GPR101 mediates the pro-resolving actions of RvD5n-3 DPA in arthritis and infections
Magdalena B. Flak, … , Francesco Palmas, Jesmond Dalli
Magdalena B. Flak, … , Francesco Palmas, Jesmond Dalli
Published January 2, 2020; First published December 3, 2019
Citation Information: J Clin Invest. 2020;130(1):359-373. https://doi.org/10.1172/JCI131609.
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Research Article Infectious disease Inflammation

GPR101 mediates the pro-resolving actions of RvD5n-3 DPA in arthritis and infections

Abstract

N-3 docosapentaenoic acid–derived resolvin D5 (RvD5n-3 DPA) is diurnally regulated in peripheral blood and exerts tissue-protective actions during inflammatory arthritis. Here, using an orphan GPCR screening approach coupled with functional readouts, we investigated the receptor(s) involved in mediating the leukocyte-directed actions of RvD5n-3 DPA and identified GPR101 as the top candidate. RvD5n-3 DPA bound to GPR101 with high selectivity and stereospecificity, as demonstrated by a calculated KD of approximately 6.9 nM. In macrophages, GPR101 knockdown limited the ability of RvD5n-3 DPA to upregulate cyclic adenosine monophosphate, phagocytosis of bacteria, and efferocytosis. Inhibition of this receptor in mouse and human leukocytes abrogated the pro-resolving actions of RvD5n-3 DPA, including the regulation of bacterial phagocytosis in neutrophils. Knockdown of the receptor in vivo reversed the protective actions of RvD5n-3 DPA in limiting joint and gut inflammation during inflammatory arthritis. Administration of RvD5n-3 DPA during E. coli–initiated inflammation regulated neutrophil trafficking to the site of inflammation, increased bacterial phagocytosis by neutrophils and macrophages, and accelerated the resolution of infectious inflammation. These in vivo protective actions of RvD5n-3 DPA were limited when Gpr101 was knocked down. Together, our findings demonstrate a fundamental role for GPR101 in mediating the leukocyte-directed actions of RvD5n-3 DPA.

Authors

Magdalena B. Flak, Duco S. Koenis, Agua Sobrino, James Smith, Kimberly Pistorius, Francesco Palmas, Jesmond Dalli

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Figure 6

GPR101 mediates the protective actions of RvD5n-3 DPA on neutrophils and monocytes.

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GPR101 mediates the protective actions of RvD5n-3 DPA on neutrophils and...
(A–C) Human neutrophils were incubated with anti-GPR101 antibody or an isotype control antibody (15 minutes at room temperature) and then with RvD5n-3 DPA at the indicated concentrations or with vehicle (PBS containing 0.1% ethanol), and chemotaxis toward LTB4 (10 nM) was assessed using the xCELLigence DP system. (A) representative traces, (B) neutrophil chemotactic rate calculated from the slope of the curve, and (C) neutrophil chemotaxis calculated from the AUC of the traces shown in A. *P < 0.05 versus the indicated control group; Kruskal-Wallis test with Dunn’s post hoc multiple comparisons test. (D) Neutrophils were isolated and incubated as detailed above, and neutrophil–endothelial cell interactions were assessed after perfusing (0.1 Pa) neutrophils over an activated endothelial cell monolayer. Results represent the mean ± SEM (n = 4 donors from 3–4 distinct experiments). *P < 0.05 and ***P < 0.001 versus the indicated control group; 2-way ANOVA with Sidak’s post hoc test. (E and F) Mice were administered 9 μg siRNA against mouse Gpr101 or a scrambled control sequence. After 72 hours, blood was collected and cells incubated with RvD5n-3 DPA (10 nM) or vehicle (20 minutes at 37°C) and then with 5 × 107 CFU fluorescently labeled bacteria (30 minutes at 37°C), and phagocytosis in (E) neutrophils and (F) monocytes was assessed by flow cytometry. Results represent the mean ± SEM (n = 4 per group from 2 distinct experiments). **P < 0.01 versus the indicated control group; Kruskal-Wallis test with Dunn’s post hoc multiple comparisons test.