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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 in M.tuberculosis-infected macrophage culture supernatants is mediated by heat-sensitive factor(s) greater than 50 kDa in molecular size.(A) Heat inactivation (95°C, 20 min) of supernatants from low MOI and high MOI-infected macrophages (shaded bars, infection + and ++ respectively) inhibited induction of Jurkat T cell death. (B) Death-inducing factor(s) in infected macrophage supernatants were retained in the >50 kDa fractions following molecular size separation, and killed Jurkat T cells in a dose-dependent manner (25% and 10% dilution). Data shown in each panel are from one representative experiment, showing fold-change in cell death ± SEM in low MOI and high MOI-infected macrophage supernatants (shaded bars, infection + and ++, respectively) relative to uninfected control (closed bars), from incubations performed in triplicate. Each experiment was repeated a minimum of three times. ** =  p<0.01, *** =  p<0.001, Student’s t test relative to uninfected control.
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pone-0038488-g006: T cell death in M.tuberculosis-infected macrophage culture supernatants is mediated by heat-sensitive factor(s) greater than 50 kDa in molecular size.(A) Heat inactivation (95°C, 20 min) of supernatants from low MOI and high MOI-infected macrophages (shaded bars, infection + and ++ respectively) inhibited induction of Jurkat T cell death. (B) Death-inducing factor(s) in infected macrophage supernatants were retained in the >50 kDa fractions following molecular size separation, and killed Jurkat T cells in a dose-dependent manner (25% and 10% dilution). Data shown in each panel are from one representative experiment, showing fold-change in cell death ± SEM in low MOI and high MOI-infected macrophage supernatants (shaded bars, infection + and ++, respectively) relative to uninfected control (closed bars), from incubations performed in triplicate. Each experiment was repeated a minimum of three times. ** =  p<0.01, *** =  p<0.001, Student’s t test relative to uninfected control.

Mentions: To investigate the composition of the signalling factor(s) that induced T cell death, supernatants from uninfected and infected macrophages were heat-denatured at 95°C for 20 min, and then applied to Jurkat T cells. Following 24 h incubation, cell death was measured by Hoechst/PI staining, and expressed as fold-change in death relative to uninfected control. Unheated supernatants from low and high MOI-infected macrophages caused a significant (p<0.01, p<0.001 respectively) fold-increase in cell death to 1.52±0.07 and 2.04±0.05 of that in the uninfected control supernatants. This effect was mitigated by heat denaturing; in Jurkats incubated in heat-treated supernatants, fold-change in cell death was 0.88±0.02 and 1.01±0.08 of that in uninfected control supernatants (fig. 6A).


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 in M.tuberculosis-infected macrophage culture supernatants is mediated by heat-sensitive factor(s) greater than 50 kDa in molecular size.(A) Heat inactivation (95°C, 20 min) of supernatants from low MOI and high MOI-infected macrophages (shaded bars, infection + and ++ respectively) inhibited induction of Jurkat T cell death. (B) Death-inducing factor(s) in infected macrophage supernatants were retained in the >50 kDa fractions following molecular size separation, and killed Jurkat T cells in a dose-dependent manner (25% and 10% dilution). Data shown in each panel are from one representative experiment, showing fold-change in cell death ± SEM in low MOI and high MOI-infected macrophage supernatants (shaded bars, infection + and ++, respectively) relative to uninfected control (closed bars), from incubations performed in triplicate. Each experiment was repeated a minimum of three times. ** =  p<0.01, *** =  p<0.001, Student’s t test relative to uninfected control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366923&req=5

pone-0038488-g006: T cell death in M.tuberculosis-infected macrophage culture supernatants is mediated by heat-sensitive factor(s) greater than 50 kDa in molecular size.(A) Heat inactivation (95°C, 20 min) of supernatants from low MOI and high MOI-infected macrophages (shaded bars, infection + and ++ respectively) inhibited induction of Jurkat T cell death. (B) Death-inducing factor(s) in infected macrophage supernatants were retained in the >50 kDa fractions following molecular size separation, and killed Jurkat T cells in a dose-dependent manner (25% and 10% dilution). Data shown in each panel are from one representative experiment, showing fold-change in cell death ± SEM in low MOI and high MOI-infected macrophage supernatants (shaded bars, infection + and ++, respectively) relative to uninfected control (closed bars), from incubations performed in triplicate. Each experiment was repeated a minimum of three times. ** =  p<0.01, *** =  p<0.001, Student’s t test relative to uninfected control.
Mentions: To investigate the composition of the signalling factor(s) that induced T cell death, supernatants from uninfected and infected macrophages were heat-denatured at 95°C for 20 min, and then applied to Jurkat T cells. Following 24 h incubation, cell death was measured by Hoechst/PI staining, and expressed as fold-change in death relative to uninfected control. Unheated supernatants from low and high MOI-infected macrophages caused a significant (p<0.01, p<0.001 respectively) fold-increase in cell death to 1.52±0.07 and 2.04±0.05 of that in the uninfected control supernatants. This effect was mitigated by heat denaturing; in Jurkats incubated in heat-treated supernatants, fold-change in cell death was 0.88±0.02 and 1.01±0.08 of that in uninfected control supernatants (fig. 6A).

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