Cholera Toxin and Heat-Labile Enterotoxin Activate Human Monocyte-Derived Dendritic Cells and Dominantly Inhibit Cytokine Production through a Cyclic AMP-Dependent Pathway

Abstract

Cholera toxin (CT) and heat-labile enterotoxin (LT) are powerful mucosal adjuvants whose cellular targets and mechanism of action are unknown. There is emerging evidence that dendritic cells (DC) are one of the principal cell types that mediate the adjuvant effects of these toxins in vivo. Here we investigate the effects of CT and LT on the maturation of human monocyte-derived DC (MDDC) in vitro. We found that an enzymatically active A domain is necessary for both CT and LT to induce the maturation of MDDC and that this activation is strictly cyclic AMP (cAMP) dependent. ADP-ribosylation-defective derivatives of these toxins failed to induce maturation of MDDC, whereas dibutyryl-cyclic-3,5-AMP and Forskolin mimic the maturation of MDDC induced by CT and LT. In addition, an inhibitor of cAMP-dependent kinases, Rp-8-Br-cAMPs, blocked the ability of CT, LT, and Forskolin to activate MDDC. CT, LT, dibutyryl-cyclic-3,5-AMP, and Forskolin also dominantly inhibit interleukin 12 and tumor necrosis factor alpha production by MDDC in the presence of saturating concentrations of lipopolysaccharide. Taken together, these results show that the effects of CT and LT on MDDC are mediated by cAMP.Cholera toxin (CT) and heat-labile enterotoxin (LT) are AB5 enterotoxins produced by Vibrio cholerae and enteropathic Escherichia coli, the primary causative agents of cholera and traveler's diarrhea, respectively. CT and LT consist of a 27-kDa catalytic A domain anchored in a ring of five identical, 11.7-kDa B subunits (18). The B pentamers of these toxins bind to gangliosides on cell membranes (12). The B pentamer of CT (CTB) binds exclusively to GM1 gangliosides, while the B pentamer of LT (LTB) binds to other gangliosides in addition to GM1 (9). These toxins exploit the host protein retention and degradation pathways to gain access to the cytoplasm, reviewed in reference 11. In the cytosol, their A1 subunits catalyze the transfer of an ADP-ribose from NAD to stimulatory ␣-subunits of G proteins (Gs␣). After ADP-ribosylation, Gs␣ binds to adenylate cyclase and constitutively activates it, leading to a sustained increase in intracellular cyclic AMP (cAMP) concentration (4).CT and LT are also powerful mucosal immunogens and adjuvants, reviewed in reference 14. In mice, antibody responses to CT and bystander antigens can last up to 2 years (15, 26). CT has been shown to induce primarily Th2 responses, characterized by CD4 ϩ T cells producing interleukin 4 (IL-4), IL-5, IL-6, and IL-10 and by the production of immunoglobulin G1 (IgG1), IgA, and IgE antibodies (17,29). By contrast, LT has been reported to induce mixed Th1 and Th2 responses (24). It has been proposed that differences in the ganglioside binding specificities of their B pentamers contribute to their discordant Th1/Th2 patterns (30, 31). Although these toxins are extensively used as adjuvants in animal models, their toxicity makes them unsuitable for human use. For this reason, a number of investigators have attempted to identify nontoxic derivatives of CT and LT that retain ...

Bordetella pertussis Adenylate Cyclase Toxin Modulates Innate and Adaptive Immune Responses: Distinct Roles for Acylation and Enzymatic Activity in Immunomodulation and Cell Death

“…This suggests that increased intracellular cAMP concentrations are involved in modulation of innate immune cell activation by CyaA. This conclusion is consistent with the observations that increases in intracellular cAMP concentration by stimuli such as cholera toxin or forskolin (23,24), lead to suppression of proinflammatory cytokines and chemokines, such as IL-12 and CXCL10 and enhancement of IL-10 and CCL17.…”

“…Likewise, acylation of the RTX domain of CyaA is considered to be necessary for its hemolytic and cytotoxic activity (2,6,12). Previous studies have suggested that acylation is required for efficient adjuvanticity of CyaA, while the increase in intracellular cAMP levels generated by the adenylate cyclase domain is responsible for its immunomodulatory effects (5,6,23). Our data demonstrate that acylation is not essential for CyaA to induce cAMP accumulation in macrophages or to modulate activation of macrophages and DC.…”

Cholera toxin promotes the generation of semi-mature porcine monocyte-derived dendritic cells that are unable to stimulate T cells

“…Ctx causes increased plasma levels of cAMP through the ADP-ribosyltransferase activity of the A-subunit after binding of the B-subunit to the cell surface via the ubiquitous GM1 ganglioside [2,9,10,46]. Consistent with our observations for Ctx, cAMP-elevating agents can directly increase IL-10 production, inhibit TNFα secretion and MHC-II and CD40 expression, and reduce stimulatory capacity in murine DC [25].…”

“…Ctx or Ctx-B-subunit (Sigma, Taufkirchen, Germany) were added to MoDC cultures for 24 to 48 h on day 3 at final concentrations of 1 µg/mL, following previous studies with human and murine DC [2,18,19,22]. To exclude the influence of LPS contamination of the toxin preparation, Ctx detoxified with Detoxi-Gel TM AffinityPak TM Columns (Perbio Science, Bonn, Germany) was used in some experiments.…”

Pharmacological Interference With Protein-protein Interactions of Akinase Anchoring Proteins as a Strategy for the Treatment of Disease

“…AKAPs have also been identified in dendritic cells, the major antigen presenting cells of the immune system [154]. Dendritic cell maturation and activity is regulated, amongst others, by PKA and cAMP [155]. Human dendritic cells express AKAP79, AKAP1, AKAP95, AKAP--Lbc and Ezrin; their expression upon maturation from a monocyte to a mature dendritic cell is differentially regulated: The expression of AKAP95 and Ezrin does not change, but that of AKAP79, AKAP1 and AKAP--Lbc increases and this is accompanied by a 1.8--fold increase of RIIα protein expression.…”