Fig 1.
Alcohol intoxication augments susceptibility to CLP and impairs cellular infiltration and bacterial clearance.
A, Enhanced mortality of acute alcohol intoxicated mice after CLP. Alcohol intoxicated and control (saline-challenged) mice were subjected to CLP or a sham procedure. Survival of mice was assessed every 12 h until 10 days after CLP. Results are expressed as a percentage of 20 animals per group from 2 separate experiments. Significance between groups was examined by Wilcoxon rank test. B, Impaired bacterial clearance in alcohol-challenged mice. CFUs were examined in blood, peritoneal lavage or homogenates obtained from livers, lungs, spleens and kidneys of alcohol-challenged and control mice at 6 and 24 h post-CLP. (n = 4-8/group). C, Acute alcohol-intoxicated mice showed reduction in total while blood cell count and neutrophil (PMN) and macrophage accumulation in the peritoneum after CLP, as measured by direct cell counts in peritoneal lavage (PL). (n = 4-8/group). D. Peritoneal cell (neutrophil) viability was not different between alcohol- and saline-challenged mice. A total of 1000 cells were examined in peritoneal fluid of each group was enumerated using trypan blue exclusion assay (n = 4-6/group). *, p<0.05, ***, p<0.001.
Fig 2.
Alcohol impairs NETosis in response to CLP.
A, Western blot of PAD-4 and neutrophil elastase expression in peritoneal cells (neutrophils) and peritoneal fluid from alcohol-administered and control (saline-challenged) mice at 24 h post-CLP. n = 3 mice/group from two independent experiments. B. Relative band densities normalized against neutrophil elastase are representative of the blots. C-D, Peritoneal cells (neutrophils) harvested from alcohol-challenged and control (saline-challenged) mice at 24 h post-CLP were washed with saline (0 h), added SYTOX Green and allowed to monitor NETosis for 8 h and then were fixed. Neutrophils were stained with DAPI to stain DNA and citrullinated H3 Ab (α-H3Cit) to visualize citrullinated histone. Double-positive cells are indicated by arrowheads as evidence of NETosis. Images presented are representative of two independent experiments (n = 5-7/group). E, Twenty random images were selected from three experiments and quantified for the presence of double-positive (SYTOX Green and H3Cit) peritoneal cells (neutrophils) from alcohol-administered and control mice. n = 5-8/group. F, Kinetic analysis of NETosis by peritoneal cells (neutrophils) harvested from alcohol-administered and control mice, washed with saline, and NETosis was measured up to 8 h. Relative fluorescence intensity (RFU) was determined to evaluate NETosis each hour up until 8 h of in vitro culture. n = 6-9/group. G, Evaluation of NETosis by SEM. Peritoneal cells (neutrophils) harvested from alcohol-administered and control mice after CLP were analyzed by SEM. Presence of long thread-like structures is evidence of NETosis. Images presented are representative of two independent experiments (n = 5-8/group). *, p<0.05. NET forming peritoneal cells (neutrophils) are indicated by the arrows on merged images and original magnification 20x. SEM original magnification 8000x.
Fig 3.
Alcohol reduces NETosis and NET-mediated bacterial killing in mouse bone marrow-derived neutrophils triggered by E. coli infection.
A, Mouse neutrophils harvested from WT mice were pretreated with either 25 or 250 mM alcohol before infection with E. coli (MOI 1), added SYTOX Green, allowed to undergo NETosis, and then fixed after 8 h. Neutrophils were stained with citrullinated H3 Ab to visualize citrullinated histones in fixed cells. DNA and Citrullinated histones are indicated by arrowheads and double positive cells are considered of NETosis. Images presented are representative of three independent experiments (each in duplicate). B, Twenty random images were selected from three experiments and quantified for the presence of NET-positive neutrophils. C, Kinetic analysis of NETosis by E. coli-infected mouse neutrophils treated with alcohol. Mouse neutrophils were pretreated with either 25 mM or 250 mM alcohol and infected with E. coli and stained with SYTOX Green DNA stain to visualize every hour over a period of 8 h. Relative fluorescent intensity (RFU) was determined to evaluate NETosis. Significant differences between infected and alcohol-treated (25 mM alcohol) are indicated by asterisks. D, Evaluation of NETosis by SEM. Mouse neutrophils were pretreated with either 25 or 250 mM alcohol before infection with E. coli and incubation for 8 h to allow for NETosis. E, Alcohol-treated mouse bone marrow neutrophils exhibited diminished NET-mediated killing activity. Bacterial killing capacity of E. coli-infected, alcohol-treated and untreated bone marrow neutrophils was determined by assessing extracellular (CFUs) at 8 h post-infection with E. coli (MOI 1) in the presence or absence of DNase. For experiments A-E, four to five mice/group were used and cells used in triplicate in 3 separate experiments, *, p<0.05. NET forming neutrophils are indicated by the arrows on merged images and original magnification 20x. SEM original magnification 8000x.
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.
Fig 5.
Attenuation of ROS production in alcohol-challenged mice and in alcohol-treated bone marrow-derived neutrophils in response to bacterial infection.
A, ROS+ neutrophils were identified by fluorescence microscopy after intracellular staining for ROS in peritoneal cells (neutrophils) derived from alcohol-administered and control mice at 24 h post-CLP. Results are representative of a microscopic view of three independent experiments (ROS+ cells are green, ROS− cells are blue). Arrows show ROS producing cells. B, Twenty random images were selected from three experiments and quantified for the presence of ROS-positive (green) neutrophils. (*, p<0.05). C, The production of total ROS in peritoneal fluid of alcohol-administered and control mice at 24 h post-CLP were evaluated as relative fluorescence units (RFUs) and normalized to total peritoneal cells. D. Total ROS production by bone marrow-derived neutrophils stimulated with E. coli were quantified as relative fluorescence units (RFU). (n = 5-8/group; *, p<0.05). E. Western blotting of GP91phox, P47phox, P67phox and GAPDH expression in peritoneal cells (neutrophils) from alcohol-administered and saline-challenged mice at 24 h post-CLP. This is a representative blot of 3 independent experiments with identical results. Original magnification 20x.
Fig 6.
Alcohol impairs extracellular bacterial killing of mouse and human neutrophils.
A-B, Bacterial killing capacity of mouse bone marrow-derived neutrophils (A) and human peripheral blood-derived neutrophils (B) pretreated without or alcohol (25 mM) and infected with an MOI of 1 was determined by assessing extracellular CFUs at 30, 60, and 120 min after infection with E. coli or S. aureus. Experiments were performed in triplicate wells and from three independent experiments and 3–5 mice/group was used. For human neutrophils, 4–5 donors were used per group (*p <0.05 between infected groups in the presence and absence of alcohol).
Fig 7.
rCXCL1 reduces susceptibility to alcohol-challenged CLP mice and augments bacterial clearance and neutrophil recruitment.
A, Enhanced mortality of alcohol intoxicated WT and CXCL1 knockout (KO) mice following CLP. Alcohol intoxicated and control (saline-challenged) mice were subjected to CLP. Survival of mice was assessed every 12 h until 10 days after CLP. Results are expressed as a percentage of 20 animals per group. Significance between groups was examined by Wilcoxon rank test. WSC versus CSC (*, p<0.05), WAC versus CAC (***, p<0.001), WAC versus WSC (***, p<0.001), and CAC versus CSC (***, p<0.001). B, Reduced mortality of alcohol treated WT mice by rCXCL1 following CLP. Alcohol intoxicated WT mice were subjected to CLP followed by rCXCL1 or BSA treatment. Survival of mice was assessed every 12 h until 10 days after CLP. Results are expressed as a percentage of 20 animals per group. Significance between groups was examined by Wilcoxon rank test. **, p<0.01. C, Impaired bacterial clearance in alcohol-challenged mice. CFUs were examined in peritoneal lavage and blood or homogenates obtained from kidneys, livers, lungs, and spleens of alcohol-challenged and control mice at 6 and 24 h post-CLP. (n = 6/group). D, Acute alcohol-intoxicated mice showed reduced total while blood cell count and neutrophil (PMN) and macrophage influx in the peritoneum after CLP, as measured by direct cell counts in peritoneal lavage (PL). (n = 4-8/group). *, p<0.05.
Fig 8.
rCXCL1 enhances NETosis in alcohol-challenged septic mice.
A, Western blotting of PAD-4, H3Cit and GAPDH in peritoneal cells (neutrophils) from alcohol-administered and control mice at 24 h post-CLP after rCXCL1 or BSA administration. This blot is representative of three independent experiments with identical results. B. Relative densities normalized against GAPDH are representative of the blots from 3 separate experiments (n = 3-5/mice/group). C, Peritoneal cells (neutrophils) harvested from alcohol-challenged and control mice after CLP were allowed to undergo NETosis after adding SYTOX Green for 8 h and then were fixed. Neutrophils were stained with citrullinated H3 Ab (α-H3Cit) to visualize citrullinated histone. Double-positive cells are indicated by arrowheads as evidence of NETosis. Images presented are representative of three independent experiments (n = 5-7/group). D, Twenty random images were selected from three experiments and quantified for the presence of NET-positive (double-positive; SYTOX Green- and H3Cit-positive) neutrophils from alcohol-administered and control mice. n = 5-8/group. E, Kinetic analysis of NETosis by peritoneal cells (neutrophils) harvested from alcohol-administered and control mice. Relative fluorescence intensity (RFU) was determined to evaluate NETosis each hour up until 8 h of culture. n = 6-9/group. Significance was calculated between rCXCL1 treated and BSA treated peritoneal cells from mice in the presence or absence of alcohol. F, Evaluation of NETosis by SEM. Peritoneal cells (neutrophils) harvested from alcohol-administered and control mice after CLP in rCXCL1 or BSA administration were analyzed by SEM. Presence of long thread-like structures (arrowhead) is evidence of NETosis. Scale bars, 20 μm. (n = 5–8 mice/group); NET forming neutrophils are indicated by the arrows and original magnification 20x; *, p<0.05. AL, alcohol. Arrow indicates NET forming cells on merged images. SEM original magnification 8000x.
Fig 9.
rCXCL1 augments extracellular bacterial killing of neutrophils.
A-B, Alcohol impairs the extracellular bacterial killing activity of mouse bone marrow-derived neutrophils which can be improved by rCXCL1. Bacterial killing of mouse neutrophils treated with alcohol (25 mM) prior to infection (MOI 1) was determined by assessing extracellular CFUs at 60 min after infection with E. coli (A) or S. aureus (B). Experiments were performed in triplicate wells and 3–5 mice/group were used (*, p <0.05).