The Study On Mechanism And Intervention Strategy Of Imbalanced CD4 T Cells In Primary Immune Thrombocytopenia

Primary immune thrombocytopenia(ITP)is the most common autoimmune hemorrhagic disease,accounting for about 1/3 of all bleeding disorders.Clinically,ITP patients are characterized by hemorrhagic tendency or bleeding symptoms caused by thrombocytopenia.Traditionally,the term ITP has been used to denote "idiopathic thrombocytopenic purpura",but ITP has been revised to refer to "primary immune thrombocytopenia" as studies have demonstrated the important role of immune imbalance in thrombocytopenia and the absence or infrequent symptoms of hemorrhage in a large number of clinical cases.As an autoimmune disease,the pathogenesis of ITP has not been completely clarified so far.Current understanding of the pathogenesis of ITP is mainly focused on thrombocytopenia caused by autoantibody-mediated platelet clearance.Platelets coated with surface antigens(glycoprotein ?b/?a)are susceptible to phagocytosis and opsonization of mononuclear-macrophage system,of which self-antigens are presented to lymphocytes,leading to activation of T and B cells.As a result,self-reactive cytotoxic T cells and self-antibodies against platelets are produced and result in platelets destruction in large amount.Meanwhile,activated CD8+ T cells and self-antibodies can also cause reduction and dysfunction of megakaryocyte.Moreover,we see a significantly reduction of thrombopoietin(TPO)level in ITP patients,and the lifespan of platelets in patients is more limited than healthy person,all of which participant in the pathogenesis of ITP.Recent studies has indicate that the imbalance of Th1/Th2 and Th17/Treg is considered as a critical factor contributing to self-antigen immune intolerance.Recommendation of ITP treatment includesimmunosuppressive agents such as glucocorticoids,B cell-targeted rituximab and intravenous immunoglobulin(IVIG).Another treatment strategy is stimulus of platelet production,such as platelet receptor agonism,TPO-RAs.Overall,non-specific drugs are the main agents in ITP therapy.Although glucocorticoids are currently used as the first-line therapy for newly-diagnosed ITP,the long-term side-effects cannot be ignored,such as osteoporosis,femoral head necrosis,hypertension,diabetes and acute gastric mucosal lesions.Despite the recommendation of new treatment strategies such as rituximab and TPO receptor agonists in recent years,there are still one-third of ITP patients that appear recurrence because of the lack of effective treatment.Many studies have shown that Th cell subsets and Treg cells are closely related to various immune diseases.Therefore,investigation of the roles and mechanisms of Th subsets and Treg cells in the development of ITP is absolutely necessary,which can provide novel targets for ITP treatment and prognosis.PART ?Aberrant Expression of MicroRNAs in CD4+ Cells May Contribute to the Imbalance of Th17/Treg Cells in Primary Immune ThrombocytopeniaBackgroundPrimary immune thrombocytopenia(ITP)is a hemorrhagic disease caused by increased platelet destruction or decreasedproduction of platelets,which is mediated by immune dysfunction.Especially in recent years,the imbalance of Th17/Treg has provided a new pointin the pathogenesis of ITP.MicroRNA(miRNA)is a type of non-coding RNAs with about 18-25 nucleotides in length,which regulate cell functions in eukaryotic cells.The miRNA usually actsas an inhibitor of mRNA transcription or induces transcript degradation by binding to the 3'UTR sequence of the target genes.Approximately 30%of the human genes are regulated by miRNAs and one miRNA may have the potential to regulate the function of hundreds of genes.It has been shown that miRNAs are involved in the regulation of immune function and homeostasis,including the activation,function and homeostasis of effector T cells and Treg cells.miR-183-96-182 clusters could regulate Th17 differentiation.miR-99 is involved in regulation ofTreg cell differentiation.Our team and other study groups have found that the abnormal expression of miRNAs in PBMCs,serum and plateletsmay be closely related to the immune imbalance in ITP patients.However,it is not clear whether miRNAs are involved in the Th17 and Treg cells imbalance of ITP patients.Therefore,it is necessary to explore the relationship between miRNAs and Th17 and Treg cells imbalance in ITP pathological mechanism.ObjectivesThis study aimed to investigate the involvement of microRNAs(miRNAs)in chronic ITP immune imbalance,especially the pathological mechanism of Th17 and Treg imbalance.The differential expression of miRNAs between chronic ITP patients and healthy controls andthe correlation between abnormally expressed miRNAs,proportions of Th 17 and Treg,and clinical featuresin chronic ITP patients was analyzed.By interfering the abnormal expression of miRNAs in CD4+ in vitro,we observed the effect of miRNAson Th17 subsets and Treg cells.Finally,the mechanisms of miRNAsin affecting Th17 subsets and Treg cells were investigated in ITP.Methods(1)In this study,peripheral blood was collected from 36 chronic ITP patients from Qilu Hospital of Shandong University,and 60 healthy controls were enrolled in the study.PBMCs and CD4+cells were isolated and the plasma was collected.(2)The relative expression of miR-17-5p,miR-99a,miR-146-5p,miR-155-5p,miR-181-5p,miR-182-5p,miR-183-5p and miR-326 in CD4+ cell wereanalyzed by RT-PCR.(3)The frequency of Th17 and Treg cell in PBMCs of ITP patients was detected by FCM.Relationship between abnormally expressed miRNAs and Th17 subsets and Treg cells was analyzed.(4)The expression of IL-17A?IL-10 and TGF-? in plasma weredetectedby ELISA.Relationship between abnormally expressed miRNAs and IL-17A?IL-10 and TGF-? was analyzed.(5)Correlation analysis between abnormally expressed miRNAs and clinical featuresof chronic ITP patients was performed.(6)miRNAsmimics and inhibitors weretransfected into CD4+ cells of chronic ITP patients.And the frequency of Th17 or Treg cells in the transfected CD4+ cells was analyzed by FCM(7)The expression of mTOR and p-mTOR in miR-99a transfected CD4+ was analyzed by western blot.Results:(1)The relative expression of miRNAs was analyzed in CD4+ cells of ITP patients and healthy controls.RT-PCR analysis showed that the expression of miR-182-5p and miR-183-5p in CD4+ cells from patients was higher than that of the healthy control,while miR-99a expression significantly decreased in ITP patients.However,there was no significant difference in the expression of miR-17-5p,miR-96-5p,miR-146-5p,miR-155-5p,miR-181-5p,and miR-326 between ITP patient and healthy control.(2)Correlation analysis between miRNAs and Th17 or Treg cells was analyzed.Flow cytometry analysis showed that the frequency of Th17 cells in peripheral blood from chronic ITP patients was significantly increased,while the frequency of Treg cells was significantly decreased.Correlation analysis showed that the expression of miR-99a in CD4+ cells was positively correlated with the frequency of Treg cells in PBMCs from chronic ITP and healthy controls.There was no correlation between miR-182-5p or miR-183-5p and Th17 cells.Compared with healthy control,the proportion of Treg/Thl 7 in patients with chronic ITP was significantly reduced,corresponding to the ratio of miR-99a/miR-182-5p or miR-99a/miR-183-5p in CD4+ cells of patients with chronic ITP.(3)Correlations between miRNAs and IL-10,IL-17A or TGF-? in plasma was analyzed.The level of IL-17A in peripheral blood of patients with chronic ITP was significantly higher than that of healthy control,while it was not associated with the relative expression of miR-182-5p or miR-183-5p in CD4+ cells.Compared with healthy control,the level of IL-10 and TGF-?decreased.IL-10 was positively correlated with the expression of miR-99a,but TGF-? was not associated with miR-99a in CD4+cells.(4)The relationship between miRNAs and clinical features self-antibody in chronic ITP patient.To determine whether miRNAs were associated with clinical characteristics of patients,such as platelet count,severity,and corticosteroid sensitivity,we analyzed the expression of miRNAs between different groups.The expression of miR-183-5p was negatively correlated with the platelet count.The expression of miR-183-5p in CD4+ T cells from severe ITP patients was significantly higher than that from non-severe ITP patients.(3)there was no significant difference in the expression of miRNAs between the corticosteroids sensitive group and the resistant group;(4)there was no significant difference in the expression of miRNAs between the positive group and the negative group of GP antibody(5)The effects of miRNAs mimics or inhibitors on the differentiation of Th17 and Treg cells of ITP patients.The frequency of Th17 was decreased after down-regulation of miR-1 83-5p,while up-regulation of miR-99a expression with mimics increased Treg proportions in CD4+ cells of ITP patients.However,there was no significant difference in the proliferation of Thl7 cell with the inhibitor of miR-182-5p.(6)miR-99a may regulate the differentiation of Treg cells of ITP patients by mTOR signaling pathway.To further investigatethe mechanism by which miR-99a is involved in the regulation of Treg cells from ITP patients,we found that miR-99a mimics significantly inhibited the level of mTOR phosphorylation and mTOR expression in comparison with vector mimics.Conclusions(1)Abnormal expression of mir-99a,mir-182-5p and mirl83-5p in CD4+ cells from patients with chronic ITP is closely related to Th17/Treg imbalance,suggesting that abnormal expression of miRNAs may be involved in the pathological mechanism of Th17/Treg immune imbalance in patients with chronic ITP.(2)Intervention of miRNAs in CD4+ cells of chronic ITP patients can regulate the differentiation of Th17 and Treg cells;especially miR-99a may participate in the regulation of Treg cell subsets through mTOR signaling pathway.(3)miR-183-5p is closely related to clinical features such as platelet count and disease severity in patients with chronic ITP,suggesting that it may provide a basis for clinical evaluation of patients with chronic ITP.PART ?The role and Mechanisms of Th17/1 subsets in Chronic ThrombocytopeniaBackgroundThl7 is an importantsubsets of effector T cells,which is characterized by the secretion of IL-17A,IL-17F,IL-21 and IL-22 for immunological function.These cytokines are considered to be key regulators of immune response,but are also important drivers of autoimmune disease.At present,several studies have shown that Th17 and itsrelated cytokines are abnormally elevated in ITP patients,but one study has also shown that Th17 is not elevated.Therefore,whether Th17 participates in the pathogenesisof ITP remains to be studied.Recently,Th17 cells have been divided into two subgroups:pathological(proinflammatory)Th17(also termed as Thl7/1)and non-pathological(non-proinflammatory)Th17.The Th17/1 secretes IL-17A and IFN-? and loses IL-10.Studies have confirmed that pathological Th17 cells are involved in a variety of autoimmune diseases,such as Graves' disease,systemic lupus erythematosus.At present,whether the proinflammatory Th17/1 is abnormally expressed in ITPand participates in the pathogenesis of ITP remains to be clarified.ObjectiveIn this study,we aimed to explore the proportion of Th17/1 and the related pathological molecular markers.We also analyzed the effect of glucocorticoid on Th17/1 subgroup and its molecular "marker" expression in the development of ITP.Methods(1)We collected peripheral blood from 36 chronic ITP patients from Qilu Hospital of Shandong Universityand 60 healthy control,from which PBMCs and CD4+ cells were isolated.(2)The frequency of proinflammatory Th17 cellswereanalyzed by flow cytometry.PBMCs isolated werestimulated by PMA,ionomycin and monensin for 4 hours,and subsequently stained with intracellular antibodies.(3)Detection of "markers" of pathological Th17 cells:The expression of pathological Th17markers in CD4+ cells was analyzed by RT-PCR.(4)To definethe effect of glucocorticoid on Th17 cells and their molecular "markers"in chronic ITP patients,we treated the PBMCs with dexamethasone and observe the changes of pathological Th17 and its characteristic markers by flow cytometry.(5)Expression analysis of transcription factors related to Th17:NF-?B/STAT signaling pathway in CD4+ cells were analyzed by Western blot.Results(1)Flow cytometry analysis showed that the proportion of Th1 and Th17 in peripheral blood of patients with chronic ITP was significantly higher than that of healthy control.More importantly,the proportion of pro-inflammatory Th17/1 in patients with chronic ITP was higher than that in control group.(2)Further analysis of pro-inflammatory Th17/1 related molecular "markers" in CD4+cells of patients with chronic ITP by RT-PCR revealed that Cxcl3,Il-22,Tbx-21 Stat4?Toso and Il-23r in CD4+ cells of ITP patients elevated compared with healthy control,while the expression of Il-10 and Ahr was significantly reduced.(3)After treatment with dexamethasone,cytokines related to pro-inflammatory Th17/1 in culture supernatant were analyzed.The proportion of th17/1 subgroup in chronic ITP patients was positively correlated with IL-17A level.The proportion of th17/1 subgroup in chronic ITP patients was not correlated with IFN-? or IL-22.The proportion of th17/1 subgroup in chronic ITP patients was positively correlated with the level of IL-23 expression in plasma.There was no correlation between th17/1 subgroup ratio and plasma IL-6 or TGF-?1 expression in patients with chronic ITP.(4)Correlation analysis between th17/1 subgroup proportion and clinical characteristics of patients with chronic ITP was analysed.The proportion of th17/1 subgroup in peripheral blood of patients with severe ITP was significantly higher than that of patients with non-severe ITP.There was no correlation between the proportion of th17/1 subgroup and the level of platelet count in ITP patients.The proportion of th17/1 subgroup in the corticosteroid-resistant group was higher than that in the corticosteroid-sensitive group,but there was no statistical significance.There was no significant difference in the level of th17/1 subgroup between patients with positive and negative antiplatelet autoantibodies.(5)The effect of rhIL-6 or rhIL23 on th 17/1 differentiation in PBMCs of chronic patients was analysed.Flow test results showed that,compared with the control group,there was no significant difference in the proportion of th17/1 subgroup in rhIL-6 group,rhIL-23 group,rhIL-6 group and rhIL-23 combined group.(6)The effect of dexamethasone on th17/1 subgroup was analysed.The proportion of th17/1 subgroup in the dexamethasone group was significantly lower than that in the control group after 96 hours of treatment with dexamethasone in vitro.(7)The effect of dexamethasone on the pathogenic molecular markers related to thl7/1 subgroup was analysed.The expressions of chemotactic factors CXCL3,CCL4 and CCL5 in the dexamethasone-treated group were significantly lower than that in the control group in vitro.The expression of effector molecules or receptors GZMB and IL23R in dexamethasone-treated group was significantly reduced,while there was no significant difference in the expression of IL22 after dexamethasone treatment.The expression of the transcriptional regulatory molecule TBX21 was significantly decreased in the dexamethasone-treated group,while the expression of IKZF3 and STAT4 was not significantly different.The expression of IL-10 and its regulatory molecule MAF was significantly increased,while the expression of AHR was not significantly changed after dexamethasone treatment.(8)Mechanisms of dexamethasone involved in the regulation of pro-inflammatory T17/1 subpopulation was analysed.The expression of pSTAT-3/STAT3 decreased after dexamethasone treatment compared with control group,while there was no significant difference in the expression of pSTAT1/STAT1 and pSTAT4/STAT4.2)There was no significant difference in p-mTOR/mTOR levels after dexamethasone treatment.The expression of pp65/p65 decreased after dexamethasone treatment when we analysed the NF-?B signaling pathway.After treatment with NF-?B inhibitor Bay 11-7082 and mTOR inhibitor rapamycin for 4 days,flow cytometry revealed that Bay 11-7082 resulted in a significant reduction of Th 17/1 while rapamycin had no significant effect on the proportion of pro-inflammatory Th17/1.Conclusions(1)Abnormal elevated proinflammatory Th17/1 and expression of closely related molecules in peripheral CD4+ cells of chronic ITP patients suggest that they may be involved in the development of chronic ITP(2)In vitro experiments showed that dexamethasone can inhibit the differentiation of pro-inflammatory Th17/1 and regulate the expression of pro-inflammatory Th17/1 related molecules.Correction of th17/1 subgroup imbalance may provide a new therapeutic direction for chronic ITP.(3)Dexamethasone may inhibit the pro-inflammatory Th17/1 cell subset by regulating the NF-?B/STAT-3 signaling pathway,which indicated that correcting the th17/1 immune imbalance in patients with chronic ITP by targeting the NF-B/STAT-3 signaling pathway may be expected as a potential target.PART ?The roles and mechanism of NLRP3Inflammasome-mediated adaptive immune imbalance in adult immune thrombocytopeniaBackgroundInnate immunity plays an important role in the initiation and activationof adaptive immunity as the first response to the invasion of foreign microorganisms.Therefore,it is necessaryto elucidate the pathogenesis of ITP from a novel point of innate immunity.As an important part of innate immunity,NLRP3 inflammasome has been well studied in the defense of exogenous microorganisms.In normal conditions,basal expression of NLRP3 is relatively low.When activated,NLRP3 inflammasome plays important biological roles through IL-1? and IL-18.NLRP3 inflammasome not only acts as an important part of innate immunity against exogenous microorganisms,but also plays a vital role in adaptive immunity.IL-1? can promote the secretion of IL-17A cytokines by T cells whichtendentiously polarize to Th17.As another effector cytokines of NLRP3 inflammasome,IL-18induces T cells to produce IFN-?[10].In addition,it was found that inthe development of gouty,NLRP3 inflammasome activated byurate crystalscaused excessive secretion of IL-1?,which is the main reason of gouty development.IL-1 receptor blocker and anti-IL-1? antibody were shown to have significant effect in the treatment of gout and arthritis.NLRP3 can also sense abnormal changes in metabolism and participate in inflammatory pathological processes related to metabolic disorders,such as decreased insulin sensitivity,pancreatic ?-cell dysfunction,and atherosclerosis.Therefore,whether NLRP3 inflammasome participates in the Th subsets and imbalance of ITP patients remains to be studied.ObjectivesThis study was designed to explore the effects and mechanisms of NLRP3 inflammasome on Th subgroups and Treg cells in patients with chronic ITP.Afterinterfering the activation of NLRP3 inflammasome in PBMC,we observed the proliferation and apoptosis of CD4+ or CD8+ T cells and analyzed proportions of Th subsets and Treg cells in peripheral blood.Cytokines and transcription factor-associated toThsubsets and Treg cell were detected after NLRP3 inflammasome activation.Further studies were made to define the key molecules involved in the regulation of Th subsets and Treg cells by NLRP3 inflammasome,thereby clarifying the roles and mechanisms of NLRP3 inflammasomein immune homeostasis imbalance of ITP.Materials and methods(1)A total of 28 patients with chronic ITP were enrolled in this study.PBMCs were isolated using Ficoll densitygradient centrifugation and the CD4+ cells were purified by positive immuno-magnetic microbeads selection.(2)NLRP3 inflammasome activation:The isolated PBMCs or CD4+ cells were divided into three groups:(1)Experimental group(LPS + ATP):LPS pre-treatment for 6 hours,plus ATP activation for 45 minutes;(2)Inhibition group:Before adding LPS,MCC950 was added and pre-treated for 1 h;(3)Control group:onlyaddDMSO and serum-free 1640 medium.(3)Cell culture after activation of NLRP3 inflammasome:cells were washed 10 mL of sterile PBS and residual PBS was discarded after NLRP3 inflammasome activation.The activated cells were resuspended in RPMI 1640 complete medium supplemented with IL-2,anti-human CD3 antibody and anti-human CD8 antibody,and were cultured in 37 ?C with5%CO2in cell culture incubator.(4)Analysis of Th subsets and Treg cell:flow cytometry(FCM)was used to detect the ratio of Thl,Th2,Th17 and Treg cells.(5)Analysis of Th subsets and Treg cell-associated cytokine and transcription factor expression:,cytokines:the relative expression of Ifn-y,Il-6,IL-10,Il-17,T-bet,Gata3,Foxp3,Ror-?t,Ctla-4,Btla,Tim3,Lag3,Pdl and Vista was detectedby RT-PCR.(6)T cell proliferation assay:the proliferation of CD4+ orCD8+ in PBMCs stained with CFSE was detected by FCM.(7)Detection of cytokines in the culture supernatant:ELISA were used to detect IFN-?,IL-17A and TNF-? cytokines in the culture supernatant.(8)Analysis of the effect of NLRP3 inflammasome on NF-?B signaling pathway:The changes of I?B?,I?B?,p105/p50 and p65 were analyzed by Western blot(WB).Results(1)The activation of NLRP3 inflammasome in PBMCs of ITP patients.Western blots showed that LPS+ATP promoted the expression of NLRP3,cleavedcaspase-1 and cleaved IL-1? in PBMCs of chronic ITP patients.ELISA showed that the expression of cleaved caspase-1 and celaved IL-1? were reduced aftertreatment of NLRP3 inhibitor MCC950.(2)The activation of NLRP3 inflammasome inhibited the proliferation of Thl in CD4+ cells fromITP patients.Compared with the control group,The activation of NLRP3 inflammasome inhibited the differentiation of Th1(CD3+CD8 IFN-?+IL-1T)inCD4+ cells from ITP patients.When the NLRP3 inhibitor MCC950 was added,Thl subset partially restored.Furthermore,the activation of NLRP3 inflammasome inhibited the differentiation of Treg cell subset in CD4+ cells.However,there was no difference in the differentiation of Th2 and Th17 subsetsin the control group.(3)The activation of NLRP3 inflammasome decreased the level of Thl-related cytokines secreted by CD4+ cells in culture supernatant.ELISA analysis showed that the expression of IFN-y and TNF-? were decreased in cell culture supernatant after activation of NRLP3 inflammasome.However,there was no significant difference in the level of IL-17 before and after activation of NRLP3 inflammasome.(4)CD4+ proliferation was inhibited after the activation of NLRP3 inflammasome.Compared with the control group,the activation of NLRP3 inflammasome significantly inhibited the proliferation of CD4+ cells,but this inhibition can be partially restored by MCC950.In addition,activation of NLRP3 inflammasome did not induce apoptosis in PBMCs.(5)The expression of inhibitory molecules was elevated by the activation of NLRP3 inflammasome.RT-PCR showed that Btla,pdcdl and Ctla-4 were up-regulated in the NLRP3-activated group compared with the control group.Flow cytometry revealed that the expression of BTLA and CTLA4 was up-regulated in CD4+ cells.(6)NLRP3/I?B? signaling pathway may be involved in the inhibition of Thl differentiation caused by NLRP3 inflammasome activation.In order to clarify that NLRP3 inflammasome activation inhibited the differentiation of Thl cells in patients with chronic ITP,western blot revealed that NLRP3 inflammasome activation could increase the expression of I?B?,especially in the nucleus,while the expression of I?B? decreased in the inhibitor groupConclusions(1)NLRP3 inflammasome inhibits CD8+ and Thl cells differentiation,which provides a new research direction for correcting T-cell immune imbalance in patients with chronic ITP.(2)The inhibition of Thl differentiation may be regulated by IL-1?/I?B?/NF-?B signaling pathway driven by NLRP3 inflammasome activation.(3)BTLA and CTLA-4 may be involved in the inhibitory effect of NLRP3 inflammasome activation on TCR-activated lymphocyte proliferation in patients with chronic ITP.