Effects Of IL-18 And IL-33 On The Expression Of Th1 And Th2 Cytokines In The Pathogenesis Of Primary Autoimmune Thrombocytopenia

Purpose and Background:Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by antibody mediated destruction of platelets, inhibition of production of platelets by megakaryocytes, and T cell dysfunction. We aimed at providing a comprehensive analysis of cytokine profiles and their dynamic changes of newly diagnosed ITP, complete response to dexamethasone, and relapsed patients after treatment of dexamethasone.Materials and Methods:10healthy controls (defined as "Control" group) and23ITP patients were included in our study; before treatment,17samples of sera were collected (classified as "New ITP" group), all of the17patients achieved complete response to dexamethasone (grouped as "Response"group).4out of17plus another6patients were relapsed after treatment of dexamethasone (grouped as "Relapse" group). Utilizing quantitative cytokine array ELISA to analyze serum cytokine levels.Results:Increased Th1associated cytokines:IFN-γ. TNF-a, were observed in newly diagnosed ITP patients compared with healthy controls; while Th2associated cytokines:IL-5, IL-6, IL-13also decreased in newly diagnosed ITP patients. After complete response to dexamethasone treatment, Th1associated cytokines:IL-2. IFN-y. TNF-a. Th2associated cytokines IL-6. IL-10were increased compared with each patient’s pre-treatment levels respectively. However, patients who relapsed after Dexamethasone showed no significant changes of these cytokines.Conclusion:Quantitative ELISA data revealed, compare to control group, that there were significant decrease of Thl and Th2associated cytokines in newly diagnosed ITP patients, which were increased after treatment with dexamethasone. PART II Role of IL-18, IL-33Regulates CD4+TCell Function and Cytokine Production in Primary Immune Thrombocytopenia through T-bet, GATA3ABSTRACTPurpose and Background:To determine the role of Thl, Th2imbalance in Immune Thrombocytopenia (ITP), we conducted in vitro culture of CD4+T lymphocytes aiming at finding its functional changes in ITP. and its response to dexamethasone; and to explore the function of interleukin18(IL-18), and interleukin33(IL-33) and its signal pathway in pathogenesis of ITP.Materials and Methods:15samples from5healthy controls (defined as "Control" group),9samples from3ITP patients without treatment (classified as "New ITP" group), and another9samples from the other3ITP patients who had complete response to dexamethasone (grouped as "Response" group) were collected. When isolated from peripheral blood and purified with magnetic beads, CD4+T lymphocytes were cultured under three different conditions of10ng/mL of IL-18,100ng/mL of IL-33and none stimulus respectively for4days. Cells were yielded on day0,1,4and underwent flow cytometry for markers of CD4.IL-13. T-bet, and GATA3. Their mRNAs were extracted on day0,1,3.4and evaluated with RT-PCR for E4BP4, IL-5, IL-10, IL-18Ra. GATA3, ST2L, and T-bet. Supernatants were collected on day0,1,3,4for testing levels of IL-5, IL-10. IL-13. TNF-α. and IFN-y using Cytometry Beaded Array (CBA).Results:In vitro CD4-T cell culture studies showed that both New and Response ITP patients had significantly higher cytometry expression of intracellular T-bet in all days, IL-13at day0. day1; but decreased expression of intracellular GATA3at day4. Intracellular mRNAs were significantly increased of E4BP4. T-bet, IL-10expressions:meanwhile, increased expressions of IL-10. GATA3. and ST2L were also noticed in Response group. Significantly lower extracellular levels of IL-5, IL-10. IL-13, and TNF-a and IFN-γ were found in both New ITP and Response ITP patient compare with Control group in all days. No significantly different expression of neither of the above-mentioned markers, mRNAs or cytokines were observed among IL-18, IL-33, and none stimulus groups. Generally speaking. Response group to dexamethasone did not showed significant changes compared with New ITP group in cytokine expression and excretions.Conclusion:Extracellular Th1/Th2associated cytokines in ITP patient showed decreased level of IL-5, IL-10, IL-13, TNF-α, and IFN-γ compared with control group, which was consistent with quantitative ELISA assay we performed earlier. Intracellular cytokines showed increased expression of Th1associated T-bet; intracellular Th2associated expression of GATA3and IL-13showed high expression during first day of culture, but low expression4days later. It revealed a potential new point of view:Th1/Th2associated cytokines were regulated at different stages of cytokine excretion, naming, post-transcriptional regulation, post-translational modification and excretion control. In vitro culture of CD4+T cell has no significance between groups with IL-18, IL-33, or none stimulus. Therefore, we conclude that IL-18and IL-33function during Th0early differentiation but not after Th1/Th2differentiation.