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, 130 (1), 359-373

GPR101 Mediates the Pro-Resolving Actions of RvD5n-3 DPA in Arthritis and Infections

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GPR101 Mediates the Pro-Resolving Actions of RvD5n-3 DPA in Arthritis and Infections

Magdalena B Flak et al. J Clin Invest.

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.

Keywords: Arthritis; Bacterial infections; G-protein coupled receptors; Infectious disease; Inflammation.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. RvD5n-3 DPA receptor candidates are expressed on human leukocytes.
(A) Activation of orphan receptors by RvD5n-3 DPA (10 nM). Results represent the percentage increase in luminescence signal over vehicle control. (B and C) Expression of the top 3 candidate receptors on human (B) peripheral blood leukocytes and (C) macrophages. Results are representative of 4 donors. FSC, forward scatter; SSC, side scatter.
Figure 2
Figure 2. Activation of GPR101 by RvD5n-3 DPA.
(A) RvD5n-3 DPA was incubated at the indicated concentrations with CHO cells expressing human GPR101 (circles), GPR84 (squares), or GPR12 (triangles) coupled with the β-arrestin reporter system, and receptor activation was measured as an increase in luminescence signal. Results represent the mean ± SEM. n = 5–7 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the respective vehicle control group; 2-way ANOVA with Tukey’s post hoc multiple comparisons test. (B) CHO cells overexpressing GPR101 were incubated with either isotype control or anti-GPR101 antibody (30 minutes at room temperature) and then with 1 nM RvD5n-3 DPA, and impedance was measured over a 20-minute period using the xCELLigence DP system. Results are representative of 3 distinct experiments. (C) CHO cells expressing GPR101 coupled with the β-arrestin reporter system were incubated with the indicated concentrations of RvD5n-3 DPA, RvD1n-3 DPA, PD1n-3 DPA, or vehicle (PBS containing 0.01% ethanol), and receptor activation was measured as an increase in luminescence signal. Results represent the mean ± SEM. n = 5–7 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the vehicle control group; 2-way ANOVA with Tukey’s post hoc multiple comparisons test. (D) RvD5n-3 DPA, RvD1n-3 DPA, and PD1n-3 DPA (10 nM) were incubated with GPR101-expressing CHO cells, and impedance was measured over a 30-minute period using the xCELLigence DP system. Results are representative of 3 distinct experiments.
Figure 3
Figure 3. RvD5n-3 DPA stereospecifically activates GPR101 and increases cAMP in human macrophages.
(A) GPR101-expressing CHO cells were incubated with RvD5n-3 DPA or DA-RvD5n-3 DPA (10 nM), and impedance was measured for 30 minutes. Results are representative of 3 distinct experiments. (B) GPR101-expressing CHO cells were incubated with the indicated concentrations of the ligands described in A, and changes in impedance from baseline values were determined at t = 10 minutes. Results represent the mean ± SEM. n = 3 from 3 distinct experiments. *P < 0.05; 1-way ANOVA with a Holm-Sidak post hoc multiple comparisons test. (C) GPR101-overexpressing CHO cells were incubated with RvD5n-3 DPA or DHA-derived RvD5 (1 nM), and cell impedance was measured over a 30-minute period using the xCELLigence DP system. Results are shown as the mean ± SEM (n = 4 in 3 independent experiments). (D) CHO cells expressing GPR101 coupled with the β-arrestin luminescence reporter system were incubated with the indicated concentrations of RvD5n-3 DPA, DHA-derived RvD5, or vehicle (cell-plating reagent containing 0.01% ethanol), and receptor activation was measured as an increase in luminescence signal. Results are shown as the mean ± SEM (n = 3 in 2 independent experiments). (E) GPR101-expressing CHO cells were incubated with CTX (1 μg/mL, 2 hours), PTX (1 μg/mL, 16 hours), or vehicle and then with RvD5n-3 DPA (10 nM), and impedance was measured over a 30-minute period. Results are representative of 3 distinct experiments. (F) Human monocyte–derived macrophages were incubated with either an siRNA against GPR101 or a control sequence (CT siRNA; 72 hours at 37°C) and then with RvD5n-3 DPA (10 nM) or vehicle (Veh) (PBS containing 0.01% ethanol) for 2 minutes, and cAMP concentrations were assessed. Results represent the mean ± SEM (n = 4 donors). *P < 0.05; 1-way ANOVA with Holm-Sidak post hoc multiple comparisons test.
Figure 4
Figure 4. Specific binding of [3H]-RvD5n-3 DPA to human GPR101.
(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.
Figure 5
Figure 5. GPR101 mediates the protective actions of RvD5n-3 DPA on human macrophages.
(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.
Figure 6
Figure 6. GPR101 mediates the protective actions of RvD5n-3 DPA on neutrophils and monocytes.
(AC) 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.
Figure 7
Figure 7. Knockdown of GPR101 reverses the antiarthritic actions of RvD5n-3 DPA.
Mice were administered 9 μg siRNA targeting mouse Gpr101 or a scrambled control sequence. After 24 hours and 72 hours, mice were administered arthritogenic serum and then treated with RvD5n-3 DPA (150 ng/mouse) or vehicle (72 hours and 96 hours after siRNA administration). (A) Clinical scores, (B) AUC for clinical scores, (C) weight loss, and (D) edema were determined throughout the disease process. (E and F) On day 7, hind paws were harvested and eicosanoid concentrations determined using LC-MS/MS–based lipid mediator profiling. (E) Prostaglandin and (F) LTB4 metabolomic concentrations. Results represent the mean ± SEM (n = 4 mice per group). *P < 0.05 versus the vehicle-treated group; Kruskal-Wallis test with Dunn’s post hoc multiple comparisons test (B, E, and F) and 2-way ANOVA (C and D).
Figure 8
Figure 8. Gpr101 knockdown limits the ability of RvD5n-3 DPA to activate host responses to promote the resolution of E. coli infections.
(A) Mice were inoculated with 105 CFU E. coli via i.p. injection and lavages collected at the indicated time intervals before (0 hours) or after inoculation. RvD5n-3 DPA concentrations were determined using lipid mediator profiling. Results represent the mean ± SEM (n = 3 mice per group). (B) Mice were administered 9 μg siRNA against mouse Gpr101 or a scrambled control sequence, and after 72 hours, they were administered RvD5n-3 DPA (100 ng/mouse) or vehicle control (PBS containing 0.1% ethanol) and then inoculated with 105 CFU E. coli via i.p. injection. (C) Fourteen-hour exudate neutrophil counts. Results represent the mean ± SEM (n = 6 mice per group from 2 distinct experiments). (D and E) Bacterial phagocytosis was determined in exudate (D) neutrophils and (E) macrophages at the 14-hour interval using flow cytometry. (F) Efferocytosis was determined at the 14-hour interval in CD64+F4/80+ macrophages using flow cytometry. (G) The expression of MHC class II and IL-10R was assessed in CD64+F4/80+ macrophages using flow cytometry. Results represent the mean ± SEM (n = 6 mice per group from 2 distinct experiments). *P < 0.05 versus the vehicle-treated group; Kruskal-Wallis test with Dunn’s post hoc multiple comparisons test (CG).

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