Investigation of Functional Activity of Cells in Granulomatous Inflammatory Lesions from Mice with Latent Tuberculous Infection in the NewEx VivoModel

Abstract

The new ex vivo model system measuring functional input of individual granuloma cells to formation of granulomatous inflammatory lesions in mice with latent tuberculous infection has been developed and described in the current study. Monolayer cultures of cells that migrated from individual granulomas were established in the proposed culture settings for mouse spleen and lung granulomas induced by in vivo exposure to BCG vaccine. The cellular composition of individual granulomas was analyzed. The expression of the leukocyte surface markers such as phagocytic receptors CD11b, CD11c, CD14, and CD16/CD32 and the expression of the costimulatory molecules CD80, CD83, and CD86 were tested as well as the production of proinflammatory cytokines (IFNγ and IL-1α) and growth factors (GM-CSF and FGFb) for cells of individual granulomas. The colocalization of the phagocytic receptors and costimulatory molecules in the surface microdomains of granuloma cells (with and without acid-fast BCG-mycobacteria) has also been detected. It was found that some part of cytokine macrophage producers have carried acid-fast mycobacteria. Detected modulation in dynamics of production of pro-inflammatory cytokines, growth factors, and leukocyte surface markers by granuloma cells has indicated continued processes of activation and deactivation of granuloma inflammation cells during the latent tuberculous infection progress in mice.

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

“…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.…”

“…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].…”

“…Any of the monolayer cultures of granuloma cells that may or may not retain cell clusters in the center of granulomatous lesions will be referred to as the "granuloma" throughout. The cellular composition of each granuloma and the mycobacterial loads in granuloma cells from different mice, the expression of various surface markers, and the production of various proinflammatory cytokines and growth factors by mouse granuloma cells were characterized previously [55][56][57].…”