The Study Of Nup98 In Th17 Cell Differentiation In Acute Viral Myocarditis

Part 1.The Nup98 expression of CD4+ T cell in patients with acute viral myocarditis.Background:Acute viral myocarditis(AVMC)is an inflammatory disease of the myocardium caused by virus.Th17 cell has been reported to play a vital role in AVMC progression.Recently,functions of nucleoporins have attracted more and more attention.Nup98 is an important nuclear protein associated with viral infection and cell differentiation.However,the expression of Nup98 in CD4+ T cells of AVMC patients has not been elucidated.Methods:A total of 21 acute viral myocarditis patients with CVB3 IgM positive were enrolled for this study in Union Hospital,Huazhong University of Science and Technology from from June 2014 to January 2015.Besides,23 healthy volunteers were recruited as controls.All of them have signed informed consent for this study.The percentage and absolute number of Thl7 cells in peripheral blood were determined by flow cytometry.Peripheral CD4+ T lymphocytes were isolated and then western blot were performed to determine the protein expression of Nup98 and RORyt.The serum IL-17 levels were measured by ELISA.Virus titers of peripheral CD4+ T cells in healthy volunteers and AVMC patients were detected by plaque assay.The expression of CVB3 mRNA in CD4+ T cells were determined by RT-PCR.Result:Compared with healthy controls,the proportion and absolute number of Thl7 cells in peripheral blood as well as the level of serum IL-17 of AVMC patients were increased(P<0.05).At the same time,Nup98 expressions of CD4+ T cells in AVMC patients were lower than that of controls,while RORyt expressions were higher(P<0.05).Virus titers and CVB mRNA expression were elevated in CD4+ T cells isolated from peripheral blood of AVMC patients.Conclusion:The Nup98 expression of CD4+ T cells was down-regulated in AVMC patients while the percentage of Th17 cells,the secretion of IL-17.Virus were detected in CD4+T cells isolated from AVMC patientsPart 2.The effect and mechanism of Nup98 in CVB3-induced Th17 cell differentiationBackground:CVB3is a kind of small RNA virus belongs to enteroviruses,which is a main pathogen leading to viral myocarditis.It is known that CVB3 can cause severe damage to cardiomyocytes in AVMC.However,the direct effect of CVB3 on immune cells,especially the CD4+ T cell,is unclear.In the previous study,we found that the Nup98 expression of CD4+T cells decreased in AVMC patients while the virus titer,RORyt(Th17 transcription factor)expression increased.Whether CVB3 can directly induce CD4+ T cells differentiate into Th17 cells via Nup98 is still under investigation.Methods:We isolated the peripheral blood CD4+ T cells of healthy volunteers and infected these cells with CVB3.Supernatant and cells were collected for further experiments such as plaque assay,flow cytometry,Western blot,RT-PCR and ELISA.In addition,293T cells and Chinese hamster ovary(CHO)cells were used as positive and negative controls to detect whether two main receptors for coxsackievirus infection:CAR and DAF were expressed on CD4+ T cells.Then,Nup98 were down-regulated or up-regulated by Nup98 siRNA or Nup98 plasmid in vitro.Percentages of Th17 cells in each group were detected by flow cytometry.The secretion of IL-17 in culture supernatant was determined by ELISA.Western blot was used to detect expressions of RORyt and Nup98 of CD4+ T cells.The gene expression of RORyt,Nup98,and IL-17 were detected by RT-PCR.Finally,Western blot was used to detect signaling pathways involved in Nup98 regulating the differentiation of Th17cells.Results:CVB3-infected CD4+ T cells(CVB3)could form plaques with a virus titer of 3.1*103PFU/ml,while mock group had no plaque(P<0.05).Western blot and ELISA showed that the proportion of Th17 cells and the secretion of IL-17 increased in CVB3-infected group compared with mock group(P<0.05).In addition,the protein and gene expression of Nup98 decreased while the expression of ROR?t increased in CVB3-infected group(P<0.05).In order to clarify the way of CVB3 entry into CD4+ T cells,we examined the expression of two common Coxsackieviruses receptors:CAR and DAF on CD4+ T cells.Results showed that almost no expression of CAR were detected in both nonactivated and activated CD4+ T cells while DAF were highly expressed on these cells.The virus titer of CVB3-infected group was decreased after antagonism with DAF monoclonal antibody(mAb)(P<0.05).To understand the direct effect of Nup98 on the differentiation of Th17 cells in vitro,Nup98 siRNA or Nup98 plasmid and corresponding controls were transfected into human CD4+ T cells.Compared with control group(NC)and no-load plasmid(pcDNA3.1)group,the protein and gene expression of Nup98 in CD4+ T cells of siRNA-Nup98 transfected group decreased while the percentage of Th17 cell,RORyt and secretion of IL-17 increased(P<0.05).The expression of p300/CBP and acetylated Stat3 in siRNA-Nup98 transfected group were significantly increased(P<0.05).On the contrary,the expression of RORyt,p300/CBP and acetylated Stat3 was down-regulated with the overexpression of Nup98 in pcDNA3.1-Nup98 transfected group(P<0.05).Total Stat3 showed no significant difference among these groups.Conclusion:CVB3 enters into human CD4+ T cells through DAF rather than CAR,leading to the cleavage of Nup98 and promoting the differentiation of Th17 cell through p300/CBP-acetylated Stat3-RORyt pathway.Part 3.The effect of Nup98 on Th17 cell differentiation in mouse model of acute viral myocarditisBackground:CVB3-induced VMC model is widely used in the research of viral myocarditis.This study focused on the effect of Nup98 on the differentiation of Th17 cells and the severity of disease in acute viral myocarditis by establishing CVB3-induced mouse model.Methods:BALB/c mice were infected with Coxsackievirus B3 for establishing VMC mouse model.The expression of Nup98 was down-regulated in Nup98 siRNA group by tail vein injection of Nup98 siRNA while the NC group was injected with NC.The expression of Nup98 was up-regulated by tail vein injection of Nup98 plasmid in Nup98 pcDNA3.1 group.The pcDNA3.1 group was injected with no-load plasmid(pcDNA3.1)as overexpression control.The activity and fur condition of these mice were observed and compared.The heart weight and body weight of mice were recorded.Cardiac histopathological examination was used to compare the degree of myocardial inflammation and morphological changes in each group.The levels of serum cTNT in the mice were measured.The level of serum IL-17 was determined by ELISA.Flow cytometry was used to detect the percentage of intracardiac Th17 cells in mice.Western blot and RT-PCR were used to detect the levels of Nup98 and RORyt in spleen CD4+ T cells.Results:The expression of Nup98 in CD4+ T in Nup98 siRNA group was significantly lower than that of NC group and VMC group while the expression of RORyt,the ratio of HW/BW,the percentage of intracardiac Th17 cells,the secretion of IL-17 were significantly higher(P<0.05).On the contrary,the expression of Nup98 in CD4+ T cells in Nup98 pcDNA3.1 group was significantly higher than that in both VMC group and pcDNA3.1 group(P<0.05).The expression of ROR?t,the ratio of HW/BW,the percentage of intracardiac Th17 cells,the secretion of IL-17 were lower in Nup98 pcDNA3.1 group than that in the pcDNA3.1 group and VMC group(P<0.05).Conclusion:Nup98 siRNA injection through tail vein can down-regualte the expression of Nup98 in mice,promote Th17 differentiation and IL-17 secretion and contribute to the development of myocarditis.Overexpression of Nup98 in vivo can limit Th17 differentiation in VMC mice and slow the progression of myocarditis.As a result,regulating Th17 cells by targeting Nup98 may be a promising therapeutic approach for A VMC.