nNIF does not inhibit macrophage pyroptosis or phagocytosis but does reduce extracellular bacterial killing. (A-D) J774 cells were preincubated with medium alone, nNIF (1 nM), or nNIF-scramble (1 nM) for 1 hour before LPS priming. (A) Representative live cell images of J774 cells after LPS (100 ng/mL, 4 hours) and ATP (3 mM, 30 minutes) stimulation obtained at 60× original magnification with confocal microscopy (cell membrane, magenta; cell nucleus, cyan; activated caspase-1, green). Yellow arrows point to cells undergoing pyroptosis. (B) Cell surface area of J774 cells was measured using Image J. Five images obtained at 60× original magnification were used to determine the mean for each group. Cell surface area is shown on the y-axis (mean ± SEM), with each treatment group shown on the x-axis. (C) Active caspase-1 fluorescence intensity of J774 cells measured using Image J. Five images obtained at 60× original magnification were used to determine the mean for each group. Fluorescence intensity is shown on the y-axis (mean ± SEM), with each treatment group shown on the x-axis. (D) Supernatant IL-1β concentration in J774 cells after LPS/ATP stimulation. Supernatant IL-1β concentration is shown on the y-axis (mean ± SEM), with each treatment group shown on the x-axis. (E-F) J774 cells were preincubated with medium alone, nNIF (1 nM), or nNIF-scramble (1 nM) for 1-hour before incubation with E coli bioparticles. (E) Representative live cell images of J774 cells after incubation with E coli bioparticles (MOI 5, 4 hours) obtained at 60× original magnification with confocal microscopy (cell membrane, purple; cell nucleus, cyan; E coli bioparticles, green). (F) Phagocytic index (PI) of J774 cells and E coli bioparticles calculated using confocal images. PI = ([percent of cells containing ≥1 particle] × [mean number of particles/cells containing particles]). PI is shown on the y-axis (mean ± SEM), with each treatment group shown on the x-axis. (G) J774 cells were preincubated in medium alone, nNIF (1 nM), or nNIF-scramble (1 nM) for 1 hour before incubation with E coli. Total bacterial cell killing in J774 cells after incubation with a pathogenic strain of E coli (MOI 2, 3 hours). One group of J774 cells was preincubated with DNase I (40 U/mL, 10 minutes) to inhibit MET-mediated bacterial killing. Percent bacterial killing compared to an untreated control group is shown on the y-axis (mean ± SEM), with each treatment group shown on the x-axis. The blue dashed line represents bacterial killing in untreated cells. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. MFI, mean fluorescence intensity.