Glycogen Synthase Kinase 3 Activation Is Important for Anthrax Edema Toxin-Induced Dendritic Cell Maturation and Anthrax Toxin Receptor 2 Expression in Macrophages

Abstract

Anthrax edema toxin (ET) is one of two binary toxins produced by Bacillus anthracis that contributes to the virulence of this pathogen. ET is an adenylate cyclase that generates high levels of cyclic AMP (cAMP), causing alterations in multiple host cell signaling pathways. We previously demonstrated that ET increases cell surface expression of the anthrax toxin receptors (ANTXR) in monocyte-derived cells and promotes dendritic cell (DC) migration toward the lymph node-homing chemokine MIP-3␤. In this work, we sought to determine if glycogen synthase kinase 3 (GSK-3) is important for ET-induced modulation of macrophage and DC function. We demonstrate that inhibition of GSK-3 dampens ET-induced maturation and migration processes of monocytederived dendritic cells (MDDCs). Additional studies reveal that the ET-induced expression of ANTXR in macrophages was decreased when GSK-3 activity was disrupted with chemical inhibitors or with small interfering RNA (siRNA) targeting GSK-3. Further examination of the ET induction of ANTXR revealed that a dominant negative form of CREB could block the ET induction of ANTXR, suggesting that CREB or a related family member was involved in the upregulation of ANTXR. Because CREB and GSK-3 activity appeared to be important for ET-induced ANTXR expression, the impact of GSK-3 on ET-induced CREB activity was examined in RAW 264.7 cells possessing a CRE-luciferase reporter. As with ANTXR expression, the ET induction of the CRE reporter was decreased by reducing GSK-3 activity. These studies not only provide insight into host pathways targeted by ET but also shed light on interactions between GSK-3 and CREB pathways in host immune cells.

Anthrax Vaccine Precipitated Induces Edema Toxin-Neutralizing, Edema Factor-Specific Antibodies in Human Recipients

“…While there have been several studies examining ET neutralization with commercially available immunoassay-based cAMP detection reagents, none of these assays generated reproducible data with human serum samples in our hands, possibly due to interference from serum or plasma constituents. To measure ET neutralization reproducibly, we employed a cAMP-inducible luciferase reporter cell line (40). This assay resulted in 95.4% (103/108) and 100% (108/108) of repeated experiments having neutralization values within 12% and 26%, respectively, of the first experiment.…”

“…Cyclic AMP production and ET neutralization were assessed using a RAW 264.7 cAMP response element (CRE) luciferase reporter cell line (40). Cells (100,000/well) were cultured in 96-well plates for 18 h at 37°C.…”

Opening moves: early events in anthrax pathogenesis and their consequences for dissemination

“…Only Voth et al have reported ET to be directly cytotoxic to both zebrafish embryos and RAW264.7 macrophage-like cells [241]. However, ET inhibits cell cycle progression in the J774.1A and RAW264.7 macrophagelike cell line [197,230]. It is unknown if this delay leads to apoptosis or whether it is unique to macrophages.…”

“…LT has been shown to inhibit chemotaxis in PBMC's; however, addition of purified LT with ET also leads to an increase in chemotaxis compared to untreated DCs [193,196]. Additionally, recombinant ET is capable of activating glycogen synthase kinase (GSK) in DCs, which in turn is able to fully activate CREB-mediated transcriptional changes shown by Kim et al to be involved with chemotaxis [194,197]. It is unknown if GSK is involved with CREB activation in either neutrophils or macrophages.…”

“…While AdsA confers a survival advantage for B. anthracis in ex vivo experiments, it is not known if this also occurs in vivo. Further, adenosine signaling to stimulating receptors leads to an increase in cAMP at the membrane, whereas edema toxin is known to increase cAMP concentrations perinuclearly [197,328]. Since cAMP production can have distinct effects based on its cellular location, adenosine stimulated cAMP may have different effects than edema toxin stimulated cAMP [197,330].…”