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Networked T cell death following macrophage infection by Mycobacterium tuberculosis.

Macdonald SH, Woodward E, Coleman MM, Dorris ER, Nadarajan P, Chew WM, McLaughlin AM, Keane J - PLoS ONE (2012)

Bottom Line: This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas.The supernatant cytotoxic signal(s) were heat-labile and greater than 50 kDa in molecular size.Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Medicine, Trinity Institute of Molecular Medicine, St James's Hospital, Dublin, Ireland. macdonaldez@gmail.com

ABSTRACT

Background: Depletion of T cells following infection by Mycobacterium tuberculosis (Mtb) impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised.

Methodology/principal findings: We found that lymphopenia (<1.5 × 10(9) cells/l) was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb) or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s) were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG)- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system.

Conclusions: Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as interfere with microbial eradication in the granuloma.

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T cell death after exposure to M.tuberculosis secreted antigens.Jurkat T cells were exposed for 24 h to a panel of secreted factors derived from Mtb. No increase in cell death occurred when T cells were incubated with 16 kDa, 45 kDa, Antigen 85 complex, GroES, ManLAM or Phos1 (A–C, E–G, respectively, shaded bars), however elevated death occurred in cells incubated with ESAT-6 at 10 µg/ml or 1 µg/ml (D). 1 µM Staurosporine was used as a positive control in each case (open bars); vehicle control was either PBS or DMSO in the case of ManLAM (closed bars). Supernatants of AMs infected with H37Ra or M.bovis BCG both significantly increased T cell death (H, shaded bars), compared with uninfected AM supernatants (H, closed bars). Data in each panel are from one representative experiment, showing mean percentage cell death ± SEM from incubations performed in triplicate. * =  p<0.05, ** =  p<0.01, *** =  p<0.001, Student’s t test relative to vehicle (A–G) or uninfected (H) control.
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pone-0038488-g004: T cell death after exposure to M.tuberculosis secreted antigens.Jurkat T cells were exposed for 24 h to a panel of secreted factors derived from Mtb. No increase in cell death occurred when T cells were incubated with 16 kDa, 45 kDa, Antigen 85 complex, GroES, ManLAM or Phos1 (A–C, E–G, respectively, shaded bars), however elevated death occurred in cells incubated with ESAT-6 at 10 µg/ml or 1 µg/ml (D). 1 µM Staurosporine was used as a positive control in each case (open bars); vehicle control was either PBS or DMSO in the case of ManLAM (closed bars). Supernatants of AMs infected with H37Ra or M.bovis BCG both significantly increased T cell death (H, shaded bars), compared with uninfected AM supernatants (H, closed bars). Data in each panel are from one representative experiment, showing mean percentage cell death ± SEM from incubations performed in triplicate. * =  p<0.05, ** =  p<0.01, *** =  p<0.001, Student’s t test relative to vehicle (A–G) or uninfected (H) control.

Mentions: To examine whether T cell death in our system was attributable to other specific secreted proteins from Mtb, and since such secreted factors have previously been shown to alter immune cell survival [25], [26], [27] and activity [28], we exposed Jurkat cells to a panel of Mtb-derived factors. In Jurkat cells incubated in the presence of ESAT-6 at concentrations of 10 µg/ml, 1 µg/ml and 0.1 µg/ml, 58.33±0.58%, 12.43±0.55% and 7.04±0.23% cell death was observed. At 10 µg/ml and 1 µg/ml, cell death was significantly (P<0.001) elevated above that of the vehicle control, where 6.35±0.16% death was observed. Staurosporine 1 µM was used as a positive cell killing stimulus, and induced 64.63±1.34% death. Elevated death was not observed in T cells incubated with other secreted antigens such as 16 kDa, 45 kDa, Antigen 85 complex, GroES, ManLAM or Phos1 (fig. 4A–G). To examine whether ESAT-6 could be the principal mediator of T cell death in our system, we performed AM infections using Mtb H37Ra in parallel with M.bovis BCG, then cultured Jurkat T cells in the resulting cell-free supernatants as described previously. In uninfected AM supernatants, T cell death was 9.56±0.22%; this significantly increased to 14.39±1.38% (p<0.05) and 18.30±0.31% (p<0.001) in low and high MOI H37Ra-infected AM supernatants, respectively. In low and high MOI BCG-infected AM supernatants, T cell death also increased significantly (p<0.01) to 12.38±0.52% and 17.1±0.91%, respectively (fig. 4H). No significant difference was found when T cell death in H37Ra-infected AM supernatants at low or high MOI was compared with that in BCG-infected AM supernatants at the same MOIs.


Networked T cell death following macrophage infection by Mycobacterium tuberculosis.

Macdonald SH, Woodward E, Coleman MM, Dorris ER, Nadarajan P, Chew WM, McLaughlin AM, Keane J - PLoS ONE (2012)

T cell death after exposure to M.tuberculosis secreted antigens.Jurkat T cells were exposed for 24 h to a panel of secreted factors derived from Mtb. No increase in cell death occurred when T cells were incubated with 16 kDa, 45 kDa, Antigen 85 complex, GroES, ManLAM or Phos1 (A–C, E–G, respectively, shaded bars), however elevated death occurred in cells incubated with ESAT-6 at 10 µg/ml or 1 µg/ml (D). 1 µM Staurosporine was used as a positive control in each case (open bars); vehicle control was either PBS or DMSO in the case of ManLAM (closed bars). Supernatants of AMs infected with H37Ra or M.bovis BCG both significantly increased T cell death (H, shaded bars), compared with uninfected AM supernatants (H, closed bars). Data in each panel are from one representative experiment, showing mean percentage cell death ± SEM from incubations performed in triplicate. * =  p<0.05, ** =  p<0.01, *** =  p<0.001, Student’s t test relative to vehicle (A–G) or uninfected (H) control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366923&req=5

pone-0038488-g004: T cell death after exposure to M.tuberculosis secreted antigens.Jurkat T cells were exposed for 24 h to a panel of secreted factors derived from Mtb. No increase in cell death occurred when T cells were incubated with 16 kDa, 45 kDa, Antigen 85 complex, GroES, ManLAM or Phos1 (A–C, E–G, respectively, shaded bars), however elevated death occurred in cells incubated with ESAT-6 at 10 µg/ml or 1 µg/ml (D). 1 µM Staurosporine was used as a positive control in each case (open bars); vehicle control was either PBS or DMSO in the case of ManLAM (closed bars). Supernatants of AMs infected with H37Ra or M.bovis BCG both significantly increased T cell death (H, shaded bars), compared with uninfected AM supernatants (H, closed bars). Data in each panel are from one representative experiment, showing mean percentage cell death ± SEM from incubations performed in triplicate. * =  p<0.05, ** =  p<0.01, *** =  p<0.001, Student’s t test relative to vehicle (A–G) or uninfected (H) control.
Mentions: To examine whether T cell death in our system was attributable to other specific secreted proteins from Mtb, and since such secreted factors have previously been shown to alter immune cell survival [25], [26], [27] and activity [28], we exposed Jurkat cells to a panel of Mtb-derived factors. In Jurkat cells incubated in the presence of ESAT-6 at concentrations of 10 µg/ml, 1 µg/ml and 0.1 µg/ml, 58.33±0.58%, 12.43±0.55% and 7.04±0.23% cell death was observed. At 10 µg/ml and 1 µg/ml, cell death was significantly (P<0.001) elevated above that of the vehicle control, where 6.35±0.16% death was observed. Staurosporine 1 µM was used as a positive cell killing stimulus, and induced 64.63±1.34% death. Elevated death was not observed in T cells incubated with other secreted antigens such as 16 kDa, 45 kDa, Antigen 85 complex, GroES, ManLAM or Phos1 (fig. 4A–G). To examine whether ESAT-6 could be the principal mediator of T cell death in our system, we performed AM infections using Mtb H37Ra in parallel with M.bovis BCG, then cultured Jurkat T cells in the resulting cell-free supernatants as described previously. In uninfected AM supernatants, T cell death was 9.56±0.22%; this significantly increased to 14.39±1.38% (p<0.05) and 18.30±0.31% (p<0.001) in low and high MOI H37Ra-infected AM supernatants, respectively. In low and high MOI BCG-infected AM supernatants, T cell death also increased significantly (p<0.01) to 12.38±0.52% and 17.1±0.91%, respectively (fig. 4H). No significant difference was found when T cell death in H37Ra-infected AM supernatants at low or high MOI was compared with that in BCG-infected AM supernatants at the same MOIs.

Bottom Line: This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas.The supernatant cytotoxic signal(s) were heat-labile and greater than 50 kDa in molecular size.Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Medicine, Trinity Institute of Molecular Medicine, St James's Hospital, Dublin, Ireland. macdonaldez@gmail.com

ABSTRACT

Background: Depletion of T cells following infection by Mycobacterium tuberculosis (Mtb) impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised.

Methodology/principal findings: We found that lymphopenia (<1.5 × 10(9) cells/l) was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb) or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s) were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG)- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system.

Conclusions: Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as interfere with microbial eradication in the granuloma.

Show MeSH
Related in: MedlinePlus