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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

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.
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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.

Mentions: M. tuberculosis can survive and replicate in the phagosomal compartment of infected cells. Following M. tuberculosis phagocytosis, several proteins, including Rab5, PI(3)P, early endosomal antigen-1 (EEA-1), Rab7, RILP and LAMP-1, are subsequently recruited to M. tuberculosis-containing phagosomes to accelerate the intracellular killing of M. tuberculosis (Zhou and Yu, 2008). A number of studies have shown that M. tuberculosis can arrest phagosome–lysosome fusion and modulate several events during phagolysosome maturation to avoid destruction by the host’s immune system (Scherr et al., 2009). To assess whether susceptibility to M. tuberculosis infection is associated with phagosomal maturation in DBA/2 BMDMs, we measured co-localization between M. tuberculosis and EEA1 or LAMP-1 using confocal microscopy in the three mouse BMDM strains. When the BMDMs were analyzed at 3 h after infection, many FITC-labeled M. tuberculosis bacteria (both H37Ra and H37Rv) were co-localized with EEA1 in all three mouse BMDM strains (Figure 3A). Furthermore, co-localization between LAMP-1- and H37Ra-containing phagosomes was significantly increased in all three of the BMDM types (Figure 3B). In marked contrast to these results, the co-localization between the virulent H37Rv strain and LAMP-1 was significantly decreased in DBA/2 BMDMs (Figure 3B). However, the level of co-localization was restored in this strain when the cells were pre-treated with IFN-γ (Figure 5).


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)

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.
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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.
Mentions: M. tuberculosis can survive and replicate in the phagosomal compartment of infected cells. Following M. tuberculosis phagocytosis, several proteins, including Rab5, PI(3)P, early endosomal antigen-1 (EEA-1), Rab7, RILP and LAMP-1, are subsequently recruited to M. tuberculosis-containing phagosomes to accelerate the intracellular killing of M. tuberculosis (Zhou and Yu, 2008). A number of studies have shown that M. tuberculosis can arrest phagosome–lysosome fusion and modulate several events during phagolysosome maturation to avoid destruction by the host’s immune system (Scherr et al., 2009). To assess whether susceptibility to M. tuberculosis infection is associated with phagosomal maturation in DBA/2 BMDMs, we measured co-localization between M. tuberculosis and EEA1 or LAMP-1 using confocal microscopy in the three mouse BMDM strains. When the BMDMs were analyzed at 3 h after infection, many FITC-labeled M. tuberculosis bacteria (both H37Ra and H37Rv) were co-localized with EEA1 in all three mouse BMDM strains (Figure 3A). Furthermore, co-localization between LAMP-1- and H37Ra-containing phagosomes was significantly increased in all three of the BMDM types (Figure 3B). In marked contrast to these results, the co-localization between the virulent H37Rv strain and LAMP-1 was significantly decreased in DBA/2 BMDMs (Figure 3B). However, the level of co-localization was restored in this strain when the cells were pre-treated with IFN-γ (Figure 5).

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