Bordetella Adenylate Cyclase Toxin Inhibits Monocyte-to-Macrophage Transition and Dedifferentiates Human Alveolar Macrophages into Monocyte-like Cells

Abstract

Monocytes arriving at the site of infection differentiate into functional effector macrophages to replenish the resident sentinel cells.Bordetella pertussis, the pertussis agent, secretes an adenylate cyclase toxin-hemolysin (CyaA) that binds myeloid phagocytes through complement receptor 3 (CD11b/CD18) and swiftly delivers its adenylyl cyclase enzyme domain into phagocytes. This ablates the bactericidal capacities of phagocytes through massive and unregulated conversion of cytosolic ATP into the key signaling molecule cAMP. We show that exposure of primary human monocytes to as low a concentration as 22.5 pM CyaA, or a low (2:1) multiplicity of infection by CyaA-producingB. pertussisbacteria, blocks macrophage colony-stimulating factor (M-CSF)-driven differentiation of monocytes. CyaA-induced cAMP signaling mediated through the activity of protein kinase A (PKA) efficiently blocked expression of macrophage markers, and the monocytes exposed to 22.5 pM CyaA failed to acquire the characteristic intracellular complexity of mature macrophage cells. Neither M-CSF-induced endoplasmic reticulum (ER) expansion nor accumulation of Golgi bodies, mitochondria, or lysosomes was observed in toxin-exposed monocytes, which remained small and poorly phagocytic and lacked pseudopodia. Exposure to 22.5 pM CyaA toxin provoked loss of macrophage marker expression onin vitrodifferentiated macrophages, as well as on primary human alveolar macrophages, which appeared to dedifferentiate into monocyte-like cells with upregulated CD14 levels. This is the first report that terminally differentiated tissue-resident macrophage cells can be dedifferentiatedin vitro. The results suggest that blocking of monocyte-to-macrophage transition and/or dedifferentiation of the sentinel cells of innate immunity through cAMP-elevating toxin action may represent a novel immune evasion strategy of bacterial pathogens.IMPORTANCEMacrophages are key sentinel cells of the immune system, and, as such, they are targeted by the toxins produced by the pertussis agentBordetella pertussis. The adenylate cyclase toxin (CyaA) mediates immune evasion ofB. pertussisby suspending the bactericidal activities of myeloid phagocytes. We reveal a novel mechanism of potential subversion of host immunity, where CyaA at very low (22 pM) concentrations could inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. Furthermore, exposure to low CyaA amounts has been shown to trigger dedifferentiation of mature primary human alveolar macrophages back into monocyte-like cells. This unprecedented capacity is likely to promote survival of the pathogen in the airways, both by preventing maturation of monocytes attracted to the site of infection into phagocytic macrophages and by dedifferentiation of the already airway-resident sentinel cells.

Adenylate Cyclase Toxin Tinkering With Monocyte-Macrophage Differentiation

“…In contrast, the more phagocytic and highly bactericidal mature macrophages are capable of self-renewal ( 6 ). We observed that exposure of monocytes to CyaA/cAMP signaling blocked M-CSF-driven formation of intracellular granules and prevented development of larger Golgi bodies and the formation of an expanded ER network typical for the mature macrophage cells ( 6 , 42 – 44 ). Hence, CyaA/cAMP-elicited signaling restricted development of organelles required for enhanced synthesis and secretion of cytokines and providing membranes for formation of phagosomes ( 45 – 47 ).…”

“…and prevented development of larger Golgi bodies and the formation of an expanded ER network typical for the mature macrophage cells (6,(42)(43)(44). Hence, CyaA/cAMP-elicited signaling restricted development of organelles required for enhanced synthesis and secretion of cytokines and providing membranes for formation of phagosomes (45)(46)(47).…”

“…The mechanism by which CyaA-elicited cAMP signaling through the PKA-regulated pathways blocks M-CSF-triggered differentiation into macrophages requires further investigation. A plausible hypothesis would be that cAMP signaling inhibits the expression of the receptor for IL-2 (IL-2R) and thus reduced IL-2 signaling downregulates expression of the M-CSF receptor, making the CyaA-exposed monocytes insensitive to pro-differentiating signals (42,48,49). Moreover, we observed that cAMP signaling of CyaA also provoked de-differentiation of mature terminally differentiated tissue-resident human alveolar macrophages back into monocyte-like cells (42).…”

“…Due to its extremely high catalytic activity, CyaA at even very low amounts generates enough intracellular cAMP to elicit a subversive “signaling storm” in host phagocytes through activation of the protein kinase A (PKA)-directed pathways. Exposure to as little as 22.5 pM (4 ng/mL) CyaA provokes a complete inhibition of the M-CSF-driven transition of human monocytes into macrophages without affecting their viability and toxin concentrations over 50 pM (10 ng/mL) trigger apoptosis of human monocyte/macrophage cells ( 40 – 42 ).…”

Pertussis Toxin Ameliorates Microglial Activation Associated With Ischemic Stroke

“…In our study, we use 50 ng/ml of PTX to treat the culture microglia, which did not change the cells into small and rounded macrophages; the microglia showed a similar morphology as control, suggesting that PTX, at least at this concentration of 50 ng/ml, is not toxic to microglia, and it can inhibit the LPS effect to microglia. In another article (Ahmad et al, 2019), at 2.75 ng/ml, PTX can inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. It is similar to our data.…”