Figure 4. RvD5_{n-3 DPA} rectifies intestinal macrophage lipid mediator profiles and upregulates macrophage IL-10 and IL-10R expression via the activation of IDO and AHR.

(A and B) Lamina propria macrophages were isolated from naive mice and incubated with vehicle (Naive), isotype control antibodies (K/BxN), or anti-CD16 and anti-CD32 antibodies (anti-Fc Ab + K/BxN) for 20 minutes (37°C). Cells were then incubated with vehicle or K/BxN serum (1:1000 dilution; 16 hours at 37°C). Incubations were quenched with ice-cold methanol and lipid mediators identified and quantified using lipid mediator profiling. (A) oPLS-DA of lipid mediator profiles. (B) Concentrations of RvD5_{n-3 DPA} (left panel), prostaglandins (PG; center), and ratio of proresolving mediators to proinflammatory eicosanoids (Resolution Index; right). Results are mean ± SEM, with horizontal bars depicting mean values. n = 4 mice per group. *P < 0.05 versus naive using Kruskall-Wallis test followed by Dunn’s post hoc test. (C and D) Lamina propria macrophages were isolated from naive mice and arthritic mice and then incubated with either vehicle (K/BxN) or RvD5_{n-3 DPA} (10 nM; K/BxN + RvD5_{n-3 DPA}) for 20 minutes (37°C; 16 hours). Incubations were quenched with ice-cold methanol, and lipid mediators were identified and quantified using lipid mediator profiling. (C) oPLS-DA of lipid mediator profiles. (D) Concentrations of prostaglandins (left panel) and resolution index (right). Results are mean ± SEM. n = 4 mice per group. *P < 0.05 versus naive using Kruskall-Wallis test followed by Dunn’s post hoc test. (E) Bone marrow–derived macrophages were incubated with 10 nM RvD5_{n-3 DPA} or vehicle (16 hours, 37°C), and IL-10R and IL-10 expression was determined using flow cytometry. (F) Bone marrow–derived macrophages were incubated with 10 nM RvD5_{n-3 DPA} or vehicle (2 hours, 37°C) and concentrations of l-kynurenine measured by LC-MS/MS. (G) Bone marrow–derived macrophages were incubated with 20 μM INCB 024360 or vehicle (30 minutes, 37°C), then with 10 nM RvD5_{n-3 DPA} or vehicle (16 hours, 37°C) and IL-10R and IL-10 expression determined using flow cytometry (lower panels). Representative histograms are shown (upper panels). Results for E–G are mean ± SEM. n = 4–5 mice per group from 2 independent experiments. *P < 0.05 versus vehicle group using Mann-Whitney U test for E and F, and Kruskall-Wallis test followed by Dunn’s post hoc test for G.