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The adenylate cyclase toxins of Bacillus anthracis and Bordetella pertussis promote Th2 cell development by shaping T cell antigen receptor signaling.

Rossi Paccani S, Benagiano M, Capitani N, Zornetta I, Ladant D, Montecucco C, D'Elios MM, Baldari CT - PLoS Pathog. (2009)

Bottom Line: This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3zeta phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation.These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4(+) T cells.Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins.

View Article: PubMed Central - PubMed

Affiliation: Department of Evolutionary Biology, University of Siena, Siena, Italy.

ABSTRACT
The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2-driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4(+) T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2-polarizing concentrations of ET and CyaA selectively inhibit TCR-dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR-signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3zeta phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4(+) T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins.

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CyaA and ET promote Th2 cell differentiation through their cAMP elevating activity.Enriched CD4+ T cells from 6 healthy donors were primed with anti-CD3 mAb, as such or following pretreatment for 2 h with 0.28 nM CyaA/CyaA-E5, or with 0.11 nM ET/EL1. Cells primed in Th2- (IL-4) or Th1- (IL-12) inducing conditions were included as controls. After 10 days cells were washed and restimulated with anti-CD3 mAb for 48 and 24 h respectively, and the levels of IL-4 and IL-13 (A) and IFN-γ and TNF-α (B) were quantified by ELISPOT. The results, obtained on duplicate samples, are expressed as % spot-forming units by CyaA or ET treated cells compared to control cells primed in the absence of either toxin (taken as 100%). ***P≤0.0001; **P≤0.001; *P≤0.01. Error bars, SD.
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ppat-1000325-g003: CyaA and ET promote Th2 cell differentiation through their cAMP elevating activity.Enriched CD4+ T cells from 6 healthy donors were primed with anti-CD3 mAb, as such or following pretreatment for 2 h with 0.28 nM CyaA/CyaA-E5, or with 0.11 nM ET/EL1. Cells primed in Th2- (IL-4) or Th1- (IL-12) inducing conditions were included as controls. After 10 days cells were washed and restimulated with anti-CD3 mAb for 48 and 24 h respectively, and the levels of IL-4 and IL-13 (A) and IFN-γ and TNF-α (B) were quantified by ELISPOT. The results, obtained on duplicate samples, are expressed as % spot-forming units by CyaA or ET treated cells compared to control cells primed in the absence of either toxin (taken as 100%). ***P≤0.0001; **P≤0.001; *P≤0.01. Error bars, SD.

Mentions: To assess the impact of low concentrations of ET and CyaA on human helper T cell polarization, enriched human CD4+ T cells from healthy donors were exposed to ET or CyaA and subsequently primed by TCR/CD3 cross-linking using immobilized anti-CD3 mAb. After 10 days, cells were washed, and restimulated for 24 h or 48 h using the same anti-CD3 mAb. The identity of the Th subset into which cells had differentiated was determined by ELISPOT analysis of cytokine production. As shown in Figure 3A, priming of cells that had been exposed to low concentrations of ET or CyaA resulted in a dramatic increase in production of the Th2 cytokines, IL-4 and IL-13, to levels close to those measured in cells primed to differentiate to the Th2 subset (TCR/CD3 cross-linking in the presence of IL-4). Conversely, consistent with the mutual antagonism of the Th1/Th2 differentiation programs, ET or CyaA had a modest inhibitory effect on production of the Th1 cytokines IFNγ and TNF-α (Figure 3B). No significant enhancement in Th2 cytokines above the levels produced by T cells primed in neutral conditions (TCR/CD3 cross-linking alone) was observed when cells were pretreated with the adenylate cyclase defective EL1 or CyaA-E5 mutants (Figure 3A), indicating that the Th2 driving activity of ET and CyaA is dependent on their capacity to produce cAMP.


The adenylate cyclase toxins of Bacillus anthracis and Bordetella pertussis promote Th2 cell development by shaping T cell antigen receptor signaling.

Rossi Paccani S, Benagiano M, Capitani N, Zornetta I, Ladant D, Montecucco C, D'Elios MM, Baldari CT - PLoS Pathog. (2009)

CyaA and ET promote Th2 cell differentiation through their cAMP elevating activity.Enriched CD4+ T cells from 6 healthy donors were primed with anti-CD3 mAb, as such or following pretreatment for 2 h with 0.28 nM CyaA/CyaA-E5, or with 0.11 nM ET/EL1. Cells primed in Th2- (IL-4) or Th1- (IL-12) inducing conditions were included as controls. After 10 days cells were washed and restimulated with anti-CD3 mAb for 48 and 24 h respectively, and the levels of IL-4 and IL-13 (A) and IFN-γ and TNF-α (B) were quantified by ELISPOT. The results, obtained on duplicate samples, are expressed as % spot-forming units by CyaA or ET treated cells compared to control cells primed in the absence of either toxin (taken as 100%). ***P≤0.0001; **P≤0.001; *P≤0.01. Error bars, SD.
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Related In: Results  -  Collection

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ppat-1000325-g003: CyaA and ET promote Th2 cell differentiation through their cAMP elevating activity.Enriched CD4+ T cells from 6 healthy donors were primed with anti-CD3 mAb, as such or following pretreatment for 2 h with 0.28 nM CyaA/CyaA-E5, or with 0.11 nM ET/EL1. Cells primed in Th2- (IL-4) or Th1- (IL-12) inducing conditions were included as controls. After 10 days cells were washed and restimulated with anti-CD3 mAb for 48 and 24 h respectively, and the levels of IL-4 and IL-13 (A) and IFN-γ and TNF-α (B) were quantified by ELISPOT. The results, obtained on duplicate samples, are expressed as % spot-forming units by CyaA or ET treated cells compared to control cells primed in the absence of either toxin (taken as 100%). ***P≤0.0001; **P≤0.001; *P≤0.01. Error bars, SD.
Mentions: To assess the impact of low concentrations of ET and CyaA on human helper T cell polarization, enriched human CD4+ T cells from healthy donors were exposed to ET or CyaA and subsequently primed by TCR/CD3 cross-linking using immobilized anti-CD3 mAb. After 10 days, cells were washed, and restimulated for 24 h or 48 h using the same anti-CD3 mAb. The identity of the Th subset into which cells had differentiated was determined by ELISPOT analysis of cytokine production. As shown in Figure 3A, priming of cells that had been exposed to low concentrations of ET or CyaA resulted in a dramatic increase in production of the Th2 cytokines, IL-4 and IL-13, to levels close to those measured in cells primed to differentiate to the Th2 subset (TCR/CD3 cross-linking in the presence of IL-4). Conversely, consistent with the mutual antagonism of the Th1/Th2 differentiation programs, ET or CyaA had a modest inhibitory effect on production of the Th1 cytokines IFNγ and TNF-α (Figure 3B). No significant enhancement in Th2 cytokines above the levels produced by T cells primed in neutral conditions (TCR/CD3 cross-linking alone) was observed when cells were pretreated with the adenylate cyclase defective EL1 or CyaA-E5 mutants (Figure 3A), indicating that the Th2 driving activity of ET and CyaA is dependent on their capacity to produce cAMP.

Bottom Line: This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3zeta phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation.These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4(+) T cells.Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins.

View Article: PubMed Central - PubMed

Affiliation: Department of Evolutionary Biology, University of Siena, Siena, Italy.

ABSTRACT
The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2-driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4(+) T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2-polarizing concentrations of ET and CyaA selectively inhibit TCR-dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR-signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3zeta phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4(+) T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins.

Show MeSH
Related in: MedlinePlus