Insufficient Generation of Mycobactericidal Mediators and Inadequate Level of Phagosomal Maturation Are Related with Susceptibility to Virulent Mycobacterium tuberculosis Infection in Mouse Macrophages

Abstract

Tuberculosis is caused by Mycobacterium tuberculosis infection, and it remains major life-threatening infectious diseases worldwide. Although, M. tuberculosis has infected one-third of the present human population, only 5-10% of immunocompetent individuals are genetically susceptible to tuberculosis. All inbred strains of mice are susceptible to tuberculosis; however, some mouse strains are much more susceptible than others. In a previous report, we showed that Th1-mediated immunity was not responsible for the differential susceptibility between mouse models. To examine whether these susceptibility differences between inbred mouse strains are due to the insufficient production of effector molecules in the early stage of innate immunity, we investigated mycobacteriostatic function of bone marrow-derived macrophages (BMDMs) in resistant (BALB/c and C57BL/6) and susceptible strains (DBA/2) that were infected with virulent M. tuberculosis (H37Rv) or attenuated M. tuberculosis (H37Ra). The growth rate of virulent M. tuberculosis in infected cells was significantly higher in DBA/2 BMDMs, whereas the growth of the attenuated strain was similar in the three inbred mouse BMDM strains. In addition, the death rate of M. tuberculosis-infected cells increased with the infectious dose when DBA/2 BMDMs were infected with H37Rv. The intracellular reactive oxygen species level was lower in DBA/2 BMDMs that were infected with virulent M. tuberculosis at an early post-infection time point. The expression levels of phagosomal maturation markers, including early endosomal antigen-1 (EEA1) and lysosome-associated membrane protein-1 (LAMP-1), were significantly decreased in DBA/2 BMDM that were infected with virulent M. tuberculosis, whereas IFNγ-treatment restored the phagosomal maturation activity. The nitric oxide (NO) production levels were also significantly lower in DBA/2 BMDMs that were infected with virulent H37Rv at late post-infection points; however, this was not observed with the attenuated H37Ra strain. Furthermore, IFNγ-treatment rescued the low NO production level and insufficient M. tuberculosis growth control of DBA/2 BMDMs to the same level as of both resistant strains. The secreted TNF-α and IL-10 level were not significantly different between strains. Therefore, our findings suggest that DBA/2 BMDMs may have defects in the phagosomal maturation process and in inflammatory mediator production, as they showed innate immune defects when infected with the virulent, but not attenuated M. tuberculosis strain.

Keywords: Mycobacterium tuberculosis; macrophage; nitric oxide (NO); phagosomal maturation; reactive oxygen species (ROS); susceptibility.

FIGURE 1

H37Rv-infected DBA/2 BMDMs fail to control M. tuberculosis growth and host cell death. (A) BMDMs obtained from each of the three mouse strains were seeded at 2 × 10⁵ cells/well and infected with virulent M. tuberculosis H37Rv or attenuated M. tuberculosis H37Ra (at MOIs of 0.1 and 1) for 4 h. The viability of intracellular bacteria was assayed based on the number of CFUs at indicated time points. The data are presented as the mean ± standard deviation of experiments that were perforemd in triplecate. (B) BMDM were seeded at 1 × 10⁴ cell/well and then infected with H37Rv or H37Ra for 4 h. BMDM viability was assessed at indicated time points using trypan-blue exclusion assays. The data are presented as the mean ± standard deviation of experiments that were perforemd in triplecate. (C) BMDMs were infected with H37Rv at an MOI of 1 for 12 or 72 h. Apoptotic and necrotic cell death were measured using Annexin-V/Propidium iodide (PI) assays by flow cytometry. Bar graphs represent the percentage of M. tuberculosis-infected BMDMs that were apoptotic or necrotic cells. (D) BMDMs waere infected with H37Rv or H37Ra at MOI of 1 for 48 h and lactate dehydrogenase (LDH) release in the media was determined using a cytotoxicity assay kit. The data are presented as the mean percentage ± standard deviation of experiments that were performed in triplicate. All of the experiments were repeated at least three times with similar results. Significant differences are indicated by ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; and n.s., not significant (P > 0.05).

FIGURE 2

H37Rv-infected DBA/2 BMDMs generate deficient levels of ROS. BMDMs were infected with virulent M. tuberculosis H37Rv or attenuated M. tuberculosis H37Ra (at an MOI of 1) for 3 or 24 h. Cells were labeled with 5-[and-6]-chloromethyl-2′,7′-dichlorodihydro fluorescein diacetate (DCFH-DA) and then measured using confocal microscopy (A–D) and flow cytometry (E,F). The bar graph indicates the ratio of DCF fluorescence-positive cells. Ratios were assessed compared to the corresponsding non-infected control cells. The mean ratio ± standard deviation of triplicate measurements are shown (B,D; n = 6). MFI means mean fluorescence intensity. The mean value ± standard deviation for triplicate measurements are shown (F). All of the experiments were repeated at least three times with similar results. Significant differences are indicated by ^∗∗∗P < 0.001.

FIGURE 3

Virulent H37Rv arrests phagosome maturation in DBA/2 BMDMs. BMDMs that were isolated from BALB/c, C57BL/6, or DBA/2 mice were infected with virulent H37Rv or attenuated H37Ra for 4 h. After then, cells were fixed and stained with anti-CD107a LAMP-1-Alexa Fluor^® 647 or anti-EEA1-Alexa Fluor^® 647 antibodies. Co-localization between M. tuberculosis and EEA1 (A) or LAMP-1 (B) was determined using confocal microscopy. The bar graphs shows the percentage of the M. tuberculosis foci that co-localized with EEA1 (A, top panel) or LAMP-1 (B, bottom panel) in the quantitative analysis. The data are expressed as the mean value ± standard deviation of five independent experiments for each condition. Experiments were performed in triplicate. (C) Cleaved cathepsin D was detected in H37Rv-infected cells using Western blot assays. Significant differences are indicated by ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 4

Treatment with IFN-γ can overcome deficient NO production in DBA/2 BMDMs to control M. tuberculosis growth. BMDMs were infected with virulent H37Rv or attenuated H37Ra for 48 or 72 h. (A) The levels of NO that were secreted into the supernatants were measured. The data are presented as the mean ± standard deviation of experiments performed in triplicate (left panel). iNOS levels were measured in the BMDMs using Western blot assays (right panel). (B,C) BMDMs were stimulated with IFN-γ (20 ng/ml) after infection with H37Rv or H37Ra. (B) The level of NO that was secreted into the supernatants was measured using an NO detection kit. The data are presented as the mean ± standard deviation of experiments that were performed in triplicate (left). iNOS levels were determined in the BMDMs using Western blot assays (right). (C) Bacterial growth was confirmed using CFU assessments. The data are presented as the mean ± standard deviation of triplicate experiments. All of the experiments were independently repeated three to five times for each condition with similar results. Significant differences are indicated by ^∗∗∗P < 0.001.

FIGURE 5

IFN-γ restores phagosome maturation in H37Rv-infected DBA/2 BMDMs. BMDMs were stimulated with IFN-γ (20 ng/ml) for 2 h and then infected with either virulent H37Rv or attenuated H37Ra. After 4 h, the cells were fixed and stained with anti-CD107a LAMP-1-Alexa Fluor^® 647 or anti-EEA1-Alexa Fluor^® 647 antibodies. Co-localization between M. tuberculosis and EEA1 (A) or LAMP-1 (B) was analyzed using confocal microscopy. The bar graphs show the percentage of M. tuberculosis foci that co-localized with EEA1 (A, top panel) or LAMP-1 (B, bottom panel) in the quantitative analysis. The data are presented as the mean ± standard deviation (n = 10).

FIGURE 6

TNF-α and IL-10 levels were not significantly different in H37Rv-infected DBA/2 BMDMs. The production of TNF-α (A,B) and IL-10 (C,D) was measured using ELISA in culture media after BMDMs obtained from three inbred mouse strains were infected with H37Ra or H37Rv. The BMDMs were infected with either attenuated M. tuberculosis H37Ra or virulent M. tuberculosis H37Rv (at an MOI of 0.1 or 1) for 4 h, and the production of cytokines was anlayzed at 72 h post-infection. Significant differences are indicated by ^∗P < 0.05 and n.s., not significant (P > 0.05).