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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

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.
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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.

Mentions: In the murine M. tuberculosis infection model, NO is generated in large amounts by macrophages via the activation of inducible nitric oxide synthase (iNOS). The generation of NO controls and restricts the growth of invading mycobacteria (MacMicking et al., 1997; Bosca et al., 2005). To investigate whether the capacity to produce NO is required for resistance to infection with virulent or attenuated M. tuberculosis strains in both susceptible (DBA/2) and resistant (C57BL/6 and BALB/c) mouse BMDM strains, we infected BMDMs obtained from BALB/c, C57BL/6, and DBA/2 mice with virulent M. tuberculosis H37Rv or attenuated M. tuberculosis H37Ra at an MOI of 1 until the indicated time points. We monitored NO production in each BMDM strain. As shown in Figure 4, when the BMDMs were infected with virulent M. tuberculosis H37Rv, the DBA/2 BMDMs produced significantly lower NO levels than were produced by the BALB/c and C57BL/6 BMDMs. However, this difference was not observed when cells were infected with the attenuated M. tuberculosis H37Ra strain. In line with these results, immunoblotting assays further demonstrated that iNOS expression was enhanced in the H37Rv-infected BALB/c and C57BL/6 BMDMs, but not in the H37Rv-infected DBA/2 BMDMs (Figure 4A). On the contrary, infection with attenuated H37Ra led to increased iNOS expression levels in all three BMDM strains (Figure 4A).


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: In the murine M. tuberculosis infection model, NO is generated in large amounts by macrophages via the activation of inducible nitric oxide synthase (iNOS). The generation of NO controls and restricts the growth of invading mycobacteria (MacMicking et al., 1997; Bosca et al., 2005). To investigate whether the capacity to produce NO is required for resistance to infection with virulent or attenuated M. tuberculosis strains in both susceptible (DBA/2) and resistant (C57BL/6 and BALB/c) mouse BMDM strains, we infected BMDMs obtained from BALB/c, C57BL/6, and DBA/2 mice with virulent M. tuberculosis H37Rv or attenuated M. tuberculosis H37Ra at an MOI of 1 until the indicated time points. We monitored NO production in each BMDM strain. As shown in Figure 4, when the BMDMs were infected with virulent M. tuberculosis H37Rv, the DBA/2 BMDMs produced significantly lower NO levels than were produced by the BALB/c and C57BL/6 BMDMs. However, this difference was not observed when cells were infected with the attenuated M. tuberculosis H37Ra strain. In line with these results, immunoblotting assays further demonstrated that iNOS expression was enhanced in the H37Rv-infected BALB/c and C57BL/6 BMDMs, but not in the H37Rv-infected DBA/2 BMDMs (Figure 4A). On the contrary, infection with attenuated H37Ra led to increased iNOS expression levels in all three BMDM strains (Figure 4A).

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