Determination Of Cellular Transformation Of THP-1 Cells Mediated By Mycobacterium Tuberculosis Spp Infection And The Involved 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 EC transformed from monocytes/ macrophages and multinucleated giant cells from EC.It is reported that MAPK,PI3K/Akt and JAK/STAT signaling pathways were activated and/or depressed in host ceils infected with M.tuberculosisis,and these signaling pathways had been involved in the transduetion process 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 EC transformation process in host cell infected with M.tuberculosisis.In our studies,we established the human monocyte cell line THP-1 as an in vitro model for infection with M.tuberculosis H37Rv,M.tuberculosis bovis and M. phlei,carried out Pathscan(?)Phospho-p38 MAP kinase alpha(Thr180/Tyr182) Sandwich ELISA Kit,Pathscan(?)Phospho-Aktl(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 CD 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.The activity of acid phosphatase was determined by methods ofpyrocatechol phosphate.Methods M.tuberculosis H37Rv,M.tuberculosis bovis and M.phlei infected with THP-1 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,6h and 12h).Signaling pathways specific inhibitors SB203580,LY294002 and AG490 were pretreated for 30min,respectively,and M.tuberculosis H37Rv,M. tuberculosis bovis and M.phlei infected with THP-1 for 72h,respectively,then we detected the expression changes of CD in monocytes/macrophages and detected whether these signaling pathways had a relationship with the transformation process using immunofluorescent staining with FITC.M.tuberculosis H37Rv,M.tuberculosis bovis and M.phlei infected with THP-1 for 2h,respectively,with negative control LATEX BEADS for 30min,acid phosphatase activity was detected by kits on different infection phase(after ending infection Oh,24h,48h,and 72h).Results The strain H37Rv or strain bovis infected THP-1 cells showed a temporal up-regulation of the p38MAP-kinase phosphorylation level but no obvious alteration of Akt and STAT3 phosphorylation levels could be found.All the THP-1 cells respectively infected with the three strains weakened their CD14,CD115 expression and strongly express CD82,CD138.The THP-1 cells infected with anyone of the three strains continuously expressedCD14,CD115 after MAPK,PI3K/Akt or JAK/STAT of the cells was blocked.Althrough JAK/STAT was blocked,the THP-1 cells respectively infected with the three different strains still expressed CD82,CD138.However, CD82 and CD138 expressed by the strain H37Rv or strain boris infected THP-1 cells was disappeared when p38MAPK and PI3K/Akt pathways of the cells were blocked.The strain H37Rv infected THP-1 cells showed an obvious down-regulation of the acid phosphatase activities.Conclusion M.tuberculosis strain H37Rv and M.bovis can induce the infected THP-1 cells transforming to EC and p38MAPK and PI3K/Akt signaling pathways participate and regulate the transformation procedure.Of the two signaling pathways p38MAPK seems to be more impoetant.Significant changes in the levels of acid phosphatase in the macrophage lysate from M.tuberculosis infected cultures also indicated the cell transformation.