Mechanisms Underlying the Mycobacterium Tuberculosis Mediated IP-10 (IFN-gamma-inducible protein 10 kD) expression in Human Monocytic cells
| Autor: | Kuo-Hsiung Huang, 黃國雄 |
|---|---|
| Rok vydání: | 2014 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 102 C-X-C chemokines, such as IP-10 (CXCL10, IFN-gamma-inducible Protein 10 kD) and IL-8 (CXCL8, Interleukin 8) play critical roles in the immunopathogenesis of pulmonary against Mycobacterium tuberculosis (M. Tb) at the pathologic site(s). NF-kappaB repressing factor (NRF) is a transcriptional silencer and has been reported to bind in negative regulatory element (NRE) site and implicated in the basal silencing of IL-8 genes. We checked and found a similar NRE site in IP-10 gene promoter region. To understand the regulatory role of NRF in IP-10 release on pulmonary TB, we studied the alveolar macrophages (AM) and peripheral blood mononuclear cells (PBMC) derived from patients with active TB and normal subjects, and THP-1 cell line, respectively. The Amplified Mycobacterium Tuberculosis Direct (AMTD) test showed that the bacterial loads in AM of pulmonary TB patients in terms of ribosomal RNA were highly compatible with sputum bacterial load of M. Tb. Using confocal image analysis, western blot and quantitative real-time PCR, we demonstrated that the protein and mRNA levels of IP-10 and NRF were significantly higher in AM, PBMC in patients with active pulmonary TB and THP-1 cells treated with heated Tuberculosis bacilli (H. TB), respectively. And the release of IP-10 is mediated via NF-kappaB (p65). In PBMC, the chromatin immunoprecipitation (ChIP) assay showed a higher binding of NRF to IP-10 promoter sites in TB patients with high bacterial load compared to low bacterial load or normal subjects. The ChIP assay in THP-1 cell treated with H. TB showed NRF binding to IP-10 promoter sites was higher in high dose H. TB group. NRF knockdown (SiNRF) significantly increased the release of IP-10 from PBMC of TB patients with high bacterial load than normal subjects. SiNRF can also increase the release of IP-10 in normal subjects’ PBMC and THP-1 cells treated with H. TB, significantly. Using p-CMV6-XL4-NRF (p-CMV-NRF), overexpression NRF inhibited NF-kappaB-mediated IP-10 synthesis and release in active pulmonary TB patients’ PBMC and THP-1 cells treated with H. TB, respectively. In active pulmonary TB patients’ AM and PBMC, the repressive effect of NRF is mediated via interference with NF-kappaB (p65) binding and recruitment to promoter sites of IP-10. NRF downregulated IP-10 basal expression and H. TB induced IP-10 synthesis via interference with NF-kappaB (p65) binding and RNA polymerase II recruitment to IP-10 promoter site in THP-1 cells. Taken together the present results, we not only reveal the mechanism in NRF modulate M. Tb induced IP-10 release for the first time, but also provide a possible therapeutic strategies in the regulation of NRF to helping pulmonary TB patients. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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