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

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 5

GPR101 mediates the protective actions of RvD5n-3 DPA on human macrophages.

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GPR101 mediates the protective actions of RvD5n-3 DPA on human macrophag...
(A) Human monocyte–derived macrophages were incubated with either an siRNA against GPR101 or a control sequence (CT siRNA) for 96 hours, and GPR101 expression was assessed using flow cytometry (n = 4 donors). (B) Cells were transfected as in A and then incubated with RvD5n-3 DPA (0.001–10 nM) or vehicle (RPMI-1640 containing 0.1% ethanol, 15 minutes, 37°C), after which (B and C) efferocytosis of pHrodo Red–conjugated apoptotic HL-60 cells and (D and E) phagocytosis of pHrodo Green–conjugated S. aureus bioparticles were measured using a Zeiss Celldiscoverer 7 high-content imager. B and D show the increase in signal over time for vehicle and 1 nM RvD5n-3 DPA groups, whereas C and E show the AUC for all tested concentrations. Results represent the mean ± SEM (n = 6 donors from 2 distinct experiments). *P < 0.05, **P < 0.01, and ***P < 0.001; 2-way ANOVA with Tukey’s post hoc multiple comparisons test. (F) Human monocyte–derived macrophages were incubated with either an siRNA against GPR101 or a control sequence and then with 10 nM RvD5n-3 DPA or vehicle (RPMI-1640 containing 0.1% ethanol; 2 hours at 37°C), and the expression of l-kynurenine was measured using LC-MS/MS. Results represent the mean ± SEM (n = 4 donors from 2 distinct experiments). *P < 0.05; Friedman’s test with Dunn’s post hoc multiple comparisons test.