Figure 5. Upregulation of 15-PGDH in the lamina propria during inflammatory arthritis promotes the inactivation of RvD5_{n-3 DPA}.

Inflammatory arthritis was initiated as detailed in Figure 1, and small intestines were collected on day 8 after initiation. (A) mRNA expression of 15-PGDH. (B) Immunofluorescence analysis of 15-PGDH expression in the small intestine. Original magnification, ×60. (C) Bone marrow macrophages were incubated with immune complexes (IC) or control IgG (16 hours, 37°C), and the expression of 15-PGDH was assessed using flow cytometry. Results for A and C are mean ± SEM. For A, n = 7 mice per group; for C, n = 4 mice per group from 2 independent experiments. Results for B are representative of n = 4 mice per group from 2 independent experiments. (D–F) RvD5_{n-3 DPA} was incubated with hr15-PGDH (0.5 U, room temperature, 45 minutes). Products were extracted and lipid mediators were identified using liquid chromatography tandem mass spectrometry and reversed-phase UV-HPLC. (D) MRM chromatograms of 361 > 199 (RvD5_{n-3 DPA}) and 359 > 215 (17-oxo-RvD5_{n-3 DPA}). (E) Online UV chromatogram for RvD5_{n-3 DPA} and 17-oxo-RvD5_{n-3 DPA}. (F) MS/MS spectrum employed in the identification of 17-oxo-RvD5_{n-3 DPA}. Results are representative of n = 4 incubations. (G) Arthritis was initiated as in Figure 1, small intestines harvested on day 8 after initiation, products were extracted, and the concentration of 17-oxo-RvD5_{n-3 DPA} was determined using LC-MS/MS. (H) Bone marrow–derived macrophages were incubated with vehicle or immune complexes (37°C, 16 hours) and 17-oxo-RvD5_{n-3 DPA} concentrations were determined using LC-MS/MS. Results are mean ± SEM n = 4–5 mice per group from 2 independent experiments. (I–K) Bone marrow–derived macrophages were incubated with vehicle or U0126 (20 μM, 37°C, 1 hour), then with either vehicle or immune complexes (37°C, 16 hours), and the expression of (I) 15-PGDH and (J) ELK1 and was investigated using flow cytometry. (K) Concentrations of 17-oxo-RvD5_{n-3 DPA} were determined using lipid mediator profiling. (L) Bone marrow–derived macrophages were incubated with RvD5_{n-3 DPA} (10 nM), 17-oxo-RvD5_{n-3 DPA} (10 nM), or vehicle (37°C, 2 hours), and concentrations of l-kynurenine determined using LC-MS/MS. (M) Cells were incubated as in K for 16 hours (37°C), and expression of IL-10R and IL-10 was assessed using flow cytometry. Results for I–M are mean ± SEM. n = 4 mice per group from 2 independent experiments. *P < 0.05 using Kruskall-Wallis test followed by Dunn’s post hoc test.