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Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy.

Joshi SK, Lang GA, Larabee JL, Devera TS, Aye LM, Shah HB, Ballard JD, Lang ML - PLoS Pathog. (2009)

Bottom Line: In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro.The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression.We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.

ABSTRACT
Exogenous CD1d-binding glycolipid (alpha-Galactosylceramide, alpha-GC) stimulates TCR signaling and activation of type-1 natural killer-like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

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LT inhibits α-GC-stimulated cytokine production.A C57BL/6 mice were treated with 100 µg of LT in PBS by the i.v. route or mock-treated with PBS alone. After 4 d, splenocytes were obtained and stimulated in vitro with α-GC at a final concentration of 50 ng/ml. Supernatants were collected after a further 24 and 48 h and stored at −80°C. B Splenocytes from Jα18−/− mice and ex vivo-expanded NKT cells from C57BL/6 mice were treated in vitro with LT at a final concentration of 1 µg/ml for 1 h before washing and culturing separately or together in the presence or absence of α-GC as described in A. IL-4 and IFNγ concentrations in the supernatants were then determined by Bio-Plex analysis. Data show mean cytokine concentration for 3 mice per group ±SD. Asterisk indicates significant difference between cytokine concentration in un-treated control and samples from LT-treated mice. C C57BL/6 mice were treated with PBS, non-functional LT mutant, or wild type LT before enrichment of NKT cells using anti-NK1.1-based magnetic isolation. Cells were stimulated with anti-CD3 and CD28 mAbs and culture supernatants collected after 48 h.
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ppat-1000588-g004: LT inhibits α-GC-stimulated cytokine production.A C57BL/6 mice were treated with 100 µg of LT in PBS by the i.v. route or mock-treated with PBS alone. After 4 d, splenocytes were obtained and stimulated in vitro with α-GC at a final concentration of 50 ng/ml. Supernatants were collected after a further 24 and 48 h and stored at −80°C. B Splenocytes from Jα18−/− mice and ex vivo-expanded NKT cells from C57BL/6 mice were treated in vitro with LT at a final concentration of 1 µg/ml for 1 h before washing and culturing separately or together in the presence or absence of α-GC as described in A. IL-4 and IFNγ concentrations in the supernatants were then determined by Bio-Plex analysis. Data show mean cytokine concentration for 3 mice per group ±SD. Asterisk indicates significant difference between cytokine concentration in un-treated control and samples from LT-treated mice. C C57BL/6 mice were treated with PBS, non-functional LT mutant, or wild type LT before enrichment of NKT cells using anti-NK1.1-based magnetic isolation. Cells were stimulated with anti-CD3 and CD28 mAbs and culture supernatants collected after 48 h.

Mentions: LT induced an inactivated but not an apoptotic phenotype. We therefore determined if prior toxin treatment affected TCR-mediated NKT activation (Figure 4). We obtained splenocytes from PBS- and LT-treated mice and stimulated them in vitro with α-GC. We observed that LT-treated mice had a substantially reduced production of IL-4 and IFNγ following toxin treatment (Figure 4A). While this experiment showed that LT treatment reduced the ability of NKT cells to be activated by α-GC, it did not determine whether the LT was acting on APCs in the mixed splenocyte culture or the NKT cells or both. Published reports show that DCs, macrophages, B cells and Th cells can all be targeted by anthrax toxins [16],[17],[18],[26], and thus it is conceivable that the target of LT in these cultures is the APC. To address this issue we separately exposed the NKT cells and the APCs (Jα18−/− splenocytes) to LT, washed the cells and then cultured them separately or together with and without α-GC (Figure 4B). For this approach we obtained splenocytes from Type I (α-GC-reactive) NKT-deficient Jα18−/− mice and in vitro-expanded NKT cells derived from C57BL/6 splenocytes. We treated Jα18−/− splenocytes and NKT cells with PBS or LT before washing and then co-cultured the cells with or without α-GC (Figure 4B). We observed that IL-4 production was not affected, but IFNγ production was inhibited when the NKT cells but not the Jα18−/− splenocytes that were treated with LT. The reason for the lack of inhibition of IL-4 production in Figure 4B is not clear, but may be attributable to the behavior of the in vitro-expanded NKT cells as compared to the whole C57BL/6 splenocyte cultures. It is possible that the Th1/Th2 balance in ex vivo-expanded NKT cells is more readily disrupted following toxin treatment than in NKT cells in freshly harvested mixed splenocytes.


Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy.

Joshi SK, Lang GA, Larabee JL, Devera TS, Aye LM, Shah HB, Ballard JD, Lang ML - PLoS Pathog. (2009)

LT inhibits α-GC-stimulated cytokine production.A C57BL/6 mice were treated with 100 µg of LT in PBS by the i.v. route or mock-treated with PBS alone. After 4 d, splenocytes were obtained and stimulated in vitro with α-GC at a final concentration of 50 ng/ml. Supernatants were collected after a further 24 and 48 h and stored at −80°C. B Splenocytes from Jα18−/− mice and ex vivo-expanded NKT cells from C57BL/6 mice were treated in vitro with LT at a final concentration of 1 µg/ml for 1 h before washing and culturing separately or together in the presence or absence of α-GC as described in A. IL-4 and IFNγ concentrations in the supernatants were then determined by Bio-Plex analysis. Data show mean cytokine concentration for 3 mice per group ±SD. Asterisk indicates significant difference between cytokine concentration in un-treated control and samples from LT-treated mice. C C57BL/6 mice were treated with PBS, non-functional LT mutant, or wild type LT before enrichment of NKT cells using anti-NK1.1-based magnetic isolation. Cells were stimulated with anti-CD3 and CD28 mAbs and culture supernatants collected after 48 h.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2742733&req=5

ppat-1000588-g004: LT inhibits α-GC-stimulated cytokine production.A C57BL/6 mice were treated with 100 µg of LT in PBS by the i.v. route or mock-treated with PBS alone. After 4 d, splenocytes were obtained and stimulated in vitro with α-GC at a final concentration of 50 ng/ml. Supernatants were collected after a further 24 and 48 h and stored at −80°C. B Splenocytes from Jα18−/− mice and ex vivo-expanded NKT cells from C57BL/6 mice were treated in vitro with LT at a final concentration of 1 µg/ml for 1 h before washing and culturing separately or together in the presence or absence of α-GC as described in A. IL-4 and IFNγ concentrations in the supernatants were then determined by Bio-Plex analysis. Data show mean cytokine concentration for 3 mice per group ±SD. Asterisk indicates significant difference between cytokine concentration in un-treated control and samples from LT-treated mice. C C57BL/6 mice were treated with PBS, non-functional LT mutant, or wild type LT before enrichment of NKT cells using anti-NK1.1-based magnetic isolation. Cells were stimulated with anti-CD3 and CD28 mAbs and culture supernatants collected after 48 h.
Mentions: LT induced an inactivated but not an apoptotic phenotype. We therefore determined if prior toxin treatment affected TCR-mediated NKT activation (Figure 4). We obtained splenocytes from PBS- and LT-treated mice and stimulated them in vitro with α-GC. We observed that LT-treated mice had a substantially reduced production of IL-4 and IFNγ following toxin treatment (Figure 4A). While this experiment showed that LT treatment reduced the ability of NKT cells to be activated by α-GC, it did not determine whether the LT was acting on APCs in the mixed splenocyte culture or the NKT cells or both. Published reports show that DCs, macrophages, B cells and Th cells can all be targeted by anthrax toxins [16],[17],[18],[26], and thus it is conceivable that the target of LT in these cultures is the APC. To address this issue we separately exposed the NKT cells and the APCs (Jα18−/− splenocytes) to LT, washed the cells and then cultured them separately or together with and without α-GC (Figure 4B). For this approach we obtained splenocytes from Type I (α-GC-reactive) NKT-deficient Jα18−/− mice and in vitro-expanded NKT cells derived from C57BL/6 splenocytes. We treated Jα18−/− splenocytes and NKT cells with PBS or LT before washing and then co-cultured the cells with or without α-GC (Figure 4B). We observed that IL-4 production was not affected, but IFNγ production was inhibited when the NKT cells but not the Jα18−/− splenocytes that were treated with LT. The reason for the lack of inhibition of IL-4 production in Figure 4B is not clear, but may be attributable to the behavior of the in vitro-expanded NKT cells as compared to the whole C57BL/6 splenocyte cultures. It is possible that the Th1/Th2 balance in ex vivo-expanded NKT cells is more readily disrupted following toxin treatment than in NKT cells in freshly harvested mixed splenocytes.

Bottom Line: In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro.The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression.We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.

ABSTRACT
Exogenous CD1d-binding glycolipid (alpha-Galactosylceramide, alpha-GC) stimulates TCR signaling and activation of type-1 natural killer-like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

Show MeSH
Related in: MedlinePlus