Research On CpG-ODN Regulation Of PD-L1 In Experimental Autoimmune Myocarditis

BackgroudMyocarditis is a combination of a viral infection,drug toxicity and other causes of acute inflammatory heart disease,accompanied by inflammatory cell infiltration and myocardial cell injury or necrosis.Part of myocarditis can be quickly developed to dilated cardiomyopathy,while serum samples of these patients can be detected different kinds of autoantibodies,which confirmed that autoimmune myocarditis plays an important role in the pathogenesis of dilated cardiomyopathy.Therefore,the establishment of an appropriate animal model of autoimmune myocarditis and the simulation of the pathogenesis is of crucial importance to the research of pathogenesis of myocarditis and autoimmune injury,treatment and prognosis.This study was to establish an appropriate mouse model of autoimmune myocarditis by PCR and immunohistochemical methods.It researched the expression of programmed death ligand 1(PD-L1)in mice with autoimmune myocarditis,finding a way to regulate cardiac endothelial cells to express PD-L1.It's expected to afford a new direction in research and clinical treatment of myocarditis.Materials and methodsMaterials and methods(1)Building two T-cells transferred induced models of autoimmune myocarditis,the expression level of PD-L1 was observed in both models by immunohistochemistry,PCR and other methods.(2)Successively using DYNAbeads of anti-PECAM-1 antibody and anti-ICAM-2 antibody to isolate and purify cardio-endothelial cell,then authenticate endothelial cell by observing anti-CD31 antibody and VE-Caherin staining.(3)Exploring the regulation of IFN on PD-L1 of mouse cardiac endothelial cells through vivo study.(4)Exploring the expression of Oligodeoxynucleotides containing Cp G motifs(Cp G-ODN)in PD-L1 of mouse cardiac endothelial cells through vivo study.(5)Establishing models of autoimmune myocarditis,calculate pathological scores by paraffin-embedded sections and observe the function of Cp G-ODN in autoimmune myocarditis treatment.(6)Detecting the expression levels of IL-1?,TNF-? in hearts by PCR.Results(1)The expression of PD-L1 of heart's m RNA in both kinds of autoimmune myocarditis is significantly higher.(2)The expression of PD-L1 is consistent with CD31 in heart through immunohistochemical method.(3)The mouse cardiac microvascular endothelial cells purified through twice sorting method get good growth,normal morphology,99.9% positive rate of endothelial cells' CD31 staining,and the 85.1% expression rate of VE-Caherin.They could be stably transferred in vitro culture and remains good activity after resuscitating from cryopreservation.(4)Real-time PCR showed that PD-L1 of mouse cardiac endothelial cells are statistically significantly raised after 8 hours' stimulation by 10 U / ml IFN-? and 100 U / ml IFN-?.(5)In vivo,it was found that IFN-? and IFN-? fail to upregulate the expression of PD-L1 m RNA as well as PD-L1 protein.(6)In vivo,it also found Cp G-ODN can increase the expression of PD-L1 m RNA,which was confirmed via immunohistochemical method.(7)Analyzed by HE staining and histopathological scores of myocarditis mice,it was found that compared to PBS intervention group,the prophylactic use of Cp G-ODN could alleviate the extent of autoimmune myocarditis.(8)By RT-PCR,,IL-1? expression of CpG-ODN group was significantly lower than the PBS control group,IL-1?(0.313 ± 0.022)vs.(1.180 ± 0.148);TNF-?(0.075 ± 0.011)vs.(0.255 ± 0.018).ConclusionPD-L1 involved in pathogenesis of autoimmune myocarditis.Cp G-ODN could upregulate the expression of PD-L1 of endothelial cells,which reduced the degree of inflammation of myocarditis.Based on this result,CpG-ODN is likely to be an effective method for the treatment of autoimmune myocarditis.