UC Irvine

Neutrophil extracellular trap (NET) formation requires chromatin decondensation before nuclear swelling and eventual extracellular release of DNA, which occurs together with nuclear and cytoplasmic antimicrobial proteins. A key mediator of chromatin decondensation is protein deiminase 4 (PAD4), which catalyzes histone citrullination. In the current study, we examined the role of PAD4 and NETosis following activation of neutrophils by A. fumigatus hyphal extract or cell wall β-glucan (curdlan) and found that both induced NET release by human and murine neutrophils. Also, using blocking antibodies to CR3 and Dectin-1 together with CR3-deficient CD18^-/- and Dectin-1^-/- murine neutrophils, we found that the β-glucan receptor CR3, but not Dectin-1, was required for NET formation. NETosis was also dependent on NADPH oxidase production of reactive oxygen species (ROS). Using an antibody to citrullinated histone 3 (H3Cit) as an indicator of PAD4 activity, we show that β-glucan stimulated NETosis occurs in neutrophils from C57BL/6, but not PAD4^-/- mice. Similarly, a small molecule PAD4 inhibitor (GSK484) blocked NET formation by human neutrophils. Despite these observations, the ability of PAD4^-/- neutrophils to release calprotectin and kill A. fumigatus hyphae was not significantly different from C57BL/6 neutrophils, whereas CD18^-/- neutrophils exhibited an impaired ability to perform both functions. We also detected H3Cit in A. fumigatus infected C57BL/6, but not PAD4^-/- corneas; however, we found no difference between C57BL/6 and PAD4^-/- mice in either corneal disease or hyphal killing. Taken together, these findings lead us to conclude that although PAD4 together with CR3-mediated ROS production is required for NET formation in response to A. fumigatus, PAD4-dependent NETosis is not required for A. fumigatus killing either in vitro or during infection.