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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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The role of ESAT-6 in macrophage necrosis.hMDMs were left uninfected or infected at the indicated MOI with H37Rv, an ESAT-6 mutant, a complemented ESAT-6 mutant (ESAT-6-compl.), or an RD1 mutant, and cell death was assessed by the three different staining kits after two days, and analyzed by flow cytometry, or by calcein staining. Alternatively, infected cells were lysed, dilutions were plated for viable count, and the number of CFU was enumerated. Cell culture supernatants were collected and IL-1β was assayed by ELISA. A) TUNEL analysis of DNA fragmentation after infection at MOI 10 (n = 6). B) MitoTracker analysis of mitochondrial membrane potential loss after infection at MOI 10 (n = 6). C) Plasma membrane integrity analysis after infection at MOI 10 (n = 6). D) Relative viability of hMDMs infected for two days with the different strains at MOI 40, as analyzed by calcein staining (n = 4). The values were normalized against uninfected controls from the same reading, which were set to 100%. E) The number of CFU per well after infection with the different strains at MOI 10 for two days, as determined by viable count analysis (n = 5). F) IL-1β concentration in cell culture supernatants from hMDMs infected for two days with the different strains at MOI 10 (n = 3). Bar graphs show the mean and SEM. The means were compared to that of uninfected hMDMs (A-D) or H37Rv-infected hMDMs (E-F) using ANOVA with Dunnett’s post-hoc test. A statistically significant difference is denoted by *(p<0.05), **(p<0.01), or ***(p<0.001).
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pone-0020302-g005: The role of ESAT-6 in macrophage necrosis.hMDMs were left uninfected or infected at the indicated MOI with H37Rv, an ESAT-6 mutant, a complemented ESAT-6 mutant (ESAT-6-compl.), or an RD1 mutant, and cell death was assessed by the three different staining kits after two days, and analyzed by flow cytometry, or by calcein staining. Alternatively, infected cells were lysed, dilutions were plated for viable count, and the number of CFU was enumerated. Cell culture supernatants were collected and IL-1β was assayed by ELISA. A) TUNEL analysis of DNA fragmentation after infection at MOI 10 (n = 6). B) MitoTracker analysis of mitochondrial membrane potential loss after infection at MOI 10 (n = 6). C) Plasma membrane integrity analysis after infection at MOI 10 (n = 6). D) Relative viability of hMDMs infected for two days with the different strains at MOI 40, as analyzed by calcein staining (n = 4). The values were normalized against uninfected controls from the same reading, which were set to 100%. E) The number of CFU per well after infection with the different strains at MOI 10 for two days, as determined by viable count analysis (n = 5). F) IL-1β concentration in cell culture supernatants from hMDMs infected for two days with the different strains at MOI 10 (n = 3). Bar graphs show the mean and SEM. The means were compared to that of uninfected hMDMs (A-D) or H37Rv-infected hMDMs (E-F) using ANOVA with Dunnett’s post-hoc test. A statistically significant difference is denoted by *(p<0.05), **(p<0.01), or ***(p<0.001).

Mentions: We established that H37Rv-induced cell death in hMDMs was not executed by any of the cell death signals tested above. However, we hypothesized that induction of cell death might be dependent on bacterial factors as well as bacterial load. To elucidate this we used ESAT-6-deficient mutants of H37Rv. hMDMs were infected at MOI 10 with wild type H37Rv, an ESAT-6 deletion mutant (H37Rv:Δ3875), an ESAT-6 complemented strain (H37Rv:Δ3875:pMH406), and an RD1 deletion mutant (H37Rv:ΔRD1) [9] and analyzed by all three cell death assays. Infection with the parental H37Rv strain induced significant DNA fragmentation (p<0.001), mitochondrial membrane permeabilization (p<0.001) and plasma membrane integrity loss (p<0.001), as compared to uninfected controls, while the ESAT-6 or RD1 mutant did not induce any cell death features. Complementation of ESAT-6 significantly restored the capability to induce DNA fragmentation (p<0.01), mitochondrial membrane permeabilization (p<0.001) and plasma membrane integrity loss (p<0.001), as compared to the uninfected controls (Fig. 5A–C). Furthermore, the increased concentration of HMGB1 in the cell culture supernatant observed after H37Rv infection was dependent on an intact ESAT-6 gene (Fig. S2). To investigate whether cell death could be triggered by the mutant strains when the bacterial load was increased further, hMDMs were infected at an MOI of 40, and cell viability was assessed by the calcein assay. Even at MOI 40, however, the ESAT-6 and RD1 mutants failed to induce cell death (Fig. 5D). To investigate growth of the different strains upon challenge of hMDMs, cells infected for two days were lysed and the number of colony forming units (CFU) was enumerated after two to three weeks. This showed that both the ESAT-6 (p<0.05) and RD1 (p<0.05) mutants were significantly deficient in total growth as compared to the parental H37Rv strain. When the ESAT-6-complemented strain was used to infect hMDMs, however, the replication ability was restored (Fig. 5E). Thus, the cell death induced by H37Rv was directly or indirectly dependent on a functional RD1 region. Furthermore, as shown in Fig. 5F, infection of hMDMs with the ESAT-6 (p<0.05) or RD1 (p<0.05) mutant strain resulted in diminished secretion of IL-1β from the cells, as compared to the wild type strain.


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)

The role of ESAT-6 in macrophage necrosis.hMDMs were left uninfected or infected at the indicated MOI with H37Rv, an ESAT-6 mutant, a complemented ESAT-6 mutant (ESAT-6-compl.), or an RD1 mutant, and cell death was assessed by the three different staining kits after two days, and analyzed by flow cytometry, or by calcein staining. Alternatively, infected cells were lysed, dilutions were plated for viable count, and the number of CFU was enumerated. Cell culture supernatants were collected and IL-1β was assayed by ELISA. A) TUNEL analysis of DNA fragmentation after infection at MOI 10 (n = 6). B) MitoTracker analysis of mitochondrial membrane potential loss after infection at MOI 10 (n = 6). C) Plasma membrane integrity analysis after infection at MOI 10 (n = 6). D) Relative viability of hMDMs infected for two days with the different strains at MOI 40, as analyzed by calcein staining (n = 4). The values were normalized against uninfected controls from the same reading, which were set to 100%. E) The number of CFU per well after infection with the different strains at MOI 10 for two days, as determined by viable count analysis (n = 5). F) IL-1β concentration in cell culture supernatants from hMDMs infected for two days with the different strains at MOI 10 (n = 3). Bar graphs show the mean and SEM. The means were compared to that of uninfected hMDMs (A-D) or H37Rv-infected hMDMs (E-F) using ANOVA with Dunnett’s post-hoc test. A statistically significant difference 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-g005: The role of ESAT-6 in macrophage necrosis.hMDMs were left uninfected or infected at the indicated MOI with H37Rv, an ESAT-6 mutant, a complemented ESAT-6 mutant (ESAT-6-compl.), or an RD1 mutant, and cell death was assessed by the three different staining kits after two days, and analyzed by flow cytometry, or by calcein staining. Alternatively, infected cells were lysed, dilutions were plated for viable count, and the number of CFU was enumerated. Cell culture supernatants were collected and IL-1β was assayed by ELISA. A) TUNEL analysis of DNA fragmentation after infection at MOI 10 (n = 6). B) MitoTracker analysis of mitochondrial membrane potential loss after infection at MOI 10 (n = 6). C) Plasma membrane integrity analysis after infection at MOI 10 (n = 6). D) Relative viability of hMDMs infected for two days with the different strains at MOI 40, as analyzed by calcein staining (n = 4). The values were normalized against uninfected controls from the same reading, which were set to 100%. E) The number of CFU per well after infection with the different strains at MOI 10 for two days, as determined by viable count analysis (n = 5). F) IL-1β concentration in cell culture supernatants from hMDMs infected for two days with the different strains at MOI 10 (n = 3). Bar graphs show the mean and SEM. The means were compared to that of uninfected hMDMs (A-D) or H37Rv-infected hMDMs (E-F) using ANOVA with Dunnett’s post-hoc test. A statistically significant difference is denoted by *(p<0.05), **(p<0.01), or ***(p<0.001).
Mentions: We established that H37Rv-induced cell death in hMDMs was not executed by any of the cell death signals tested above. However, we hypothesized that induction of cell death might be dependent on bacterial factors as well as bacterial load. To elucidate this we used ESAT-6-deficient mutants of H37Rv. hMDMs were infected at MOI 10 with wild type H37Rv, an ESAT-6 deletion mutant (H37Rv:Δ3875), an ESAT-6 complemented strain (H37Rv:Δ3875:pMH406), and an RD1 deletion mutant (H37Rv:ΔRD1) [9] and analyzed by all three cell death assays. Infection with the parental H37Rv strain induced significant DNA fragmentation (p<0.001), mitochondrial membrane permeabilization (p<0.001) and plasma membrane integrity loss (p<0.001), as compared to uninfected controls, while the ESAT-6 or RD1 mutant did not induce any cell death features. Complementation of ESAT-6 significantly restored the capability to induce DNA fragmentation (p<0.01), mitochondrial membrane permeabilization (p<0.001) and plasma membrane integrity loss (p<0.001), as compared to the uninfected controls (Fig. 5A–C). Furthermore, the increased concentration of HMGB1 in the cell culture supernatant observed after H37Rv infection was dependent on an intact ESAT-6 gene (Fig. S2). To investigate whether cell death could be triggered by the mutant strains when the bacterial load was increased further, hMDMs were infected at an MOI of 40, and cell viability was assessed by the calcein assay. Even at MOI 40, however, the ESAT-6 and RD1 mutants failed to induce cell death (Fig. 5D). To investigate growth of the different strains upon challenge of hMDMs, cells infected for two days were lysed and the number of colony forming units (CFU) was enumerated after two to three weeks. This showed that both the ESAT-6 (p<0.05) and RD1 (p<0.05) mutants were significantly deficient in total growth as compared to the parental H37Rv strain. When the ESAT-6-complemented strain was used to infect hMDMs, however, the replication ability was restored (Fig. 5E). Thus, the cell death induced by H37Rv was directly or indirectly dependent on a functional RD1 region. Furthermore, as shown in Fig. 5F, infection of hMDMs with the ESAT-6 (p<0.05) or RD1 (p<0.05) mutant strain resulted in diminished secretion of IL-1β from the cells, as compared to the wild type strain.

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