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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 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

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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 4

Specific binding of [3H]-RvD5n-3 DPA to human GPR101.

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Specific binding of [3H]-RvD5n-3 DPA to human GPR101.
(A and B) Characte...
(A and B) Characterization of [10, 11, 13, 14 3H]-RvD5n-3 DPA ([3H]-RvD5n-3 DPA). (A) RP-UV-HPLC chromatogram for RvD5n-3 DPA and [3H]-RvD5n-3 DPA. (B) RP-UV-HPLC chromatogram of [3H]-RvD5n-3 DPA and online radioactivity monitoring. (C) GPR101-overexpressing CHO cells (0.5 × 106 cells in 100 μL) were incubated with [3H]-RvD5n-3 DPA at the indicated concentrations in the presence or absence of 10 μM RvD5n-3 DPA (60 minutes at 4°C). Cell incubations were transferred to a vacuum manifold, unbound radioligand was removed, and activity was measured. Results represent the mean ± SEM (n = 4 from 2 distinct experiments). Inset shows a Scatchard plot. (D) To assess competition binding, GPR101-expressing CHO cells (0.5 × 106 cells in 100 μL) were incubated with 3 nM [3H]-RvD5n-3 DPA in the presence or absence of increasing concentrations of RvD5n-3 DPA for 60 minutes at 4°C.
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