Diminished neutrophil extracellular trap (NET) formation is a novel innate immune deficiency induced by acute ethanol exposure in polymicrobial sepsis, which can be rescued by CXCL1
Fig 4
Alcohol decreases NETosis and NET-mediated bacterial killing in mouse bone marrow-derived neutrophils in response to S. aureus infection.
A, Mouse bone marrow-derived neutrophils treated with 25 or 250 mM alcohol exhibit diminished NETosis in response to Gram-positive bacterial (S. aureus) infection. Mouse neutrophils harvested from C57BL/6 mice were pretreated with either 25 or 250 mM alcohol before infection with S. aureus (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, Twenty images in a random manner were selected from three experiments and quantified for the presence of NET-positive neutrophils. C, Kinetics of NETosis by S. aureus-infected mouse neutrophils treated with alcohol. Mouse neutrophils were pretreated with either 25 mM or 250 mM alcohol and infected with S. aureus 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. D, Assess NETosis by SEM. Mouse neutrophils were pretreated with either 25 or 250 mM alcohol before infection with S. aureus and incubation for 8 h to allow for NETosis. E, Alcohol-treated mouse bone marrow neutrophils show diminished NET-mediated killing activity. Bacterial killing capacity of S. aureus-infected, alcohol-treated and untreated bone marrow-derived neutrophils was determined by assessing extracellular (CFUs) at 8 h post-infection with S. aureus (MOI 1) in the DNase treated and untreated groups. Experiments A-E were performed in triplicate and 4–5 mice/group were used. *, p<0.05. NET forming neutrophils are indicated by the arrows on merged images and original magnification 20x. SEM original magnification 8000x.