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Mitochondrial Damage and Activation of the STING Pathway Lead to Renal Inflammation and Fibrosis.

ABSTRACT: Fibrosis is the final common pathway leading to end-stage renal failure. By analyzing the kidneys of patients and animal models with fibrosis, we observed a significant mitochondrial defect, including the loss of the mitochondrial transcription factor A (TFAM) in kidney tubule cells. Here, we generated mice with tubule-specific deletion of TFAM (Ksp-Cre/Tfamflox/flox). While these mice developed severe mitochondrial loss and energetic deficit by 6 weeks of age, kidney fibrosis, immune cell infiltration, and progressive azotemia causing death were only observed around 12 weeks of age. In renal cells of TFAM KO (knockout) mice, aberrant packaging of the mitochondrial DNA (mtDNA) resulted in its cytosolic translocation, activation of the cytosolic cGAS-stimulator of interferon genes (STING) DNA sensing pathway, and thus cytokine expression and immune cell recruitment. Ablation of STING ameliorated kidney fibrosis in mouse models of chronic kidney disease, demonstrating how TFAM sequesters mtDNA to limit the inflammation leading to fibrosis.

SUBMITTER: Chung KW

PROVIDER: S-EPMC7054893 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Mitochondrial Damage and Activation of the STING Pathway Lead to Renal Inflammation and Fibrosis.

Chung Ki Wung KW Dhillon Poonam P Huang Shizheng S Sheng Xin X Shrestha Rojesh R Qiu Chengxiang C Kaufman Brett A BA Park Jihwan J Pei Liming L Baur Joseph J Palmer Matthew M Susztak Katalin K

Cell metabolism 20190829 4

Fibrosis is the final common pathway leading to end-stage renal failure. By analyzing the kidneys of patients and animal models with fibrosis, we observed a significant mitochondrial defect, including the loss of the mitochondrial transcription factor A (TFAM) in kidney tubule cells. Here, we generated mice with tubule-specific deletion of TFAM (Ksp-Cre/Tfam<sup>flox/flox</sup>). While these mice developed severe mitochondrial loss and energetic deficit by 6 weeks of age, kidney fibrosis, immune ...[more]

PMID: 31474566

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Project description:Diabetic cardiomyopathy (DCM) is a serious cardiac complication of diabetes that currently lacks specific treatment. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway has been suggested to contribute to the pathogenesis of cardiovascular diseases. However, whether cGAS-STING is involved in the development of DCM has not been established. Our study aimed to determine the role of cGAS-STING in the initiation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome-induced cardiac pyroptosis and chronic inflammation during the pathogenesis of DCM. C57BL/6J mice were preinjected with adeno-associated virus 9 (AAV9) intravenously via the tail vein to specifically knock down myocardial STING. After four weeks, mice with myocardium-specific knockdown of STING received injections of streptozotocin (STZ; 50 mg/kg) and a high-fat diet to induce diabetes. Measurements included echocardiography, immunohistochemical analyses, wheat germ agglutinin (WGA) staining, and western blotting. Here, we showed that the cGAS-STING signaling pathway was activated in diabetic hearts, which was indicated by the increased phosphorylation of TANK-binding kinase 1 (TBK1) and interferon (IFN) regulatory factor 3 (IRF3), leading to the activation of the NLRP3 inflammasome in the hearts of diabetic mice and proinflammatory cytokine release into serum. Moreover, STING knockdown via adeno-associated virus-9 (AAV9) in diabetic mouse heart alleviated cardiac pyroptosis and the inflammatory response, prevented diabetes-induced hypertrophy, and restored cardiac function. Mechanistically, we showed that palmitic acid (PA)-induced lipotoxicity impairs mitochondrial homeostasis, producing excessive mitochondrial reactive oxygen species (mtROS), which results in oxidative damage to mitochondrial DNA (mtDNA) and its release into the cytoplasm while switching on cGAS-STING-mediated pyroptosis in cardiomyocytes, thereby worsening the progression of diabetic cardiomyopathy. Our study demonstrated that activation of the cGAS-STING pathway caused by mitochondrial oxidative damage and mtDNA escape induced by free fatty acids promoted pyroptosis and proinflammatory responses in cardiomyocytes in a NLRP3 inflammasome-dependent manner, thus promoting myocardial hypertrophy during the progression of DCM.

Project description:BackgroundPathological stimuli cause mitochondrial damage and leakage of mitochondrial DNA (mtDNA) into the cytosol, as demonstrated in many cell types. The cytosolic mtDNA then drives the activation of noninfectious inflammation. Retinal microvascular endothelial cells (RMECs) play an important role in the inner endothelial blood-retinal barrier (BRB). RMEC dysfunction frequently occurs in posterior-segment eye diseases, causing loss of vision. In this study, we investigated the involvement of cytosolic mtDNA in noninfectious immune inflammation in RMECs under pathological stimuli.MethodsRMECs were stimulated with 100 ng/ml lipopolysaccharide (LPS), 200 μM hydrogen peroxide (H2O2), or 25 mM D-glucose. After 24 h, immunofluorescent staining was used to detect the opening of the mitochondrial permeability transition pore (MPTP). Cytosolic mtDNA was detected with immunofluorescent staining and PCR after stimulation. mtDNA was then isolated and used to transfect RMECs in vitro, and the protein levels of cGAS were evaluated with western blotting. Real-time PCR was used to examine cGAS mRNA expression levels at different time points after mtDNA stimulation. The activation of STING was detected with immunofluorescent staining 6 h after mtDNA stimulation. Western blotting was used to determine the expression of STING and IFNβ, the phosphorylation status of TBK1, IRF3, and nuclear factor-κB (NF-κB) P65, and the nuclear translocation of IRF3 and NF-κB P65 at 0, 3, 6, 12, and 24 h. The mRNA expression of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were determined with real-time PCR, together with the concentrations of intercellular adhesion molecule 1 (ICAM-1) mRNA.ResultsPathological stimuli caused mtDNA to leak into the cytosol by opening the MPTP in RMECs after 24 h. Cytosolic mtDNA regulated the expression of cGAS and the distribution of STING in RMECs. It promoted ICAM-1, STING and IFNβ expression, TBK1, IRF3, and NF-κB phosphorylation and the nuclear translocation in RMECs at 12 and 24 h after its transfection. The mRNAs of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were significantly elevated at 12 and 24 h after mtDNA stimulation.ConclusionsPathological stimulation induces mtDNA escape into the cytosol of RMECs. This cytoplasmic mtDNA is recognized by the DNA sensor cGAS, increasing the expression of inflammatory cytokines through the STING-TBK1 signaling pathway. Video Abstract. (MP4 37490 kb).

Project description:Background & aimsXBP1 modulates the macrophage proinflammatory response, but its function in macrophage stimulator of interferon genes (STING) activation and liver fibrosis is unknown. X-box binding protein 1 (XBP1) has been shown to promote macrophage nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) activation in steatohepatitis. Herein, we aimed to explore the underlying mechanism of XBP1 in the regulation of STING signalling and the subsequent NLRP3 activation during liver fibrosis.MethodsXBP1 expression was measured in the human fibrotic liver tissue samples. Liver fibrosis was induced in myeloid-specific Xbp1-, STING-, and Nlrp3-deficient mice by carbon tetrachloride injection, bile duct ligation, or a methionine/choline-deficient diet.ResultsAlthough increased XBP1 expression was observed in the fibrotic liver macrophages of mice and clinical patients, myeloid-specific Xbp1 deficiency or pharmacological inhibition of XBP1 protected the liver against fibrosis. Furthermore, it inhibited macrophage NLPR3 activation in a STING/IRF3-dependent manner. Oxidative mitochondrial injury facilitated cytosolic leakage of macrophage self-mtDNA and cGAS/STING/NLRP3 signalling activation to promote liver fibrosis. Mechanistically, RNA sequencing analysis indicated a decreased mtDNA expression and an increased BCL2/adenovirus E1B interacting protein 3 (BNIP3)-mediated mitophagy activation in Xbp1-deficient macrophages. Chromatin immunoprecipitation (ChIP) assays further suggested that spliced XBP1 bound directly to the Bnip3 promoter and inhibited the transcription of Bnip3 in macrophages. Xbp1 deficiency decreased the mtDNA cytosolic release and STING/NLRP3 activation by promoting BNIP3-mediated mitophagy activation in macrophages, which was abrogated by Bnip3 knockdown. Moreover, macrophage XBP1/STING signalling contributed to the activation of hepatic stellate cells.ConclusionsOur findings demonstrate that XBP1 controls macrophage cGAS/STING/NLRP3 activation by regulating macrophage self-mtDNA cytosolic leakage via BNIP3-mediated mitophagy modulation, thus providing a novel target against liver fibrosis.Lay summaryLiver fibrosis is a typical progressive process of chronic liver disease, driven by inflammatory and immune responses, and is characterised by an excess of extracellular matrix in the liver. Currently, there is no effective therapeutic strategy for the treatment of liver fibrosis, resulting in high mortality worldwide. In this study, we found that myeloid-specific Xbp1 deficiency protected the liver against fibrosis in mice, while XBP1 inhibition ameliorated liver fibrosis in mice. This study concluded that targeting XBP1 signalling in macrophages may provide a novel strategy for protecting the liver against fibrosis.

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Mitochondrial Damage and Activation of the STING Pathway Lead to Renal Inflammation and Fibrosis.

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Mitochondrial Damage and Activation of the STING Pathway Lead to Renal Inflammation and Fibrosis.

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