The Effect On The Transformation Of Monocytes/macrophages Infected With Mycobacterium Tuberculosis By MAPK,PI3K/Akt And JAK/STAT Signaling Pathways

Background and Objective Mycobacterium tuberculosis is one of the most important infectious agents yet identified. Approximately one-third of the world'population is infected with M. tuberculosis. One lesson learned from the resurgence of tuberculosis and multidrug-resistant tuberculosis is the important of tuberculosis control strategies.M. tuberculosisis is an intracellular pathogen which survives within macrophages. Tuberculous nodules is the most important pathological characteristic of tuberculosis. Fibroblasts proliferlation consists of granuloma. Later, tuberculous nodules consist of ECS transformed from monocytes/ macrophages and multinucleated giant cells from ECS.Many studies had reported that MAPK , PI3K/Akt and JAK/STAT signaling pathways were activated and/or depressed in host cells infected with M. tuberculosisis, and these signaling pathways had been involved in the transductionprocess of cell transformation signals. But at present there's no study having reported that these signaling pathways had been involved in the monocytes/macrophages to ECS transformation process in host cell infected with M. tuberculosisis.In our studies, we established the human monocyte cell line THP-1 and fibroblast cell line WI-38 as an in vitro model for infection with M. tuberculosis H37Rv, M. tuberculosis bovis and M. phlie, carried out Pathscan? Phospho-p38 MAP kinase alpha (Thr180/Tyr182) Sandwich ELISA Kit, Pathscan? Phospho-Akt1 (Ser473) Sandwich ELISA Kit and Pathscan? Phospho-Stat3 (Tyr705) Sandwich ELISA Kit to detect the phosphorylated p38MAPK, phosphorylated Akt and phosphorylated STAT3 in cells on different infection phase. Immunofluorescent staining with Annexin V-fluorescein isothiocyanate (FITC) was carried out to detect expression changes of CD115 and CD82 in monocytes/macrophages infected with M. tuberculosis , and specific inhibitors SB203580, LY294002 and AG490 were used to detect whether these signaling pathways had been involved in the transformation process .Methods M. tuberculosis H37Rv, M. tuberculosis bovis and M. phlie infected with THP-1 and WI-38 for 2h, respectively, with negative control LATEX BEADS for 30min, phosphorylated p38MAPK, phosphorylated Akt and phosphorylated STAT3 were detected by kits on different infection phase(after ending infection Oh, 0.5h, 3h and 12h).Signaling pathways specific inhibitors SB203580 , LY294002 and AG490 were pretreated for 30min, respectively, and M. tuberculosis H37Rv, M. tuberculosis bovis and M. phlie infected with THP-1 for 72h, respectively, then we detected the expression changes of CD115 and CD82 in monocytes/macrophages and detected whether these signaling pathways had a relationship with thetransformation process using immunofluorescent staining with FITC.Results Compared to control LATEX BEADS, phosphorylated p38MAPK had significant down-regulation during the phase 0~0.5h in THP-1 infected with M. tuberculosis bovis, while phosphorylated Aktl had up-regulation during the phase 0~0.5h and phosphorylated STAT3 had no significant chang at the same treatment Phosphorylated Aktl had a peak at the point of 0.5h in WI-38 infected with M tuberculosis H37Rv and M. tuberculosis bovis, respectively.Monocytes/macrophages had an expression of CD115 but no CD82. After infected with M. tuberculosis H37Rv and M. tuberculosis bovis, respectively, We detected significant expression of CD82 in monocytes/macrophages. Specific inhibitors SB203580 and LY294002 pretreated , we found the loss of the significant expression of CD82, while no change after AG490 were pretreated.Conclusion MAPK and PDK/Akt signaling pathways were activated and/or depressed in human monocyte cell line THP-1 and fibroblast cell line WI-38 infected with Afycobacterium, but there's no JAK/STAT signaling pathway involved.M. tuberculosis H37Rv and M. tuberculosis bovis play an important role in the monocytes/macrophages to ECS transformation process, respectively, which can be inhibited by specific inhibitors SB203580 and LY294002, respectively. MAPK and PI3K/Akt signaling pathways have great relationship with the transformation process of tuberculous nodules.