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

Lee HJ, Ko HJ, Jung YJ - Front Microbiol (2016)

Bottom Line: 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.The secreted TNF-α and IL-10 level were not significantly different between strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and BIT Medical Convergence Graduate Program, Kangwon National University Chuncheon, South Korea.

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.

No MeSH data available.


Related in: MedlinePlus

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).
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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).

Mentions: Several studies have shown that the DBA/2 mouse strain is very susceptible to infection with virulent M. tuberculosis, whereas the BALB/c and C57BL/6 mouse strains are much more resistant (Medina and North, 1999; Jung et al., 2002; Beamer and Turner, 2005). To investigate differences in M. tuberculosis growth between M. tuberculosis-infected susceptible and resistant mouse BMDMs, each of the mouse BMDM strains was infected with different infectious doses of virulent H37Rv or attenuated H37Ra, and bacterial growth was measured using CFU assays. After infection with virulent H37Rv, the DBA/2 mouse BMDMs contained approximately twofold more M. tuberculosis than was contained in the BALB/c and C57BL/6 mouse BMDMs. However, this was not observed after infection with H37Ra (Figure 1A). There was no difference between mouse strains in ingesting bacteria at 4 h after infection when cells were infected with either H37Rv or H37Ra at an MOI of 0.1 or 1 (Supplementary Figure S1).


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

Lee HJ, Ko HJ, Jung YJ - Front Microbiol (2016)

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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4834433&req=5

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).
Mentions: Several studies have shown that the DBA/2 mouse strain is very susceptible to infection with virulent M. tuberculosis, whereas the BALB/c and C57BL/6 mouse strains are much more resistant (Medina and North, 1999; Jung et al., 2002; Beamer and Turner, 2005). To investigate differences in M. tuberculosis growth between M. tuberculosis-infected susceptible and resistant mouse BMDMs, each of the mouse BMDM strains was infected with different infectious doses of virulent H37Rv or attenuated H37Ra, and bacterial growth was measured using CFU assays. After infection with virulent H37Rv, the DBA/2 mouse BMDMs contained approximately twofold more M. tuberculosis than was contained in the BALB/c and C57BL/6 mouse BMDMs. However, this was not observed after infection with H37Ra (Figure 1A). There was no difference between mouse strains in ingesting bacteria at 4 h after infection when cells were infected with either H37Rv or H37Ra at an MOI of 0.1 or 1 (Supplementary Figure S1).

Bottom Line: 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.The secreted TNF-α and IL-10 level were not significantly different between strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and BIT Medical Convergence Graduate Program, Kangwon National University Chuncheon, South Korea.

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.

No MeSH data available.


Related in: MedlinePlus