The Neuroprotective Effect Of IL-33/ST2 Axis Activation Of Treg Cells On Ischemic Stroke

Background:After cerebral ischemia,the infiltration of inflammatory cells in the central nervous system and the overexpression of pro-inflammatory cytokines can cause irreversible damage to brain tissue and seriously affect the recovery of nerve function.The inflammatory cascade reaction lasts for a long time and occurs in the late stage of ischemia,which can cause the second injury after cerebral ischemia and aggravates the degree of brain injury after cerebral ischemia.Therefore,the inflammatory response is an important part of the pathophysiological mechanism leading to neurological function defects which have become a new target of current studies for inhibiting the inflammatory response and promoting the recovery of neurological function.CD4~+CD25~+Foxp3~+Regulatory T(Treg)cells are a kind of T cell subsets with unique immunomodulatory functions.They can inhibit the expression of self-reactive T cells,prevent the occurrence of autoimmune diseases,and maintain autoimmune tolerance.The most specific marker of this group of cells is Foxp3,which plays an important role in inducing the generation and maintenance of Treg cells.In addition,Treg cells can also exert their immunomodulatory function by secreting transforming growth factor(TGF)-?,interleukin(IL)-10 and other cytokines.Recent studies have found that the number and function of Treg cells are changed after acute ischemic stroke.Our previous studies have shown that the number and function of Treg cells are also changed after cerebral hemorrhage.These results provide evidence that Treg cells may be involved in the development of brain injury.Current studies focus on the neuroprotective effect of exogenous Treg cell transplantation or exogenous application of Treg cell-derived cytokines on acute cerebral ischemic stroke.It is still unclear whether a signaling pathway exists in vivo to enhance the activity of Treg cells and the activated Treg cells can better exert their neuroprotective effects.As a member of the IL-1 cytokine family,IL-33 is involved in the inflammatory process after ischemic stroke by binding its specific receptor ST2.Exogenous up-regulation of IL-33/ST2 axis can regulate the function of CD4~+T cell subgroup Th2 after stroke in the model of middle cerebral artery occlusion(MCAO)in mice,thereby reducing the degree of neurological injury.Recent studies have shown that ST2 receptors can be expressed on the surface of Treg cells,and IL-33/ST2 axis can regulate the number and function of Treg cells through multiple ways.Matta et al.found that IL-33/ST2 axis can directly or indirectly promote the proliferation of Treg cells in vivo and in vitro.Therefore,it is expected to explore the signaling pathway that can activate Treg cells,and further up-regulate the function of endogenous Treg cells to find new therapeutic strategies for neuroprotection after stroke.Objective:Clarify the relationship between IL-33/ST2 axis and Treg cells in acute ischemic stroke,and further reveal the role and mechanism of IL-33/ST2 axis activation of Treg cells in acute ischemic stroke.Methods:1.The oxygen and glucose deprivation(OGD)model of neurons was constructed at the cell level.The Treg cells were cultured with ordinary Treg cell culture medium(CM),IL-33+sST2 CM,and sST2 CM for 24 h.The expression levels of related cytokines(IL-10,IL-35,and TGF-?)were analyzed by ELISA method.The medium supernatant was applied to the neurons in the process of OGD,and the neuronal survival rate and apoptosis-related protein(Caspase-3 and Bcl-2)expression levels were analyzed by MTT and Western Blot.2.In an animal study,transient middle cerebral artery occlusion(tMCAO)model and Treg cell-deficient mice were created by regular intraperitoneal injection of anti-CD25 antibody.By regularly intraperitoneal injection anti-ST2 antibody to create ST2 blocking tMCAO mice.Recorded and analyzed the weight and survival rate of mice was calculated.The behavior of mice was analyzed by mNSS,balance beam test,and rotarod test.The size of the infarct was analyzed by TTC staining.The degree of cerebral edema was analyzed by dry and wet weight method.Western Blot and TUNEL staining were used to analyze the degree of apoptosis,and flow cytometry and Western Blot were used to analyze the number of Treg cells and the expression level of related cytokines.Results:1.The percentage of CD3~+CD4~+Foxp3~+Treg cells in the brain tissue of the anti-CD25 group were significantly lower than that of the vehicle group(P<0.001),which was detected by flow cytometry.The mNSS score of the anti-CD25 group was continuously increased within 14 days,and the incubation period of wheel drop was significantly shortened.TTC staining showed that the infarct volume of the anti-CD25 group was larger than that of the vehicle group(P=0.016).TUNEL staining showed that the anti-CD25 group had a higher degree of cell death.The dry-wet weight method showed that the brain water content of the anti-CD25 group was significantly increased than that of the vehicle group(P=0.023).2.MTT showed that compared with other OGD groups,IL-33 conditioned Treg cell culture medium significantly increased the survival rate of neurons after OGD(P=0.004),and there was no statistical difference in the survival rate of neurons between the other three groups(P=0.997).In the tMCAO model,after exogenous administration of IL-33,flow cytometry showed that the percentage of Treg cells in the brain tissue of the anti-CD25+IL-33 group was significantly higher than that of the anti-CD25 group(P<0.001).The percentage of Treg cells in the brain tissue of the IL-33+anti-ST2 group was 4.48±0.78%,which significantly lower than that of the IL-33 group(P<0.001).3.TTC staining showed that after exogenous IL-33 administration the infarct volume was 25.9±2.21%,which was significantly lower than that of the anti-CD25group(P=0.002),and TUNEL staining results were consistent with it.The edema level of the anti-CD25+IL-33 group was lower than that of the anti-CD25 group(P=0.002).The mNSS score of the anti-CD25+IL-33 group was significantly lower than that of the anti-CD25 group(P=0.005),which was consistent with the results of the wheel experiment and balance beam experiment.4.Western blot was used to detect the expression levels of apoptosis-related proteins(Caspase-3,Bcl-2,and Bax).The results showed that in the OGD model,IL-33 significantly down-regulated the expression levels of apoptosis-related protein caspase-3(P=0.072)and up-regulated the expression levels of Bcl-2 protein.There was no statistical difference in the expression levels of apoptosis-related proteins among the other three groups(P=0.997).In tMCAO model,Caspase-3 and Bax levels were significantly up-regulated in the anti-CD25 group compared with the blank operation group(P<0.001)and the expression level of Bcl-2 protein was significantly down-regulated(P=0.002).The expression levels of Caspase-3 and Bax were down-regulated in the anti-CD25+IL-33 group compared with the anti-CD25group(P=0.003)and Bcl-2 protein expression was up-regulated(P=0.013).5.Western blot was used to detect the expression levels of apoptosis-related proteins(Caspase-3,Bcl-2,and Bax).The results showed that compared with the sham group,the levels of pro-apoptotic proteins Caspase-3 and Bax were significantly up-regulated(P<0.001),and the anti-apoptotic proteins Bcl-2 were significantly down-regulated(P=0.027).Comparing with the vehicle group,Caspase-3 and Bax levels in the anti-CD25 group were significantly up-regulated(P<0.001),and Bcl-2 protein expression was significantly down-regulated(P=0.002).The expression levels of Caspase-3 and Bax were down-regulated in the anti-CD25+IL-33 group compared with the anti-CD25 group(P=0.003).Bcl-2 protein expression was up-regulated(P=0.013).6.In the cell study,the concentration of Treg-related cytokines(IL-10,IL-35,TGF-?)in the supernatant of culture medium was detected by ELISA.Comparing with the other three groups,the concentrations of IL-10(P<0.001),IL-35(P=0.004)and TGF-?(P<0.001)in the IL-33 group were significantly higher than those in the other three groups.In the animal study,the changes of the three cytokines in each group were consistent.The level of IL-10 in the anti-CD25 group was significantly lower than that in the vehicle group(P=0.004),and IL-10 in the anti-CD25+IL-33group was significantly higher than that in the anti-CD25 group(P=0.036).IL-33significantly increased the level of IL-10 after ischemia-reperfusion injury(P=0.015),and the level of IL-10 significantly decreased after the injection of anti-ST2 antibody(P=0.025).Conclusion:1.Further define Treg cells have a neuroprotective effect in ischemic stroke;2.IL-33 induces Treg cells to recruit into ischemic brain tissue through the ST2pathway;3.IL-33/ST2 axis actives Treg cells to protect the ischemic stroke with an anti-apoptotic protein and cytokine-dependent way.