Medium-chain fatty acid-sensing receptor, GPR84, is a proinflammatory receptor

Abstract

G protein-coupled receptor 84 (GPR84) is a putative receptor for medium-chain fatty acids (MCFAs), whose pathophysiological roles have not yet been clarified. Here, we show that GPR84 was activated by MCFAs with the hydroxyl group at the 2- or 3-position more effectively than nonhydroxylated MCFAs. We also identified a surrogate agonist, 6-n-octylaminouracil (6-OAU), for GPR84. These potential ligands and the surrogate agonist, 6-OAU, stimulated [(35)S]GTP binding and accumulated phosphoinositides in a GPR84-dependent manner. The surrogate agonist, 6-OAU, internalized GPR84-EGFP from the cell surface. Both the potential ligands and 6-OAU elicited chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and amplified LPS-stimulated production of the proinflammatory cytokine IL-8 from PMNs and TNFα from macrophages. Furthermore, the intravenous injection of 6-OAU raised the blood CXCL1 level in rats, and the inoculation of 6-OAU into the rat air pouch accumulated PMNs and macrophages in the site. Our results indicate a proinflammatory role of GPR84, suggesting that the receptor may be a novel target to treat chronic low grade inflammation associated-disease.

FIGURE 1.

Potential ligands for GPR84. Membranes from Sf9 cells expressing human GPR84-fused with bovine Gα_i protein were incubated with various FFAs for 1 h at room temperature. The data represent the means ± S.D. for quadruple determinations.

FIGURE 2.

Effect of FFAs in PI assay. HEK293 cells were transfected with human GPR84 or control vector, pcDNA3.1, in the presence of G_qi5 plasmid. Cells were incubated with FFAs for 2 h at 37 °C. The data represent the means ± S.D. for triplicate determinations.

FIGURE 3.

Identification and characterization of a surrogate agonist for GPR84. A, effect of 6-OAU on the [³⁵S]GTPγS binding assay. Membranes from Sf9 cells expressing human GPR84 (hGPR84), human GPR109A (hGPR109A), or OXER1 fused with bovine Gα_i protein were incubated with 6-OAU for 1 h at room temperature. The data represent the means ± S.D. for quadruple determinations. B, effect of 6-OAU on GPR84 in the PI assay. HEK293 cells were transfected with human GPR84 or control vector, pcDNA3.1, in the presence of G_qi5 plasmid. Cells were incubated with various concentrations of 6-OAU or 3-OH-C12 for 2 h at 37 °C. The data represent the means ± S.D. for triplicate determinations. C, effect of 6-OAU on GPR40 in the PI assay. HEK293 cells were transfected with human GPR40 or control vector, pcDNA3.1. Cells were incubated with various concentrations of 6-OAU or C14 for 2 h at 37 °C. The data represent the means ± S.D. for triplicate determinations. D, receptor internalization assay. Internalization of stably expressing human GPR84-EGFP fusion protein in HEK293 cells was observed by fluorescence microscopy. Cells were treated without or with 0, 6.25, or 200 μm 6-OAU for 30 min.

FIGURE 4.

Cellular functions of human GPR84. A, chemotaxis of human peripheral PMNs. PMNs isolated from human peripheral blood were applied onto the upper chamber of a Transwell permeable support. Various concentrations of 6-OAU or 3-OH-C12 were added in the lower chambers of the Transwell. Transwell permeable supports were incubated at 37 °C for 1 h. The chemotaxis activities were assessed as a percentage of migrated cells to a lower chamber across the membrane from an upper chamber. The data were expressed as the means ± S.D. of quadruple determinations. B, chemotaxis of human GPR84 stable transfectant (CHO-GPR84). CHO-GPR84 was generated as described under “Experimental Procedures.” CHO-GPR84 cells were applied onto the upper chambers of the 96-well chemotaxis chamber. Various concentrations of 6-OAU or FFAs were added into the lower chambers. The chemotaxis activities were expressed as a migration index: (A₅₉₅ of experimental well − A₅₉₅ of background well)/(A₅₉₅ of medium control well − A₅₉₅ of background well). The data were expressed as means ± S.D.; n = 5. C, chemotaxis of U937 cell lines. U937 cells were used for the experiment after differentiation into macrophage-like cells as described under “Experimental Procedures.” Differentiated U937 cells were applied onto the upper chambers of the 96-well chemotaxis chamber. Various concentrations of 6-OAU or FFAs were added into the lower chambers. The chemotaxis activities were expressed as the same as B. The data were expressed as the means ± S.D. of triplicate determinations. *, p < 0.001 when compared with the medium control group (M). #p < 0.001 when compared with the PTX-untreated group.

FIGURE 5.

Effects of 6-OAU or 3-OH-C12 on cytokine production. A, cytokine production from human peripheral PMNs. PMNs isolated from human peripheral blood were stimulated by LPS in the presence or absence of 6-OAU or 3-OH-C12. Culture supernatant was collected 4 h after stimulation. IL-8 concentration in the supernatant was measured with a commercial ELISA kit. The data were expressed as the means ± S.D. of triplicate determinations. *, p < 0.05, **, p < 0.01, and ***, p < 0.001 when compared with the agonist untreated group. B, negative control siRNA or GPR84 siRNA-transfected U937 macrophages were stimulated by 100 ng/ml LPS in the presence or absence of 6-OAU or 3-OH-C12. Culture supernatant was collected 16 h after stimulation. TNFα concentration in the supernatant was measured with a commercial ELISA kit. The data were expressed as the means ± S.D. of triplicate determinations. *, p < 0.05, **, p < 0.01, and ***, p < 0.001 when compared with the agonist untreated group. #, p < 0.01 when compared with the negative control siRNA-transfected group.

FIGURE 6.

Proinflammatory effects of 6-OAU in vivo. A, 6-OAU (10 mg/kg) suspended in 1% rat serum or 1% rat serum as control was injected into the jugular vein in rats. Blood was collected from the jugular vein 3 h after 6-OAU injection. CXCL1 concentration in the serum was measured with a commercial ELISA kit. The data were expressed as the means ± S.D.; n = 5. **, p < 0.01 when compared with the 1% rat serum-injection group. B, 6-OAU suspended in 0.3% BSA or 0.3% BSA alone as control was injected into the dorsal air pouch prepared on rats. Infiltrated cells into the air pouch were collected with PBS 4 h after 6-OAU inoculation. The cell number was counted with the XT-2000i. The data were expressed as the means ± S.D.; n = 6. **, p < 0.01 and *, p < 0.05 when compared with the 0.3% BSA injection group.