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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 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.
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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.

Mentions: Reactive oxygen species generation controls mycobacterial infection and intracellular signaling pathways (Robinson, 2009; Voskuil et al., 2011). To examine whether ROS generation differs between the susceptible and resistant mouse BMDM strains, intracellular ROS levels were monitored using flow cytometry and confocal microscopy as a function of the observed reduction of the redox-sensitive dye, DCFH-DA. As shown in Figures 2A,B, infection with virulent H37Rv increased intracellular DCFH-DA fluorescence at 4 h, and this response was maintained at a higher level for up to 24 h in the BALB/c and C57BL/6 BMDMs, but not in the DBA/2 BMDMs. In contrast, during attenuated H37Ra infection, intracellular DCFH-DA fluorescence intensity was increased in all three mouse BMDM strains (Figures 2C,D). In line with this result, the flow cytometric analysis also showed that the BALB/c and C57BL/6 BMDMs yielded a stronger DCFH-DA fluorescence signal than the DBA/2 BMDMs after infection with virulent H37Rv (Figures 2E,F). These data suggest that the early induction of intracellular ROS determines susceptibility to infection with virulent H37Rv in BMDMs.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: Reactive oxygen species generation controls mycobacterial infection and intracellular signaling pathways (Robinson, 2009; Voskuil et al., 2011). To examine whether ROS generation differs between the susceptible and resistant mouse BMDM strains, intracellular ROS levels were monitored using flow cytometry and confocal microscopy as a function of the observed reduction of the redox-sensitive dye, DCFH-DA. As shown in Figures 2A,B, infection with virulent H37Rv increased intracellular DCFH-DA fluorescence at 4 h, and this response was maintained at a higher level for up to 24 h in the BALB/c and C57BL/6 BMDMs, but not in the DBA/2 BMDMs. In contrast, during attenuated H37Ra infection, intracellular DCFH-DA fluorescence intensity was increased in all three mouse BMDM strains (Figures 2C,D). In line with this result, the flow cytometric analysis also showed that the BALB/c and C57BL/6 BMDMs yielded a stronger DCFH-DA fluorescence signal than the DBA/2 BMDMs after infection with virulent H37Rv (Figures 2E,F). These data suggest that the early induction of intracellular ROS determines susceptibility to infection with virulent H37Rv in BMDMs.

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