IL-37 Alleviates Myocardial Injury Induced By CVB3 Via Inhibiting Neutrophil Extracellular Traps Formation

BackgroundViral myocarditis(VMC)is an inflammatory cardiomyopathy related to a lot of viral infections,of which coxsackievirus B3(CVB3)is the most common.Direct viral damage induced by CVB3 and subsequent immune response determine the severity of VMC.Since the pathogenesis of VMC is still unclear,symptomatic supportive therapy is still used in clinical VMC patients,such as arrhythmia,heart failure and so on.Therefore,it is very important to explore a more effective treatment for myocarditis to improve the prognosis of patients with myocarditis.Neutrophils are the most abundant leukocytes in the body and play a crucial role in both innate immunity and acute inflammation response.After being stimulated by aseptic and bacterial inflammation,neutrophils trap pathogens and limit the spread of inflammation by triggering the release of neutrophil extracellular traps(NETs).However,excessive NETs are closely associated with inflammation,autoimmune diseases,thrombosis,and tumor.Recent studies have shown that a lot of NETs were detected in myocardial tissue from both patients and mice with myocarditis.Therefore,the formation of NETs may play an important role in the disease progression of myocarditis,and blocking the process is promising as a new target for the treatment of VMC.Interleukin-37(IL-37)is a natural protector of innate and adaptive immunity which plays.a protective role in a variety of diseases,such as inflammation,cancer,autoimmune diseases aging and so on.A mass of studies has shown that IL-37 can reduce inflammatory cell infiltration,inhibit cell apoptosis and immune response by inhibiting the signal pathway of nuclear factor kappa-B(NFκB).And NFκB plays a pro-inflammatory role in a variety of diseases,triggering inflammatory responses and stimulating the secretion of pro-inflammatory cytokines(IL-1β,IL-6,and TNF-α)to accelerate disease progression.Therefore,we hypothesize that IL-37 can inhibit the formation of neutrophil extracellular traps by regulating the NFκB signal pathway,to reduce the damage of cardiomyocyte in viral myocarditis induced by CVB3 and improve cardiac function.Objective1.Stable expression of IL-37 in mice was induced by caudal vein injection of adeno-associated virus-9(AAV9)-IL-37.2.The VMC model was constructed by intraperitoneal injection of CVB3.After the intervention of AAV9-IL-37 or DNASE1,observing the changes of body weight,survival state and mortality in each group.3.By detecting the expression of inflammatory factors in serum,evaluating myocardial histopathological changes by H&E staining and evaluating cardiac function by echocardiography,exploring the regulatory effects of IL-37 or DNASE1 on myocardial inflammatory response and cardiac function in VMC.4.Exploring the effect of IL-37 on the formation of NETs by detecting NETs in serum and myocardial tissue of mice in each group.5.Neutrophils were isolated from the peripheral blood of healthy individuals.NETs induced by Phorbol 12-myristate 13-acetate(PMA)was detected after the intervention of IL-37 to explore the function of IL-37 on the formation of NETs.6.Exploring the expression of NFκB signaling pathway in acute VMC and the formation of NETs,and elucidating the mechanism of IL-37 on myocardial inflammatory injury and formation of NETs in VMC.MethodsBALB/c male mice aged 4-6 weeks were divided into 4 groups randomly:In VMC+AAV9-NC group and VMC+AAV9-IL-37 group,mice were injected with AAV9-NC or AAV9-IL-37 through the tail vein 7 days in advance.On day 0,mice were intraperitoneal injected CVB3(103TCID50)to induce acute VMC.On days 0 to 7,mice in VMC+DNASE1 group were given DNASE1(50μg/per mouse,twice a day)by intraperitoneal injection,while those in control group were given the isovolumetric solvent.The body weight and general condition of mice were monitored daily,and the survival rate was recorded every day.On day 7,the cardiac function of mice was evaluated by echocardiogram.After the mice were euthanized,the expression level of inflammatory factors in serum of mice was detected by ELISA,and the inflammation of myocardium were evaluated by H&E staining,the dsDNA concentration in serum was determined by picogreen reagent and the compound of MPO-DNA in myocardium was assessed by immunofluorescence staining.The protein levels(IκBα,p-IκBα,NFκB,p-NFκB)of related signaling pathways in VMC were detected by western blot.In vitro,neutrophils were isolated from peripheral blood of healthy individuals,stimulated by PMA(100nM)to release NETs,and interfered with IL-37(0.1ng/ml)or BAY11-7082(2.5μM).After 4h of stimulation,the dsDNA concentration in cell supernatant was determined by picogreen reagent and the compound of MPO-DNA in coverslips was assessed by immunofluorescence staining.Besides,the expression level of related proteins(IκBα,p-IκBα,NFκB,p-NFκB)in neutrophils was detected by western blot.Results1.IL-37 improve general condition and survival rate in mice with acute VMC in the early phase.2.IL-37 improve the cardiac function of CVB3-induced acute VMC in the early phase.3.IL-37 can alleviate the inflammation response of acute VMC induced by CVB3 in the early stage.4.IL-37 inhibit the formation of NETs in the heart of acute VMC mice at early stage.5.IL-37 and NFκB inhibitor(BAY11-7082)inhibits the formation of NETs induced by PMA in neutrophils.6.IL-37 inhibits the phosphorylation of NFκB in acute VMC mice induced by CVB3.7.IL-37 inhibits the phosphorylation of NFκB in neutrophils stimulated by PMA.ConclusionsThis study shows that NETs participate in the early development of acute VMC,Meanwhile,IL-37 inhibits the formation of NETs in VMC mice,thus reducing the inflammatory reaction and myocardial damage in the early stage by NFκB signal pathway.At the same time,we obtained further verification in neutrophils isolated from peripheral blood.Therefore,this study confirmed that IL-37 alleviates CVB3 induced myocardial injury partially by inhibiting the formation of NETs.And this study provides preliminary evidence for IL-37 targeting NETs to treat related diseases.In addition,inhibiting the formation of NETs can also be a new target for the treatment of early myocarditis.