Invasion of Dendritic Cells, Macrophages and Neutrophils by the Bordetella Adenylate Cyclase Toxin: A Subversive Move to Fool Host Immunity

Abstract

Adenylate cyclase toxin (CyaA) is released in the course of B. pertussis infection in the host's respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC), macrophages and neutrophils are professional phagocytes and key players of the innate immune system that provide a first line of defense against invading pathogens. Recent findings revealed the capacity of B. pertussis CyaA to intoxicate DC with high concentrations of 3',5'-cyclic adenosine monophosphate (cAMP), which ultimately skews the host immune response towards the expansion of Th17 cells and regulatory T cells. CyaA-induced cAMP signaling swiftly incapacitates opsonophagocytosis, oxidative burst and NO-mediated killing of bacteria by neutrophils and macrophages. The subversion of host immune responses by CyaA after delivery into DC, macrophages and neutrophils is the subject of this review.

Keywords: T-helper cells; immune response; intracellular pathways; phagocytosis.

Figure 1

Mode of action of adenylate cyclase toxin (CyaA) on phagocyte membrane. CyaA is secreted from the bacterium into the calcium-containing serous fluid on mucosal surfaces through the Type I secretion system (T1SS). It binds calcium ions and co-secretionally folds into a conformation capable to bind the complement receptor 3 (CR3), known as the α_Mβ₂ integrin, CD11b/CD18 or Mac-1, on the surface of myeloid phagocytic cells [49,50,51]. Concentration and positioning of the toxin on the surface of the cell enables its insertion into the lipid bilayer of cellular membrane. The translocation precursor of CyaA generates a path for influx of extracellular calcium ions into cells [52], which leads to activation of calpain-mediated cleavage of talin [52] and mobilization of the CyaA–CR3 complex into lipid rafts [53]. From there the AC domain translocates across cellular membrane into the submembrane region of cells, where signaling complexes including protein kinase A are clustered [53,54]. The AC enzyme binds cytosolic calmodulin and catalyzes unregulated conversion of ATP into the key second messenger molecule 3′,5′-cyclic adenosine monophosphate (cAMP). In parallel, the RTX hemolysin part of CyaA is functionally independent of the invasive AC domain and the pore-forming precursors form oligomeric cation-selective pores that permeabilize cellular membrane for efflux of cytosolic potassium ions from cells [54]. See parallel review by Novak et al. in this issue for further mechanistic details on CyaA action (adapted from Masin et al. [55]).

Figure 2

Hijacking of cellular signaling cascades blocks the bactericidal activities of phagocytes. Elevation of cAMP concentration in the cytosol of CR3-expressing myeloid phagocytes activates a wealth of signaling cascades that converge towards ablation of bactericidal activities of phagocytes, such as oxidative burst and phagocytic uptake and killing of opsonized bacteria. By an as-yet unknown mechanism, CyaA/cAMP signaling inhibits the activity of the Syk kinase and blocks, thereby, CR3 signaling triggered by binding of iC3b-opsonized bacteria to the open (extended) CR3 receptor [59]. In parallel, cAMP activates the protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) signaling pathways, interfering with numerous physiological signaling processes (see parallel in this issue the in-depth review of CyaA-triggered signaling by Novak et al.). In particular, PKA mediates by an as yet undefined mechanism the activation of the Src homology domain 2 containing protein tyrosine phosphatase (SHP) 1 [63]. This provokes inhibition of ERK1/2 and together with Epac-mediated inhibition of phospholipase C (PLC) inhibits the assembly of the NADPH oxidase, production of reactive oxygen species (ROS) and oxidative burst of neutrophils and macrophages [6,61]. In macrophages, the activation of SHP-1 yields dephosphorylation of the c-Fos subunit of the transcription factor AP-1 and blocks expression of TLR-inducible NO synthase (iNOS), which enables the internalized non-opsonized B. pertussis bacteria to evade NO-mediated intracellular killing [45,63]. Lastly, activation of SHP-1, inhibition of the pro-survival kinase Akt/PKB and inhibition of ERK1/2 act together to abrogate degradation of BimEL. Enhanced BimEL levels then activate Bax and trigger apoptosis of macrophages through the mitochondrial pathway [64].

Figure 3

3′,5′-cyclic adenosine monophosphate (cAMP) signaling of CyaA subverts the functions of dendritic cells and fools the induction of T cell immune responses. CyaA/cAMP signaling in CR3-expressing (CD11b⁺) dendritic cells (DC) of the submucosa of the airways triggers an array of counteracting immunomodulatory signaling processes that fool induction of antigen-specific T cell immune responses by TLR-activated DC. cAMP signaling interferes with the proper maturation of DC and downregulates their capacity to process and present antigens to both CD4⁺ and CD8⁺ T cells. On the other hand, cAMP signaling enhances the migratory capacity of potentially tolerogenic DC that are impaired in IL-12 and TNFα secretion, while secreting enhanced amounts of IL-10 and exhibiting reduced expression of the MHC class II molecules and of the CD40 co-receptor. At least in vitro, CyaA-exposed DC can expand antigen-specific CD4⁺CD25⁺Foxp3⁺ T regulatory cells [71,72,73,74,75,76]. In parallel, concurrent cAMP signaling and permeabilization of DC by the CyaA pores and activation of NALP3 inflammasome due to potassium efflux-driven activation of JNK and p38 MAPK contributes secretion of IL-1β and of IL-17 [34]. As a result, the CyaA-hijacked DC are likely to contribute suppression of pro-inflammatory responses and delay activation of adaptive T- and B-cell mediated immunity to B. pertussis infection.

Figure 4

The CyaA toxin of B.pertussis inhibits the TRIF-dependent signaling pathways in human monocyte derived dendritic cells (MDDC). B. pertussis triggers TLR2 and TLR4 signaling in MDDC. In normal conditions, the MyD88-dependent signaling pathway downstream of TLR2 and TLR4 leads to the transcription of pro- and anti-inflammatory cytokines, including the p40 subunit of IL-12p70, while the TRIF-dependent signaling pathway downstream of TLR4 leads to the phosphorylation of interferon regulatory factor (IRF)3 and thereafter to the transcription of IFNβ and, through STAT1 phosphorylation, of IFN-inducible genes, including IRF1 and IRF8. A second wave of gene transcription is then activated, including the p35 subunit of IL-12p70 [77,78,79]. Upon encounter with B. pertussis, cellular intoxication with CyaA blocks the TRIF-dependent pathway in MDDC by inhibiting IRF3 phosphorylation and IFN-β, IRF-1, and IRF-8 expression [74,75].