| Meningeal tuberculosis(MTB), caused by Mycobacterium tuberculosis(Mtb) bacilli, is a serious, often fatal form of TB and disproportionately affects children and HIV-infected patients, and charactered with high mortality. In order for meningitis to develop, Mtb bacilli must transit through the blood to the central nervous system that protected by a tighty brain microvascular endothelial cells(BMECs) formed blood-brain barrier(BBB). In in vitro and in vivo BBB models, Mtb bacilli were shown to successfully invade and traverse the BBB, but the cellular and molecular mechanisms involved are poorly understood.In this study, we investigated the interaction of mycobacterial bacilli with BMECs to illuminate the role of BMECs in controlling bacilli. Alos, we constructed an in vitro BBB using BMECs and astrocytes, and then infected with pathogenic and non-pathogenic mycobacterial bacilli to illuminate the mechanisms involved in the bacilli traverse BBB. The contents in this study are including:1. The analysis of receptors involved in mycobacterial bacilli infection in murine BMECs. To investigate if CD44 involved in mycobacterial bacilli infection, the murine BMECs were infected with M. bovis BCG and Mtb H37 Rv bacilli, and then examined at indicated time points using transmission electron microscopy(TEM), confocal microscopy and flow cytometry and CD44 antibody neutralizing experiment. The TEM results show that both M. bovis BCG and Mtb H37 Rv bacilli could internalize murine BMECs in absence of significantly difference. The confocal microscopy results show that CD44 was mainly expressed on the surface of murine BMECs, and little expressed inside the cell. Meanwhile, we found that CD44 was recruited at the site of mycobacterial bacilli infection, but the bacilli infection did not regulate the CD44 expression. Interestingly, CD44 neutralization antibody could not block the attachment or/and internalization of mycobacterial bacilli in murine BMECs. The data indicated that mycobacterial bacilli could infect and internalize murine BMECs, but CD44 does not involved in the process.2. The survival assay of mycobacterial bacilli in murine BMECs. To investigate if mycobacterial bacilli replicate in murine BMECs, the cells were infected with M. bovis BCG and Mtb H37 Rv bacilli, and then examined the viable bacilli by colony formation units and counted the intracellular bacilli by microscopy. The results show that the number of viable bacilli were light decreased and then increased over time in absence of antibiotic; but that significantly decreased over time in presence of antibiotic. The bacilli from cultural supernatant were also detected at indicated time points. The results show that significant increased number of bacilli was detected in absence of antibiotic, but not in presence of antibiotic. In addition, the intracellular bacilli counted by microscopy were used to further confirm the previously results. The data show that the number of infected cells, the number of cells including more than ten bacilli and the total bacilli were significantly decreased. All results indicated that neither M. bovis BCG nor Mtb H37 Rv bacilli replicate in murine BMECs.3. The effects of mycoabcterial bacilli infection on murine BMECs. To investigate if mycoabcterial bacilli infection causes murine BMECs cytotoxicity, the cells were infected with M. bovis BCG and Mtb H37 Rv bacilli. Monolayer visualization; LDH detection and percentage of trypan blue positive cells were performed at indicated time points. The results show that neither M. bovis BCG nor Mtb H37 Rv bacilli infection or persistence disrupt BMECs monolayer. In addition, the LDH released from infected cells and trypan blue positive cells did not significantly increase when compare to uninfected cells, indicating that both examined species did not induce cytotoxicity to BMECs.4. The trafficking of mycoabcterial bacilli in murine BMECs. To investigate if the failure of intracellular replication caused by degradation in BMECs, the cells were infeted with M. bovis BCG and Mtb H37 Rv bacilli, and the bacilli-containing comparmts were labeled with endosomal markers, Rab5 and Rab7, and lysosomal markers, LAMP2 and cathepsin L, and then analyzed using confocal microscopy. The results show that both Mtb H37Rv- and M. bovis BCG bacilli-containing compartments could lebel with the markers, and the colocalizion of bacilli with Rab5 alone were significantly decreased, but the colocalization of bacilli with both Rab7 and LAMP2 were significantly over time. In addition, Mtb H37 Rv bacilli-, but not M. bovis BCG-containing compartments are weak staining with cathepsin L, indicating that Mtb H37 Rv bacilli could delay endolysosomes acidification in some way. All the results indicate that bacilli-endosomes mature into endolysosomes and then degraded by the cell.5. The analysis of mycoabcterial bacilli escape into cytosol in murine BMECs. To investigate if mycoabcterial bacilli escape from lysosomes into cytosol to avoid the fast killing, the infected murine BMECs were labeled with SpiOC18 and Phalloidin, and examined using confocal microscopy. The results show that few Mtb H37 Rv bacilli could not be labeled with SpiOC18, but not M. bovis BCG bacilli, indicating that the pathogenic bacilli are capable to escape into cytosol. In addition, we detected if Mtb H37 Rv bacilli could traffic out of the infected cells. The results show that neither Mtb H37 Rv nor M. bovis BCG bacilli are form plaques in presence of antibiotic, but plaques are observed in absence of antibiotic, indicating that both examined species are incapable to traffic out of infected cells. All the data indicated that Mtb H37 Rv bacilli could avoid the rapid killing by escaoping into cytosol in murine BMECs, but could not traffic out of the infected cells.6. The survival assay of mycobacterial bacilli in human BMECs and human astrocytes. To investivate if mycobacterial bacilli replicate in human BMECs and astrocytes, the cells were infected with M. bovis BCG and Mtb virulent strain H37 Rv, and then the cells were lyased at indicated time points for survival assay. The results show that the number of viable bacilli from examined species was significantly decreased in both human BMECs and astrocytes over time. In addition, we found that both M. bovis BCG and Mtb H37 Rv could traffick out of the infected cells and infect adjacent cells. All the data show that all examined species could not replicate in human BMECs and astrocytes, and also could not traffic out of the infected cells.7. The analysis of mycibacoterial bacilli traversees an in vitro BBB bilayer system. To investigate if mycibacoterial bacilli traverse the BBB by paracellular pathway and the role of monocytes in traversal of BBB by Mtb bacilli, an in vitro BBB was constructed using human BMECs and astrocytes, and then infected with free mycobacterial bacilli and bacilli infected monocytes. The results show that the pathogenic Mtb bacilli, but not non-pathogenic bacilli, could traverse the BBB in vitro model with an enhancement of permeability, as shown decreased Transendothelial electrical resistance(TEER) and increased permeability for fluorescent sodium(Na-F). Interestingly, the infected monocytes infection enhanced the permeability of the BBB in vitro model, but only pathogenic bacilli could traverse the BBB in vitro model.Taken together, all the results indicate that the traversal of BBB by Mtb bacilli may via paracellular pathway with an enhancement of permeability. In addition, the monocytes might also involve in this process. |
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