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Human macrophages infected with a high burden of ESAT-6-expressing M. tuberculosis undergo caspase-1- and cathepsin B-independent necrosis.

Welin A, Eklund D, Stendahl O, Lerm M - PLoS ONE (2011)

Bottom Line: The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response.It was, however, dependent on mycobacterial expression of ESAT-6.We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

View Article: PubMed Central - PubMed

Affiliation: Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.

ABSTRACT
Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1β release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

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Features of cell death induced by Mtb.hMDMs were left uninfected, or infected with H37Rv at MOI 1 or 10, or H37Ra at MOI 10 over two days. Three different staining procedures were used to investigate cell death features on each day, and the cells were analyzed by flow cytometry. A) TUNEL analysis of DNA fragmentation. The first histogram shows representative flow cytometry data for hMDMs negative for TUNEL staining (untreated), and the second one for hMDMs positive for TUNEL staining (DNase-treated). The bar graph shows the percentage of hMDMs that were positive for TUNEL staining in uninfected or infected samples over two days (n≥5). B) MitoTracker analysis of mitochondrial membrane potential loss. The first histogram shows representative flow cytometry data for hMDMs with high MitoTracker staining (untreated), and the second one for hMDMs with low MitoTracker staining (UV-treated). The bar graph shows the percentage of hMDMs that had low MitoTracker staining (i.e. compromised mitochondria) in uninfected or infected samples over two days (n≥3). C) Plasma membrane integrity analysis. The first histogram shows representative flow cytometry data for hMDMs negative for plasma membrane leakiness staining (untreated), and the second one for hMDMs positive for plasma membrane leakiness staining (heat-treated). The bar graph shows the percentage of hMDMs that were positive for plasma membrane leakiness staining in uninfected or infected samples over two days (n≥3). D) HMGB1 concentration in supernatants of uninfected hMDMs or hMDMs infected with H37Rv for two days at the indicated MOI (n = 3), as measured by ELISA. Bar graphs show the mean and SEM. The means were compared to that of uninfected hMDMs on each day using ANOVA with Dunnett’s post-hoc test. A statistically significant difference from uninfected cells is denoted by *(p<0.05), **(p<0.01), or ***(p<0.001).
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pone-0020302-g003: Features of cell death induced by Mtb.hMDMs were left uninfected, or infected with H37Rv at MOI 1 or 10, or H37Ra at MOI 10 over two days. Three different staining procedures were used to investigate cell death features on each day, and the cells were analyzed by flow cytometry. A) TUNEL analysis of DNA fragmentation. The first histogram shows representative flow cytometry data for hMDMs negative for TUNEL staining (untreated), and the second one for hMDMs positive for TUNEL staining (DNase-treated). The bar graph shows the percentage of hMDMs that were positive for TUNEL staining in uninfected or infected samples over two days (n≥5). B) MitoTracker analysis of mitochondrial membrane potential loss. The first histogram shows representative flow cytometry data for hMDMs with high MitoTracker staining (untreated), and the second one for hMDMs with low MitoTracker staining (UV-treated). The bar graph shows the percentage of hMDMs that had low MitoTracker staining (i.e. compromised mitochondria) in uninfected or infected samples over two days (n≥3). C) Plasma membrane integrity analysis. The first histogram shows representative flow cytometry data for hMDMs negative for plasma membrane leakiness staining (untreated), and the second one for hMDMs positive for plasma membrane leakiness staining (heat-treated). The bar graph shows the percentage of hMDMs that were positive for plasma membrane leakiness staining in uninfected or infected samples over two days (n≥3). D) HMGB1 concentration in supernatants of uninfected hMDMs or hMDMs infected with H37Rv for two days at the indicated MOI (n = 3), as measured by ELISA. Bar graphs show the mean and SEM. The means were compared to that of uninfected hMDMs on each day using ANOVA with Dunnett’s post-hoc test. A statistically significant difference from uninfected cells is denoted by *(p<0.05), **(p<0.01), or ***(p<0.001).

Mentions: DNA fragmentation and increased permeability of the cell membrane are well-characterized features of pyroptosis and pyronecrosis, while contradicting results concerning changes in the mitochondrial membrane potential have been shown [16], [21], [26]. In our system, the higher bacterial burden of H37Rv inside macrophages caused cell death and release of IL-1β. In order to characterize the features of cell death induced by H37Rv, we analyzed hMDMs infected for up to two days using three different assays. Cells infected with avirulent H37Ra were analyzed for comparison since H37Ra has previously been shown to induce macrophage apoptosis, with caspase-dependent DNA fragmentation [4], [6], [27], [28]. TUNEL staining and flow cytometry analysis showed that H37Rv infection at MOI 10 caused extensive DNA fragmentation (p<0.001 at Day 1 and 2, Fig. 3A). In contrast, infection with a lower MOI of H37Rv did not induce any DNA fragmentation. H37Ra at MOI 10 induced less DNA fragmentation than H37Rv after one and two days of infection (Fig. 3A). TUNEL staining of infected cells on glass cover slips and fluorescence microscopy analysis confirmed this result (Fig. S1). Cell samples were also stained using MitoTracker to assess mitochondrial integrity. The results showed a similar pattern to the DNA fragmentation data, where H37Rv at the higher but not lower MOI led to an increase in the number of cells with compromised mitochondrial integrity (p<0.05 at Day 1 and p<0.01 at Day 2, Fig. 3B). Infection with H37Ra at MOI 10 also led to an increase in the number of cells with compromised mitochondrial potential as compared to uninfected cells (p<0.05 at Day 2, Fig. 3B). Finally, a time-dependent damage to macrophage plasma membranes could be clearly seen in H37Rv infection at MOI 10 (p<0.01 at Day 1 and p<0.001 at Day 2), while no increase in plasma membrane permeability was observed in cells with a low burden of H37Rv. Infection with H37Ra resulted in moderate cell damage (Fig. 3C). Thus, all three cell death-related features were induced by the higher bacterial burden of H37Rv. Furthermore, H37Rv infection at MOI 10 led to elevated levels of HMGB1 in the cell culture supernatant (p<0.01, Fig. 3D).


Human macrophages infected with a high burden of ESAT-6-expressing M. tuberculosis undergo caspase-1- and cathepsin B-independent necrosis.

Welin A, Eklund D, Stendahl O, Lerm M - PLoS ONE (2011)

Features of cell death induced by Mtb.hMDMs were left uninfected, or infected with H37Rv at MOI 1 or 10, or H37Ra at MOI 10 over two days. Three different staining procedures were used to investigate cell death features on each day, and the cells were analyzed by flow cytometry. A) TUNEL analysis of DNA fragmentation. The first histogram shows representative flow cytometry data for hMDMs negative for TUNEL staining (untreated), and the second one for hMDMs positive for TUNEL staining (DNase-treated). The bar graph shows the percentage of hMDMs that were positive for TUNEL staining in uninfected or infected samples over two days (n≥5). B) MitoTracker analysis of mitochondrial membrane potential loss. The first histogram shows representative flow cytometry data for hMDMs with high MitoTracker staining (untreated), and the second one for hMDMs with low MitoTracker staining (UV-treated). The bar graph shows the percentage of hMDMs that had low MitoTracker staining (i.e. compromised mitochondria) in uninfected or infected samples over two days (n≥3). C) Plasma membrane integrity analysis. The first histogram shows representative flow cytometry data for hMDMs negative for plasma membrane leakiness staining (untreated), and the second one for hMDMs positive for plasma membrane leakiness staining (heat-treated). The bar graph shows the percentage of hMDMs that were positive for plasma membrane leakiness staining in uninfected or infected samples over two days (n≥3). D) HMGB1 concentration in supernatants of uninfected hMDMs or hMDMs infected with H37Rv for two days at the indicated MOI (n = 3), as measured by ELISA. Bar graphs show the mean and SEM. The means were compared to that of uninfected hMDMs on each day using ANOVA with Dunnett’s post-hoc test. A statistically significant difference from uninfected cells is denoted by *(p<0.05), **(p<0.01), or ***(p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102687&req=5

pone-0020302-g003: Features of cell death induced by Mtb.hMDMs were left uninfected, or infected with H37Rv at MOI 1 or 10, or H37Ra at MOI 10 over two days. Three different staining procedures were used to investigate cell death features on each day, and the cells were analyzed by flow cytometry. A) TUNEL analysis of DNA fragmentation. The first histogram shows representative flow cytometry data for hMDMs negative for TUNEL staining (untreated), and the second one for hMDMs positive for TUNEL staining (DNase-treated). The bar graph shows the percentage of hMDMs that were positive for TUNEL staining in uninfected or infected samples over two days (n≥5). B) MitoTracker analysis of mitochondrial membrane potential loss. The first histogram shows representative flow cytometry data for hMDMs with high MitoTracker staining (untreated), and the second one for hMDMs with low MitoTracker staining (UV-treated). The bar graph shows the percentage of hMDMs that had low MitoTracker staining (i.e. compromised mitochondria) in uninfected or infected samples over two days (n≥3). C) Plasma membrane integrity analysis. The first histogram shows representative flow cytometry data for hMDMs negative for plasma membrane leakiness staining (untreated), and the second one for hMDMs positive for plasma membrane leakiness staining (heat-treated). The bar graph shows the percentage of hMDMs that were positive for plasma membrane leakiness staining in uninfected or infected samples over two days (n≥3). D) HMGB1 concentration in supernatants of uninfected hMDMs or hMDMs infected with H37Rv for two days at the indicated MOI (n = 3), as measured by ELISA. Bar graphs show the mean and SEM. The means were compared to that of uninfected hMDMs on each day using ANOVA with Dunnett’s post-hoc test. A statistically significant difference from uninfected cells is denoted by *(p<0.05), **(p<0.01), or ***(p<0.001).
Mentions: DNA fragmentation and increased permeability of the cell membrane are well-characterized features of pyroptosis and pyronecrosis, while contradicting results concerning changes in the mitochondrial membrane potential have been shown [16], [21], [26]. In our system, the higher bacterial burden of H37Rv inside macrophages caused cell death and release of IL-1β. In order to characterize the features of cell death induced by H37Rv, we analyzed hMDMs infected for up to two days using three different assays. Cells infected with avirulent H37Ra were analyzed for comparison since H37Ra has previously been shown to induce macrophage apoptosis, with caspase-dependent DNA fragmentation [4], [6], [27], [28]. TUNEL staining and flow cytometry analysis showed that H37Rv infection at MOI 10 caused extensive DNA fragmentation (p<0.001 at Day 1 and 2, Fig. 3A). In contrast, infection with a lower MOI of H37Rv did not induce any DNA fragmentation. H37Ra at MOI 10 induced less DNA fragmentation than H37Rv after one and two days of infection (Fig. 3A). TUNEL staining of infected cells on glass cover slips and fluorescence microscopy analysis confirmed this result (Fig. S1). Cell samples were also stained using MitoTracker to assess mitochondrial integrity. The results showed a similar pattern to the DNA fragmentation data, where H37Rv at the higher but not lower MOI led to an increase in the number of cells with compromised mitochondrial integrity (p<0.05 at Day 1 and p<0.01 at Day 2, Fig. 3B). Infection with H37Ra at MOI 10 also led to an increase in the number of cells with compromised mitochondrial potential as compared to uninfected cells (p<0.05 at Day 2, Fig. 3B). Finally, a time-dependent damage to macrophage plasma membranes could be clearly seen in H37Rv infection at MOI 10 (p<0.01 at Day 1 and p<0.001 at Day 2), while no increase in plasma membrane permeability was observed in cells with a low burden of H37Rv. Infection with H37Ra resulted in moderate cell damage (Fig. 3C). Thus, all three cell death-related features were induced by the higher bacterial burden of H37Rv. Furthermore, H37Rv infection at MOI 10 led to elevated levels of HMGB1 in the cell culture supernatant (p<0.01, Fig. 3D).

Bottom Line: The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response.It was, however, dependent on mycobacterial expression of ESAT-6.We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

View Article: PubMed Central - PubMed

Affiliation: Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.

ABSTRACT
Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1β release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

Show MeSH
Related in: MedlinePlus