The A subunit of type IIb enterotoxin (LT-IIb) suppresses the proinflammatory potential of the B subunit and its ability to recruit and interact with TLR2

The type IIb heat-labile enterotoxin of Escherichia coli (LT-IIb) and its nontoxic pentameric B subunit (LT-IIb-B₅) display different immunemodulatory activities, the mechanisms of which are poorly understood. We investigated mechanisms whereby the absence of the catalytically active A subunit from LT-IIb-B₅ renders this molecule immunostimulatory through TLR2. LT-IIb-B₅, but not LT-IIb, induced TLR2-mediated NF-κB activation and TNF-α production. These LT-IIb-B₅ activities were antagonized by LT-IIb; however, inhibitors of adenylate cyclase or protein kinase A reversed this antagonism. The LT-IIb antagonistic effect is thus likely dependent upon the catalytic activity of its A subunit, which causes elevation of intracellular cAMP and activates cAMP-dependent protein kinase A. Consistent with this, a membrane-permeable cAMP analog and a cAMP-elevating agonist, but not catalytically defective point mutants of LT-IIb, mimicked the antagonistic action of wild-type LT-IIb. The mutants moreover displayed increased proinflammatory activity compared with wild-type LT-IIb. Additional mechanisms for the divergent effects on TLR2 activation by LT-IIb and LT-IIb-B₅ were suggested by findings that the latter was significantly stronger in inducing lipid raft recruitment of TLR2 and interacting with this receptor. The selective use of TLR2 by LT-IIb-B ₅ was confirmed in an assay for IL-10, which is inducible by both LT-IIb and LT-IIb-B₅ at comparable levels; TLR2-deficient macrophages failed to induce IL-10 in response to LT-IIb-B₅ but not in response to LT-IIb. These differential immunomodulatory effects by LT-IIb and LT-IIb-B₅ have important implications for adjuvant development and, furthermore, snggest that enterotoxic E. coli may suppress TLR-mediated innate immunity through the action of the enterotoxin A subunit.