The Role Of Regulatory T Cells And Cytokines In The Pathogenesis Of Immune Thrombocytopenia

Background and aim Immune thrombocytopenia(ITP)is an autoimmune hemorrhagic disease characterized by thrombocytosis and deficiency of platelet formation.It has been thought that plate let autoantibody mediated humoral immunity plays an important role in the pathogenesis of ITP.In recent years,with the development of research,the role of cellular immunity,especially the abnormality of T cell immunity in the pathogenesis of ITP has been paid more and more attention.The regulatory T cells(Treg)were thought to play an important role in maintaining immune balance.Many studies have shown that the number of CD4+Tregs in bone marrow,spleen,and peripheral blood of patients with ITP decreased significantly,and the immunosuppressive function also decreased significantly.The abnormality of number and function of CD8+Tregs were also associated with the pathogenesis of autoimmune diseases in some basic researches.However,the changes in the number and function of CD8+Tregs subsets and the corresponding CD8+CD28+T(cytotoxic T lymphocyte,CTL)cell levels in patients with ITP have not been reported in detail.It has been reported that Th cells can secrete many kinds of cytokines to mediate a series of immune responses.The cytokines secreted by Th1 cells were mainly IL-2,IFN-?,TNF-?,etc.The cytokines secreted by Th2 cells were mainly IL-4,IL-5,IL-10,etc.Th17 cells secreted IL-17 specifically.In addition,antigen-presenting cells(APC)can also secrete many cytokines,such as IL-12,IL-23,IL-27 and so on.The role of various cytokines in the pathogenesis of ITP remains to be confirmed.The aim of this study were:(1)To explore the role of CD4+ Tregs and CD8+Tregs subsets in the pathogenesis of ITP through comparing the number of CD4+Tregs(CD4+CD25+Foxp3),CD8+Tregs(CD8+CD28-Tregs,CD8+CD103+Tregs,CD8+CD25+Tregs)subsets,CTL in peripheral blood circulation of patients with ITP and controls;(2)To explore the mechanism of ITP immune imbalance through comparing the levels of IL-2,IFN-?,TNF,IL-10,IL-17 A,IL-12 and IL-21 secreted by Th1,Th2 and Th17 in ITP patients and controls,;(3)To explore the therapeutic effect of hormone therapy on cellular immune abnormalities in ITP patients,HD-DXM treatment were used to treat patients with ITP,and the changes of CD4+Tregs,CD8+Tregs subsets,CTL and cytokines levels were detected before and after treatment.Methods 30 patients newly diagnosed ITP were admitted to the Department of Hematology,the first affiliated Hospital of Anhui Medical University from April 2015 to September 2016,.At the same time,20 healthy adults were enrolled as normal control.1.EDTA anticoagulants were added in blood samples to isolate peripheral blood mononuclear cells(PBMCs)and plasma.PBMCs were labeled with monoclonal antibodies,and the proportion of CD4+CD25+Foxp3,CD8+CD28-Tregs,CD8+CD103+Tregs,CD8+CD25+Tregs and CD8+CD28+T cells(CTL)in lymphocytes of ITP patients and normal controls were detected by flow cytometry.2.The levels of serum IL-2,TNF,IFN-?,IL-10,IL-17 A,IL-12 and IL-21 in patients with ITP and normal controls were detected by flow cytometry.3.All patients with ITP were treated with continuous high-dose dexamethasone regimen(HD-DXM 40mg/d)for four days.The peripheral blood of patients with EDTA was collected before treatment,and the blood routine examination were performed on the 7th and 14 th day after the treatment.The clinical curative effect and side effect were evaluated according to the platelet count of blood routine examination on the 14 th day.The peripheral blood samples of EDTA anticoagulant were collected on the 14 th day after treatment.Flow cytometry were used to detect the changes of regulatory T cells and cytokines before and after treatment.Results 1.The percentage of CD4+CD25+Foxp3 in lymphocytes in patients with ITP was 1.500+0.813%,which was significantly lower than that in normal controls(p<0.05).And the percentage of CD8+CD25+ in lymphocytes was 0.213+ 0.106%,while CD8+CD103+ was 2.127+0.858%,both of them were significantly lower than that in normal controls(p <0.05).The percentage of CD8+CD28-in CD8+ T lymphocytes was 4.832+0.531% in the patient group and 8.313+0.841% in the normal control group,which was significantly lower in the patient group than that in the control group(p<0.05),and the proportion of CD8+CD28+ in lymphocytes was 40.853+12.178% in the patient group and 4.765+1.000% in the normal control group,which were significantly different between the two groups(p<0.05).2.The level of IL-2 in serum of ITP patients was 3.460+ 1.323 pg / ml,TNF was 37.494+8.013 pg/ml;IFN-?was 12.371+ 2.875 pg/ml,IL-12P70,was 27.106+ 7.364 pg/ml,IL-17 A was 51.319+9.329 pg/ml,IL-21 was 34.280+ 5.838 pg/ml.All of above were significantly higher than that of the control group (p<0.05).While the level of IL-10 was1.647+0.566 pg/ml,which was significantly lower than that of the control group(p < 0.05).3.On the 14 th day after treatment with high-dose dexamethasone,the efficiencies of treatment were divided into complete response(complete response,CR),response (response,R)and non-response(no response,NR).The ratios of CR,R and NR were 60.00%,33.33% and 6.67%,respectively.No serious complications and side effects were found.4.The proportions of CD4+CD25+Foxp3,CD8+CD25+Tregs and CD8+CD103+Tregs in lymphocytes of ITP patients were 2.287+0.834%,2.702+ 0.748% and 3.640 +1.058% respectively,which were significantly higher than those before treatment(p<0.05).The proportions of CD8+CD28-Tregs in lymphocytes was 6.560+0.704%,which was significantly higher than that before treatment(p < 0.05),while the percentage of CD8+CD28+(CTL)in CD8+T lymphocytes was 11.052+ 2.549%,which was significantly lower than that before treatment(p < 0.05). 5.After high dose dexamethasone treatment,the level of IL-2 was 1.363 + 0.649pg/mL,TNF was 15.383 +7.132 pg/mL,IFN-? was 3.705+ 1.447 pg/mL,IL-12P70 was 13.495+2.961 pg/mL,IL-17 A was 19.430 + 5.310 pg/mL,IL- 21 was 9.208+ 1.505 pg/mL,all of above were lower than those before treatment (p<0.05),but still higher than those before treatment.The level of IL-10 was 5.437 + 0.978 pg/mL,which was higher than that before treatment(p <0.05),but still lower than that in control group.Conclusion.(1)CD4+CD25+Foxp3 and CD8+Tregs subsets may be involved in the pathogenesis of ITP;(2)Various cytokines were involved in the pathogenesis of ITP.(3)HD-DXM can significantly improve the levels of T lymphocytes and many kinds of cytokines,participating the cellular immunity and maintaining the immune balance,which may be one of the mechanisms of its therapeutic effect of ITP.