The Unbalanced T Cell Homeostasis In The Pathogenesis Of Immune Thrombocytopenia

Part Ⅰ:Aberrant levels of Treg subsets are involved in the mechanism of immune thrombocytopeniaImmune thrombocytopenia(ITP)is an acquired autoimmune disease,and its clinical features are decreased platelet count and increased risk of bleeding.The main pathogenesis of ITP includes abnormal reduction of platelets produced by megakaryocytes and increased platelet destruction.Among them,T cell immune intolerance is one of the important factors of increased platelet destruction in ITP patients.Treg cells play an important role in maintaining immune homeostasis,mediating immune tolerance,controlling autoimmune diseases,and suppressing excessive immune responses against microorganisms or allergens.Recent studies have shown that Treg cells are composed of heterogeneous subsets with different functional characteristics.Under certain inflammatory conditions,some Treg cells can acquire the helper T cell phenotype and have the ability to secrete inflammatory cytokines.For example,Thl-like Treg cells and Th17-like Treg cells can secrete IFN-y and IL-17A respectively.In physiological conditions and various autoimmune diseases,the existence of Thl-like Treg and Th17-like Treg cells has been observed.Whether these two Treg subsets are related to the occurrence and development of ITP has not been reportedDexamethasone treatment is one of the first-line treatment options in ITP and is also the commonly used glucocorticoid for clinical use.Application of high-dose dexamethasone in autoimmune diseases can inhibit the secretion of inflammatory factors.We have found that the treatment of dexamethasone can inhibit the phenotype and function of M1 splenic macrophages in ITP,polarize them to M2 cells,and also promote the production of myeloid-derived suppressor cells in ITP patients.However,how dexamethasone continuously corrects the immune microenvironment of patients is unclear,and whether dexamethasone treatment plays a therapeutic role through regulating Treg subsets in ITP has not been reported.This study used flow cytometry to find that the proportion of Th1-like Treg and Th17-like Treg cells in Treg cells in peripheral blood of ITP patients was higher than that of healthy controls.It has been reported that Thl-like Treg and Th17-like Treg cells have their specific molecular signatures.For example,Thl-like Treg cells highly express CXCR3 and TBX21,while Th17-like Treg cells highly express CCR6 and RORC.We sorted the Treg cells from healthy controls and ITP patients and performed real-time quantitative PCR detection.The results showed that the relative expression of specific molecular signatures of Thl-like Treg and Th17-like Treg cells in Treg cells of ITP patients increased,which further validates the results of flow cytometry.In addition,we found that the proportion of Thl-like Treg cells in Treg cells was negatively correlated with platelet count in ITP patients through correlation analysis.We also found that the proportion of Thl-like Treg cells in Treg cells was related to the severity and glucocorticoid sensitivity in ITP.In order to explore the causes of the imbalance of Treg cell subsets in ITP patients,we detected related inflammatory factors in the plasma of ITP patients.We found increased expression of various inflammatory cytokines,including IFNy,IL-12,TNF-α,IL-6 and IL-23.We further found that the inflammatory factors IFNy,IL-12,TNF-α are related to the differentiation of Thl-like Treg,and IL-6,IL-23 are related to the differentiation of Th17-like Treg cells in vitro.In order to explore the effect of dexamethasone treatment on Treg subsets,after cultivating CD4+T cells of ITP patients in vitro and adding dexamethasone to stimulate for 4 days,Treg cells were sorted and tested by real-time quantitative PCR to analyze the specific molecular signatures of Treg cells.The results showed that the relative expression of CXCR3 and TBX21 was significantly decreased compared with control group.Besides,the relative expression of CCR6 and RORC was not statistically different compared with control group.These results suggested that dexamethasone treatment inhibited the differentiation of Thl-like Treg cells.We detected that the expression of T-bet in Treg cells decreased by flow cytometry after treatment with dexamethasone,and the expression of RORyt remained unchanged.These results verified the conclusion that dexamethasone treatment can inhibit the differentiation of Thl-like Treg cells.This study found that dexamethasone treatment in vitro increased the percentage of Treg cells to CD4+T cells in ITP patients by flow cytometry,while reduced the percentage of Th1-like Treg cells to Treg cells.To further explore the molecular mechanism of dexamethasone regulating Thl-like Treg cells,we found that dexamethasone treatment significantly inhibited the phosphorylation level of STAT1 by Western blot,which plays an important role in Thl phenotype.In view of the fact that STAT1 is located downstream of the IFN-γreceptor,we also examined the effect of dexamethasone treatment in vitro on PBMCs secretion of IFN-γ and exogenous IFN-y-induced phosphorylation of STAT1.We found that dexamethasone treatment significantly inhibited the production and secretion of IFN-γ,but did not affect the exogenous IFN-y-induced phosphorylation and activation of STAT1,indicating that dexamethasone may regulate the differentiation of Thl-like Treg cells in ITP patients by regulating the production of IFN-γ.Our research not only found the proportion of Thl-like Treg and Thl 7-like Treg cells in Treg cells of ITP patients was increased,but also found high levels of inflammatory cytokines in ITP patients are conducive to the differentiation of Thl-like Treg and Th17-like Treg cells.In addition,this study found that dexamethasone treatment can inhibit the differentiation of Thl-like Treg cells by inhibiting the IFN-γ/Jak-STAT1 axis.Our results indicate that the differentiation of Treg cells is related to the occurrence and development of ITP,and also suggest the potential mechanism of dexamethasone treatment of ITP.Part Ⅱ:C5a is involved in the pathogenesis of immune thrombocytopenia by regulating the balance of Th17/TregImmune thrombocytopenia(ITP)is an autoimmune disease whose main clinical manifestation is the reduction of platelet count caused by bleeding of the skin and mucous membranes or the tendency to bleeding.More and more studies have shown that immune homeostasis is one of the important pathogenesis of ITP,in which the abnormal functions and proportions of helper T cells(Th cells)and regulatory T cells(Treg cells)play an important role.Th17 cells have the function of inducing inflammation and autoimmune diseases,stimulating a variety of cells to recruit neutrophils to the infection site,and mediating immune response against extracellular bacteria and fungi,while the main function of Treg cells is to suppress inflammation and immunity to maintain immune homeostasis.Therefore,exploring the mechanisms that affect Th17/Treg cell balance is of great significance for us to understand the pathogenesis of autoimmune diseases.As the first line of defense against microbial invasion,complement has also been found to be involved in many physiological activities in recent years such as synaptic maturation,immune complex clearance,angiogenesis,hematopoietic stem cell/progenitor cell mobilization,tissue regeneration,lipid metabolism and so on.It can also participate in the pathogenesis of many autoimmune diseases by affecting a variety of immune cells.However,with regard to the role of the complement system in the pathogenesis of ITP,the current relevant research is relatively limited.C5a is one of the important products of complement activation.Three complement activation pathways(classical pathway,alternative pathway,and lectin pathway)can produce C5a.C5a receptor(C5aR)is widely distributed on a variety of immune cells,such as neutrophils,monocytes/macrophages,mast cells and so on.Many studies have shown that C5a can indirectly regulate the proliferation and function of T cells by regulating antigen presenting cells.C5a can also induce the production of IL-6 and IL-23 by peritoneal macrophages,and cooperate with granulocyte/macrophage colony stimulating factors secreted by activated T cells to participate in the regulation of Th17 cell differentiation.In addition to being widely distributed on granulocytes and antigen-presenting cells,C5aR is also highly expressed on CD4+ T cells.However,there are few studies on how C5a directly regulates the differentiation of CD4+T lymphocytes.In this study,the level of C5a in the plasma of ITP patients was first detected by ELISA.The results showed that the level of C5a in the plasma of ITP patients was higher than that of the healthy controls.C5aR expression in CD4+T cells of ITP patients detected by real-time quantitative PCR was not significantly different from that of healthy controls.Subsequently,we analyzed the correlation between the level of plasma C5a and clinical characteristics in ITP patients.The results showed that the level of plasma C5a in ITP patients was negatively correlated with platelet count.The level of plasma C5a in severe ITP patients was significantly higher than that in non-severe patients,but had no correlation with the sensitivity of glucocorticoid treatment.These results suggested that C5a has a potential correlation with the occurrence and development of ITP.In order to verify whether the balance of Th17/Treg cells is unstable in the peripheral blood of the ITP patient,we found that the percentage of Th17 cells in CD4+cells of ITP patients was increased,and the percentage of Treg cells in CD4+cells of ITP patients decreased by flow cytometry.At the same time,the level of IL-17A in plasma of ITP patients increased while the levels of IL-10 and TGF-β in plasma of ITP patients decreased by ELISA.To investigate whether C5a in ITP regulates Th17/Treg balance,we first performed a correlation analysis.The results showed that the level of plasma C5a in ITP patients was positively correlated with the percentage of Th17 cells in CD4+cells,but not correlated with the percentage of Treg cells in CD4+cells.We further found that rhC5a treatment can increase the level of Th17 cells and decrease the level of Treg cells through in vitro cell culture and flow cytometry.C5aR inhibitors can block this regulatory effect.In order to further explore the molecular mechanism of C5a regulating Th17/Treg cell balance,we detected the activation levels of C5aR downstream of PI3K/Akt signaling pathway and NF-κB signaling pathway by western blot.The experimental results showed that the phosphorylation levels of PI3K and Akt in CD4+T cells after rhC5a treatment increased,and the phosphorylation levels of NF-κB activation did not change.But the addition of C5aR inhibitors can significantly inhibit this regulatory effect.The above results suggest that C5a can directly regulate Th17/Treg balance in ITP patients through the PI3K/Akt signaling pathway.In order to verify whether C5a regulates Th17/Treg cell balance through the PI3K/Akt signaling pathway,we used rhC5a in combination with PI3K inhibitor LY294002 to treat CD4+ T cells from ITP patients in vitro and then detected the ratio of Th17/Treg cells by flow cytometry.The results showed that the addition of PI3K inhibitors can inhibit the regulatory effect of C5a on Th17 and Treg cells,verifying the conclusion that C5a regulates Th17/Treg balance in ITP patients through the PI3K/Akt signaling pathway.In summary,we found that the level of C5a is significant increased in peripheral blood of ITP patients and the balance of Th17/Treg cells is unstable.The percentage of Th17 cells to CD4+T cells is increased,and the proportion of Treg cells is decreased.Correlation analysis reveals that the level of C5a is correlated with the percentage of Th17 cells.In terms of mechanism,C5a can influence the CD4+T cell surface C5aR downstream PI3K/Akt signaling pathway to regulate the balance of Th17/Treg cells,and then participate in the pathogenesis of ITP.This regulatory effect of C5a on Th17/Treg cell balance reveals the potential causes of the imbalance of T cell homeostasis in ITP,and also provides new ideas and potential targets for clinical ITP treatment.