Fig. 4.

Influenza virus particles are trapped and inactivated by NETs released via suicidal NETosis. (A) Primary human neutrophils were stimulated with PMA, IgG–IAV, or IgA–IAV ICs for 0.5, 1.5, 3, or 6 h prior to fixation and staining for DNA (DAPI) and neutrophil elastase to quantify NETosis. (B) Primary human neutrophils were incubated with DPI, a NOX inhibitor, prior to 3 h of simulation with IgA–IAV ICs (n = 3). (C) Neutrophils were stimulated with PMA for 90 min before the addition 10⁵ PFU per well of IAV. The virus was incubated with the NETs for 3 h and then fixed and stained with anti-hemagglutinin antibodies (6F12), DNA (Hoechst), and neutrophil elastase. Immunofluorescence microscopy was used to measure the colocalization of viral particles (green) with NETs composed of DNA (blue) coated with neutrophil elastase (red) (n = 3) (D) Quantification of the raw integrated GFP density was measured using ImageJ and normalized to the number of cells per field. (E) Neutrophils were stimulated with PMA prior to the addition of an IAV expressing an mNeon reporter. The virus was incubated on intact NETs or NETs that had been digested with DNase (n = 3). The contents of wells were collected, and MDCK cells were infected for 8 h to measure residual infectivity. Mean and ± SEM are shown. Statistical significance was evaluated using one-way ANOVA with Tukey post hoc test. *P < 0.05; **P < 0.01.