Requirement for Cyclic AMP/Protein Kinase A-Dependent Canonical NFκB Signaling in the Adjuvant Action of Cholera Toxin and Its Non-toxic Derivative mmCT.

Manuela Terrinoni,Maximilian Larena,Jan Holmgren,Michael Lebens

doi:10.3389/fimmu.2019.00269

Manuela Terrinoni, Maximilian Larena + Show 2 more

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https://doi.org/10.3389/fimmu.2019.00269

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Abstract

Cholera toxin (CT) is widely used as an effective adjuvant in experimental immunology for inducing mucosal immune responses; yet its mechanisms of adjuvant action remain incompletely defined. Here, we demonstrate that mice lacking NFκB, compared to wild-type (WT) mice, had a 90% reduction in their systemic and mucosal immune responses to oral immunization with a model protein antigen [Ovalbumin (OVA)] given together with CT. Further, NFκB−/− mouse dendritic cells (DCs) stimulated in vitro with CT showed reduced expression of MHCII and co-stimulatory molecules, such as CD80 and CD86, as well as of IL-1β, and other pro-inflammatory cytokines compared to WT DCs. Using a human monocyte cell line THP1 with an NFκB activation reporter system, we show that CT induced NFκB signaling in human monocytes, and that inhibition of the cyclic AMP—protein kinase A (cAMP-PKA) pathway abrogated the activation and nuclear translocation of NFκB. In a human monocyte-CD4+ T cell co-culture system we further show that the strong Th17 response induced by CT treatment of monocytes was abolished by blocking the classical but not the alternative NFκB signaling pathway of monocytes. Our results indicate that activation of classical (canonical) NFκB pathway signaling in antigen-presenting cells (APCs) by CT is important for CT's adjuvant enhancement of Th17 responses. Similar findings were obtained using the almost completely detoxified mmCT mutant protein as adjuvant. Altogether, our results demonstrate that activation of the classical NFκB signal transduction pathway in APCs is important for the adjuvant action of both CT and mmCT.

Highlights

Cholera toxin (CT) is a potent enterotoxin produced by Vibrio cholerae bacteria that, through its action on the intestinal epithelium in infected individuals, can cause the severe, often life-threatening diarrhea and fluid loss characteristic of cholera disease [1]
Previous work by numerous groups has shown that CT promotes both cellular and humoral immune responses via its action mainly on antigen-presenting cells (APCs) in which it activates intracellular cyclic AMP—protein kinase A—and inflammasome-dependent pathways associated with expression, maturation, and release of IL-1β [5,6,7,8,9,10,11,12,13]
Transcriptomic analyses of BMDCs from WT mice exposed for different time periods to either OVA plus CT or for comparisons to OVA alone demonstrated that the transcripts for a large number of cytokines and other immunological activation markers were strongly upregulated by CT (Supplementary Figure S1)

Summary

Introduction

Cholera toxin (CT) is a potent enterotoxin produced by Vibrio cholerae bacteria that, through its action on the intestinal epithelium in infected individuals, can cause the severe, often life-threatening diarrhea and fluid loss characteristic of cholera disease [1]. Previous work by numerous groups has shown that CT promotes both cellular and humoral immune responses via its action mainly on antigen-presenting cells (APCs) in which it activates intracellular cyclic AMP—protein kinase A (cAMPPKA)—and inflammasome-dependent pathways associated with expression, maturation, and release of IL-1β [5,6,7,8,9,10,11,12,13] This in turn indirectly, enhances both humoral and effector T cell responses [5, 13,14,15,16] and promotes Th17 as well as, Th2 and Th1 responses, the latter being more pronounced in mice than in humans. IL-1β is an important pro-inflammatory cytokine known to be induced via NFκB signaling by various well-established adjuvants, such as lipopolysaccharide (LPS), aluminum hydroxide, and saponins [17,18,19]

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