CONICET | Buscador de Institutos y Recursos Humanos

Previous studies have shown that in the absence of opsonic antibodies B. parapertussis (Bpp) largely avoids neutrophil uptake. Still, neutrophils can control extracellular bacteria through non-phagocytic bactericidal mechanisms known as degranulation and neutrophil extracellular traps (NETs) that kill and/or immobilize extracellular bacteria. In this study we investigated whether non-phagocytosed Bpp is inactivated by any of these extracellular bactericidal mechanisms upon neutrophil encounter. β-glucuronidase activity in the supernatant of infected neutrophils was used as degranulation marker. Our results showed that Bpp inhibits degranulation during the innate interaction with neutrophils. Next, by mean of DNA labeling with propidium iodide, antibodies against NET-associated proteins, and fluorescence microscopy we observed that Bpp can inhibit NETs formation. However, since NETs can be prompted by the simultaneous presence of cytokines and bacteria, we next investigated whether Bpp can preclude NETs formation in a proinflammatory environment. Fluorescence microscopy analyses revealed that Bpp prevents NETs formation in IL-8 pre-primed neutrophils. NETs can also be induced by a cytokine combination (i.e.TNF-α+IL-8) that leads to NADPH oxidase (NOX) activation. To examine whether Bpp is also able to interfere with NOX-dependent NETs induction we used phorbol-myristate-acetate, PMA, a well-known NET inductor by NOX activation. Fluorescence microscopy showed that Bpp prevents PMA-induced NETs formation. Additional luminometry assays showed that this PMA-induced NETs prevention occurs through Bpp virulence factors mediated inhibition of the oxidative burst. These results suggest that Bpp subverts most of the innate bactericidal mechanism of neutrophils eventually precluding the efficient clearance of bacteria in non-immune hosts.