The Role Of CaN/NFAT Pathway In Kawasaki Disease-induced Immune Coronary Artery Lesions

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The role of CaN-NFAT signaling pathway in macrophages on coronary artery injury induced by LCWEObjective:Kawasaki disease is an acute self-limiting systemic vasculitis syndrome that mainly causes coronary dilatation and coronary aneurysm.Serious complications may cause coronary artery stenosis,occlusion,myocardial ischemia or coronary aneurysm rupture.At present,Kawasaki disease has become the most common cause of acquired heart disease in children in developed countries.KD induced coronary arteritis is characterized by granulomatous inflammation involving severe accumulation of monocytes/macrophages infiltration.Abnormal activation of macrophages and lymphocytes is thought to be closely related to the formation of coronaty artery lesions.Previous studies have found that by intraperitoneal injection of Lactobacillus casei cell-wall extract(LCWE)in C57BL/6 mice,the coronary artery injury which was similar with the histopathological finding of KD patients,can be induced in mice.Therefore,it is of great significance to study the pathogenesis of Kawasaki disease-induced immune coronary injury by constructing LCWE-induced mouse model of immune coronary artery injury with intraperitoneal injection of LCWE.Calcineurin(CaN/Cn)is a serine/threonine protein phosphatase enzyme and is a heterodimer that is dependent on the regulation of Ca2+/calmodulin(CaM).Current studies have found that CaN is involved in the regulation of a variety of cellular functions,particularly in the regulation of innate immunity and acquired immune function.The nuclear factor of activated T cell(NFAT)is an important inducible transcription factor that regulates intracellular signal transduction.It was first discovered in T cells.Until now it has been found in most immune cells including dendritic cells,macrophages,NK cells,etc,which is involved in the differentiation and regulation of immune cells.As the main substrate of CaN,NFAT's activity can be regulated by CaN,which is activated mainly by the receptor-Ca2+-CaN signaling pathway.Activated CaN can cause NFAT dephosphorylation of multiple residues in the N-terminal regulatory region NHR,enhance the affinity of NFAT to the target DNA,and translocate NFAT into the nucleus to induce transcription of the target gene.By using GWAS and linkage disequilibrium analysis to investigate the susceptibility genes of Kawasaki disease,it is found that CaN/NFAT signaling pathway plays an important role in the pathogenesis of Kawasaki disease,but the mechanism is still unclear.Therefore,the main purpose of this study is to investigate the role of Ca2+/CaN/NFAT signaling pathway in macrophages on coronary artery lesions induced by LCWE.Methods:Construct the NFAT-luciferase reporter system and use LCWE to stimulate RAW264.7 cell in vitro to detect whether LCWE can induce the activation of NFAT signaling pathway.And detect the expression of NFAT1,NFAT2 and related cytokines by using flow cytometry,ELISA,WB and qPCR.Using LCWE to induce mouse model of immune coronary artery lesions,study NFAT 1,NFAT2 and related cytokines expression by WB,qPCR and ELISA.By extracting cytoplasm and nuclear protein separately,study whether LCWE could induce NFAT nuclear translocation in macrophages.Study the effects of Ca2+/CaN-NFAT signaling pathway by knocking down or overexpressing NFAT1 or NFAT2 in macrophages.Using NFAT-luciferase reporter system,ELISA,WB,ICC and qPCR to investagate the effects of calcineurin inhibitor CsA on CaN/NFAT signaling pathway in LCWE-induced macrophages.Results:1.The microphotographs showed significant phenotypic changes of LCWE-treated RAW 264.7 cells when compared with control group.The ELISA and qPCR results showed that the IL-6,IL-1(3 and TNF-a in the supernatant of LCWE-treated macrophages increased signifantly(P<0.05),which is suggested that the macrophages can be activated when treated with LCWE in vitro.2.WB and qPCR results showed that after treated by LCWE,the expression of NFAT1 and NFAT2 is up-regulated(P<0.05).By using NFAT-luciferase reporter system and detecting nuclear levels by WB,the results showed that LCWE can promote NFAT pathway activation and induce NFAT nuclear translocation.All of these results suggested that LCWE can induce the activation of NFAT signaling pathway in macrophages in vitro.3.By using LCWE to induce KD vasculitis mouse model,the WB results showed that the expression of NFAT1 and NFAT2 increased significantly in myocardial tissue and bone marrow-derived macrophages on the 14th day after LCWE injected(P<0.05).The serum levels of IL-6,IL-1?,TNF-a,MCP-1 and MIP-la on the 14th day and 28th day increased significantly(P<0.05).It is suggested that LCWE can induce the activation of NFAT signaling pathway in LCWE-induced coronary artery injury model.4.After overexpressing NFAT1 or NFAT2,the IL-6,IL-1 p and TNF-a produced by LCWE-treated macrophages was elevated significantly(P<0.01).After knocking down NFAT1 or NFAT2,the IL-6,IL-1? and TNF-a decreased significantly(P<0.01),which indicated that NFAT signaling pathway played an important regulatory role in the synthesis and secretion of IL-6,IL-1? and TNF-a in LCWE-induced macrophage activation.5.By pretreatment with CsA,the NFAT-luciferase reporter system showed that NFAT-luc activity decreased significantly in CsA+LCWE group than that in LCWE group(P<0.05).The WB and qPCR results showed that CsA could down-regulate the expression of NFAT1 and NFAT2 induced by LCWE(P<0.05).The ELISA and qPCR results showed that the expression of IL-6,IL-1?and TNF-a decreased significantly after treated with CsA(P<0.01).The ICC results showed that CsA can effectively inhibit the nuclear translocation of NFAT induced by LCWE.All of these results indicated that CsA could effectively inhibit the CaN/NFAT pathway activation in LCWE-treated macrophages,which was enrolled in the regulation of IL-6,IL-1?,TNF-a production.Conclusions:1.LCWE can induce the activation of NFAT signaling pathway in macrophages in vitro.2.LCWE can induce the activation of NFAT signaling pathway in LCWE-induced coronary artery injury model,and the expression of related pro-inflammatory cytokines and chemokines are up-regulated.3.By Overexpression and knockdown of NFAT1 and NFAT2,it is suggested that CaN/NFAT signaling pathway plays an important regulatory role in the synthesis and secretion of proinflammatory cytokines such as IL-6,IL-1? and TNF-a in LCWE-induced macrophage activation.4.As a calcineurin inhibitor,CsA could down-regulate the expression of NFAT1 and NFAT2,and inhibit the activation of NFAT signaling pathway in macrophages induced by LCWE,and regulate expression of related pro-inflammatory cytokines and chemokines such as IL-6,IL-1? and TNF-a.Part ? The elevated serum levels of calcineurin and nuclear factor of activated T-cells 1 in children with Kawasaki diseaseObjective:Kawasaki Disease(KD)is an acute self-limiting immune vasculitis syndrome while its etiology and pathogenesis are still unclear.The main complications caused by KD are coronary artery dilatation and coronary aneurysm.Serious complications may cause coronary artery stenosis,occlusion,myocardial ischemia or coronary aneurysm rupture.By using GWAS and linkage disequilibrium analysis to investigate the susceptibility genes of Kawasaki disease,it is found that CaN/NFAT signaling pathway plays an important role in the pathogenesis of Kawasaki disease,but the mechanism is still unclear.The first part of this study has found that LCWE can induce activation of CaN/NFAT signaling pathway in macrophages,indicating that CaN/NFAT pathway played an important role in KD-induced immune coronary lesions.Therefore,the main purpose of this study is to evaluate serum calcineurin(CaN)and nuclear factor of activated T-cells(NFAT1)levels in KD patients at different stages in order to further explore the role of CaN/NFAT signaling pathway in Kawasaki diseaseMethods:Serum levels of CaN and NFAT1 were measured by enzyme-linked immunosorbent assay method in 66 healthy children and 74 KD patients at acute,afebrile and subacute stage.Compare the differences of CaN and NFAT1 serum levels between IVIG responding and non-responding KD patients and also in KD patients with or without CALs.Results:The serum levels of CaN and NFAT1 increased significantly in the acute stage,and decreased progressively in the afebrile and subacute stage,along with the reduction of C-reactive protein,white blood cells and neutrophil.And in the acute stage,the afebrile stage and the subacute stage,the expression of CaN and NFAT1 was upregulated significantly in KD patients compared to that in the healthy control.After treatment,the CaN serum levels of IVIG responders decreased significantly when compared with that in acute stage of KD(P<0.05).However,there is no significantly decrease in the CaN serum levels of IVIG non-responders after treatment(P>0.05).The serum levels of CaN and NFAT1 had no difference between intravenous immunoglobulin responders and non-responders.There is no difference in CaN serum levels with or without coronary artery lesions.Nevertheless,in the afebrile stage,the NFAT1 levels were lower in KD patients with coronary artery lesions(CALs)(269±12 pg/ml)than those without CALs(286 ± 25 pg/ml).Conclusions:CaN and NFAT1 may be involved in the pathophysiology of KD,which brings novel treatment evidence of CNIs for Kawasaki disease.After treatment,the CaN serum levels of IVIG responders decreased significantly when compared with that in acute stage of KD.But there is no significantly decrease in the CaN serum levels of IVIG non-responders,which is suggested that IVIG non-responders may benefit from CsA treatment by precisely suppressing abnormal activation of CaN/NFAT signal pathway.