Interleukin-37 Ameliorates Coxsackievirus B3-induced Viral Myocarditis By Modulating The Th17/Regulatory T Cell Immune Response

BackgroundViral myocarditis is a heterogeneous group of disorders defined by inflammation of the heart muscle associated with an excessively activated immune response.Numerous interventions have been investigated for the treatment of myocarditis;however,success has been limited.IL-37.a novel member of the IL-1 cytokine family,is a natural inhibitor of innate immunity associated with autoimmune diseases.Although no murine homologue of IL-37 has been identified,human IL-37 is functional in mice.Emerging studies have found that the balance of Thl7 cells and regulatory T cells(Tregs)is involved in the pathogenesis of myocarditis and can be regulated by IL-37 to ameliorate the inflammatory response in autoimmune diseases.However,the modulatory effect of IL-37 on the Thl7/Treg immune response during myocarditis is unknown.ObjectiveIn this study,we investigated the immunological regulation of IL-37 in the coxsackievirus B3(CVB3)-induced model of murine viral myocarditis.By observing the general manifestation changes,myocardial histopathology,echocardiographic indexes,spleen Th17 cells and Tregs ratio and serum concentrations of inflammatory factors,we studied the therapeutic effect and therapeutic mechanism of IL-37 on viral myocarditis,and found a new breakthrough for the treatment of viral myocarditis.MethodsFour-week-old male BALB/c mice were randomly divided into 3 groups:(1)Control mice were injected i.p.with 100 ?l of PBS and then received i.p.injections of 200 ?l of normal saline.(2)Virus mice were injected i.p.with 100 ?l of CVB3 diluted in PBS and then received an equivalent volume of saline.(3)IL-37 mice received both 100 ?l of CVB3 and 2 ?g of recombinant human IL-37 suspended in 200 ?l of saline.The injection was given on day 0.The general condition of each group of mice was observed,such as body weight change,survival rate,eating situation,coat color change,mental state and so on.Mice were sacrificed on day 7 and day 10:(1)The inflammatory cell infiltration and interstitial fibrosis were observed by H&E staining and Masson trichrome staining,respectively,and quantified by quantitative method.(2)The cardiac function of mice was quantitatively evaluated by echocardiography measurement.(3)The proportion of Th17 cells and Tregs in spleen was evaluated by flow cytometric analysis and the mechanism of IL-37 in improving the progression of myocarditis was also deduced.(4)The concentrations of Th17 cells and Tregs related inflammatory factors such as IL-17A,IL-6 and IL-10 were determined by ELISA assay.(5)Real-time PCR analysis was used to detect the expression of ROR?t,IL-17A,IL-6,Foxp3 and IL-10,which were related to Th17 cells and Tregs,in order to further clarify the mechanism of IL-37.ResultsThe results show that IL-37 significantly ameliorates the symptoms of myocarditis with improved histological changes,increased survival rate and bodyweight,and a decreased proportion of Th17 cells and increased numbers of Tregs in the spleen.Moreover.IL-37 down-regulates retinoic acid-related orphan receptor ?t(RORyt)expression and the levels of IL-6 and IL-17A,while promoting Treg-related forkhead box p3(Foxp3)expression and IL-10 levels in the heart and serum.ConclusionTherefore,IL-37 may exhibit anti-inflammatory activity in the murine model of myocarditis by regulating the balance between Th17 and Treg cells,thereby providing a possible novel therapeutic target in myocarditis.