Reactive oxygen species elevations in human immune cell subsets during sepsis are mitigated by norepinephrine and N-acetylcysteine

Figure 1 Gating strategy utilized for flow cytometry analysis. Representative gating strategy used to identify peripheral blood mononuclear cell subsets. Singlet events were gated first, followed by identification of CD14+ monocytes, CD15+ neutrophils, and CD14-CD-15 lymphocytes. Lymphocyte populations were further subdivided into CD3+ T cells and CD19+ B cells. CD3+ and CD14+ populations were prioritized for reactive oxygen species (ROS) analyses in subsequent figures due to consistently elevated ROS levels observed in sepsis.

Figure 2 Reactive oxygen species levels are increased septic patients in CD3+ and CD14+ cells. A–D: Representative histograms of intracellular reactive oxygen species (ROS) fluorescence in CD3+ T cells, CD14+ monocytes, CD15+ granulocytes and CD19+ B cells, from 1 control and 1 septic patient; E–H: Quantification of ROS levels (mean ± SD) in each subset from 6 control and 6 septic patients. While CD3+ and CD14+ cells exhibited significantly elevated ROS levels in sepsis, CD19+ and CD15+ cells did not show statistically significant differences; I–K: Plasma concentrations of interleukin (IL)-6, tumor necrosis factor-α, and IL-10 measured in 13 control and 12 septic patients. Statistical analysis: Unpaired two-tailed Student’s t-test. ^aP < 0.05, ^cP < 0.001. IL: Interleukin; ROS: Reactive oxygen species.

Figure 3 Norepinephrine reduces Reactive oxygen species levels in patients stimulated with lipopolysaccharide and hydrogen peroxide in CD3+ and CD14+ cells. A–F: Representative histograms of reactive oxygen species (ROS) fluorescence intensity in CD3+ and CD14+ peripheral blood mononuclear cells from control patients following in vitro stimulation with lipopolysaccharide (100 ng/mL) or hydrogen peroxide (10 μmol/L), with or without co-treatment with norepinephrine (5 mmol/L); G and H: Quantification of ROS levels (mean ± SD) in CD3+ and CD14+ cells from 7 independent control donors. All experimental conditions were performed in biological duplicate. Statistical analysis: Two-way analysis of variance with Bonferroni post hoc correction. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. H₂O₂: Hydrogen peroxide; LPS: Lipopolysaccharide; NE: Norepinephrine; ROS: Reactive oxygen species.

Figure 4 N-acetylcysteine reduces reactive oxygen species levels in patients stimulated with lipopolysaccharide and hydrogen peroxide in CD3+ and CD14+ cells. A–F: Representative histograms of reactive oxygen species (ROS) fluorescence intensity in CD3+ and CD14+ peripheral blood mononuclear cells from control patients following stimulation with lipopolysaccharide (100 ng/mL) or hydrogen peroxide (10 μmol/L), with or without co-treatment with N-acetylcysteine (10 mmol/L); G and H: Quantification of ROS levels (mean ± SD) in CD3+ and CD14+ cells from 3 independent control donors. All experiments were conducted in biological duplicate. Statistical analysis: Two-way analysis of variance with Bonferroni post hoc correction. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. H₂O₂: Hydrogen peroxide; LPS: Lipopolysaccharide; NAC: N-acetylcysteine; ROS: Reactive oxygen species.