The Efficiency of the Translocation ofMycobacterium tuberculosisacross a Bilayer of Epithelial and Endothelial Cells as a Model of the Alveolar Wall Is a Consequence of Transport within Mononuclear Phagocytes and Invasion of Alveolar Epithelial Cells

Abstract

The mechanism(s) by which Mycobacterium tuberculosis crosses the alveolar wall to establish infection in the lung is not well known. In an attempt to better understand the mechanism of translocation and create a model to study the different stages of bacterial crossing through the alveolar wall, we established a two-layer transwell system. M. tuberculosis H37Rv was evaluated regarding the ability to cross and disrupt the membrane. M. tuberculosis invaded A549 type II alveolar cells with an efficiency of 2 to 3% of the initial inoculum, although it was not efficient in invading endothelial cells. However, bacteria that invaded A549 cells were subsequently able to be taken up by endothelial cells with an efficiency of 5 to 6% of the inoculum. When incubated with a bicellular transwell monolayer (epithelial and endothelial cells), M. tuberculosis translocated into the lower chamber with efficiency (3 to 4%). M. tuberculosis was also able to efficiently translocate across the bicellular layer when inside monocytes. Infected monocytes crossed the barrier with greater efficiency when A549 alveolar cells were infected with M. tuberculosis than when A549 cells were not infected. We identified two potential mechanisms by which M. tuberculosis gains access to deeper tissues, by translocating across epithelial cells and by traveling into the blood vessels within monocytes.Infection caused by M. tuberculosis represents one of the great tragedies in world history. Approximately 3 million people die annually of the disease (7), despite the availability of cheap, efficacious, and curative therapy for tuberculosis.Once more, it seems clear that the improvement of the knowledge about the mechanisms employed by M. tuberculosis to infect the host will certainly offer new opportunities for the development of both effective therapy and vaccine.M. tuberculosis is inhaled into the respiratory tract, eventually reaching the alveolar space. It has been assumed that the bacterium is ingested by alveolar macrophages and subsequently gains access to the bloodstream by being transported by the alveolar macrophages and blood monocytes across the alveolar wall (10). Recently, however, it was demonstrated by several groups that M. tuberculosis invades and survives within human type II alveolar epithelial cells in vitro (3, 14, 17), and a possible role for alveolar epithelial cells in vivo has been postulated. In fact, the chance that M. tuberculosis would encounter an alveolar epithelial cell (the average human male has 1,500 type II and 28,000 type I alveolar epithelial cells [22]) is significantly greater than encountering an alveolar macrophage (50 macrophages per alveolus [8]). Therefore, the participation of type II alveolar epithelial cells, alveolar macrophages, and blood monocytes in the translocation of M. tuberculosis across the alveolar wall is currently poorly understood. Previous work has established the use of an in vitro model with a bilayer with alveolar epithelial cells and human lung endothelial cells (6). Using this model, i...

Granulocyte–macrophage colony stimulating factor-mediated innate responses in tuberculosis

“…On the other hand, previous studies showed that alveolar Type II epithelial cells and macrophages collaborate in the translocation of mycobacteria into distal lung parenchyma. 55 In addition, alveolar epithelial cell lines in culture are subject to necrosis by virulent but not attenuated mycobacterial organisms. 56,57 The attenuation of BCG is related to the loss of the RD1 locus encoding ESAT-6, which is responsible in part for the cytolysis of pneumocytes by virulent mycobacteria.…”

Insights into early mycobacterial pathogenesis from the zebrafish

“…Cell culture studies using transwells have suggested that mycobacteria traverse epithelial barriers both within macrophages as well as directly [1,30]. In fact, the ESX-1/RD1 virulence determinant has been implicated in the ability of pathogenic mycobacteria to directly cross epithelial barriers [31].…”

“…However, a direct examination of mycobacterial transport in the zebrafish embryo has revealed that infecting mycobacteria traverse both endothelial and epithelial barriers mainly within macrophages in vivo; very little transit occurs in embryos lacking phagocyte lineages (created by morpholino knockdown of the myeloid transcription factor pu.1) [15]. The signals causing these infected macrophages to migrate back into deeper tissue to initiate granuloma formation are poorly understood [1] and this area is ripe for exploration in the zebrafish. For example, the function of chemokines important in migration of myeloid cells to and from the lung in mouse models may be dissected in greater detail in the zebrafish [32].…”

“…The discernible steps of mycobacterial pathogenesis suggest the engagement of a wide range of immune response pathways. In human pulmonary TB, infecting mycobacteria traverse the alveolar epithelium in macrophages and dendritic cells [1,2]. Mycobacteria replicate within these cells by subverting host endocytic trafficking mechanisms [3].…”

Mycobacterium tuberculosis adhesins: potential biomarkers as anti-tuberculosis therapeutic and diagnostic targets

“…This is further complicated by the lack of an effective vaccine (Russell et al, 2010), prolonged chemotherapy regimens (Mitchison & Davies, 2012) and adverse TB/HIV drug interactions (Luetkemeyer et al, 2011). Knowledge on the mechanisms utilized by M. tuberculosis to infect the host would offer novel perspective and define new targets to facilitate the design and development of drugs that are effective against both sensitive and resistant organisms (Ginsberg & Spigelman, 2007), efficacious vaccines (Bermudez et al, 2002), as well as crucially needed rapid, accurate and cheap point-of-care tests (Wallis et al, 2010).…”