Bordetella pertussis Adenylate Cyclase Toxin Modulates Innate and Adaptive Immune Responses: Distinct Roles for Acylation and Enzymatic Activity in Immunomodulation and Cell Death 1 Aoife P. Boyd, 2,3 Pa ´ draig J. Ross, 2 Helen Conroy, Nicola Mahon, Ed C. Lavelle, and Kingston H. G. Mills 4 Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the repeat in toxin family of pore-forming toxins, which require posttranslational acylation to lyse eukaryotic cells. CyaA modulates dendritic cell (DC) and macrophage function upon stimulation with LPS. In this study, we examined the roles of acylation and enzymatic activity in the immunomodulatory and lytic effects of CyaA. The adenylate cyclase activity of CyaA was necessary for its modulatory effects on murine innate immune cells. In contrast, acylation was not essential for the immunomodulatory function of CyaA, but was required for maximal caspase-3 activation and cytotoxic activity. The wild-type acylated toxin (A-CyaA) and nonacylated CyaA (NA-CyaA), but not CyaA with an inactive adenylate cyclase domain (iAC-CyaA), enhanced TLR-ligand-induced IL-10 and inhibited IL-12, TNF-, and CCL3 production by macrophages and DC. In addition, both A-CyaA and NA-CyaA, but not iAC-CyaA, enhanced surface expression of CD80 and decreased CpG-stimulated CD40 and ICAM-1 expression on immature DC. Furthermore, both A-CyaA and NA-CyaA promoted the induction of murine IgG1 Abs, Th2, and regulatory T cells against coadministered Ags in vivo, whereas iAC-CyaA had more limited adjuvant activity. In contrast, A-CyaA and iAC-CyaA induced caspase-3 activation and cell death in macrophages, but these effects were considerably reduced or absent with NA-CyaA. Our findings demonstrate that the enzymatic activity plays a critical role in the immunomodulatory effects of CyaA, whereas acylation facilitates the induction of apoptosis and cell lysis, and as such, NA-CyaA has considerable potential as a nontoxic therapeutic molecule with potent anti-inflammatory properties. The Journal of Immunology, 2005, 175: 730 –738. B ordetella pertussis, the causative agent of whooping cough, and Bordetella bronchiseptica, which causes chronic respiratory disease in a variety of mammals, both express adenylate cyclase toxin (CyaA) 5 (1). This toxin is encoded by the cyaA gene and is an essential virulence factor for both Bordetella species. CyaA specifically binds to the  M  2 integrin (CD11b/CD18) on the surface of macrophages and dendritic cells (DC) (2, 3). Upon entry into target eukaryotic cells, CyaA is ac- tivated by binding calmodulin and converts ATP into cAMP, gen- erating supraphysiological concentrations of this powerful signal- ing molecule. Treatment of neutrophils with CyaA leads to inhibition of chemotaxis, phagocytosis, and superoxide generation (4). CyaA has also been shown to induce apoptosis in macrophages (5, 6). We and others have previously shown that CyaA of B. pertussis is an effective vaccine adjuvant in vivo, enhancing Ab responses to coadministered Ags (7–10). Our studies also demonstrated that CyaA promotes Th2 and type 1 regulatory T (Tr1) cell responses to coadministered Ags (9). Studies with innate immune cells in vitro indicated that these adaptive immune responses were mediated via the modulation of DC and macrophage activation. Treatment of LPS-stimulated macrophages or murine bone marrow- derived DC with CyaA suppressed production of the proinflam- matory cytokines and chemokines IL-12p70, TNF-, and CCL3 (MIP-1) and enhanced IL-10 production (9). Furthermore, CyaA enhanced surface expression of the costimulatory molecule CD80 on immature DC, but decreased expression of LPS-induced ICAM-1 (CD54) and CD40, indicating that CyaA selectively mod- ulates DC activation and maturation. The enzymatic domain of CyaA is located in the 400 NH 2 - terminal amino acids, while interaction of CyaA with CD11b/ CD18 and with eukaryotic membranes is mediated through the 1306 residue COOH-terminal region of the protein, that has sim- ilarity with the repeat in toxin (RTX) family of bacterial pore- forming toxins (3). This family of toxins requires amide-linked fatty acylation by a toxin-specific accessory protein for function, as originally demonstrated for the Escherichia coli hemolysin, HlyA (11). Likewise, acylation of CyaA by its specific accessory protein, CyaC, is necessary for the toxin to lyse RBC, J774 macrophages, and Jurkat T cells (6, 12). Differences in the acylation pattern have been demonstrated to alter the hemolytic and pore-forming activity of CyaA, but not its capacity to deliver the enzymatic domain into the cell cytosol (13, 14). The channel-forming activity of CyaA Immune Regulation Research Group, Department of Biochemistry, Trinity College, Dublin, Ireland Received for publication December 21, 2004. Accepted for publication May 3, 2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Science Foundation Ireland under Grant No. 00/PI.I/ B045. P.J.R. also receives support from Enterprise Ireland. 2 A.P.B. and P.J.R. contributed equally to this work. 3 Current address: Department of Microbiology, National University of Ireland, Gal- way, Ireland. 4 Address correspondence and reprint requests to Dr. Kingston Mills, Immune Reg- ulation Research Group, Department of Biochemistry, Trinity College, Dublin 2, Ireland. E-mail: kingston.mills@tcd.ie 5 Abbreviations used in this paper: CyaA, adenylate cyclase toxin; RTX, repeat in toxin (protein family); DC, dendritic cell; A-CyaA, acylated CyaA; NA-CyaA, non- acylated CyaA; iAC-CyaA, CyaA with an inactive adenylate cyclase domain; Tr1, type 1 regulatory T; LDH, lactate dehydrogenase; KLH, keyhole limpet hemocyanin; CpG-ODN, oligodeoxynucleotide-containing CpG motif The Journal of Immunology Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00