Limits...
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

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).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4834433&req=5

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).

Mentions: Macrophages are important effector cells that contribute to the production of proinflammatory cytokines, ROS, and NO. These cells therefore contribute to innate immune responses during the early phase of M. tuberculosis infection (Raja, 2004). In particular, TNF-α production contributes to the initiation of protective immunity during the early post-infection period (Marino et al., 2010), whereas the increase in IL-10 production that occurs in response to M. tuberculosis infection suppresses the protective Th1 response (Redford et al., 2010). To determine whether the observed differences in susceptibility following infection with different M. tuberculosis strains might reflect differences in the production of TNF-α or IL-10, we measured the amount of TNF-α or IL-10 that was secreted into the supernatant of each type of BMDM at 24 h after the cells were infected with the virulent M. tuberculosis strain H37Rv or the attenuated H37Ra strain at an MOI of 0.1 or 1. As shown in Figure 6A, in cells infected with the virulent H37Rv strain at an MOI of 0.1, slightly higher levels of TNF-α were secreted by the DBA/2 BMDMs following infection with H37Rv compared with the BALB/c and C57BL/6 BMDMs, whereas when cells were infected with this strain at an MOI of 1, slightly lower levels of TNF-α were secreted by the DBA/2 BMDMs than the BALB/c and C57BL/6 BMDMs. There was no difference in the amount of TNF-α that was secreted by the three BMDM strains following infection with the attenuated H37Ra strain (Figure 6B). Therefore, while the amount of secreted TNF-α appears to be dependent on the infectious dose when cells are infected with virulent H37Rv, this does not appear to be the case when cells are infected with attenuated H37Ra infection. In addition, when cells were infected with the H37Rv strain at MOI of 1, lower IL-10 levels were detected in the DBA/2 BMDM supernatants than in the C57BL/6 BMDM supernatants (Figure 6C). In contrast, among the mouse BMDM types, IL-10 levels were slightly higher in the DBA/2 BMDMs following infection with the attenuated H37Ra strain at an MOI of 0.1 (Figure 6D). These results suggest that the differential induction of either pro-inflammatory or anti-inflammatory cytokines is not correlated with differential susceptibility to virulent and attenuated M. tuberculosis strains in M. tuberculosis-resistant and -susceptible mouse macrophages.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

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).
Mentions: Macrophages are important effector cells that contribute to the production of proinflammatory cytokines, ROS, and NO. These cells therefore contribute to innate immune responses during the early phase of M. tuberculosis infection (Raja, 2004). In particular, TNF-α production contributes to the initiation of protective immunity during the early post-infection period (Marino et al., 2010), whereas the increase in IL-10 production that occurs in response to M. tuberculosis infection suppresses the protective Th1 response (Redford et al., 2010). To determine whether the observed differences in susceptibility following infection with different M. tuberculosis strains might reflect differences in the production of TNF-α or IL-10, we measured the amount of TNF-α or IL-10 that was secreted into the supernatant of each type of BMDM at 24 h after the cells were infected with the virulent M. tuberculosis strain H37Rv or the attenuated H37Ra strain at an MOI of 0.1 or 1. As shown in Figure 6A, in cells infected with the virulent H37Rv strain at an MOI of 0.1, slightly higher levels of TNF-α were secreted by the DBA/2 BMDMs following infection with H37Rv compared with the BALB/c and C57BL/6 BMDMs, whereas when cells were infected with this strain at an MOI of 1, slightly lower levels of TNF-α were secreted by the DBA/2 BMDMs than the BALB/c and C57BL/6 BMDMs. There was no difference in the amount of TNF-α that was secreted by the three BMDM strains following infection with the attenuated H37Ra strain (Figure 6B). Therefore, while the amount of secreted TNF-α appears to be dependent on the infectious dose when cells are infected with virulent H37Rv, this does not appear to be the case when cells are infected with attenuated H37Ra infection. In addition, when cells were infected with the H37Rv strain at MOI of 1, lower IL-10 levels were detected in the DBA/2 BMDM supernatants than in the C57BL/6 BMDM supernatants (Figure 6C). In contrast, among the mouse BMDM types, IL-10 levels were slightly higher in the DBA/2 BMDMs following infection with the attenuated H37Ra strain at an MOI of 0.1 (Figure 6D). These results suggest that the differential induction of either pro-inflammatory or anti-inflammatory cytokines is not correlated with differential susceptibility to virulent and attenuated M. tuberculosis strains in M. tuberculosis-resistant and -susceptible mouse macrophages.

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