Immunoregulatory Effects Of Treg Cells Mediated By Cx43 And Gap Junction In Mice Undergone Cerebral Ischemia And Reperfusion Injury

After cerebral ischemia and reperfusion,a variety of immune cells and inflammatory mediators interact with the focal area,triggering an inflammatory cascade reaction.Regulatory T cells(Treg)can play a protective role after cerebral ischemia and reperfusion.Exogenous injection or endogenous growth of Treg cells has been found to reduce infarct foci and neurological impairment in mice with cerebral ischemia and reperfusion.However,the underlying mechanism remains poorly understood.The transport of cAMP from Treg cells to target cells to exert inhibitory effects has been shown to involve the Connexin 43(Cx43)and gap junctions.In a prior study,we discovered that Cx43 on astrocytes,as well as the hemichannels and gap junctional communication they form,can stimulate microglia to produce inflammatory factors via ATP release,resulting in severe neuroinflammatory injury.In addition,we found that Cx43 and gap junctions were altered on peripheral Treg cells after cerebral ischemia in patients.To further investigate the underlying mechanism,we hypothesized that after cerebral ischemia and reperfusion,the peripheral Treg cells suppressed the inflammatory response of effector T cells through Cx43 and gap junction,which was modulated by the cAMP/PKA pathway.The mechanisms involved have not been reported before.Part 1 Changes of Treg cells and Cx43 and gap junction in mice undergone cerebral ischemia and reperfusion injuryObject:Our study aims to investigate the changes in splenic Treg cells,Cx43 protein expression,and immunosuppressive function following cerebral ischemia and reperfusion in mice.Methods:C57BL/6 mice were used to create a reperfusion model(Transient MCAO,tMCAO)following 60 minutes of middle cerebral artery occlusion(MCAO).There were Sham group,tMCAO 1d group,3d group and 5d group.Longa score,corner test,and cylinder test were used to assess neurological deficits in mice.TTC staining was used to assess the volume of brain infarction.The flow cytometry was used to detect splenic Treg cells and Cx43 protein expression,and immunofluorescence staining was used to observe the localization and expression of Cx43 protein in Treg cells.To detect the gap junction intercellular communication(GJIC),immunofluorescence tracing dye Calcein AM was employed.CFSE staining was used to identify the proliferation inhibition rate of Treg cells to Teff cells.To assess the activation of Teff cells,the IL-2 level in the cell supernatant was evaluated by ELISA.Results:Behavioral experiments and TTC staining showed that the Id,3d,and 5d groups after cerebral ischemia/reperfusion had higher levels of neurological deficits and larger volumes of cerebral infarcts compared to the sham-operated group(P<0.05).However,there were no differences among these three groups(P>0.05).Flow cytometry examination showed that the ratio of Treg/CD4+T cells was elevated at all time points,but only the Id group made statistical differences(P<0.05).At all time points,the Cx43 protein expression in Treg cells was significantly higher(P<0.05),and there was no difference between the groups(P>0.05).Immunofluorescence staining showed that the Cx43 fluorescence intensity on the surface of Treg cells was higher in all groups compared to the sham-operated group(P<0.01),but there was no significant difference between groups(P>0.05),and gap junction spots were observed in adjacent Treg cells.After Treg and Teff cells were co-cultured in vitro,the GJIC function was significantly elevated at Id(P<0.001)and reduced at 3d and 5d compared to 1d(P3d<0.05,P5d<0.01).The inhibition rate was highest in the 1d group(P<0.01)when Treg cells and Teff cells were mixed in 1:1,and the 3d and 5d groups showed no difference from the Sham group(P>0.05).The IL-2 secretion was significantly decreased at all time points(P<0.01).Conclusion:During cerebral ischemia and reperfusion injury,Treg cells are activated,the expression of Cx43 protein in Treg cells is up-regulated,GJIC is enhanced,and the immunosuppressive effect of Treg cells is intensified.Part 2 The function of gap junctions in the immunosuppressive activity of Treg cells following cerebral ischemia and reperfusionObject:This part aims to investigate the role of Cx43-mediated gap junction in the immunosuppressive activity of Treg cells following cerebral ischemia and reperfusion in mice.Methods:C57BL/6 mice were used in the tMCAO model.By using the magnetic selection,spleen Treg cells and Teff cells were obtained and co-cultured together.There were control group(Control),vehicle group(Vehicle),Cx43 mimic peptide Gap27 treatment group(Gap27),Gap27 and exogenous cAMP treatment group(Gap27+cAMP),and Transwell chamber group(Transwell),which could separate Treg cells and Teff cell.CFSE staining was used to evaluate the inhibition rate of Treg cells to Teff cells.ELISA was used to measure IL-2 in the supernatant to assess Teff cells activation,and immunofluorescence tracing dye Calcein AM was used to detect GJIC.Teff cells in the co-culture suspension were sorted by flow cytometry sorting and cAMP in Teff was tested by ELISA.Results:Gap27 is used to inhibit gap junctions formed by Cx43.After Gap27 treatment,the inhibitory effect of Treg cells on the proliferation of Teff cells was weakened(P<0.01),and Teff cells were significantly activated,which was manifested by an increase in IL2(P<0.01).At the same time,the GJIC was inhibited(P<0.01),resulting in cAMP decrease(P<0.01).Exogenous cAMP was added to the Gap27+cAMP group.The proliferation inhibitory effect of Treg cells in the Gap27+cAMP group was stronger than the Gap27 group(P<0.01);compared with the Gap27 group,the activation of Teff cells was inhibited and IL-2 was decreased(P<0.01).Compared with the Gap27 group,the function of gap junction and cAMP level in Teff cells were elevated,but the difference was not statistically significant(P>0.05).Transwell chamber culture can isolate direct contact between cells and avoid the formation of gap junctions.The Transwell group completely eliminated the proliferation inhibitory effect of Treg cells(P<0.01),and the secretion of IL-2 was increased significantly(P<0.01),as there was no GJIC between Treg cells and Teff cells,cAMP cannot be transmitted between cells,and the cAMP was significantly lower than that in the Control group(P<0.01).Conclusion:During cerebral ischemia and reperfusion,suppression of Cx43 and GJIC can diminish the anti-proliferation and anti-activation effects of Treg cells on Teff cells.Part 3 The impact of modulating Cx43 expression on the immunological control of Treg cells after cerebral ischemia and reperfusionObject:This part aims to clarify the protective effect of exogenous Treg cells on cerebral ischemia and reperfusion injury in mice,and investigate the particular mechanism underlying Cx43 expression.Methods:Treg cells from healthy mice were transfected with Cx43 siRNA to knock down the expression of Cx43.Two hours after the tMCAO model,exogenous Treg cells were injected through the tail vein.There were control group(Control),PBS injection group(Vehicle),Treg cell treatment group(Treg group),Cx43 knockdown Treg cell treatment group(siCx43-Treg group)and transfect negative control RNA Treg cell treatment group(siNC-Treg group).At three time points including 1d,3d,and 7d,Longa score,corner test,and cylinder test were used to evaluate neurological deficits.TTC staining was used to evaluate the volume of cerebral infarction in mice,and flow cytometry was used to detect changes of Treg cells and Cx43 protein expression.Immunofluorescence staining was used to observe the apoptosis of neurons and the infiltration of microglia in the brain at 1 day.The flow cytometry was used to detect the proportion of Thl,Th2 and Th17 cells in the spleen,and CBA method was applied to measure IL-2,IL-4,IL6,IL-10,IL-17A,TNF-α,and IFN-γ levels in peripheral blood.Results:After Treg cells treatment,the neurological deficit showed no difference at 1d(P>0.05),but was significantly relieved at 3d and 7d compared with the Control group(P<0.05).TTC staining showed that infarct volume was decreased at 3d and 7d(P<0.05),and no difference at 1d(P>0.05).The ratio of Treg/CD4+T cells in the Treg group increased significantly 1 day after Treg cells treatment(P<0.01).While there was no significant change in the expression of Cx43 at Id,3d,and 7d(P<0.05).In the brain,Treg cell treatment decreased neuronal apoptosis and microglial infiltration after cerebral ischemia/reperfusion.In the peripheral immune system,Treg cell treatment decreased the proportion of Thl and Th17(P<0.01),and increased the proportion of Th2(P<0.01),and elevated the concentration of IL-10(P<0.01),and decreased IL-6 and TNF-α secretion(P<0.05).However,treatment of Cx43-knockdown Treg cells(Si-Cx43 Treg group)abolished the neuroprotective effect of Treg cells on cerebral ischemia and reperfusion injury.Compared with the Treg group,the neurological damage was aggravated at 3d and 7d in the Si-Cx43 Treg group(P<0.05),and the infarct volume was significantly increased at 7d(P<0.05).The ratio of Treg/CD4+T cells was extremely higher than that of the Control group at 1 day(P<0.01),but lower than the Treg group(P<0.05).In addition,the expression of Cx43 in the Si-Cx43 Treg group was significantly lower than the Treg group at 1d and 3d(P<0.05).In the brain,neuronal apoptosis and microglial infiltration in the Si-Cx43 Treg group were more severe than the Treg group.In the peripheral immune system,the siCx43-Treg group increased Thl and Th17(P<0.001),and decreased Th2 significantly(P<0.01).At the same time.It decreased the secretion of IL-10(P<0.05).There was no significant difference in IL-2,IL-4,IL-17A and IFN-γ among the groups(P>0.05).Conclusion:Knocking down the Cx43 gene eliminates the protective effect of Treg treatment on cerebral ischemia and reperfusion injury,demonstrating that Treg cells regulate the immune inflammatory response to minimize ischemic injury via Cx43 expression.Part 4 Effects of the cAMP/PKA pathway on Cx43,gap junctions and the immune-suppressive function of Treg cellsObject:This part aims to clarify the effect of cAMP/PKA pathway in Treg cells on Cx43,gap junction and its immunosuppressive function after cerebral ischemia and reperfusion in mice.Methods:C57BL/6 mice were used in tMCAO model.There were control group(Control),vehicle group(Vehicle),Forskolin group(Forskolin),and Rp-cAMP group(Rp-cAMP).Magnetic selection was used to separate Treg cells and Teff cells at Id after tMCAO.Treg cells were pre-treated in different groups,and the expression of PKA,p-PKA,Cx43,p-Cx43-Ser373,and p-Cx43-Ser368 was detected by flow cytometry.On the other hand,pre-treated-Treg cells and Teff cells were co-cultured together to detect GJIC function by flow cytometry and IL-2 by ELISA.Results:Following treatment with Forskolin(a cAMP/PKA pathway activator),the GJIC function was enhanced between Treg cells and Teff cells(P<0.01),IL-2 secretion was decreased significantly(P<0.05).Rp-cAMP(a PKA antagonist)reduced GJIC function(P<0.01),and elevated IL-2 secretion(P<0.01).The expressions of PKA and p-PKA were up-regulated in the Forskolin group,and the p-PKA was increased significantly(P<0.01).Cx43 and p-Cx43-Ser373 were significantly increased(P<0.05),while the expression of p-Cx43-Ser368 showed no difference in the Forskolin group(P>0.05).In the Rp-cAMP group,PKA and p-PKA expression was decreased compared to the Forskolin group(P<0.05).And Cx43 and p-Cx43-Ser373 expression was reduced(P<0.01),while p-Cx43-Ser368 expression was up-regulated(P<0.01).Conclusion:The cAMP/PKA pathway in Treg cells modulated the phosphorylation of Cx43 Ser373 to promote GJIC function,enhancing the immunosuppressive function of Treg.