internalized bacterial endotoxin lipopolysaccharide (LPS) activated the pore-forming protein Gasdermin D, which formed mitochondrial pores and induced mitochondrial DNA (mtDNA) release into the cytosol of endothelial cells. mtDNA was recognized by the DNA sensor cGAS and generated the second messenger cGAMP, which suppressed endothelial cell proliferation by downregulating YAP1 signaling. This indicated that the surviving endothelial cells in the penumbrium of the inflammatory injury were compromised in their regenerative capacity. In an experimental model of inflammatory lung injury, deletion of cGas in mice restored endothelial regeneration. The results suggest that targeting the endothelial Gasdermin D activated cGAS-YAP signaling pathway could serve as a potential strategy for restoring endothelial function after inflammatory injury.

Pigment epithelium‑derived factor facilitates NLRP3 inflammasome activation through downregulating cytidine monophosphate kinase 2: A potential treatment strategy for missed abortion.

A number of conditions may underlie the occurrence of missed abortion (MA), including inflammation. Pigment epithelium-derived factor (PEDF) is a novel mediator of the inflammation-related nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, which is associated with several human diseases. However, the association between MA and NLRP3 inflammasome, and whether PEDF is reduced in MA, remain unknown. In the present study, the decidua and chorion tissues of patients who had suffered a MA were examined, and a lipopolysaccharide (LPS)-induced human chorionic trophoblast HTR8/SVneo cell model was established to mimic MA in vitro. The results revealed that cytidine monophosphate kinase 2 (CMPK2) expression and NLRP3 inflammasome activation, downstream pro-IL-18 and pro-IL-1β expression, and IL-18 and IL-1β release, were all significantly increased in MA tissues or LPS-induced HTR8/SVneo cells. PEDF reversed the increase in CMPK2 expression and activation of the NLRP3 inflammasome axis and, thus, downregulated the production of mitochondrial reactive oxygen species and mitochondrial DNA release, resulting in reduced lactate dehydrogenase release, and a resultant decrease in cell viability. Recovery of CMPK2 expression abolished all the effects of PEDF, indicating that CMPK2 may be an effector downstream of PEDF. PEDF reduced CMPK2 protein levels but did not affect the mRNA levels, and treatment with the proteasomal inhibitor MG132 significantly reversed this reduction in CMPK2 protein levels. Furthermore, a ubiquitination assay of immunoprecipitation demonstrated that CMPK2 was polyubiquitinated in the presence of LPS, PEDF and MG132. These results indicated that the NLRP3 inflammasome is implicated in the pathogenesis of MA, and PEDF may reduce MA through ubiquitin-dependent proteasomal degradation of CMPK2 to inhibit NLRP3 activation, which may serve as a novel strategy for preventing or reducing the risk of MA.

Desmin mutations result in mitochondrial dysfunction regardless of their aggregation properties

Desmin, being a major intermediate filament of muscle cells, contributes to stabilization and positioning of mitochondria. Desmin mutations have been reported in conjunction with skeletal myopathies accompanied by mitochondrial dysfunction. Depending on the ability to promote intracellular aggregates formation, mutations can be considered aggregate-prone or non-aggregate-prone. The aim of the present study was to describe how expression of different desmin mutant isoforms effects mitochondria and contributes to the development of myocyte dysfunction. To achieve this goal, two non-aggregate-prone (Des S12F and Des A213V) and four aggregate-prone (Des L345P, Des A357P, Des L370P, Des D399Y) desmin mutations were expressed in skeletal muscle cells. We showed that all evaluated mutations affected the morphology of mitochondrial network, suppressed parameters of mitochondrial respiration, diminished mitochondrial membrane potential, increased ADP/ATP ratio, and enhanced mitochondrial DNA (mtDNA) release. mtDNA was partially secreted through exosomes as demonstrated by GW4869 treatment. Dysfunction of mitochondria was observed regardless the type of mutation: aggregate-prone or non-aggregate-prone. However, expression of aggregate-prone mutations resulted in more prominent phenotype. Thus, in this comparative study of six pathogenic desmin mutations that cause skeletal myopathy development, we confirmed a role of mitochondrial dysfunction and mtDNA release in the pathogenesis of desmin myopathies, regardless of the aggregation capacity of the mutated desmin.

Lipid restriction enhances suppression

Immunometabolism Regulatory T cells (Tregs) suppress immune responses to maintain tolerance and limit autoimmunity. Because lipid metabolism is crucial for the activity of Tregs, Field et al. explored the role of the lipid chaperone fatty-acid binding protein 5 (FABP5) in mouse and human Treg function. Targeting FABP5 through either genetic or pharmacological means caused mitochondrial dysfunction, which depressed oxidative phosphorylation and promoted a switch to glycolysis. FABP5 inhibition enhanced the suppressive activity of Tregs through a mechanism involving mitochondrial DNA release and subsequent cGAS-STING–dependent type I interferon signaling. The researchers found that the lipid-restrictive nature of the tumor microenvironment influenced Fabp5 gene expression and facilitated Treg suppressor function. Cell Metab. 31 , 422 (2019).

Hypoxia differently modulates the release of mitochondrial and nuclear DNA

BackgroundWe investigated the influence of hypoxia on the concentration of mitochondrial and nuclear cell-free DNA (McfDNA and NcfDNA, respectively).MethodBy an ultra-sensitive quantitative PCR-based assay, McfDNA and NcfDNA were measured in the supernatants of different colorectal cell lines, and in the plasma of C57/Bl6 mice engrafted with TC1 tumour cells, in normoxic or hypoxic conditions.ResultsOur data when setting cell culture conditions highlighted the higher stability of McfDNA as compared to NcfDNA and revealed that cancer cells released amounts of nuclear DNA equivalent to the mass of a chromosome over a 6-h duration of incubation. In cell model, hypoxia induced a great increase in NcfDNA and McfDNA concentrations within the first 24 h. After this period, cfDNA total concentrations remained stable in hypoxia consecutive to a decrease of nuclear DNA release, and noteworthy, to a complete inhibition of daily mitochondrial DNA release. In TC1-engrafted mice submitted to intermittent hypoxia, plasma NcfDNA levels are much higher than in mice bred in normoxia, unlike plasma McfDNA concentration that is not impacted by hypoxia.ConclusionThis study suggests that hypoxia negatively modulates nuclear and, particularly, mitochondrial DNA releases in long-term hypoxia, and revealed that the underlying mechanisms are differently regulated.

The Mycobacterium marinum ESX-1 system mediates phagosomal permeabilization and type I interferon production via separable mechanisms

Following mycobacterial entry into macrophages the ESX-1 type VII secretion system promotes phagosomal permeabilization and type I IFN production, key features of tuberculosis pathogenesis. The current model states that the secreted substrate ESAT-6 is required for membrane permeabilization and that a subsequent passive leakage of extracellular bacterial DNA into the host cell cytosol is sensed by the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) pathway to induce type I IFN production. We employed a collection of Mycobacterium marinum ESX-1 transposon mutants in a macrophage infection model and show that permeabilization of the phagosomal membrane does not require ESAT-6 secretion. Moreover, loss of membrane integrity is insufficient to induce type I IFN production. Instead, type I IFN production requires intact ESX-1 function and correlates with release of mitochondrial and nuclear host DNA into the cytosol, indicating that ESX-1 affects host membrane integrity and DNA release via genetically separable mechanisms. These results suggest a revised model for major aspects of ESX-1-mediated host interactions and put focus on elucidating the mechanisms by which ESX-1 permeabilizes host membranes and induces the type I IFN response, questions of importance for our basic understanding of mycobacterial pathogenesis and innate immune sensing.

Nickel induces inflammatory activation via NF-κB, MAPKs, IRF3 and NLRP3 inflammasome signaling pathways in macrophages.

Nickel (Ni), an environmental hazard, widely causes allergic contact hypersensitivity worldwide. Despite that Ni-stimulated pro-inflammatory response is vital in allergy, the underlying molecular mechanisms remain largely unclear. Here, we demonstrated that NiCl2 activated nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPKs) and interferon regulatory factor 3 (IRF3) signaling pathways in primary bone marrow-derived macrophages (BMDMs), leading to the altered transcription levels of interleukin-1β (IL-1β), -6, -8, -18, tumor necrosis factor-α (TNF-α) and interferon β (INF-β). We also found that nickel chloride (NiCl2) activated Nod-like receptor 3 (NLRP3) inflammasome pathway, resulting in the proteolytic cleavage and release of IL-1β. NiCl2 induced the accumulation of mitochondrial reactive oxygen species (mtROS) and the release of mitochondrial DNA (mtDNA), thus activating NLRP3 inflammasome pathway. Additionally, NiCl2-induced apoptosis was dependent on the generation of mtROS, and caspase-1 activation might also partly contribute to the apoptotic process. Altogether, abovementioned results indicate that NiCl2 induces inflammatory activation in BMDMs via NF-κB, MAPKs, IRF3 signaling pathways as well as NLRP3 inflammasome pathway, which provides a mechanism to improve the efficiency of treatment against Ni-induced allergic reactions.

Transcriptomic, epigenetic, and functional analyses implicate neutrophil diversity in the pathogenesis of systemic lupus erythematosus

Neutrophil dysregulation is implicated in the pathogenesis of systemic lupus erythematosus (SLE). SLE is characterized by elevated levels of a pathogenic neutrophil subset known as low-density granulocytes (LDGs). The origin and phenotypic, functional, and pathogenic heterogeneity of LDGs remain to be systematically determined. Transcriptomics and epigenetic assessment of lupus LDGs, autologous normal-density neutrophils, and healthy control neutrophils was performed by bulk and single-cell RNA sequencing and assay for transposase-accessible chromatin sequencing. Functional readouts were compared among neutrophil subsets. SLE LDGs display significant transcriptional and epigenetic heterogeneity and comprise 2 subpopulations of intermediate-mature and immature neutrophils, with different degrees of chromatin accessibility and differences in transcription factor motif analysis. Differences in neutrophil extracellular trap (NET) formation, oxidized mitochondrial DNA release, chemotaxis, phagocytosis, degranulation, ability to harm the endothelium, and responses to type I interferon (IFN) stimulation are evident among LDG subsets. Compared with other immune cell subsets, LDGs display the highest expression of IFN-inducible genes. Distinct LDG subsets correlate with specific clinical features of lupus and with the presence and severity of coronary artery disease. Phenotypic, functional, and pathogenic neutrophil heterogeneity are prevalent in SLE and may promote immune dysregulation and prominent vascular damage characteristic of this disease.

Bacterial Type I CRISPR-Cas systems influence inflammasome activation in mammalian host by promoting autophagy.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (CRISPR-Cas) systems in prokaryotes function at defending against foreign DNAs, providing adaptive immunity to maintain homeostasis. CRISPR-Cas may also influence immune regulation ability in mammalian cells through alterations of pathogenic extent and nature. Recent research has implied that Type I CRISPR-Cas systems of Pseudomonas aeruginosa strain UCBPP-PA14 impede recognition by Toll-like receptor 4, and decrease pro-inflammatory responses both in vitro and in vivo. However, the molecular mechanism by which CRISPR-Cas systems affect host immunity is largely undemonstrated. Here, we explored whether CRISPR-Cas systems can influence autophagy to alter the activation of inflammasome. Using the wild-type PA14 and total CRISPR-Cas region deletion (∆TCR) mutant strain, we elucidated the role and underlying mechanism of Type I CRISPR-Cas systems in bacterial infection, and showed that CRISPR-Cas systems impacted the release of mitochondrial DNA and induction of autophagy. CRISPR-Cas deficiency led to an increase of mitochondrial DNA release, a decrease in autophagy, an increase of inflammasome activation and, ultimately, an elevation of pro-inflammatory response. Our findings illustrate a new important mechanism by which Type I CRISPR-Cas systems control their virulence potency to evade host defense.

Abstract P193: A Novel Mechanism for the Pathogenesis of Preeclampsia: Cell-Free Mitochondrial DNA Mediated Toll Like Receptor 9 Activation

Background: Preeclampsia (PreE) is a significant global health burden which impacts 10 million pregnancies annually resulting in over a half million lost lives. Mitochondrial damage has previously been demonstrated to be present in PreE. A novel relationship may exist between preeclampsia and mitochondrial damage via the release of cell-free mitochondrial DNA (cf-mtDNA). Previous work in models of systemic hypertension shows cf-mtDNA mediated activation of TLR9 to be a key part of disease development. We aim to evaluate the level of TLR9 activation and ROS production in patients with and without PreE. Methods: To investigate whether PreE plasma contains increased levels of cell free DNA that can activate TLR9, HEK-Blue TM -hTLR9 reporter cell line (Invivogen) was treated with 3% plasma from either control or PreE patients. The TLR9 activator ODN2006 served as positive control (Fig.1, A&B). To investigate whether PreE plasma can stimulate production of ROS, HUVECs were treated in similar fashion and ROS was evaluated using Mitosox TM (Fig. 1, C&D). Results: Cells treated with PreE plasma demonstrated a 1.7-fold increase in TLR9 activation vs control (p=0.005, Fig. 1A&B). In addition, Mitosox TM signal indicated a 1.8-fold increase in ROS in PreE vs. control (p=0.066, Fig. 1C&D). Conclusion: We conclude that plasma from PreE patients contains higher levels of cf-mtDNA that stimulates inflammation response via TLR9 that promotes mitochondrial ROS production. This mechanism may present a novel target for treatment of Pre-E.

Manassantin B shows antiviral activity against coxsackievirus B3 infection by activation of the STING/TBK-1/IRF3 signalling pathway

Coxsackievirus B3 (CVB3) is an important human pathogen associated with the development of acute pancreatitis, myocarditis, and type 1 diabetes. Currently, no vaccines or antiviral therapeutics are approved for the prevention and treatment of CVB3 infection. We found that Saururus chinensis Baill extract showed critical antiviral activity against CVB3 infection in vitro. Further, manassantin B inhibited replication of CVB3 and suppressed CVB3 VP1 protein expression in vitro. Additionally, oral administration of manassantin B in mice attenuated CVB3 infection-associated symptoms by reducing systemic production of inflammatory cytokines and chemokines including TNF-α, IL-6, IFN-γ, CCL2, and CXCL-1. We found that the antiviral activity of manassantin B is associated with increased levels of mitochondrial ROS (mROS). Inhibition of mROS generation attenuated the antiviral activity of manassantin B in vitro. Interestingly, we found that manassantin B also induced cytosolic release of mitochondrial DNA based on cytochrome C oxidase DNA levels. We further confirmed that STING and IRF-3 expression and STING and TBK-1 phosphorylation were increased by manassantin B treatment in CVB3-infected cells. Collectively, these results suggest that manassantin B exerts antiviral activity against CVB3 through activation of the STING/TKB-1/IRF3 antiviral pathway and increased production of mROS.

169 – Interleukin-13-Mediated Release of Mitochondrial Dna from Esophageal Epithelial Cells: A Novel Biomarker for Eosinophilic Esophagitis

169 – Interleukin-13-Mediated Release of Mitochondrial Dna from Esophageal Epithelial Cells: A Novel Biomarker for Eosinophilic Esophagitis

Tranexamic acid suppresses the release of mitochondrial DNA, protects the endothelial monolayer and enhances oxidative phosphorylation

Damage-associated molecular patterns, including mitochondrial DNA (mtDNA) are released during hemorrhage resulting in the development of endotheliopathy. Tranexamic acid (TXA), an antifibrinolytic drug used in hemorrhaging patients, enhances their survival despite the lack of a comprehensive understanding of its cellular mechanisms of action. The present study is aimed to elucidate these mechanisms, with a focus on mitochondria. We found that TXA inhibits the release of endogenous mtDNA from granulocytes and endothelial cells. Furthermore, TXA attenuates the loss of the endothelial monolayer integrity induced by exogenous mtDNA. Using the Seahorse XF technology, it was demonstrated that TXA strongly stimulates mitochondrial respiration. Studies using Mitotracker dye, cells derived from mito-QC mice, and the ActivSignal IPAD assay, indicate that TXA stimulates biogenesis of mitochondria and inhibits mitophagy. These findings open the potential for improvement of the strategies of TXA applications in trauma patients and the development of more efficient TXA derivatives.

Inhibition of Mitochondrial Respiratory Chain Complex I Induces Vascular Endothelial Cell Apoptosis and Release of Mitochondrial DNA

BackgroundVascular endothelial oxidative stress is a common feature of preeclampsia, a pregnancy specific hypertensive syndrome with high incidence of maternal and fetal mortality and morbidity. Cellular oxidative stress can lead to cell death, which promotes the release of cellular constituents (e.g. mitochondrial fragments) into the extracellular space. Circulating cell‐free mitochondrial DNA (mtDNA) concentrations are increased in pregnant women with preeclampsia. The main objective of this study was to determine the mechanisms by which vascular endothelial cells may contribute to this increase in cell‐free mtDNA.HypothesisMitochondrial complex I inhibition results in extrusion of mtDNA from vascular endothelial cells via cell death‐dependent mechanisms.Material and MethodsHuman umbilical vein endothelial cells (HUVEC, Lonza) were grown to 80–90% confluency before treatment with a mitochondrial complex I inhibitor (Rotenone: 5, 10, 25 μM ‐ 4 h). Immunocytochemistry was used to confirm that HUVEC maintained endothelial cell characteristics. Cell death (apoptotic and non‐apoptotic) was quantified using flow cytometry (staining for Annexin V and propidium iodide). mtDNA was measured on total nucleic acid extracts from cell culture supernatants using absolute real‐time PCR techniques.ResultsTreatment of HUVECs with rotenone increased early apoptosis (Fig. A) and late apoptosis/necrosis (Fig. B; 1‐way ANOVA followed by Sidak's post‐hoc test). Concentrations of mtDNA in HUVEC supernatant were increased in HUVECs treated with 5 μM of Rotenone (Veh (n=5): 2.45 ± 0.05 pg/mL vs. Rotenone (n=6): 3.65 ± 0.39 pg/mL, p=0.07; Sidak's post‐hoc test). Higher concentrations of Rotenone had no effect on concentrations of extracellular mtDNA (p≥0.84).ConclusionsMitochondrial oxidative stress due to inhibition of mitochondrial respiratory chain complex I induces vascular endothelial cell death. Extrusion of mtDNA from apoptotic and necrotic endothelial cells may contribute to increased circulating mtDNA concentrations in preeclamptic pregnancies. Future studies will test this hypothesis using integrative pharmacological and physiological approaches.Support or Funding InformationResearch support: NIH (R25HL125447)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Placental Exposure to Hypoxia and Oxidative Stress Causes Mitochondrial DNA Release into the Extracellular Space

BackgroundIn preeclampsia, a severe hypertensive disorder of pregnancy, placentae experience reduced perfusion, increased cell death, and oxidative stress, with an increase in circulating cell‐free mitochondrial DNA (mtDNA). Our lab has shown that a synthetic mimetic of mtDNA during rodent pregnancy increases maternal blood pressure and vascular reactivity, features of human preeclampsia. The main objective of this study was to determine the role of hypoxia and oxidative stress in mtDNA release from placental cells, and to examine the effects of soluble factors from hypoxia‐exposed placentae on vascular reactivity.Hypotheses Exposure to hypoxia and oxidative stress will result in mtDNA release via cell‐death dependent mechanisms in human trophoblast cells. Soluble factors from hypoxia‐exposed placentae will result in reduced vasodilation in rat maternal arteries. MethodsTo examine the effects of preeclampsia‐related placental stressors on mtDNA release, we treated human trophoblast cells (BeWo cell line) with: 1) hypoxia (1% O2) vs. normoxia (21% O2) for 15 h, or 2) a mitochondrial complex I inhibitor (Rotenone, 10 μM) vs. vehicle for 4 h. mtDNA in cell culture supernatant was measured using absolute qPCR and cell death was quantified using flow cytometry. To test the effects of hypoxic placenta‐derived factors on maternal vascular function, we used mesenteric arteries and placenta‐conditioned media (PLmedia) from pregnant rats. Placentae were incubated in physiological salt solution (37°C) for 3 h in either 1% or 21% O2, while arteries were mounted on a wire myograph and underwent a baseline [(−) PLmedia] concentration‐response curve (CRC) to acetylcholine (ACh, 10−9 – 3×10−5 M) followed by 30‐min incubation with PLmedia, after which the CRC was repeated.ResultsExposure of trophoblast cells to rotenone resulted in cell death (Vehicle: 28.17 ± 2.67% vs. Rotenone: 48.43 ± 1.22%, n = 3, P = 0.002) and mtDNA release (Vehicle: 1.69 ± 0.12 ng/uL vs. Rotenone: 2.39 ± 0.10 ng/uL, n = 5, P = 0.002). Hypoxia did not induce trophoblast cell death (Normoxia: 24.7 ± 0.50% vs. Hypoxia: 24.25 ± 0.45%, n = 2, P = 0.6), but increased release of mtDNA (Normoxia: 14.22 ± 1.20 pg/uL vs. Hypoxia: 20.64 ± 0.39 pg/uL, n = 3, P = 0.007). PLmedia from normoxic and hypoxic placentae reduced sensitivity to ACh (−logEC50, Normoxia: −)PLmedia: 7.48 ± 0.03 vs. (+)PLmedia: 6.96 ± 0.10, n = 4, P = 0.02; Hypoxia: (−)PLmedia: 7.35 ± 0.35 vs. (+)PLmedia: 6.70 ± 0.29, n = 3, P = 0.08).ConclusionsA placental cell model of mitochondrial stress results in cell death and release of mtDNA, while a hypoxic model of stress results in release of mtDNA without cell death. Placental factors decrease resistance artery sensitivity to vasodilators in both normoxic and hypoxic conditions, indicating that the placenta contributes to maternal vascular tone in healthy pregnancies and in pregnancies complicated with reduced perfusion. Ongoing studies investigate the vasoactive potential of placenta‐derived cell‐free mtDNA.Support or Funding InformationAmerican Heart Association (13SDG17050056, 18PRE33960162)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial fission-induced mtDNA stress promotes tumor-associated macrophage infiltration and HCC progression

Tumor-associated macrophages (TAMs) contribute to hepatocellular carcinoma (HCC) progression. However, the molecular mechanism underlying the infiltration of TAMs into HCC microenvironment is largely unclear. Recent studies have reported that alteration of mitochondrial nucleoid structures induces mitochondrial DNA (mtDNA) release into the cytosol, which is recognized as mtDNA stress, and consequently regulates innate immunity. Here we aimed to investigate whether mitochondrial fission induces mtDNA stress and then promotes TAM infiltration and HCC progression. Confocal microscopy and real-time PCR were used to detect cytosolic mtDNA content in HCC cells. The relationship between the expression of mitochondrial fission key regulator dynamin-related protein 1 (Drp1) and the percentage of CD163 (a marker of TAMs)-positive cells was investigated in HCC tissues using immunohistochemistry. Finally, the effect of Drp1 overexpression in HCC cells on recruitment and polarization of TAMs was investigated. Our data showed that increased Drp1 expression was positively correlated with the infiltration of TAMs into HCC tissues. Drp1-mediated mitochondrial fission induced the cytosolic mtDNA stress to enhance the CCL2 secretion from HCC cells by TLR9-mediated NF-κB signaling pathway, and thus promoted the TAM recruitment and polarization. Depleting cytosolic mtDNA using DNase I or blocking TLR9 pathway by TLR9 antagonist, siRNA for TLR9 or p65 in HCC cells with Drp1 overexpression significantly decreased the recruitment and polarization of TAMs. Blocking CCR2 by antagonist significantly reduced TAM infiltration and suppressed HCC progression in mouse model. In conclusion, our findings reveal a novel mechanism of TAM infiltration in HCC by mitochondrial fission-induced mtDNA stress.

Induction of neutrophil extracellular traps during tissue injury: Involvement of STING and Toll-like receptor 9 pathways.

ObjectivesNeutrophils are thought to release neutrophil extracellular traps (NETs) to form in response to exogenous bacteria, viruses and other pathogens. However, the mechanisms underlying NET formation during sterile inflammation are still unclear. In this study, we would like to identify neutrophil extracellular traps formation during sterile inflammation and tissue injury and associated pathways and its mechanism.Materials and methodsWe identified different injuries such as chemical‐induced and trauma‐induced formation of NETs and investigated mechanism of the formation of NETs in vitro and in vivo during the treatment of mtDNA.ResultsHere, we find the release of mitochondrial DNA (mtDNA) and oxidized mtDNA in acute peripheral tissue trauma models or other chemically induced lung injury, and moreover, endogenous mtDNA and oxidized mtDNA induce the formation of NETs and sterile inflammation. Oxidized mtDNA is a more potent inducer of NETs. Mitochondrial DNA activates neutrophils via cyclic GMP‐AMP synthase (cGAS)‐STING and the Toll‐like receptor 9 (TLR9) pathways and increases the production of neutrophil elastase and extracellular neutrophil‐derived DNA in NETs. Mitochondrial DNA also increases the production of reactive oxygen species (ROS) and expression of the NET‐associated proteins Rac 2 and peptidylarginine deiminase 4 (PAD4).ConclusionsAltogether, these findings highlight that endogenous mitochondrial DNA inducted NETs formation and subsequent sterile inflammation and the mechanism associated with NET formation.

Untangling "NETosis" from NETs.

Neutrophil extracellular trap (NET) formation is a cellular function of neutrophils that facilitates the immobilization and killing of invading microorganisms in the extracellular milieu. To form NETs, neutrophils release a DNA scaffold consisting of mitochondrial DNA binding granule proteins. This process does not depend on cell death, but requires glycolytic ATP production for rearrangements in the microtubule network and F-actin. Such cytoskeletal rearrangements are essential for both mitochondrial DNA release and degranulation. However, the formation of NETs has also been described as a distinct form of programed, necrotic cell death, a process designated "NETosis." Necrotic cell death of neutrophils is associated with the permeabilization of both plasma and nuclear membranes resulting in a kind of extracellular cloud of nuclear DNA. The molecular mechanisms eliciting necrotic neutrophil death have been investigated and appear to be different from those responsible for NET formation following mitochondrial DNA release. Here, we discriminate between the mechanisms responsible for the release of mitochondrial versus nuclear DNA and address their respective functions. Our aim is to clarify existing differences of opinion in the fields of NET formation and neutrophil death.

FUN14 Domain-Containing 1-Mediated Mitophagy Suppresses Hepatocarcinogenesis by Inhibition of Inflammasome Activation in Mice.

Mitochondria lie at the heart of innate immunity, and aberrant mitochondrial activity contributes to immune activation and chronic inflammatory diseases, including liver cancers. Mitophagy is a selective process for removing dysfunctional mitochondria. The link between mitophagy and inflammation in tumorigenesis remains largely unexplored. We observed that FUN14 domain-containing 1 (FUNDC1), a previously characterized mitophagy receptor, accumulates in most human hepatocellular carcinomas (HCCs), and we thus explored the role of FUNDC1-mediated mitophagy in HCC initiation and progression in a mouse model in which HCC is induced by the chemical carcinogen, diethylnitrosamine (DEN). We showed that specific knockout of FUNDC1 in hepatocytes promotes the initiation and progression of DEN-induced HCC, whereas FUNDC1 transgenic hepatocytes protect against development of HCC. Hepatocyte-specific FUNDC1 ablation results in the accumulation of dysfunctional mitochondria and triggers a cascade of events involving inflammasome activation and hyperactivation of Janus kinase/signal transducer and activator of transcription signaling. Specifically, cytosolic mitochondrial DNA (mtDNA) release and caspase-1 activation are increased in FUNDC1-depleted hepatocytes. This subsequently results in the elevated release of proinflammatory cytokines, such as interleukin-1β (IL1β) and hyperproliferation of hepatocytes. Conclusion: Our results suggest that FUNDC1 suppresses HCC initiation by reducing inflammasome activation and inflammatory responses in hepatocytes, whereas up-regulation of FUNDC1 expression at the late stage of tumor development may benefit tumor growth. Our study thus describes a mechanistic link between mitophagic modulation of inflammatory response and tumorigenesis, and further implies that FUNDC1-mediated mitophagy and its related inflammatory response may represent a therapeutic target for liver cancer.