Cardiac Fibroblast NLRP3 Inflammasome Activation In Sepsis: Role Of Mitochondrial Reactive Oxygen Species

ObjectiveSepsis is a common disease in intensive care unit in hospital.It is reported that the morbidity of sepsis has reached 437 per 100,000 person-years,and the incidence has more than doubled in the past decade.At the same time,the cost of treatment for sepsis is high.In 2011,the cost of treatment for sepsis in the United States exceeded $20 billion,accounting for 5.2% of the total hospital expenses in that year.Sepsis is induced by exaggerated host responses to infection with high mortality.Heart is one of the important organs affected by sepsis.It has been reported that myocardial dysfunction plays an important role in induction of multiple organ failure and subsequent high mortality of patients with sepsis.To date,however,the underlying mechanism that leads to myocardial dysfunction in sepsis remains unclear.Inflammasome is an intracellular protein complex that serves as a platform for the maturation and activation of caspase-1,leading to proteolytic maturation and secretion of IL-1? and IL-18.It can be activated through intracellular sensor proteins which interact with a diverse set of inflammatory stimuli including pathogen-associated molecular patterns(PAMPs)and damage-associated molecular patterns(DAMPs).Nod-like receptor(NLR)family(NLRP1,NLRP3,and NLRP4)and AIM2 are sensor proteins of inflammasomes.The NLRP3 is a unique sensor which responds to diverse physical and chemical stimuli,as well as cell stress signals,such as reactive oxygen species(ROS),extracellular adenosine triphosphate(ATP),etc.It is reported that there is a dual signaling pathway in the activation of NLRP3 inflammasome.Signal 1(priming)is provided by microbial molecules or endogenous cytokines and leads to the upregulation of NLRP3 and pro-IL-1? through the activation of the transcription factor NF-?B.Signal 2(activation)is provided by a plethora of stimuli,such as ATP,particulate matter,viral RNA,and pore-forming toxins,and activates the NLRP3 inflammasome.Activation of the inflammasome triggers proteolytic cleavage of dormant procaspase-1 into active caspase-1,which converts the cytokine precursors pro-IL-1? and pro-IL-18 into mature and biologically active IL-1? and IL-18,respectively.The NLRP3 is predominantly expressed in cardiac fibroblast(CF)and endothelial cell within the heart,while the levels of NLRP3 in the cardiac myocytes are limited.Our previous studies demonstrated that activation of NLRP3 inflammasome in cardiac fibroblast contributes to sepsis-induced myocardial dysfunction.The CF releases IL-1? that mediates interaction of the CF and cardiomyocytes that leads to decrease in myocardial contractility.However,the activation mechanism of the CF NLRP3 inflammasome in sepsis remains to be further investigated.To this end,we evaluate the role of mitochondria derived reactive oxygen species(mt ROS)in activation of the NLRP3 inflammasome of CF in sepsis.MethodsThe CFs were isolated from hearts of 3-4 weeks old mice.Cultured CFs were challenged with LPS(1 ng/m L)for 6 h(priming),followed by ATP(3 mmol/L)treatment for 0.5 h(activation).Some of LPS-primed CF were pretreatmented with mito-TEMPO(a mitochondrial ROS scavenger,25 ?mol/L)30 min prior to ATP challenge.Mitochondrial dynamic was determined by mitochondrial fragmentation counts and CF mt ROS was detected with Mito SOX reagent.CF NLPR3 inflammasome activation was assessed with Western blot for NLRP3,caspase-1,ASC and ELISA for detection of IL-1?.The interaction between NLRP3 and ASC was determined by Co-immunoprecipitation.ResultsIn cardiac fibroblasts challenged with LPS and ATP,the mitochondrial fragments counts increased(p < 0.05)and subsequently intracellular mitochondrial ROS production increased(p < 0.05).After adding Mito-TEMPO,mitochondrial reactive oxygen species decreased in NLRP3 inflammasome activation in cardiac fibroblasts(p < 0.05),furthermore reducing the interaction between NLRP3 and ASC proteins,inhibiting the activation of NLRP3 inflammasome,and reducing IL-1? production(p < 0.05).Conclusion1? Mitochondrial fission and ROS production in CF increased after LPS and ATP treatment.2? Mitochondrial ROS activates NLRP3 inflammasome in CF via promotes the NLRP3 and ASC interaction.3? Inhibition of mitochondrial ROS prevents the sepsis-induced NLRP3 inflammasome activation and IL-1? production by the cardiac fibroblast.Taken together,our data indicate that mitochondria of cardiac fibroblast incurred increased mitochondrial fission and subsequently mitochondrial ROS production.The increase in mt ROS result in the NLRP3-ASC interaction and consequently activated the NLRP3 inflammasome and iled to the IL-1? production by the cardiac fibroblasts in sepsis.