The Effects Of The Interaction Between Gata3 And Nfat1 On Il-13 Gene Expression In Human T Cells

The effects of the interaction between GATA3 and NFAT1 on IL-13 gene expression in human T cellsThe First Affiliated Hospital of Nanjing Medical UnversityAsthma is a complex inflammatory disease of the lung characterized by airway hyperresponsiveness (AHR), eosinophilic inflammation and mucus hypersecretion. The inflammatory process, which is the basis of AHR and mucus hypersecretion, is believed to be a result of inappropriate immune responses to common aeroallergens in genetically susceptible individuals. It has been hypothesized that CD4+ T cells which produce a Th2 pattern of cytokines (IL-4, IL-5, IL-13) play a pivotal role in the pathogenesis of asthma. However, the therapy targeted at IL-4 and IL-5 proved little efficacy. IL-13 has been recognized as a critical regulator of the allergic response. Blockade of IL-13 markedly inhibits allergen-induced airway eosinophilia , hyperresponsiveness, mucus production and airway remodeling. However, the molecular mechanisms regulating IL-13 production are still largely unknown. GATA3 and NFAT1 both play an important role in the expression of IL-13 gene. To investigate the interaction between GATA3 and NFAT1 on IL-13 gene expression, human T cell lines (Hut-78 cells) were stimulated with anti-CD3 and anti-CD28 antibodies to mimic antigen-mediated co-stimulation in vivo. PART I: RNA interference inhibits the transcription factor expression of GATA-3 in T cellsObjective: To use the flow cytometer detect the transfection efficiency after chemosynthesis siRNA transfected T cells and investigate the interference effect on the transcription factor expression of GATA-3.Methods:Hut 78 cells were transfected with FAM-Negative siRNA .Six hours later, transfection efficiency was evaluated by flow cytometer.Hut 78 cells were transfected with GATA-3siRNA .Forty-eight hours later, the effect of GATA-3 siRNA on the expression of GATA-3 was identified by Western blot and real-time PCR.Results: The result of Western blot showed that the expression of GATA-3 was decreased in Hut 78 cells due to RNA interference. The result from real-time PCR showed the same pattern as Western blot.Conclusion: The chemosynthesis siRNA can transfect Hut 78 cells successfully and could effectively inhibit the expression of GATA-3.PART II: Regulation of IL-13 gene expression by GATA3-siRNA in T cellsObjective: To study the interleukin 13 (IL-13) gene expression in T cells due to CD3/CD28 co-stimulation and the effect of GATA3-siRNA on subsequent NFAT1 binding to IL-13 promoter and the interaction between GATA3 and NFAT1 on IL-13 gene expression.Methods: Hut-78 cells were stimulated with anti-CD3/CD28 monoclonal antibodies (10μg/ml and 5μg/ml, respectively). IL-13 mRNA level in Hut-78 cells was measured by Rael-time PCR. Chromatin immunoprecipitation assays were used to investigate the binding of NFAT1 to the promoter of IL-13. Results: The level of IL-13 mRNA increased in Hut-78 cells due to CD3/CD28 co-stimulation. However GATA3-siRNA inhibited the gene expression of IL-13. GATA3-siRNA inhibited the binding of NFAT1 and IL-13 promoter in actived human T cells in 30min.Conclusions: GATA3-siRNA would inhibit the gene expression of IL-13 and the binding of NFAT1 to IL-13 promoter in activated T cells. GATA3 might be partially contribute to the binding of NFAT1 to the IL-13 promoter.PART III: The role of FP on the gene expression of IL-13 in active human T cellsObjective: To study the effect of FP on the IL-13 gene expression and subsequent GATA3 binding to IL-13 promoter in human T cells due to CD3/CD28 co-stimulation and investigate the anti-inflammatory mechanism of FP.Methods: Hut-78 cells were stimulated with anti-CD3/CD28 monoclonal antibodies (10μg/ml and 5μg/ml, respectively). IL-13 mRNA and GATA3 level in Hut-78 cells were measured by Rael-time PCR. Chromatin immunoprecipitation assays were used to investigate the binding of GATA3 to the promoter of IL-13.Results: The level of IL-13 mRNA increased in Hut-78 cells due to CD3/CD28 co-stimulation. FP inhibited the gene expression of IL-13 but had no effect on the expression of GATA3. The binding GATA3 to the promoter of IL-13 was inhibited by FP at the concentration of 10-8M.Conclusion: FP would inhibit the gene expression of IL-13 and had no effect on the expression of GATA3. Glucocorticoids inhibit GATA3 binding to IL-13 promoter through other ways than inhibiting the gene transcription of GATA3.