IL-37 Alleviates Viral Myocarditis Via Inhibiting NLRP3 Inflammasome-mediated Pyroptosis

Background:Viral myocarditis(VMC)is a kind of a non-specific inflammatory disease in myocardium,which is an important cause of heart failure and sudden death in young people.It is mainly caused by viral infection.The clinical manifestations including fatigue,chest pain,congestive heart failure,cardiogenic shock and even cardiac arrest.Although most viral myocarditis is self-limited,some patients developed into dilated cardiomyopathy,malignant arrhythmia and heart failure,which seriously affect the quality of life.There is still no specific treatment for VMC.Antiviral and symptomatic supportive treatment is not significant.For a long time,scholars have devoted themselves to the study of the pathogenesis of viral myocarditis,including direct viral damage,immune response and various cytokine-mediated myocardial damage,but the mechanism is still unclear.Pyroptosis refers to inflammatory cell programmed death,which is mediated by inflammasomes.Pyroptosis is accompanied by the release of many inflammatory cytokines,such as IL-1β and IL-18.The inflammasome is an intracellular protein complex,and it is composed of three parts:pattern recognition receptor in cytoplasm,adapted protein ASC and cysteine Caspase-1 inflammasome activates GSDMD,causing the cell membrane to rupture and release IL-1β and IL-18.Inflammasomes are related to many inflammation-related diseases,such as myocardial infarction and ischemia-reperfusion injury.Recent studies have shown that there are over-activated inflammasomes in VMC patients and mice.Therefore,cardiomyocyte pyroptosis may play an important role in the pathogenesis of VMC,and blocking this process may become a new target for the treatment of VMC.Interleukin 37(IL-37)belongs to the IL-1 family and is a potential inhibitor of inflammation.It plays an anti-inflammatory effect in ankylosing spondylitis,ulcerative colitis,small cell lung cancer and other diseases.Previous studies have shown that IL-37 can reduce the inflammatory response of viral myocarditis in mice and reduce mortality.However,the effect of IL-37 on myocardial cell pyroptosis in viral myocarditis and its mechanism are still unclear.Therefore,we established a viral myocarditis model in this study and applied IL-37 to intervene in model mice to uncover the role of IL-37 in the pathological process of viral myocarditis,and further explored the involvement of IL-37 in myocardial cell pyroptosis.This study is divided into two parts:Part I:Establishment of an Animal Model of Viral Myocarditis in MiceBackgroundMyocarditis refers to an inflammatory disease of the myocardium,which is diagnosed by non-invasive imaging techniques such as electrocardiogram,serum virological detection,echocardiography,and endocardial biopsy(EMB).Approximately 1.5 million people worldwide suffer from myocarditis each year,which can manifest as acute,subacute or chronic myocarditis.Myocarditis can affect people of any age,and is most common in adolescents.Viruses are the most common pathogen that causes myocarditis.Among the most common viruses are enteroviruses,including Coxsackie virus.Parvovirus B-19 and human herpesvirus 6 are also common pathogens.Bacteria,fungi,rickettsia,etc.can also cause myocarditis,but it is relatively rare.Patients with viral myocarditis have different symptoms,depending on the extent of the cardiomyopathy.Mild patients may have no conscious symptoms.Some patients have a prodromal period of about 7-20 days before the onset,which manifests as fever,fatigue,and muscles.Pain and other infection symptoms.The subsequent clinical manifestations include chest pain,arrhythmia,cardiogenic shock and sudden cardiac arrest.It is generally believed that myocardial damage in patients with viral myocarditis is mainly caused by four aspects:direct damage of the virus;immune response triggered by the virus;the role of cytokines in the process of inflammation and the apoptosis of myocardial cells induced by various reasons.After the virus invades,it replicates in the myocardium and activates the immune system.Its pathophysiological progress usually has three stages:acute stage,subacute stage and chronic stage.The acute stage refers to the direct toxic effect of the virus on the myocardium.The subacute stage is mainly manifested by the activation of T cells and B cells and the autoimmune response triggered by it.The chronic stage is manifested by diffuse myocardial fibrosis and cardiac dysfunction,such as dilated myocardium.disease.A large amount of pathological evidence indicates that viral myocarditis is manifested by myocardial inflammatory cell infiltration,mainly including monocytes,including lymphocytes,macrophages,dendritic cells(DC)and natural killer(NK)cells.The prognosis in VMC patients depends on the pathogenisis,clinical symptoms,and whether the initial treatment is timely.Poor prognostic factors include decreased ejection fraction and left bundle branch block,etc.Patients with mild myocarditis usually have a good prognosis.However,the most common cause of death in severe myocarditis is cardiogenic shock,with a 1-year mortality rate of 20%and a 5-year mortality rate of 50%.However,there is still no specific treatment at this stage.Only antiviral and supportive treatment is given,and the therapeutic effect is not significant.In the first part of this experiment,we established the model of viral myocarditis by intraperitoneal injection of viral myocarditis into balb/c mice,observed the pathological characteristics of viral myocarditis,explored its pathological mechanism,and provided some new ideas for treatment of viral myocarditis.Objective1.To establish a model of viral myocarditis in mice by intraperitoneal injection of CVB3 virus and observe the pathological changes of myocardial injury in mice.2.To observe the general state of mouse,including vitality,hair,etc.And to detect the cardiac function and cardiac pathological changes.To evaluate the feasibility of establishment of VMC model induced by CVB3.Methods1.Establishment of an animal modelTwelve male 6-week-old balb/c mice were reared in the SPF animal room of Shandong Academy of Medical Sciences.The mice were randomly divided into the control group(n=6)and the VMC group(n=8).On Day 0,mice in the control group had intraperitoneal injection with 100gL PBS,and mice in the VMC group were injected with 25μl 109TICD50(Add 75μl PBS to dilute it to 100μl).An animal model of acute myocarditis was established on day 9.2.EchocardiographyOn Day 9,mice were anesthetized with 3%isoflurane.We collected parasternal long-axis images by Visual Sonics high resolution Vevo 2100 system(Visualsonics Inc.,Toronto,Canada).The left ventricular ejection fraction(LVEF),left ventricular shortening fraction(LVFS),systolic septal thickness(IVSs),diastolic septal thickness(IVSd),systolic left ventricular diameter(LVIDs),diastolic left ventricular diameter(LVIDd),the systolic left ventricular posterior wall thickness(LVPWs)and the diastolic left ventricular posterior wall thickness(LVPWd)of each group were recorded.3.Histological examinationAll 12 balb/c mice were sacrificed on the day 9.The heart tissue was immersed in 4%paraformaldehyde for 24 to 48 hours.After being embedded in paraffin,tissues were cut into 5 μm thick sections.Then the sections were stained with hematoxylin and eosin(H&E)and Masson.4.ELISA testCollect peripheral blood in each group.And collect the upper serum after centrifugation.The specific ELISA kits were used to detect the myocardial injury marker cTnI and the inflammatory factors IL-1β and IL-18.Results1.General statusThe mice in the control group were vigorous and had bright hair.Compared with the mice in the control group,the mice in the VMC group had poor vigor,and did not like to move.Mice in VMC group and had obvious hair loss and had no obvious response when grabbing.The weight of the mice in the VMC group were lower than the control group,and the heart weight/body weight ratio was increased in the VMC group.2.Decreased left heart function in VMC groupThe IVSs(P<0.01)and IVSd(P<0.05)of the VMC group mice were significantly lower than the control group.The values of LVEF and LVFS decreased in the VMC group,indicating that the ventricular septum was significantly thinner after intraperitoneal injection of CVB3 in mice.The contractile function of myocardium may be impaired.3.Myocardial inflammatory injury in the VMC group was obviousHE staining showed regular arrangement of myocardial fibers in the control group,without obvious inflammation infiltration.HE staining of mice in the VMC group showed some cell necrosis and inflammatory lesions,with obvious interstitial inflammation infiltration.The levels of serum cTnI(P<0.01),IL-1β(P<0.05)and IL-18(P<0.05)in the VMC group was significantly higher than the control group.4.Myocardial fibrosis was obvious in the VMC groupMasson staining showed obvious collagen fiber distribution in myocardial interstitium in the control group.The VMC group showed much areas of focal necrosis and collagen fibers.Conclusion1.Intraperitoneal injection of CVB3 into balb/c mice can successfully establish a mouse viral myocarditis model.2.Myocardial damage in mice with viral myocarditis includes increased levels of serum inflammatory cytokines,inflammatory infiltration and myocardial fibrosis in myocardial tissue.Part II:Study on the role and mechanism of IL-37 in viral myocarditis myocardial cell pyrolysisBackgroundViral myocarditis(VMC)refers to the non-specific inflammation of myocardium caused by virus infection,including CVB3.Its clinical manifestations vary from person to person.Although some patients have mild clinical manifestations and even self-limiting symptoms,viral myocarditis can still cause a series of serious complications,including arrhythmia,dilated cardiomyopathy,and heart failure.It is reported that VMC can account for about 10%of sudden deaths among young people.The pathogenesis of VMC is not fully clear.Recent studies have confirmed that the excessive activation of inflammasome plays an important role in diseases such as myocarditis,colitis and atherosclerosis.Inflammasome is a kind of intracellular protein complex,which is composed of cytoplasmic pattern recognition receptor,aptamer protein ASC and cysteine protease-1.Among them,the most widely studied pattern recognition receptor is NOD-like receptor.NLRP3 of the body family.When NLRP3 is activated by cytotoxin,ATP or cholesterol,ASC and Caspasel are further recruited,the inflammasome is activated and further cut IL-1β and IL-18.At the same time,NLRP3 inflammasome activates the lysis of Gasdermin D,releases its N-terminal domain,forms cell membrane pores,releases inflammatory factors,and causes a series of immune responses.Therefore,inhibiting the expression of inflammasomes may provide new ideas for the treatment of viral myocarditis.IL-37 is a potential inflammation inhibitor of the IL-1 family,which can inhibit the body’s innate immunity and adaptive immunity.IL-37 is low expressed in human peripheral blood monocytes and dendritic cells,while IL-37 is not expressed in mice.IL-37 has been proven to ameliorate viral myocarditis in mice caused by CVB3,but its specific mechanism is not clear.Therefore,this part of the experiment uses IL-37 to intervene in viral myocarditis mice to observe its effect on inflammatory infiltration and NLRP3 inflammasome expression in myocarditis mice,and to explore the effect of IL-37 on viral myocarditis and the specific mechanism.Objective1.To establish a viral myocarditis model,and use IL-37 to intervene in mice with viral myocarditis,and observe the effects of IL-37 on the general state,cardiac function and myocardial pathology of mice with viral myocarditis.2.To explore the specific mechanism of IL-37 to alleviate viral myocarditis in mice by detecting the expression of pyroptosis in the cardiomyocytes of each group of mice.Methods1.Intervention of IL-37 in mice with viral myocarditisThirty-two male 6-week-old balb/c mice were divided into 4 groups randomly:control group(n=6),IL-37 control group(n=6),VMC group(n=10),and IL-37 group(n=10).On Day 0,mice in the control group and IL-37 control group were injected with 100μl PBS,and mice in the VMC group and IL-37 group undergone intraperitoneal injection of 25μl 109TICD50(Add 75μl PBS to dilute it to 100μl).On Day3 and Day7,mice in the control group and VMC group were injected with 200μl of PBS,and the IL-37 control group and IL-37 group were intraperitoneally injected with 2μg IL-37(with 200μl PBS diluted).After 9 days,an animal model of acute myocarditis was established.2.Measurement of cardiac functionThe left ventricular ejection fraction(LVEF),left ventricular shortening fraction(LVFS),systolic septal thickness(IVSs),diastolic septal thickness(IVSd),systolic left ventricular diameter(LVIDs),diastolic left ventricular diameter(LVIDd),the systolic left ventricular posterior wall thickness(LVPWs)and the diastolic left ventricular posterior wall thickness(LVPWd)of each group were recorded.3.Histology and immunohistochemistryWe collect heart specimens of each group after all mice were sacrificed on Day 10,and the myocardial tissue was fixed and embedded in paraffin.5μm-thick sections were then stained with hematoxylin and eosin(H&E)and Masson.And GSDMD was detected by immunofluorescence staining.4.ELISA5.Collect peripheral blood in each group.And collect the upper serum aftercentrifugation.The specific ELISA kits were used to detect the myocardial injury marker cTnI and the inflammatory factors IL-1β and IL-18.5.Western blotWe collected the myocardial tissue of each group and extracted the total protein.NLRP3,ASC dimer,cleavaged caspase 1,NFκB P65 and phospho-P65 were detected by Western blo.5.RT-PCRThe myocardial tissue of each group of mice was collected,and total RNA of myocardial tissue was extracted.Real-time fluorescent quantitative PCR technology was used to detect the mRNA expression levels of NFκB P65.Results1.IL-37 improves the general state of mice in VMC groupCompared with the mice in VMC group,mice in IL-37 group were more vigorous with smooth hair,and had obvious response when grasped.Compared with the VMC group,mice in the IL-37 group lost less weight,and the heart weight/body weight ratio was lower(P<0.05).2.IL-37 improves heart function in mice with viral myocarditisCompared with the VMC group mice,the LVEF(P<0.05),LVFS(P<0.05),IVSs(P<0.01)and IVSd(P<0.01)in experimental group mice were all improved to varying degrees,indicating that the ventricular septum was thicker in the experimental group,and the left ventricular systolic function was improved.3.IL-37 reduces myocardial damage in VMC groupHE staining showed that compared with VMC group,IL-37 group had less infiltration of myocardial inflammation,less necrosis of myocardial cells,and less interstitial inflammation infiltration.Elisa examination showed that the expression of cTnI(P<0.01)and IL-18(P<0.05)in serum of IL-37 group was lower.4.The expression of NLRP3 inflammasome was increased in the VMC group of mice and decreased in the IL-37 groupWestern blot detection showed that the protein expression levels of NLRP3(P<0.01),ASC(P<0.05)and cleaved-caspase 1(P<0.05)in the VMC group were higher than control group.In the IL-37 group,the expressions of NLRP3(P<0.05),ASC dimer(P<0.001)and cleaved-caspase 1(P<0.05)were lower.The expression of GSDMD in the VMC group was higher the control group in immunofluorescence staining(P<0.01).The expression of GSDMD in the IL-37 group was lower(P<0.01)than VMC group.5.IL-37 reduces NLRP3 inflammasome expression in mice with myocarditis by inhibiting NFκBWestern blot detection showed the expression of NFκB P65(P<0.05)and phospho-P65(P<0.01)were significantly increased in the VMC group.NFκB P65(P<0.01)and phospho-P65(P<0.01)was lower in the experimental group than the VMC group.The expression levels of P65(P<0.05)and phospho-P65(P<0.05)were significantly lower in the IL-37 control group than the control group(P<0.05).RT-PCR detection showed that the expression of P65 in the VMC group was higher than that in the control group(P<0.05),and higher than the experimental group(P<0.05).And the expression level of P65 mRNA in the IL-37 control group was lower than the control mice(P<0.05).Conclusion1.IL-37 reduced myocardial damage and improved the general state of mice in VMC group.2.NLRP3 inflammasome is involved in CVB3-induced viral myocarditis,and IL-37 inhibits the expression of NLRP3 inflammasome.3.IL-37 may alleviate viral myocarditis in mice by inhibiting NLRP3 inflammasome via NFκB pathway.