Limits...
Infection of Primary Bovine Macrophages with Mycobacterium avium Subspecies paratuberculosis Suppresses Host Cell Apoptosis.

Kabara E, Coussens PM - Front Microbiol (2012)

Bottom Line: Our results demonstrate that, indeed, populations of MAP-infected macrophages contain fewer apoptotic cells than similar populations of control cells, and that MAP infection reduces the sensitivity of infected macrophages to induction of apoptosis by H(2)O(2).This reduction in caspase activity is accompanied by a pronounced reduction in transcription of caspase genes encoding caspases 3, 7, and 8, but not for caspase 9, when compared to control, uninfected cells.Together these data demonstrate that MAP specific factors may prevent caspase activity and caspase gene transcription as well as apoptosis signaling protein expression, resulting in decreased spontaneous host cell apoptosis and decreased sensitivity to apoptosis inducing agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Center for Animal Functional Genomics, Michigan State University East Lansing, MI, USA.

ABSTRACT
Mycobacterium avium subspecies paratuberculosis (MAP) is able to survive intracellularly in macrophages by preventing normal phagosome maturation processes utilized to destroy bacteria. Infected macrophages often undergo apoptotic cell death to efficiently present bacterial antigens to the host adaptive immune system in a process known as efferocytosis. Recent studies with Mycobacterium tuberculosis (MTB) showed that macrophages infected with MTB are less likely to undergo apoptosis than control, uninfected cells. It is proposed that regulation of macrophage apoptosis is an important immune evasion tactic for MTB. Based on the similarity of MAP and MTB, we hypothesized that MAP-infected macrophages would be resistant to apoptosis compared to uninfected cells within the same culture and to cells from uninfected cultures. Our results demonstrate that, indeed, populations of MAP-infected macrophages contain fewer apoptotic cells than similar populations of control cells, and that MAP infection reduces the sensitivity of infected macrophages to induction of apoptosis by H(2)O(2). We further demonstrate that MAP-infected cells contain reduced caspase activity for caspases 3/7, 8, and 9. Reduced caspase activity in MAP-infected macrophages is also maintained after H(2)O(2) induction. This reduction in caspase activity is accompanied by a pronounced reduction in transcription of caspase genes encoding caspases 3, 7, and 8, but not for caspase 9, when compared to control, uninfected cells. Furthermore, MAP infection drastically effects the expression of several host cell proteins important for regulation of apoptosis. Studies using mutant MAP strains demonstrate the importance of bacterial specific factors in the control of host macrophage apoptosis. Together these data demonstrate that MAP specific factors may prevent caspase activity and caspase gene transcription as well as apoptosis signaling protein expression, resulting in decreased spontaneous host cell apoptosis and decreased sensitivity to apoptosis inducing agents.

No MeSH data available.


Related in: MedlinePlus

Cell status post apoptotic induction. Percentage of apoptotic macrophages in populations of MAP-infected, bystander, and uninfected control cells was determined by flow cytometry after 20 min of 100 μM H2O2 treatment. Bars represent the average results of MDM cultured from six healthy Holstein cattle. Gray bars represent the percentage of apoptotic cells pre-treatment, while black bars represent the percentage of apoptotic macrophages after treatment in the three cell groups. Error bars represent Standard Error of the mean (SEM) between the six biological replicates. A +indicates significantly different from control, uninfected cells at p < 0.05 and *indicates significantly different from bystander macrophages at p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3400940&req=5

Figure 2: Cell status post apoptotic induction. Percentage of apoptotic macrophages in populations of MAP-infected, bystander, and uninfected control cells was determined by flow cytometry after 20 min of 100 μM H2O2 treatment. Bars represent the average results of MDM cultured from six healthy Holstein cattle. Gray bars represent the percentage of apoptotic cells pre-treatment, while black bars represent the percentage of apoptotic macrophages after treatment in the three cell groups. Error bars represent Standard Error of the mean (SEM) between the six biological replicates. A +indicates significantly different from control, uninfected cells at p < 0.05 and *indicates significantly different from bystander macrophages at p < 0.05.

Mentions: Based on differences in spontaneous apoptosis observed between MAP-infected, bystander, and control cell populations, we studied the relative resistance of these three cell groups to induction of apoptosis. Hydrogen peroxide (H2O2) is a well-known apoptotic inducing agent (Ryter et al., 2007). We chose to use 100 μM H2O2 for 20 min, based on time course and dose-response curve studies with uninfected MDM (data not shown). All cell populations exposed to H2O2 displayed a higher percentage of apoptotic macrophages than their untreated sister cultures (p < 0.05). When H2O2-treated macrophages were compared across the infection groups, H2O2-treated MAP-infected MDM cell populations contained a significantly lower relative percentage of apoptotic cells than either bystander or control macrophage populations (p = 0.0132 and 0.0064, respectively; Figure 2). No other significant differences were observed between the three groups (data not shown).


Infection of Primary Bovine Macrophages with Mycobacterium avium Subspecies paratuberculosis Suppresses Host Cell Apoptosis.

Kabara E, Coussens PM - Front Microbiol (2012)

Cell status post apoptotic induction. Percentage of apoptotic macrophages in populations of MAP-infected, bystander, and uninfected control cells was determined by flow cytometry after 20 min of 100 μM H2O2 treatment. Bars represent the average results of MDM cultured from six healthy Holstein cattle. Gray bars represent the percentage of apoptotic cells pre-treatment, while black bars represent the percentage of apoptotic macrophages after treatment in the three cell groups. Error bars represent Standard Error of the mean (SEM) between the six biological replicates. A +indicates significantly different from control, uninfected cells at p < 0.05 and *indicates significantly different from bystander macrophages at p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3400940&req=5

Figure 2: Cell status post apoptotic induction. Percentage of apoptotic macrophages in populations of MAP-infected, bystander, and uninfected control cells was determined by flow cytometry after 20 min of 100 μM H2O2 treatment. Bars represent the average results of MDM cultured from six healthy Holstein cattle. Gray bars represent the percentage of apoptotic cells pre-treatment, while black bars represent the percentage of apoptotic macrophages after treatment in the three cell groups. Error bars represent Standard Error of the mean (SEM) between the six biological replicates. A +indicates significantly different from control, uninfected cells at p < 0.05 and *indicates significantly different from bystander macrophages at p < 0.05.
Mentions: Based on differences in spontaneous apoptosis observed between MAP-infected, bystander, and control cell populations, we studied the relative resistance of these three cell groups to induction of apoptosis. Hydrogen peroxide (H2O2) is a well-known apoptotic inducing agent (Ryter et al., 2007). We chose to use 100 μM H2O2 for 20 min, based on time course and dose-response curve studies with uninfected MDM (data not shown). All cell populations exposed to H2O2 displayed a higher percentage of apoptotic macrophages than their untreated sister cultures (p < 0.05). When H2O2-treated macrophages were compared across the infection groups, H2O2-treated MAP-infected MDM cell populations contained a significantly lower relative percentage of apoptotic cells than either bystander or control macrophage populations (p = 0.0132 and 0.0064, respectively; Figure 2). No other significant differences were observed between the three groups (data not shown).

Bottom Line: Our results demonstrate that, indeed, populations of MAP-infected macrophages contain fewer apoptotic cells than similar populations of control cells, and that MAP infection reduces the sensitivity of infected macrophages to induction of apoptosis by H(2)O(2).This reduction in caspase activity is accompanied by a pronounced reduction in transcription of caspase genes encoding caspases 3, 7, and 8, but not for caspase 9, when compared to control, uninfected cells.Together these data demonstrate that MAP specific factors may prevent caspase activity and caspase gene transcription as well as apoptosis signaling protein expression, resulting in decreased spontaneous host cell apoptosis and decreased sensitivity to apoptosis inducing agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Center for Animal Functional Genomics, Michigan State University East Lansing, MI, USA.

ABSTRACT
Mycobacterium avium subspecies paratuberculosis (MAP) is able to survive intracellularly in macrophages by preventing normal phagosome maturation processes utilized to destroy bacteria. Infected macrophages often undergo apoptotic cell death to efficiently present bacterial antigens to the host adaptive immune system in a process known as efferocytosis. Recent studies with Mycobacterium tuberculosis (MTB) showed that macrophages infected with MTB are less likely to undergo apoptosis than control, uninfected cells. It is proposed that regulation of macrophage apoptosis is an important immune evasion tactic for MTB. Based on the similarity of MAP and MTB, we hypothesized that MAP-infected macrophages would be resistant to apoptosis compared to uninfected cells within the same culture and to cells from uninfected cultures. Our results demonstrate that, indeed, populations of MAP-infected macrophages contain fewer apoptotic cells than similar populations of control cells, and that MAP infection reduces the sensitivity of infected macrophages to induction of apoptosis by H(2)O(2). We further demonstrate that MAP-infected cells contain reduced caspase activity for caspases 3/7, 8, and 9. Reduced caspase activity in MAP-infected macrophages is also maintained after H(2)O(2) induction. This reduction in caspase activity is accompanied by a pronounced reduction in transcription of caspase genes encoding caspases 3, 7, and 8, but not for caspase 9, when compared to control, uninfected cells. Furthermore, MAP infection drastically effects the expression of several host cell proteins important for regulation of apoptosis. Studies using mutant MAP strains demonstrate the importance of bacterial specific factors in the control of host macrophage apoptosis. Together these data demonstrate that MAP specific factors may prevent caspase activity and caspase gene transcription as well as apoptosis signaling protein expression, resulting in decreased spontaneous host cell apoptosis and decreased sensitivity to apoptosis inducing agents.

No MeSH data available.


Related in: MedlinePlus