Date Published: March 6, 2009
Publisher: Public Library of Science
Author(s): Silvia Rossi Paccani, Marisa Benagiano, Nagaja Capitani, Irene Zornetta, Daniel Ladant, Cesare Montecucco, Mario M. D’Elios, Cosima T. Baldari, William Bishai.
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 CD3ζ 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.
Development of an effective humoral immune response is crucially dependent on T cell help. The last step of B cell differentiation, involving immunoglobulin affinity maturation and isotype switching, occurs in peripheral lymphoid organs under the guidance of a specialized CD4+ T cell subset, known as T helper 2 (Th2). These cells provide both soluble (IL-4) and membrane-bound (CD40L) factors essential for terminal differentiation of antigen specific B cells . Th2 cells are characterized by expression of a unique complement of cytokines, including IL-4, IL-5, IL-10 and IL-13, which are expressed through a complex transcriptional program involving chromatin remodelling at the Th2 cytokine locus control region and de novo expression of the lineage specific transcription factors c-maf and GATA-3 .
The B. anthracis ET and B. pertussis CyaA adenylate cyclase toxins act as potent suppressors of T cell activation and proliferation in the 10−9–10−6 molar range of concentrations –. In the absence of systemic intoxication, these high concentrations are likely to be reached only locally through accumulation of the toxins at the primary site of infection. However, there are anatomical districts and localized infections (e.g. cutaneous anthrax) where low amount of toxins may be released and might modulate the host immune response. We found that both ET and CyaA are potent promoters of naive CD4+ T cell differentiation to Th2 effectors when used at subnanomolar concentrations (0.1–0.3 nM). Interestingly, distinct effects of high vs low concentrations of CyaA have also been observed in neutrophils and other phagocytes, ranging from cytolysis to apoptosis to impairment of effector functions , suggesting the biological outcome of host cell exposure to the toxin is likely to be dictated by its proximity to the bacterium. The sensitivity of T cells to such low ET concentration can be accounted for by the fact that human leukocytes express the high affinity CMG2 receptor for protective antigen (PA), the receptor binding subunit of ET . Furthermore, although T cells lack CD11b/CD18, the only known CyaA receptor, CyaA can effectively insert into cell membranes or artificial lipid bilayers in the absence of CD11b/CD18, albeit with a reduced efficacy . The presence on T cells of a putative alternative CyaA receptor cannot however be ruled out.