Cyclic AMP Response Element-Binding Protein Regulates Transcription And Production Of IFN-γ

BackgroundTuberculosis remains a major health problem worldwide. Two billion people are infected with the pathogenic agent, Mycobacterium tuberculosis. Approximately 8-10 million people are infected with this pathogen every year. China was one of the highest TB epidemic countries, according to the results of the national epidemiological sampling surveys on TB conducted in 2000, it's characterized by higher TB prevalence (the prevalence of pulmonary tuberculosis was 367/100,000 and smear-positive prevalence was 122/100,000) and higher drug resistance rate(the primary resistance rate was 18.6% ). Development of multi-drug resistant TB (MDR-TB) and HIV-TB coinfection are greatly increased worldwide, posing a threat to the existing therapeutic possibilities. Hence, the reach of new prophylactic and therapeutic strategies are urgent to control TB.TB is a disease caused by Mycobacterium tuberculosis whose interaction with the host may lead to a cell-mediated protective immune response. Interferon- gamma (IFN-γ) is essential to the response. IFN-γ, a key cytokine in control of M. tuberculosis infection is produced by both CD4+ and CD8+ T cells, as well as by NK cells. IFN-γmight augment antigen presentation, leading to recruitment of CD4+ T-lymphocytes and/or cytotoxic T-lymphocytes, which might participate in mycobacterial killing. Although IFN-γproduction alone is insufficient to control M. tuberculosis infection, it is required for the protective response to this pathogen. IFN-γis the major activator of macrophages, such as generation of reactive oxygen intermediates (ROI), reactive nitrogen intermediates (RNI), so it helps macrophages to inhibit the growth of M. tuberculosis.Transcription of the IFN-γgene in activated T cells is controlled by the proximal promoter element (-73 bp to -48 bp). Binding of specific proteins to these regulatory regions markedly affects IFN-γpromoter activity and the production of IFN-γ. Cyclic adenosine monophosphate response element binding protein (CREB), as a transcription factor, binds to the IFN-γproximal promoter to adjust the transcription of the IFN-γ, and the binding is enhanced by phosphorylation of CREB.At present, the study of CREB protein regulatory of IFN-γtranscription is in an infancy stage. Some questions need to be elucidated such as CREB protein is a positive or negative regulator of IFN-γtranscription, the function of CREB in immune protection. Knowledge about the functioning of CREB protein during IFN-γtranscription and production provides the basis for rational tuberculosis treatment and prevention.Purpose1. Study binding of CREB protein to the proximal IFN-γpromoter in vitro and in vivo; discuss the function of CREB protein in IFN-γtranscription.2. Compare the level of CREB protein in tuberculosis patients and PPD-positive healthy donors; investigate the functions of CREB protein in the immunity against tuberculosis.MethodsPart one:Venous blood samples were obtained from 18 PPD-positive healthy donors and from 25 HIV-seronegative patients with smear-positive pulmonary tuberculosis, all of whom had received < 4 weeks of antituberculosis therapy. PBMC were isolated by differential centrifugation over Ficoll-Paque. Freshly isolated PBMC were incubated with magnetic beads conjugated to anti-CD3 and a magnetic cell separator was used to positively select CD3+ cells. The purity of CD3+ cells was measured by flow cytometer. After Extraction of nuclear proteins, EMSA was performed to determine nuclear proteins binding to the proximal IFN-γpromoter in vitro, and the specificity of binding complex was tested by competitive EMSA. Western blotting was performed to compare the difference of expression of CREB protein in tuberculosis patients and PPD-positive healthy donors.Part two:Venous blood samples were obtained from 18 PPD-positive healthy donors and from 25 HIV-seronegative patients with smear-positive pulmonary tuberculosis. PBMC were isolated by differential centrifugation over Ficoll-Paque,Freshly isolated PBMC were incubated with magnetic beads conjugated to anti-CD3 and a magnetic cell separator was used to positively select CD3+ cells. The purity of CD3+ cells was measured by flow cytometer. CD3+ cells were cultured with heat-killed mycobacterial Ags for 24h. Chromatin immunoprecipitation with anti-CREB Ab was used to determine whether CREB binds to the chromosomal IFN-γproximal promoter in vivo in live T cells exposed to microbial Ags. Western blotting with Abs specific for serine 133-phosphorylated CREB was performed to determine whether M. tubtuberculosis Ags elicited phosphorylation of CREB.ResultsPart one:The results of EMSA showed a low-mobility complex binding to the IFN-γpromoter, the binding pattern observed was similar for T cells from all 18 PPD-positive healthy donors. However, in T cells from 18 of 25 tuberculosis patients, the low-mobility complex binding to the IFN-γpromoter was absent. It suggested that DNA-binding proteins to the IFN-γpromoter reduced in tuberculosis patients. The results of competitive EMSA suggested these nuclear proteins specifically bound to the IFN-γpromoter region and contained CREB. CREB protein expression markedly decreased in tuberculosis patients compared with PPD-positive healthy donors detected by Western blotting.Part two: The results of ChIP showed a 204bp product yielded in CD3+T cell from 10 PPD-positive healthy donors cultured with heat-killed M. tuberculosis for 24h. However, CD3+T cell from 12 tuberculosis patients didn't yield a 204bp product. The results elicited that CREB protein can bind to the chromosomal IFN-γproximal promoter in vivo in live T cells from PPD-positive healthy donors exposed to microbial Ag, but no binding was seen in live T cells from tuberculosis patients. Furthermore, M. tubtuberculosis Ags also elicited phosphorylation of CREB in CD3~+T cell from PPD-positive healthy donors, but not in CD3+T cell from tuberculosis patients.conclusions1. CREB protein binds to IFN-γproximal promoter to regulate the transcription and production of IFN-γ.2. CREB protein binding to IFN-γproximal promoter reduced in tuberculosis patients compared with PPD-positive healthy donors. Tuberculosis patients had diminished CREB protein levels, reduced ability of binding to the IFN-γpromoter, diminished IFN-γpromoter activity, and low IFN-γproduction.3. CREB positively regulates the production of IFN-γby human T cells in response to M. tuberculosis.