Alcohol decreases NETosis and NET-mediated bacterial killing in mouse bone marrow-derived neutrophils in response to <i>S</i>. <i>aureus</i> infection.

<p><b>A</b>, Mouse bone marrow-derived neutrophils treated with 25 or 250 mM alcohol exhibit diminished NETosis in response to Gram-positive bacterial (<i>S</i>. <i>aureus</i>) infection. Mouse neutrophils harvested from C57BL/6 mice were pretreated with either 25 or 250 mM alcohol before infection with <i>S</i>. <i>aureus</i> (MOI 1), added SYTOX Green, monitored NETosis, and then fixed after 8 h. Neutrophils were stained with citrullinated H3 Ab to visualize citrullinated histones after the cells were fixed with 4% paraformaldehyde. Double-positive cells are indicated by arrowheads as evidence of NETosis. <b>B</b>, Twenty images in a random manner were selected from three experiments and quantified for the presence of NET-positive neutrophils. <b>C</b>, Kinetics of NETosis by <i>S</i>. <i>aureus</i>-infected mouse neutrophils treated with alcohol. Mouse neutrophils were pretreated with either 25 mM or 250 mM alcohol and infected with <i>S</i>. <i>aureus</i> and stained with SYTOX Green DNA stain to visualize NET formation every hour over a 8 h period. Relative fluorescent intensity (RFU) was determined to determine NETosis. Significant differences between infected and alcohol-treated (25 mM alcohol) are indicated by asterisks. <b>D</b>, Assess NETosis by SEM. Mouse neutrophils were pretreated with either 25 or 250 mM alcohol before infection with <i>S</i>. <i>aureus</i> and incubation for 8 h to allow for NETosis. <b>E</b>, Alcohol-treated mouse bone marrow neutrophils show diminished NET-mediated killing activity. Bacterial killing capacity of <i>S</i>. <i>aureus</i>-infected, alcohol-treated and untreated bone marrow-derived neutrophils was determined by assessing extracellular (CFUs) at 8 h post-infection with <i>S</i>. <i>aureus</i> (MOI 1) in the DNase treated and untreated groups. Experiments A-E were performed in triplicate and 4–5 mice/group were used. *, <i>p</i><0.05. NET forming neutrophils are indicated by the arrows on merged images and original magnification 20x. SEM original magnification 8000x.