Monocyte-Astrocyte Networks Regulate Matrix Metalloproteinase-9Gene Expression And Secretion In Central Nervous System Tuberculosis

Objective: Tuberculous meningitis (TBM) is the most common diseases of thenervous system tuberculosis, also is the highest mortality rate of extrapulmonarytuberculosis. In the central nervous system tuberculosis, MMP-9secretion increased,and MMP-9levels was associated with complications and death. However, HowMycobacterium tuberculosis caused central nervous system tissue over-expressed andsecreted MMP-9did not know much about. In this study, through Mycobacteriumtuberculosis H37Rv strain infecting human peripheral blood mononuclear cells asconditioned medium to stimulate the human star glioma cell line U251, andunderstanding the characteristics of the brain tissue major cells expression andsecretion MMP-9in TBM, as well as interaction control of tissue cells in theregulation of MMP-9secretion, in order to further understand the pathogenesis oftuberculous meningitis.Methods：1.0×10~6U251cells per hole were inoculated in Six-well plates, andMycobacterium tuberculosis H37Rv were add into wells to stimulate for72haccording to1:1,10:1,100:1multiplicity of infection. Cells supernatant werecollected and MMP-9was detected by Elisa. Extracting total RNA and MMP-9geneexpression was detected by RT-PCR. Human peripheral blood mononuclear cells(PBMCs) were isolated by density gradient centrifugation, than diluted withRPMI-1640medium, inoculated onto six-well plates (1.0×10~6cells/hole), andinfected by Mycobacterium tuberculosis H37Rv cells with10:1multiplicity ofinfection at37°C for24h. The supernatant was collected and sterilized by0.2μmmembranefilter, which was called as a conditioned medium-CoMTB((PBMC+1640+H37Rv). The monocytes medium which were not infected by H37Rv calledCoMCON (PBMC+1640). Incubation Mycobacterium tuberculosis in monocyte-free1640was called as CoTB (1640+H37Rv). The conditioned medium in the above werediluted according to titre of1:5, than added into6or24well plates cultured starglioma cell line U251. Collected12h,24h,48h and72h supernatant after stimulation, MMP-9content was tested by ELISA, and cytokines of IL-1β, IL-6, IL-8, IL-10,IL-12, TNF-a content changes were detected by Cycometric Breads Array (CBA).Collected72h cells, extraction of total RNA, MMP-9and above inflammatorycytokine mRNA expression were further detected by quantitative RT-PCR. MMP-9activity in24h,48h,72h CoMTB group and72h CoMCON, CoTB group wereexaminated using Gelatin zymography. MMP-9protein expression of12h,24h,48h,72h CoMTB group and72h CoMCON, CoTB group were analyzed by Western Blot.Collection of0,0.5h,1h,2h and6h CoMCON and CoMTB groups cell nuclearprotein, NF-κB p65protein level was detected also by Western Blot.Results：1,The MOI=1,10, and100of Mycobacterium tuberculosis infection of U251cellsafter72h, MMP-9were not detected by Elisa and RT-PCR.2,The results of Elisa showed that MMP-9in the CoMTB group was significantlyincreased. It began to increase at12h, significantly increased at24h, then continued toincrease until72h, The longer stimulate, the higher content, while MMP-9inCoMCON and CoTB group were not detected. The difference was statisticallysignificant (P<0.01) among them；3,The TNF-a, IL-1β in CoMCON group were not detected, and low contentexpression in CoTB group, while were increased significantly in CoMTB group. Thedifference was statistically significant (P<0.01). IL-6, IL-8,IL-12could be detected inthree groups, but increased obviously in CoMTB group (P <0.05). IL-10level did notincrease in three group.4,The expression of MMP-9mRNA was found only in CoMTB group by RT-PCR.No expression in CoMCON and CoTB group. The cytokines of TNF-a, IL-1β did notexpress in CoMCON group and a low expression in CoTB group, while highexpression in group CoMTB. IL-6, IL-8, IL-12were all expressed in the three groups,but the most expression in CoMTB group. There was statistically significantdifference in the three groups. IL-10was not detected by RT-PCR, maybe IL-10content was rarely.5,The results of Gelatin zymography showed the92KDa blank translucent bandwere visible from24h,48h, and72h supernatants in CoMTB group. With the extend of stimulation time, the level of MMP-9expression was increased, while no bandswere visible from72h supernatants in CoMCON and CoTB group.6,The proteins of MMP-9expression can be detected from12h,24h,48h, and72hcell supernatant in CoMTB group by Western-Blot, while the proteins of MMP-9expression can not be detected in CoMCON and CoTB group.7,The levels of nucleo-protein NF-κB p65was detected by Western-Blot. The resultsshowed that the expression of NF-κB p65began to increase after30min of CoMTBstimulating U251cells. Compared with CoMCON group, the levels of NF-κB p65expression increased two-fold after1h, increased five-fold after2h, and continued toincrease until6h.Conclusion：Using Mycobacterium tuberculosis to stimulate directly U251cells doesnot cause MMP-9secretion and gene expression.Mycobacterium tuberculosisinfection in peripheral blood mononuclear cells(PBMCs)conditioned medium(CoMTB group) can stimulate glioma cells U251to secrete MMP-9. The longerstimulate, the higher content. MTB without PMBCs conditioned medium (CoTBgroup) and PBMCs without MTB conditioned medium (CoMCON group) stimulatingastrocytes did not cause the secretion of MMP-9. CoMTB caused inflammatorycytokines like TNF-a, IL-1β, IL-6, IL-8, IL-12to increase significantly. Conditionedmedium may increase the expression and secretion of MMP-9gene through activateof NF-κB signaling pathway. The results suggested that monocyte-dependentcytokine network plays an important role in matrix degradation in the central nervoussystem TB.