An Enzymatically Active A Domain Is Required for Cholera-Like Enterotoxins To Induce a Long-Lived Blockade on the Induction of Oral Tolerance: New Method for Screening Mucosal Adjuvants

Abstract

The cholera-like enterotoxins (CLETS), cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), are powerful mucosal adjuvants. Here we show that these toxins also induce a long-lived blockade (of at least 6 months) on the induction of oral tolerance when they are coadministered with the antigen ovalbumin. Strikingly, only enzymatically active CLETS induced this blockade on the induction of oral tolerance. In this regard, the enzymatically inactive mutants of CT and LT, CTK63 and LTK63, and their recombinant B pentamers, rCTB and rLTB, failed to block the induction of oral tolerance, demonstrating a stringent requirement for an enzymatically active A domain in this phenomenon. Together with the results of other recent studies, these results indicate that the enzymatic activity of CLETS, most likely cyclic AMP elevation, is responsible for their adjuvant effects. The results of this study also indicate that measuring the ability of putative mucosal adjuvants to block the induction of oral tolerance may be a superior method for measuring mucosal adjuvanticity.Oral tolerance is a state of systemic hyporesponsiveness that occurs after the ingestion of protein antigens and is thought to be important for the maintenance of homeostasis between the immune system and food antigens (33). Systemic challenge with an antigen in Freund's adjuvant following prior oral administration of that antigen results in diminished antibody and cytolytic responses (36). Feeding of a single high dose or multiple low doses of a protein antigen induces oral tolerance to that antigen (33). The mechanism of oral tolerance is poorly understood; however, at least two independent mechanisms appear to be involved. First, feeding of a high dose of antigen induces clonal T-cell anergy or deletion, as evidenced by reduced interleukin-2 production and a lack of T-cell proliferation (8, 27, 37). Second, feeding of multiple low doses of antigen induces suppressor T cells that secrete transforming growth factor ␤ (9, 22). Interestingly, mucosal adjuvants such as the cholera-like enterotoxins (CLETS), cholera toxin (CT), and Escherichia coli heat-labile enterotoxin (LT) block the induction of oral tolerance when coadministered with an antigen. In the present study, we determined whether the blockade on the induction of oral tolerance to antigens induced by CLETS is transient or long-lived and what role the enzymatic activity of the toxins plays in this phenomenon.CT and LT are AB5 enterotoxins consisting of a 27-kDa catalytic A domain anchored in a pentamer of identical 11.7-kDa B subunits (28). The B pentamer of CT (CTB) binds exclusively to GM1 gangliosides on the surfaces of cells, while the B pentamer of LT (LTB) binds to other gangliosides in addition to GM1 (16,20). These toxins exploit the host protein retention and degradation pathways to gain access to the cytoplasm (reviewed in reference 19). In the cytosol, their A1 subunits catalyze the transfer of an ADP-ribose from NAD to stimulatory alpha subunits of G proteins. After ADP ribosylatio...

Cholera Toxin Indirectly Activates Human Monocyte-Derived Dendritic Cells In Vitro through the Production of Soluble Factors, Including Prostaglandin E2and Nitric Oxide

“…In support of this, CT and the related toxins Escherichia coli heat-labile enterotoxin and pertussis toxin were found to induce the maturation of DC (2,3,21). With further study, the elevated cAMP levels induced by these toxins were found to be responsible for DC maturation (2, 3), and their enzymatic activity has been shown to play a dominant role in adjuvanticity (5,6,23,34,37).Prostaglandins, most notably prostaglandin E 2 (PGE 2 ), have gained notoriety for their ability to synergize with other agonists to activate DC (42). Interestingly, CT has been shown to increase the production of PGE 2 in many cell types, including…”

Mucosal Immunization with a Staphylococcus aureus IsdA-Cholera Toxin A2/B Chimera Induces Antigen-Specific Th2-Type Responses in Mice

“…Spleens were mashed through 70-m-pore-size strainers in serum-free DMEMϩ10. Pelleted cells were resuspended in erythrocyte lysis buffer (144 mM NH 4 Cl, 17 mM Tris, pH 7.4, and Pen-Strep) and washed in serum-free DMEMϩ10. Splenocytes in PBS plus 5% BGS were incubated with 5 M carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) for 10 min at 37°C and washed after the reaction was quenched with 10 volumes of cold DMEMϩ10 (43).…”

“…A number of studies have reported that nontoxic CTB by itself can act as an antigen carrier and is highly immunostimulatory (17,27,47). The ability of nontoxic CTB to effectively block oral tolerance in the absence of enzymatic activity from CTA remains controversial, however, and toxigenic CT is clearly a potent adjuvant even in the absence of CTB (1,4,38). Toxigenic CT however is unsuitable for use in humans, and thus there has been much effort to separate the toxigenicity and adjuvanticity of this molecule.…”

Macrophages – Balancing Tolerance and Immunity

“…In the latter role it has been described as an apoptoic cell pattern recognition receptor' and could help terminate proinflammatory responses through activating antiinflammatory pathways [130]. Antiinflammatory macrophage phenotypes are already known to be induced by some microbial-related ligands, including the receptor-binding B unit of cholera toxin [131][132][133], the similar non-toxic AB complex of E. coli [134] excretory/secretory material from live adult filarial parasites [135][136][137] and Gram-negative flagellin [138]. Both extracellular and intracellular infections may elicit innate tolerance.…”