Limits...
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.

Show MeSH

Related in: MedlinePlus

M.tuberculosis-infected macrophage supernatants are toxic to T cells.(A) Jurkat T cells underwent significantly increased cell death in cell-free supernatants of low MOI and high MOI Mtb-infected human primary alveolar macrophages (AMs) (shaded bars; infection + and ++, respectively), compared to those of uninfected AMs (closed bars, infection –). Staurosporine 1 µM was used as a positive control to induce cell death (open bars). Similar effects were observed on PBTL and Jurkat T cells exposed to the cell-free supernatants of uninfected/infected THP-1 macrophages. (B) Jurkat cell death in infected THP-1 macrophage supernatant occurred in a dose-dependent manner; infected macrophage supernatants were diluted to 50% and 20% in uninfected supernatant, reducing the level of T cell killing. (C) Example cropped fluorescence intensity images for Hoechst and PI staining from Jurkats incubated in uninfected and infected AM supernatants. Images were processed using automated INCell image analysis to detect apoptotic condensed and fragmented nuclei (arrow 1 and 2, respectively) and PI-positive cells (arrow head). Data in each panel are from one representative experiment, showing mean percentage cell death ± SEM from incubations performed in triplicate. ** =  p<0.01, *** =  p<0.001, Student’s t test relative to uninfected control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3366923&req=5

pone-0038488-g002: M.tuberculosis-infected macrophage supernatants are toxic to T cells.(A) Jurkat T cells underwent significantly increased cell death in cell-free supernatants of low MOI and high MOI Mtb-infected human primary alveolar macrophages (AMs) (shaded bars; infection + and ++, respectively), compared to those of uninfected AMs (closed bars, infection –). Staurosporine 1 µM was used as a positive control to induce cell death (open bars). Similar effects were observed on PBTL and Jurkat T cells exposed to the cell-free supernatants of uninfected/infected THP-1 macrophages. (B) Jurkat cell death in infected THP-1 macrophage supernatant occurred in a dose-dependent manner; infected macrophage supernatants were diluted to 50% and 20% in uninfected supernatant, reducing the level of T cell killing. (C) Example cropped fluorescence intensity images for Hoechst and PI staining from Jurkats incubated in uninfected and infected AM supernatants. Images were processed using automated INCell image analysis to detect apoptotic condensed and fragmented nuclei (arrow 1 and 2, respectively) and PI-positive cells (arrow head). Data in each panel are from one representative experiment, showing mean percentage cell death ± SEM from incubations performed in triplicate. ** =  p<0.01, *** =  p<0.001, Student’s t test relative to uninfected control.

Mentions: To investigate whether soluble mediators released during Mtb infection could alter the viability of T cells in a contact-independent manner, we first infected primary human alveolar macrophages for 48 h with Mtb H37Ra bacilli, then harvested culture supernatants and applied them to Jurkat T cells for 24 h before assessing cell death. In Jurkats incubated in cell-free supernatants from uninfected AMs, 5.85±0.13% cell death was observed, whereas in supernatants from low and high MOI Mtb-infected AMs, this increased significantly (p<0.001) to 18.12±0.24% and 21.13±0.25%, respectively. 1 µM Staurosporine used as a positive cell-killing control caused 67.33±0.85% death (fig. 2A). In this, and subsequent experiments, an automated image analysis strategy (figure S1) was used to clearly identify apoptotic cells that had condensed/fragmented nuclei and/or PI staining, and discriminate them from live cells (fig. 2C).


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)

M.tuberculosis-infected macrophage supernatants are toxic to T cells.(A) Jurkat T cells underwent significantly increased cell death in cell-free supernatants of low MOI and high MOI Mtb-infected human primary alveolar macrophages (AMs) (shaded bars; infection + and ++, respectively), compared to those of uninfected AMs (closed bars, infection –). Staurosporine 1 µM was used as a positive control to induce cell death (open bars). Similar effects were observed on PBTL and Jurkat T cells exposed to the cell-free supernatants of uninfected/infected THP-1 macrophages. (B) Jurkat cell death in infected THP-1 macrophage supernatant occurred in a dose-dependent manner; infected macrophage supernatants were diluted to 50% and 20% in uninfected supernatant, reducing the level of T cell killing. (C) Example cropped fluorescence intensity images for Hoechst and PI staining from Jurkats incubated in uninfected and infected AM supernatants. Images were processed using automated INCell image analysis to detect apoptotic condensed and fragmented nuclei (arrow 1 and 2, respectively) and PI-positive cells (arrow head). Data in each panel are from one representative experiment, showing mean percentage cell death ± SEM from incubations performed in triplicate. ** =  p<0.01, *** =  p<0.001, Student’s t test relative to uninfected control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366923&req=5

pone-0038488-g002: M.tuberculosis-infected macrophage supernatants are toxic to T cells.(A) Jurkat T cells underwent significantly increased cell death in cell-free supernatants of low MOI and high MOI Mtb-infected human primary alveolar macrophages (AMs) (shaded bars; infection + and ++, respectively), compared to those of uninfected AMs (closed bars, infection –). Staurosporine 1 µM was used as a positive control to induce cell death (open bars). Similar effects were observed on PBTL and Jurkat T cells exposed to the cell-free supernatants of uninfected/infected THP-1 macrophages. (B) Jurkat cell death in infected THP-1 macrophage supernatant occurred in a dose-dependent manner; infected macrophage supernatants were diluted to 50% and 20% in uninfected supernatant, reducing the level of T cell killing. (C) Example cropped fluorescence intensity images for Hoechst and PI staining from Jurkats incubated in uninfected and infected AM supernatants. Images were processed using automated INCell image analysis to detect apoptotic condensed and fragmented nuclei (arrow 1 and 2, respectively) and PI-positive cells (arrow head). Data in each panel are from one representative experiment, showing mean percentage cell death ± SEM from incubations performed in triplicate. ** =  p<0.01, *** =  p<0.001, Student’s t test relative to uninfected control.
Mentions: To investigate whether soluble mediators released during Mtb infection could alter the viability of T cells in a contact-independent manner, we first infected primary human alveolar macrophages for 48 h with Mtb H37Ra bacilli, then harvested culture supernatants and applied them to Jurkat T cells for 24 h before assessing cell death. In Jurkats incubated in cell-free supernatants from uninfected AMs, 5.85±0.13% cell death was observed, whereas in supernatants from low and high MOI Mtb-infected AMs, this increased significantly (p<0.001) to 18.12±0.24% and 21.13±0.25%, respectively. 1 µM Staurosporine used as a positive cell-killing control caused 67.33±0.85% death (fig. 2A). In this, and subsequent experiments, an automated image analysis strategy (figure S1) was used to clearly identify apoptotic cells that had condensed/fragmented nuclei and/or PI staining, and discriminate them from live cells (fig. 2C).

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