Mycobacterium tuberculosis uptake by recipient host macrophages is influenced by environmental conditions in the granuloma of the infectious individual and is associated with impaired production of interleukin-12 and tumor necrosis factor alpha

Abstract

Transmission of Mycobacterium tuberculosis from one individual to another usually is associated with episodes of coughing. The bacteria leave the environment of the lung cavity of the infected person and travel in droplets to reach the recipient's respiratory tract. Therefore, at the time that the bacteria encounter alveolar cells (macrophages and epithelial cells) in the new host, they express virulence determinants that are regulated by the environmental conditions in the infected person. To determine if those environmental conditions encountered in the lung cavity (hyperosmolarity, acidic pH, and low oxygen tension, among others) would influence the uptake of M. tuberculosis by the recipient's alveolar macrophages, M. tuberculosis H37Rv was incubated under several conditions for different periods of time, washed at 4 degrees C, and used to infect human monocyte-derived macrophages. While increased osmolarity had no effect on M. tuberculosis uptake compared to the uptake of bacteria grown on 7H10 Middlebrook medium, both acidic pH and anaerobiosis increased the uptake of the H37Rv strain four- to sixfold. Using anti-CD11b receptor blocking antibodies or mannoside to inhibit the uptake of M. tuberculosis by macrophages, we determined that while uptake of M. tuberculosis cultured on 7H10 medium was inhibited 77% +/- 6% in the presence of anti-CD11b antibody, the antibody had no effect on the uptake of M. tuberculosis incubated at pH 6.0 and was associated with 27% inhibition of M. tuberculosis previously exposed to anaerobic conditions. The mannose receptor was also not involved with invasion after exposure to acidic conditions, and mannoside resulted in only 32% inhibition of uptake by macrophages of M. tuberculosis exposed to anaerobiosis. Uptake by macrophages also resulted in the secretion of significantly lower amounts of interleukin-12 and tumor necrosis factor alpha than that by macrophages infected with a strain cultured under laboratory conditions. M. tuberculosis cultured under the pH and oxygen concentration found in the granuloma expresses a large number of proteins that are different from the proteins expressed by bacteria grown under laboratory conditions. The results suggest that M. tuberculosis in vivo may be adapted to gain access to the intracellular environment in a very efficient fashion and may do so by using different receptors from the complement and mannose receptors.

FIG. 1.

Effect of blocking integrins as well as mannose receptors on the uptake of M. tuberculosis grown under anaerobiosis by macrophages cultured in the presence of 10% autologous serum. Laboratory conditions were defined as 37°C, 20% O₂, pH 7.2, and isoosmolarity; anaerobiosis was obtained by using an anaerobic jar as described in Materials and Methods. Antibodies were added to monolayers 1 h prior to the experiment at 23°C at concentrations of 30 μg/ml. Anti-P. aeruginosa lipid A monoclonal antibody (IgG) was used at 30 μg/ml as a nonrelevant antibody. No inhibition of uptake was observed. α-Methyl-mannoside was used at a concentration of 1 μg/ml. *, P < 0.05 compared with control.

FIG. 2.

Effect of blocking agents to the CR3 receptor and mannose receptor on the uptake of M. tuberculosis grown under pH 6.0 by macrophages cultured in the presence of 10% autologous serum. *, P < 0.05 compared with control. The antibodies used were anti-CD11b (30 μg/ml), nonrelevant IgG2b (30 μg/ml), and α-methyl-mannoside (1 μg/ml).

FIG. 3.

Production of IL-12 and TNF-α by macrophages upon uptake of M. tuberculosis. Lab, laboratory conditions; Anaero, anaerobiosis; Hyperos, hyperosmolarity; pH 6, acidic pH; control, uninfected macrophage control; *, P < 0.05 compared to laboratory conditions; **, production at 24 h. Numbers represent the means ± standard deviations of four different experiments.

FIG. 4.

Comparative evaluation of the protein profile by resolving M. tuberculosis proteins in a two-dimensional gel following exposure to pH 6 and anaerobiosis (A) or pH 7 and aerobiosis (B). At least 10 proteins showed upregulation of expression.