DRP1-dependent MtDNA Release Mediates The Activation Of STING Signaling Pathway In Kupffer Cells Leading To Septic Liver Injury

Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection.As an important metabolic and immune defense organ,liver plays a critical role in the pathogenesis of sepsis.Due to abundant blood supply and unique anatomical location,liver,as the target of intestinal endotoxin and excessive inflammatory response,is one of the most vulnerable organs in sepsis.It has been reported that up to 46%of sepsis patients are complicated with liver dysfunction,which has a great impact on the prognosis of sepsis.Kupffer cells(KCs),the liver-resident macrophages,are the main places for bacteria phagocytosis and endotoxin clearance.At the same time,KCs might initiate local inflammatory cascades by producing a large number of pro-inflammatory mediators,reactive oxygen species(ROS)and lipid peroxides,and recruiting other inflammatory cells to the liver as well,leading to the irreversible injury of hepatocytes and liver damage.Studies have confirmed that KCs activation plays a key role in septic liver injury.During sepsis,macrophages are activated by recognizing the pathogenassociated molecular patterns(PAMPs)of pathogenic microorganisms and the damage-associated molecular patterns(DAMPs)released by the host through pattern recognition receptors(PRRs),then triggered inflammatory response.Plasma levels of cell-free DNA(cf DNA)are significantly elevated in sepsis patients,mainly from PAMPs,such as cyclic dinucleotides,and DAMPs,such as nuclear DNA(nDNA)and mitochondrial DNA(mtDNA).These cf DNAs are specifically recognized by PRRs on the surface of the macrophages,and then they bind to the double-stranded DNA recognition receptors-cyclic GMP-AMP synthase(cGAS),which are widely expressed in the cytoplasm.Then the complex activates the downstream stimulator of interferons genes(STING)signaling pathway,mediates interferon regulatory factor 3(IRF3)and NF-κB into the nucleus,and promotes the expression of type Ⅰinterferons and inflammatory factors.Abnormal activation of the STING signaling pathway plays a crucial role in the pathogenesis of various inflammatory diseases,and STING knockout can alleviate multiple organ damage in septic mice.Our preliminary data indicated that the expression of STING protein and the phosphorylation level of downstream IRF3 and p65 protein were significantly increased in the liver tissue from LPS-challenged mice.However,the role of STING signaling in septic liver injury has not been reported.We wondered whether the activation of STING signaling pathway was involved in the process of septic liver injury.Mitochondria are not only the energy factory of cells,but also widely involved in cell proliferation,differentiation and signal transduction.Mitochondria continuously remodel their shape,size and function by alternating processes of fission and fusion.In sepsis,persistent inflammatory stimulation leads to excessive fission of mitochondria in parenchymal cells,damage of mitochondrial respiratory chain,reduction of ATP production,and mitochondrial dysfunction leading to dysfunction in multiple organs.Dynamin-related protein 1(DRP1),the important cytosolic GTPase,is the major pro-fission regulator.Phosphorylation at serine 616 site drives DRP1 translocating to the mitochondrial outer membrane,which promotes mitochondrial fission.Down-regulation of DRP1 expression significantly inhibited excessive mitochondrial fission-induced oxidative stress injury,systemic inflammatory response and apoptosis,which protect multi organ function.While mitochondrial fission is required for macrophages pro-inflammatory differentiation upon LPS stimulation,while inhibition of mitochondrial fission through knock-down DRP1 could effectively reduce the activation of NF-κB and the subsequent release of pro-inflammatory factors.Thus we speculated that inflammatory activation of KCs during sepsis was associated with mitochondrial fission.Excessive mitochondrial fission leads to mitochondrial outer membrane permeabilization,which is required for mtDNA releasing from mitochondria to the cytosol.There was a report demonstrated that the release of mtDNA could also be facilitated by the macropores formed by Bcl-2 family proteins-BAX/BAK in the mitochondrial outer membrane during the process of intrinsic apoptosis.At this time,mtDNA could not activate STING signaling pathway.Nucleases activated by pro-apoptotic caspases degraded mtDNA and then prevented mtDNA binding to cGAS,which inhibited inflammatory response induced by STING signaling.There was also a report demonstrated that the release of mtDNA into cytosol via VDAC 1 oligomer pores activated STING signaling in mouse embryo fibroblasts(MEFs)under moderate stress.Therefore,whether mitochondrial fission caused by persistent inflammatory stimulation during sepsis triggering the inflammatory activation of KCs or promoting cell apoptosis was also the focus of this study.Objective:This study would explore the relationship between the activation of STING signaling pathway in KCs and liver injury during sepsis on two levels of LPS-challenged septic mice model and LPS-treated primary Kupfffer cell model,and clarify the specific mechanism of mitochondrial fission-mediated the activation of STING signaling pathway in KCs.Methods:Part Ⅰ:STING signaling pathway activation in KCs leads to septic liver injury.LPS(10 mg/kg)-induced septic model was used to observe the expression and distribution of STING protein in liver tissue by immunohistochemistry.Primary KCs and hepatocytes were isolated by portal vein perfusion of type IV collagenase,gradient centrifugation and differential adhesion.Western blot was used to observe the expression of STING protein and the phosphorylation levels of downstream IRF3 and p65 proteins in primary KCs after LPS treatment.q-PCR was used to detect the mRNA levels of IFN-β,TNF-α and IL-1 in KCs.To confirm that the STING signaling pathway is activated in KCs during sepsis.At animal experimental level,using STING gene mutant mice(STINGgt,STING protein synthesis deficiency)or WT mice treated with STING-specific agonist DMXAA(abbreviated as DMX),after LPS treatment,the hepatocytes in each group mice were isolated,then the expression of apoptosis-related protein cleaved-caspase3 and the phosphorylation level of necroptosis-related protein MLKL were detected by western blot.The pathological changes of liver tissue were observed by HE staining,liver function(ALT/AST)and the levels of IFN-β,TNF-α and IL-1 in serum were detected.To examine the effects of STING activation in KCs on hepatocytes,KCs were isolated from WT mice or STINGgt mice.WT KCs treated with DMX(25 μg/mL)for 12 h were labeled as DMX WT KCs.Then WT,STINGgt and DMX WT KCs were treated with PBS or LPS(100 ng/mL)for 12 h.The supernatant of KCs(KC-CM)was then collected and used to incubate hepatocytes from WT mice for 12 h.The percentage of hepatocyte death was detected by flow cytometry with AnnexinV/PI staining,and the culture supernatant of hepatocytes was collected to detect the level of ALT and AST.The detection results confirmed that the activation of the STING signaling pathway in KCs led to liver injury in mice with sepsis.Part Ⅱ:Mitochondrial fission-induced mtDNA release mediates the activation of STING signaling pathway in KCs.The expression of DRP1 protein and its phosphorylation level of Ser616 in KCs isolated from LPS-challenged mice were detected by western blot.Then,KCs isolated from WT mice were transfected with shDRP1 before LPS stimulation.The morphology of mitochondria as well as the co-localization of mitochondria and s616p-DRP1 were observed by immunofluorescence.The expression of STING protein and the phosphorylation levels of IRF3 and p65 in KCs were detected by western blot to verify whether LPS-induced STING signaling pathway activation in KCs was related to DRP1-mediated mitochondrial fission.Immunofluorescence was used to detect cytosolic dsDNA in KCs and q-PCR was used to test the content of cytosolic mtDNA fragments of D-loop regulatory region to verify the effect of knocking down the expression of DRP1 on the mtDNA release into the cytosol.After transfection with adenoviral vectors for DRP1-overexpression,the KCs were treated with DNaseI using PULSin transfected regent and then treated with LPS.q-PCR was used to detect the content of cytosolic mtDNA fragments of D-loop regulatory region and the mRNA level of inflammatory factor IFN-β downstream of STING signaling to demonstrate whether DRP1-dependent mitochondrial fission-induced the release of mtDNA activated STING signaling pathway.Mitochondrial ROS(mtROS)was measured by immunofluorescence to verify the effect of knock-down DRP1 expression on mitochondria oxidative stress in LPS-treated KCs.To assess the effects of mtROS on mtDNA release and the subsequent STING signaling activation,mitochondria-targeted antioxidant-mitoquinone(MitoQ)was pre-treatment followed by LPS stimulation in KCs and then the content of cytosolic mtDNA fragments of D-loop regulatory region and the mRNA level of inflammatory factor IFN-βdownstream of STING signaling was detected by q-PCR.The viability of KCs under different LPS stimulation time and concentrations was evaluated by Cell Counting Kit-8(CCK-8),and the oligomerization of VDAC1 was detected by EGS cross-linking and western blot to explore the mtDNA release pores in LPS-treated KCs.Finally,at the animal level,mice treated with DRP1 GTPases inhibitor-Mdivi-1(20 mg/ml)were stimulated with LPS,and the expression of STING protein and IRF3,p65,and DRP1 proteins in KCs of mice in each group were detected by western blot.The levels of IFN-β,TNF-α and IL-1 in serum were detected by ELISA,the percentage of liver cell death was detected by AnnexinV/PI staining,the pathological changes of liver tissue were observed by HE staining,and the liver function was detected by biochemical kits.The above tests were used to explore the mechanism of mitochondrial fission mediated KCs inflammatory activation and the effect of inhibiting mitochondrial fission on sepsis liver injury.Results:Part Ⅰ:STING signaling pathway activation in KCs leads to septic liver injury.1.STING signal pathway is activated in KCs in LPS-induced septic mice.STING positive areas were concentrated in sinusoids and STING protein was expressed in KCs,but not expressed in hepatocytes.The expression of STING protein and the phosphorylation levels of IRF3 and p65 protein in KCs from LPS-induced septic mice were significantly increased,compared with KCs isolated from control mice.Consistent with the activation of STING signaling,downstream inflammatory cytokines including IFN-β,TNF-α and IL-β mRNA levels were also obviously upregulated in KCs from LPS-treated mice.2.STING activation enhanced liver injury in LPS-induced septic mice.Compared with WT mice,STINGgt mice challenged with LPS showed less swelling hepatocytes and lighter inflammation in liver tissues.The serum levels of ALT and AST,as well as IFN-β,TNF-α and IL-1 β were all significantly reduced in STINGgt mice.Compared with WT mice treated with LPS alone,the addition of STING agonist DMX worsened liver injury with more severe morphology changes and higher serum concentrations of ALT,AST and IFN-β,TNF-α,IL-1β as well.Furthermore,STING activation markedly decreased the survival rate of LPS-challenged septic mice.3.STING signaling pathway activation enhances hepatocyte death in LPS-induced septic mice.The apoptotic and necroptotic events in isolated hepatocytes from WT mice were upregulated after LPS treatment,as indicated by the increased cleaved-caspase3 and the phosphorylation of mixed lineage kinase domain-like(MLKL).Comparatively,apoptotic and necroptotic levels in hepatocytes were significantly reduced in LPS-treated STINGgt mice.The addition of STING agonist DMX further worsened hepatocyte death from WT mice treated with LPS.Compared with those incubated with LPS-treated WT KC-CM,hepatocytes incubated with LPS-treated STINGgt KC-CM showed lower death rate and released less ALT and AST,while hepatocytes incubated with DMX+LPS-treated WT KC-CM showed significantly higher death rate and released more ALT and AST.Part Ⅱ:Mitochondrial fission-induced mtDNA release mediates the activation of STING signaling pathway in KCs.1.DRP1-mediated mitochondrial fission triggers STING signaling activation in LPS-treated KCsMitochondrial fission was significantly increased in KCs isolated from LPS-challenged mice,as indicated by the up-regulated phosphorylation of DRP1 at serine 616 site.Knocking down of DRP1 with shRNA significantly inhibited LPS-induced mitochondrial fission and STING signaling pathway activation in KCs.2.DRP1-mediated mtDNA release activates STING signaling pathway in LPS-treated KCsCompared with the NC shRNA+LPS group,transfection of shDRP1 markedly decreased the number of dsDNA in cytosol and the copy numbers of cytosolic D-loops in LPS-treated KCs.While the over-expression of DRP1 enhanced LPS-induced mtDNA release,the application of DNase I significantly reduced the level of cytosolic mtDNA,and inhibited the expression of inflammatory factor(IFN-β)downstream of STING signaling pathway.3.LPS promotes VDAC1 oligomerization in KCsThe cell viability was not significantly affected in KCs treated with 100 ng/mL LPS for up to 24 h in the time-dependent test,and was decreased by up to 500 ng/mL LPS in 12 h in a does-dependent detection,indicating that there was no apoptosis in our experimental setting of KCs treated with 100 ng/mL LPS for 12 h.Compared with the control group,VDAC1 dimers and trimers were markedly increased upon LPS stimulation.These results indicated that cytosolic mtDNA in LPS-treated KCs was not driven by apoptosis but might be released through VDAC1 oligomer pores.4.DRP1-dependent mitochondrial oxidative stress enhances the leak of mtDNA and the subsequent STING signaling activationCompared with the NC shRNA+LPS group,mtROS was down-regulated in shDRP1-transfected KCs.Compared with DMSO+LPS group,the application of mitochondria-targeted antioxidant MitoQ prevented the increase of mtROS,resulting in the amelioration of cytosolic mtDNA release and the attenuation of STING downstream cytokine(IFN-β)expression.5.Mdivi-1 inhibits mitochondrial fission and down-regulates LPS-induced activation of STING signaling pathway in hepatic Kupffer cells of mice with sepsis to alleviate liver injury.After administration of Mdivi-1,the STING signaling pathway in liver Kupffer cells of LPS-induced sepsis mice was inhibited,the levels of inflammatory factors IFN-β、TNF-α、IL-1β in serum were significantly decreased,and the proportion of liver cell death,liver pathology scores,and liver enzyme(ALT and AST)levels were significantly reduced.Conclusion:1.STING signaling activation in KCs played an important role in septic liver injury by promoting hepatocyte death.2.DRP1-mediated mitochondrial fission promoted mtROS generation and mtDNA release into cytosol,which activated STING signaling activation in KCs from LPS-challenged mice.