Cytolethal distending toxin inhibits macrophage phagocytosis and subverts cytokine production

Objective: this study evaluated the effect of Cdt of A. actinomycetemcomitans (AaCdt) on macrophages phagocytosis and cytokines and nitric oxide (NO) production.

Method: A. actinomycetemcomitans D7S-SA strain (wild type) or its Cdt-deficient mutant D7S-SA CHE001 was co-cultured with Raw 264.7 macrophages. To restore Cdt activity, recombinant AaCdtABC (rCdtABC) was added to Raw 264.7 macrophage co-cultures along with the Cdt deficient strain. Intracellular bacteria levels were accessed by viable counts and by qPCR. Levels of NO and cytokines were determined in culture supernatants. The effects of rCdtABC on cell viability, NO production, cell proliferation and cell cycle were also evaluated. 

Result: intracellular viable and total bacteria levels were lower for Cdt producing A. actinomycetemcomitans wild type strain (D7S-SA) than for the Cdt deficient. The addition of rCdtABC to the co-culture with the Cdt deficient strain leads to the phagocytic activity similar to levels of the wild type. NO production was modulated by the presence of Cdt, although the production of the cytokines IL-1β, IL-12 and IL-10 were increased, and the production of TNF-α was not affected. Moreover, the different concentrations of rCdtABC did not lead to cell cycle arrest, despite about 25% of cells were dead. 

Conclusion: these data suggest that Cdt could regulate macrophage function in the infected sites, by impairing phagocytosis and modifying pro-inflammatory/anti-inflammatory cytokine balance.