Imbalance Of CD4~+ T Cell Subsets In Bone Marrow In The Pathogenesis And Immune Regulation Of Primary Immune Thrombocytopenia

Background:Primary immune thrombopenia(ITP)is an autoimmune hemorrhagic disease with an overall incidence of 2.0?5.3/100000[1-31 The clinical manifestations of chronic ITP are quite different.Most patients have asymptomatic or only mild skin and mucous purpura,while some patients may have serious visceral bleeding,such as gastrointestinal bleeding,renal hemorrhage and even intracranial hemorrhage.Addition to the severity of thrombopenia,the risk of bleeding in ITP is also affected by other factors,such as lifestyle and working environment.The pathogenesis of ITP patients is very complex.At present,it mainly involves the following aspects:?Destruction of platelets mediated by antiplatelet autoantibodies or related complements in monocyte-macrophage system;?Direct fragmentation of platelets by cytotoxicity T lymphocytes(CTLs)through perforin and granzyme;?Thrombopenia induced by helper T cell mediated cellular immune disorder;?Decreased number of regulatory T cells(Tregs)and functional deficiencies lead to decreased platelets;? Megakaryocyte differentiation and maturation disorder,apoptosis obstruction cause poor platelet production.Recent years,the imbalance,dysfunction and abnormal cytokine network of CD4+T cell subsets has played an important role in the occurrence and development of ITP[4-6].Th22 cells,as a newly discovered subset of CD4+T cells,are an important marker for evaluating the activity of ITP[4,7-11].Studies have proved the balance disorder of peripheral blood(PB)CD4+T cell subpopulation in the ITP patients[5].However,the relationship between the Th22 cells and the cytokines secreted as well as the other cell subpopulations has not been reported in the bone marrow(BM)of ITP.BM is the main part of the immune cell development.It is also the place where the platelet is generated.Identifying the state of the immune microenvironment and screening the key factors with functions is helpful to further clarify the pathogenesis of the ITP and to explore the new specific therapeutic target of the ITP.Objectives:By detecting the expression of Th22,Thl/Th2,Thl7/Tregs and Tfh cells and their secreted cytokines in BM and PB,understand the immune status of the above cells and cytokines,and explore the role and mechanism of this immune disorder in the pathogenesis of primary immune thrombocytopenia.Research method:Flow cytometry was used to analyze the expression of Thl/Th2,Th17/Tregs,Th22 and Tfh cell subsets in CD4+T cells in BM and PB;immunofluorescence labeling was used to verify the difference between CD4+T cell subsets and flow cytometry;ELISA method was used to detect the concentration of IFN-?,IL-17A and IL-22 factors;real-time fluorescent quantitative PCR was performed to detect the expression of IL-4 mRNA in BMMCs and PBMCs;gene chip technology was used to detect the content of each chemokine;Monoclonal antibody immobilization of platelet antigens assay(MAIPA)was used to determine the anti-GP ?b/?a and anti-GPIb/IX antibodies on the surface of platelet membrane;the correlation between T cell subsets and corresponding cytokines and the correlation between ITP patients and HCs group was analyzed by Pearson test.Results:1.The proportion of Th22 cells in BM and PB of ITP patients were significantly higher than those of healthy controls(HCs)(BM:2.18±0.80%vs.0.84 ± 0.17%,P<0.001;PB:1.39 ± 0.61%vs.0.83±0.16%,P=0.001);The proportion of Th22 cells in BM of ITP patients was significantly higher than paired PB Th22 cells(2.18±0.80%vs.1.39±0.61%,P<0.001).The level of IL-22 cytokine in BM also show the same pattern(33.26 ± 16.77 pg/ml vs.28.04±12.96 pg/ml,P=0.007).Th22 cells in BM and PB of ITP patients were significantly positively correlated with IL-22 factor levels(BM:r=0.796,P<0.001;PB:r=0.737,P<0.001).2.Th17 cells in BM and PB of ITP patients were significantly higher than their matched HCs(BM:3.38 ± 1.18%vs.1.39±0.17%,P<0.001;PB:2.13 ± 0.90%vs.1.32 ± 0.22%,P=0.001);Tregs cells in BM and PB of ITP patients were significantly lower than HCs(BM:1.73 ± 0.66%vs.6.12 ± 0.30%,P<0.001;PB:4.05 ± 1.05%vs.6.21 ± 0.18%,P<0.001).The ratio of Thl7/Tregs in BM and PB in ITP patients was significantly increased and the balance was unbalanced(BM:2.27± 1.18 vs.0.67± 0.64,P<0.001;PB:0.67 ± 0.64 vs.0.18 ± 0.03,P=0.006).In addition,the level of IL-17A in BM of ITP patients was higher than that of HCs(16.41±2.43 pg/ml vs.13.05± 3.27 pg/ml,P=0.001).And the level of IL-17A in PB was also slightly higher than that of HCs.However,there was no statistical difference(15.96±2.93 pg/ml vs.14.77 ± 2.85 pg/ml,P=0.232).3.Thl cells in BM and PB of ITP patients were significantly higher than matched HCs(BM:24.62±6.37%vs.7.70 ± 1.12%,P<0.001;PB:15.81 ± 3.47%vs.7.11 ± 1.33%,P<0.001).The proportion of Thl cells in BM was significantly higher than paired PB Thl cells in the patients(24.62 ± 6.37%vs.15.81 ± 3.47%,P<0.001).And the IFN-y level in BM was significantly higher than that in the paired PB in patients(5.40 ± 2.50 pg/ml vs.3.98±1.65 pg/ml,P<0.001).4.There was no significant difference between Th2 cells in BM and HCs group in ITP patients(1.47 ± 0.51%vs.1.49±0.41%,P=0.949),while Th2 cells in PB of ITP patients were significantly lower than HCs(0.81 ± 0.30%vs.1.40±0.33%,P=0.007);the expression of IL-4 mRNA in BM of ITP patients was not significantly different from that in HCs group(0.000345±0.000107 vs.0.000369 ± 0.000099,P=0.630),while the expression level of IL-4 mRNA in PB was significant lower than that of HCs(0.000206±0.000038 vs.0.00033 ± 0.000071,P=0.017).Th2 cells in BM of ITP patients were significantly higher than PB(1.47 ± 0.51%vs.0.81 ±0.30%,P=0.012),and the expression of IL-4 mRNA in BM was also significantly higher than that of PB(0.000345±0.000107 vs.0.000206±0.000038,P=0.012).5.Thl cells in BM of ITP patients measured by immunofluorescence microscope were significantly higher than that of HCs(t=1.936.P=0.048);Tregs cells were significantly lower than that of HCs(t=2.387,P=0.015);Although there is a trend that Th17 and Th22 cells are higher than HCs,it has not reached obvious statistical difference(Th17:t=1.028,P?0.190;Th22:t=1.553,P=0.082).6.The expression of CD4+CXCR5+Tfh cells in BM of ITP patients was significantly higher than that in paired PB(21.43±3.94%vs.17.01±4.47%,P=0.016).The proportion of CD4+CXCR5+ICOS+Tfh cells also showed consistent results(5.42±2.56%vs.3.21±1.75%,P=0.018).7.The CXCR3 level of CD4+IFN-?+T cells in BM and PB of ITP patients is significantly higher than that of matched HCs(BM:1527.3 ± 216.1%vs.1063.5±217.5%,P=0.001;PB:1705.5±235.1%vs.1293.7 ± 141.6%,P=0.002);CCR4 levels of CD4+IL-22+T cells in BM and PB were significantly higher than HCs(BM:2584.3 ± 824.5%vs.1624.8 ± 217.4%,P=0.005;PB:2402.8 ± 607.3%vs.1496.2± 155.4%,P?0.008).CXCR3 levels of CD4+IFN-y+T cells in BM of ITP group were significantly lower than those in PB(1527.3 ± 216.1%vs.1705.5±235.1%,P=0.033).8.The results of Quantibody(?)array chemokine chip showed that the levels of CCL27,osteopontin(OPN)and CCL18 in BM of ITP patients were significantly higher than those of HCs(CCL27:3218.39 vs.1190.38,P=0.041;OPN:8175.97 vs.2293.69,P=0.016;CCL18:807.64 vs.628.95,P=0.039),while there was no statistical difference between the 40 chemokines/cytokines measured in the PB of ITP patients and the HCs(all P>0.05).The level of macrophage migration inhibitory factor(MIF)in BM of ITP group was significantly higher than that in paired PB(11814.48 vs.2412.42,P=0.025),while the CCL23 was significantly lower than that in paired PB(590.24 vs.745.20,P=0.018).9.In patients with grade 2 and 3 bleeding,Th22 cells in BM were slightly higher than those in grade 1 bleeding(2.54±0.65%vs.2.03±0.72%;P=0.053),but the difference was not statistically significant,and those in PB was significantly higher than those in grade 1 bleeding(1.78±0.50%vs.1.31±0.53%;P=0.017);in patients with grade 2 and 3 bleeding,Thl cells in BM were higher than those with grade 1 bleeding(30.28 ± 6.72%vs.24.19 ± 6.00%;P=0.006);while there was no statistical difference in PB(19.13 ± 7.94%vs.17.37±5.02%;P=0.404).Conclusion:1.The balance of Th22 cell subsets in BM and PB of ITP patients was disordered,and the abnormal CD4+ T cells was more significant in BM2.There was imbalance of Thl/Th2,Th17/Tregs and Tfh cell subsets in BM and PB of ITP patients,and these imbalance of T cells was more obvious in BM than that in PB3.In patients with severe ITP,Th22 cells secrete more cytokines and chemokines,and the patients have a more pronounced inflammatory response4.The abnormal distribution of cytokines and chemokines in BM and PB in ITP patients suggested that the migration and differentiation of CD4+T cells was involved in the disorder of bone marrow microenvironment in ITPBackground: Primary immune thrombocytopenia(ITP)is an autoimmune hemorrhagic disease characterized by increased platelet destruction and decreased production.The therapeutic target of ITP is not to normalize the platelet count[1],but to elevate the platelet count to a safe range to minimize the risk of bleeding.Therefore,it is necessary to avoid unnecessary overtreatment in clinical practice.The first-line treatment includes glucocorticoid and immunoglobulin [2] Thrombopoietin receptor agonist(TPO-RA)and rituximab(anti-CD20 monoclonal antibody)are used for ITP as second-line therapy.What9 s more,other drugs such as areazathioprine,cyclosporine,dalazol,vinca alkaloids and so on can be applied for ITP For patients with the platelet count > 30 x 109/L? if there is no bleeding symptoms and no risk factors for bleeding,it is recommended for temporary observation and follow-up.If there is no symptom but risk factors for bleeding,platelet counts should be improved to more than 50 x 109/L.Treatment is necessary when ITP patients have any bleeding symptom no matter the number of platelets.Research purpose:By studying the immune regulation of dexamethasone on CD4+ T cell subsets such as Th22,Thl/Th22,Thl7/Tregs in ITP patients,in order to provide theoretical basis for ITP immunotherapy.Research method:Density gradient centrifiigation was used to isolate PBMCs in PB,and the expression of Th22,Thl7/Tregs and Thl in PB before and after treatment of ITP patients was analyzed by flow cytometry;ELISA was used to detect the concentration of IL-22,IL-17 A,IFN-y and TGF-p in PB;blood cell analyzer was used to detect the number of platelets;the Pearson correlation test was used to analyze the correlation between each T cell subset and platelet number after treatment in ITP patients,and the correlation between each cytokine and platelet.Results:1.After a course of treatment with dexamethasone,Th22,Thl7 and Thl cells in peripheral blood(PB)of ITP patients were significantly reduced(Th22: 0.97 ±0.49 % vs.1.39 ± 0.61 %,p=0.0282;Thl7: 1.61 ± 0.85 % vs.2.13 ± 0.90 %,P =0.0387;Thl: 11.38 ± 3.43 % vs.15.81 ± 3.47 %,p=0.0007);while Tregs cells increased significantly(5.95 ± 2.11 % vs.4.05 ± 1.05 %,P = 0.0028),the Thl7/Tregs ratio tends to recover.2.After treatment in ITP patients,compared with before treatment,the levels of IFN-y in PB were significantly reduced(2.84 ± 0.82 pg/ml vs 3.98 ± 1.65 pg/ml,P =0.0418);while TGF-p factor was significantly increased(1525.0 ± 661.3 pg/ml vs1077.3 ± 408.4 pg/ml,P = 0.0418);IL-22 and IL-17 A factors have a tendency to decrease compared with before treatment,but have not reached a statistical difference(IL-22: 20.13 ± 8.89 pg/ml vs.28.04 ± 12.96 pg/ml,P=0.0564;IL-17A: 14.13 ±4.39 pg/ml vs 15.96 ± 2.93 pg/ml,P=0.1180).3.Platelet counts were significantly increased in ITP patients after dexamethasone treatment(69.88 ± 76.60 x 109/L vs.12.80 ± 13.58 x 109/L,P=0.0073).4.There was a significant negative correlation between Th22 cells and Thl cells and platelet numbers after ITP treatment(Th22: r =-0.5330,P = 0.0335;Thl: r =-0.5423,P = 0.0300),and there was no correlation between Thl 7 cells and platelets(r=-0.4285,P = 0.0978),and Tregs cells were significantly positively correlated with platelets(r = 0.6979,P = 0.0026).After treatment,there was a significant negative correlation between IL-22 and IL-17 A factors and platelet number in patients with ITP(IL-22: r =-0.6662,P = 0.0048;IL-17A: r =-0.7585,P = 0.0007),there was no correlation between IFN-y factor and platelet(r =-0.0233,P = 0.9317),while TGF-p factor was significantly positively correlated with platelet (r = 0.8871,P <0.0001).Conclusion:1.The immune disorder of CD4+ T cell subsets is involved in the pathogenesis of ITP.After treatment with dexamethasone,the T cell subsets and cytokines of the patient gradually balance.2.By adjusting the balance of CD4+ T cell subsets,we hope to provide new targets for ITP immunotherapy.