Figure 3.
RvTs reduce NETs with isolated human PMNs. Freshly isolated human PMNs were incubated with test compounds RvT1, RvT2, RvT3, RvT4, and RvD2 (1-100 nM) or vehicle control (0.01% ethanol v/v) at 37°C for 15 minutes, followed by the addition of IL-1β (50 ng/mL) with Sytox Green (5 µM). Fluorescence was monitored from 0 to 4 hours. (A-B) Dose response and time course of NETosis. The fluorescence of extracellular DNA in the presence of IL-1β alone was taken as 100%. The percentages of extracellular DNA in the presence of test compounds (1-100 nM) at 4 hours are indicated in parentheses. (A) One representative from 7 separate donors. (B) Mean ± SEM. n = 3 separate donors. *P < .05, vs PMN + vehicle. (C) Dose response of each RvT and RvD2. Results are expressed as percent reduction compared with IL-1β alone at 4 hours. Mean ± SEM. n = 7 separate donors. *P < .05, **P < .01, ***P < .001, ****P < .0001, vs 0 nM; #P < .05, vs 1 nM (two-way analysis of variance with Tukey’s multiple comparisons). (D) Comparison of SPMs (10 nM) and a PAD4 inhibitor (10 μM). Numbers shown on top of each bar are percentages compared with the PAD4 inhibitor that was taken as 100%. Mean ± SEM. n = 7 (SPMs) or n = 3 (inhibitor) separate donors. *P < .05, **P < .01, vs PAD4 inhibitor; #P < .05, vs RvD2 (one-way analysis of variance with Tukey’s multiple comparisons). (E) GPR18-dependent RvD2 action. PMNs were incubated with a GPR18 antagonist. O-1918 (20 μM) for 10 minutes before the addition of RvD1 (1-100 nM) and/or IL-1β. Mean ± SEM. n = 3 separate donors. *P < .05, two-tailed Student t test. (F) MPO levels. PMNs were incubated with RvT1 (10 nM) or vehicle control at 37°C for 15 minutes, followed by the addition of IL-1β (50 ng/mL) for 4 hours. DNA (NET)-bound MPO was determined. MPO levels from the same donors are connected by black lines. n = 3 separate donors. *P < .05. (G) LTB4-stimulated NETosis. PMNs were incubated with RvT1 (1-100 nM) or vehicle control at 37°C for 15 minutes, followed by the addition of LTB4 (10 nM) with Sytox Green (5 µM). Fluorescence was monitored at 4 hours. Mean ± SEM. n = 3 separate donors. *P < .05, **P < .01, vs LTB4 alone and LTB4 + 1 nM RvT1.

RvTs reduce NETs with isolated human PMNs. Freshly isolated human PMNs were incubated with test compounds RvT1, RvT2, RvT3, RvT4, and RvD2 (1-100 nM) or vehicle control (0.01% ethanol v/v) at 37°C for 15 minutes, followed by the addition of IL-1β (50 ng/mL) with Sytox Green (5 µM). Fluorescence was monitored from 0 to 4 hours. (A-B) Dose response and time course of NETosis. The fluorescence of extracellular DNA in the presence of IL-1β alone was taken as 100%. The percentages of extracellular DNA in the presence of test compounds (1-100 nM) at 4 hours are indicated in parentheses. (A) One representative from 7 separate donors. (B) Mean ± SEM. n = 3 separate donors. *P < .05, vs PMN + vehicle. (C) Dose response of each RvT and RvD2. Results are expressed as percent reduction compared with IL-1β alone at 4 hours. Mean ± SEM. n = 7 separate donors. *P < .05, **P < .01, ***P < .001, ****P < .0001, vs 0 nM; #P < .05, vs 1 nM (two-way analysis of variance with Tukey’s multiple comparisons). (D) Comparison of SPMs (10 nM) and a PAD4 inhibitor (10 μM). Numbers shown on top of each bar are percentages compared with the PAD4 inhibitor that was taken as 100%. Mean ± SEM. n = 7 (SPMs) or n = 3 (inhibitor) separate donors. *P < .05, **P < .01, vs PAD4 inhibitor; #P < .05, vs RvD2 (one-way analysis of variance with Tukey’s multiple comparisons). (E) GPR18-dependent RvD2 action. PMNs were incubated with a GPR18 antagonist. O-1918 (20 μM) for 10 minutes before the addition of RvD1 (1-100 nM) and/or IL-1β. Mean ± SEM. n = 3 separate donors. *P < .05, two-tailed Student t test. (F) MPO levels. PMNs were incubated with RvT1 (10 nM) or vehicle control at 37°C for 15 minutes, followed by the addition of IL-1β (50 ng/mL) for 4 hours. DNA (NET)-bound MPO was determined. MPO levels from the same donors are connected by black lines. n = 3 separate donors. *P < .05. (G) LTB4-stimulated NETosis. PMNs were incubated with RvT1 (1-100 nM) or vehicle control at 37°C for 15 minutes, followed by the addition of LTB4 (10 nM) with Sytox Green (5 µM). Fluorescence was monitored at 4 hours. Mean ± SEM. n = 3 separate donors. *P < .05, **P < .01, vs LTB4 alone and LTB4 + 1 nM RvT1.

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