Differences betweenMycobacterium-Host Cell Relationships in Latent Tuberculous Infection of MiceEx Vivoand Mycobacterial Infection of Mouse CellsIn Vitro

Abstract

The search for factors that account for the reproduction and survival of mycobacteria, including vaccine strains, in host cells is the priority for studies on tuberculosis. A comparison of BCG-mycobacterial loads in granuloma cells obtained from bone marrow and spleens of mice with latent tuberculous infection and cells from mouse bone marrow and peritoneal macrophage cultures infected with the BCG vaccine in vitro has demonstrated that granuloma macrophages each normally contained a single BCG-Mycobacterium, while those acutely infected in vitro had increased mycobacterial loads and death rates. Mouse granuloma cells were observed to produce the IFNγ, IL-1α, GM-CSF, CD1d, CD25, CD31, СD35, and S100 proteins. None of these activation markers were found in mouse cell cultures infected in vitro or in intact macrophages. Lack of colocalization of lipoarabinomannan-labeled BCG-mycobacteria with the lysosomotropic LysoTracker dye in activated granuloma macrophages suggests that these macrophages were unable to destroy BCG-mycobacteria. However, activated mouse granuloma macrophages could control mycobacterial reproduction in cells both in vivo and in ex vivo culture. By contrast, a considerable increase in the number of BCG-mycobacteria was observed in mouse bone marrow and peritoneal macrophages after BCG infection in vitro, when no expression of the activation-related molecules was detected in these cells.

The Control of Apoptotic Death in the Cells of Granulomatous Inflammatory Lesions from Mice with Latent Tuberculous Infection in the Ex Vivo Model

“…Isolation of granulomas from the spleens, bone marrow and lungs of mice after BCG infection was performed as previously described [55][56][57]. In brief, bone marrow was flushed from femurs with RPMI 1640 medium with 50 µg/ml gentamicin (BioloT, St. Petersburg, Russia).…”

“…Granulomas were isolated from mice 1 and 2 on day 20 following intraperitoneal infection; mice 1 and 2÷5 after one month following intraperitoneal and intravenous infection, respectively; mice 1÷16 and 21÷24 after two months following intravenous infection; and mouse 25 after two months following intraperitoneal infection. The mouse nomenclature used is explained [55][56][57]. Peritoneal macrophages were isolated from mice after 20 days and mouse 25 after two months following intraperitoneal infection and cultured under the same conditions as the granuloma cells.…”

“…The preparations of cultured, as granuloma cells, peritoneal macrophages of normal BALB/c mice without BCG infection were used as the control groups. Isolation of control fibroblasts from fragments of the splenic capsule or lungs from intact BALB/c mice was performed as previously described [55,57].…”

“…Isolation of peritoneal and bone marrow cells from four intact mice and BCG infection of each cell culture were performed as previously described in detail [57]. Three independent experiments on obtaining and studying cells in the control (uninfected) mouse bone marrow and peritoneal cultures and in cell monolayers after 4-120 h of acute BCG infection in vitro have been conducted.…”

“…Similarly, in the liver of mice treated in vivo with antibodies against TNFα protein, a gradual loss of granuloma structure, decreased granuloma size, decreased granuloma macrophage populations, and increased intracellular BCG mycobacteria reproduction were observed [54]. We have previously demonstrated in our model system of monolayer cultures of cells that migrated ex vivo from individual granulomas of mice with latent BCG infection [55] that although mouse granuloma macrophages with an increased production of proinflammatory cytokines, growth factors, and CD receptors for cell activation could not eliminate intracellular BCG mycobacteria entirely, they could still control mycobacterial reproduction in host cells both in vivo and in ex vivo culture, while the mouse bone marrow and peritoneal macrophages that were not activated after BCG infection in vitro had increased mycobacterial loads and death rates [55][56][57]. Nevertheless, the in vitro, ex vivo and in vivo studies performed to date are not enough to completely understand the mechanisms by which mycobacteria persist in cells for long periods of time, particularly, at the latent stage of tuberculous infection.…”