GPR37 regulates macrophage phagocytosis and resolution of inflammatory pain

Abstract

The mechanisms of pain induction by inflammation have been extensively studied. However, the mechanisms of pain resolution are not fully understood. Here, we report that GPR37, expressed by macrophages (MΦs) but not microglia, contributes to the resolution of inflammatory pain. Neuroprotectin D1 (NPD1) and prosaptide TX14 increase intracellular Ca2+ (iCa2+) levels in GPR37-transfected HEK293 cells. NPD1 and TX14 also bind to GPR37 and cause GPR37-dependent iCa2+ increases in peritoneal MΦs. Activation of GPR37 by NPD1 and TX14 triggers MΦ phagocytosis of zymosan particles via calcium signaling. Hind paw injection of pH-sensitive zymosan particles not only induces inflammatory pain and infiltration of neutrophils and MΦs, but also causes GPR37 upregulation in MΦs, phagocytosis of zymosan particles and neutrophils by MΦs in inflamed paws, and resolution of inflammatory pain in WT mice. Mice lacking Gpr37 display deficits in MΦ phagocytic activity and delayed resolution of inflammatory pain. Gpr37-deficient MΦs also show dysregulations of proinflammatory and antiinflammatory cytokines. MΦ depletion delays the resolution of inflammatory pain. Adoptive transfer of WT but not Gpr37-deficient MΦs promotes the resolution of inflammatory pain. Our findings reveal a previously unrecognized role of GPR37 in regulating MΦ phagocytosis and inflammatory pain resolution.

Keywords: G-protein coupled receptors; Inflammation; Macrophages; Neuroscience; Pain.

Figure 1. GPR37 is expressed by MΦs, not microglia.

(A and B) IHC showing the colocalization of GRP37 and CD68 in the hind paw dermis of WT (A) but not Gpr37^–/– (B) mice. Boxes in A indicate an enlarged cell. Blue DAPI staining labels all nuclei in the skin. Scale bar: 100 μm; 45 μm (original scale in inset). (C and D) Flow cytometry showing GPR37 expression in F4/80⁺ MΦs from hind paw skin and pMΦs from WT and KO (Gpr37^–/–) mice. n = 3–5 mice/group. Data represent the mean ± SEM. *P < 0.05; unpaired t test. (E) Double staining showing GPR37 IR in F4/80⁺ pMΦs. Scale bar: 100 μm. (F) Confocal microscopic images showing cytoplasm (arrows) and surface (arrowheads) localization of GPR37 IR in pMΦs. Scale bar: 20 μm. (G) Western blot showing a single band of GRP37 in lysates of brain, spinal cord (SC), hind paw skin, and pMΦs. Note that the band is absent in Gpr37^–/– mice. (H) Double staining showing no colocalization of GPR37 with CX3CR1 in the spinal cords of Cx3cr1-GFP mice. Scale bars: 250 μm and 20 μm (inset). (I) β-Gal staining showing no colocalization of LacZ (Gpr37) expression with IBA1 in brain sections from Gpr37^+/− mice. Scale bars: 100 μm and 20 μm (inset). CC, corpus callosum.

Figure 2. NPD1 induces iCa²⁺ increases in HEK293 cells and MΦs via GPR37.

(A–E) Ca²⁺ imaging with the Fura-2AM indicator in HEK293 cells transfected with GPR37 cDNA or empty vector (mock transfection). (A) Representative images showing calcium responses (color changes) after TX14 (1 μM, 3 minute treatment) and NPD1 (30 nM, 3 minute [duration of treatment]) treatment. Scale bar: 50 μm. The pseudo-color scale (0–2) shows the possible range of calcium signaling. (B) Combined traces from 100 cells showing time-dependent iCa²⁺ increases induced by NPD1 and TX14 after GPR37 but not mock transfection. Arrows show the time points at which the images in A were collected. (C) Comparison of iCa²⁺ levels after 3 minutes of treatment with NPD1 (30 nM), TX14 (1 μM), RvD1 (100 nM), RvD2 (100 nM), RvE1 (100 nM), lipoxin (100 nM), DHA (1 μM), EPA (1 μM), and ionomycin (2 μM). *P < 0.05 versus baseline (vehicle); 2-way ANOVA followed by Bonferroni’s post hoc test. n = 3–4 cultures, with 73 to 340 cells analyzed for each condition. (D) Dose-response curves of NPD1- and TX14-induced iCa²⁺ increases. n = 3–4 cultures, with 72 to 250 cells analyzed for each condition. Note the different EC₅₀ values for these 2 compounds. (E) Inhibition of NPD1-induced (30 nM) iCa²⁺ increases by PTX (1 μg/ml, 16 h before treatment), thapsigargin (1 μM, 3 min), and EGTA (10 mM, 3 min). *P < 0.05; 1-way ANOVA. n = 3–4 cultures, with 131 to 186 cells analyzed per treatment. (F) Dot blots showing a dose-dependent binding of NPD1 and TX14, but not RvE1, to GPR37. The blots were coated with NPD1, TX14, and RvE1 and then incubated with cell lysates from HEK293 cells with GPR37 cDNA or mock transfection. (G–J) Ca²⁺ imaging with the Fluo-4AM Ca²⁺ indicator in WT and Gpr37^−/− pMΦ cultures. (G) Representative images showing Ca²⁺ responses (color changes) after NPD1 treatment (30 nM) in WT but not Gpr37^−/− mice. Pseudo-color scale (0–5) shows the possible range of calcium signaling. Scale bar: 50 μm. (H) Combined traces from 100 cells showing time-dependent iCa²⁺ responses after NPD1 treatment (100 nM, 3 min) in WT and Gpr37^–/– mice. (I) Comparison of iCa²⁺ levels after treatment with NPD1 (30 nM, 3 min), TX14 (1 μM, 3 min), PTX (1 μg/ml, 16 h), RvD1 (100 nM, 3 min), RvE1 (100 nM, 3 min), and ATP (100 μM, 3 min) in WT and Gpr37^–/– pMΦ cultures. *P < 0.05 versus Gpr37^–/–; ^#P < 0.05 (with PTX vs. without PTX); 2-way ANOVA followed by Bonferroni’s post hoc test. n = 3–4 cultures, with more than 300 cells analyzed for each condition. (J) Dose-response curves of NPD1- and TX14-induced iCa²⁺ increases in pMΦ cultures. n = 3 cultures, with 80–500 cells analyzed for each condition. Note the different EC₅₀ values for NPD1 and TX14. Data represent the mean ± SEM. F0,relative basal intensity; Fmax, relative peak intensity.

Figure 3. NPD1 enhances MΦ phagocytic activity in vitro via GPR37.

(A) NPD1 enhanced phagocytosis in WT pMΦs, as revealed by fluorescence-labeled zymosan particles. Note the reduction in NPD1-induced phagocytosis in Gpr37^–/– mice. Scale bars: 10 μm. (B) Quantification of pMΦ phagocytic activity according to the number of zymosan particles (top) and percentage of cells (bottom) with phagocytic activity (>1 particle/cell). Note the dose-dependent phagocytic activity induced by NPD1. ^#P < 0.05 versus control (vehicle, PBS); *P < 0.05 versus Gpr37^–/–; 2-way ANOVA followed by Bonferroni’s post hoc test. n = 4–5 cultures/group. (C) Phagocytic activity in pMΦs from WT and Gpr37^–/– mice following treatment with RvD1 (100 nM), RvE1 (100 nM), TX14 (100 nM), and ionomycin (2 μM), as revealed by the number of zymosan particles (top) and percentage of cells with phagocytosis (bottom). ^#P < 0.05 versus vehicle; *P < 0.05 versus Gpr37^–/–; 2-way ANOVA followed by Bonferroni’s post hoc test. n = 3–5 cultures/group. (D) Effects of LY294002 (50 μM), U0126 (10 μM), PTX (1 μg/ml), BAPTA-AM (10 μM), and ionomycin (2 μM) on basal and NPD1-induced (30 nM) phagocytosis. *P < 0.05 versus vehicle (with NPD1); ^#P < 0.05, NPD1 versus control; 2-way ANOVA followed by Bonferroni’s post hoc test. n = 3–5 cultures/group. For each culture, 113–503 cells were analyzed. Data represent the mean ± SEM.

Figure 4. GPR37 is necessary for MΦ phagocytosis in inflamed hind paw skin.

(A) Schematic illustration of MΦ phagocytosis of pH-sensitive and dye-conjugated zymosan (pH-R-zymosan) particles. Note that only phagocytized zymosan particles show red fluorescence. (B) Experimental diagram showing the timeline of i.pl. injection of pH-R-zymosan, FACS analysis, immunostaining, and edema tests. (C) Edema in a hind paw following zymosan (20 μg/20 μl) injection, as measured by paw volume. *P < 0.05 versus baseline (BL); 1-way ANOVA. n = 5 mice/group. (D) IHC showing the time courses of zymosan-induced changes in neutrophils (Gr-1⁺), MΦs (CD68⁺), GPR37, and phagocytized zymosan in inflamed hind paw skins. *P < 0.05 versus baseline in naive animals; 1-way ANOVA. n = 4 mice/group. (E) Images of phagocytized zymosan particles in skins of naive mice and inflamed mice 4 hours, 1 day, and 5 days after zymosan injection. Scale bar: 50 μm. (F) Quadruple staining of CD68 (green), DAPI (blue), GRP37 (purple), and zymosan particles (red) in inflamed skin 5 days after zymosan injection. Note that phagocytized zymosan particles are present inside GPR37⁺ MΦs. Scale bars: 20 μm and 5 μm (enlarged images). (G) Phagocytized zymosan levels (revealed by staining intensity) in naive and inflamed paws of WT and Gpr37^−/− mice. *P < 0.05; 2-way ANOVA. n = 4 mice/group. (H) Quantification of CD68 IR in hind paw skin. n = 4 mice/group. (I) Flow cytometry showing the percentage of GPR37-expressing MΦs in WT and Gpr37^–/– mice at different time points of zymosan injection. *P < 0.05; Student’s t test. n = 4–5 mice/group. Data represent the mean ± SEM.

Figure 5. GPR37 is required to regulate cytokine expression in inflamed skin.

(A–F) mRNA expression (revealed by quantitative RT-PCR in A, C, and E) and protein expression (revealed by ELISA in B, D, and F) of the proinflammatory cytokine IL-1β (B) and the antiinflammatory cytokines IL-10 (D) and TGF-β (F) in noninflamed skins (naive) and inflamed skins of WT and Gpr37^–/– mice. mRNA expression levels were normalized to Gapdh mRNA. *P < 0.05 versus Gpr37^–/–; ^#P < 0.05 versus naive; 2-way ANOVA. n = 3–5 mice/group. Data represent the mean ± SEM.

Figure 6. GPR37 is necessary for the resolution of inflammatory pain.

(A) Experimental diagram showing the timeline of i.pl. injection of zymosan and behavioral tests. (B) Zymosan-induced inflammatory pain symptoms of heat hyperalgesia and mechanical allodynia in WT and Gpr37^–/– mice. Note that baseline (BL) pain and the onset of inflammatory pain were normal, but the resolution of inflammatory pain was impaired in Gpr37^–/– mice. *P < 0.05 versus Gpr37^–/–; 2-way ANOVA. n = 10 mice/group. (C) Heat hyperalgesia and mechanical allodynia induced by i.pl. IL-1β (1 ng) in WT and Gpr37^–/– mice. Diagram shows the experimental timeline of i.pl. injection of IL-1β and behavioral tests. *P < 0.05 versus Gpr37^–/–; 2-way ANOVA. n = 5 mice/group. (D) Baseline pain for heat sensitivity (hot plate), mechanical sensitivity (Randall-Selitto and pinprick tests), and cold sensitivity (acetone test) in WT and Gpr37^–/– mice. n = 5 mice/group. (E) Rotarod test in WT and Gpr37^–/– mice. The speed of rotation was accelerated from 4 to 40 rpm over a 5-minute period. n = 5 mice/group. (F) Acute inflammatory pain induced by capsaicin (i.pl., 5 μg) in WT and Gpr37^–/– mice. n = 5 mice/group. (G) Zymosan-induced edema (paw swelling, revealed by paw volume) before and after zymosan injection in WT and Gpr37^–/– mice. n = 5–10 mice/group.

Figure 7. MΦs and MΦ GPR37 are critical for the resolution of inflammatory pain.

(A) Experimental diagram showing the timeline of drug treatments, FACS analysis, and behavioral tests. (B) MΦ depletion with clodronate (i.p., 15 μl/g, 2 and 48 h prior to the zymosan injection) delayed the resolution of heat hyperalgesia and mechanical allodynia. *P < 0.05 versus control; 2-way ANOVA. n = 5–8 mice/group. (C) Experimental diagram showing the timeline of zymosan injection, adoptive transfer of MΦs, and behavioral tests for D and E. (D and E) Adoptive transfer of WT MΦs (i.pl., 50,000 cells, D) but not Gpr37-deficient MΦs (KO MΦs) (E) promoted the resolution of heat hyperalgesia and mechanical allodynia in Gpr37^–/– mice. Note that the differences between Gpr37^–/– and WT mice (revealed in Figure 6B) were abolished after the treatment (D). *P < 0.05 versus Gpr37^–/–; 2-way ANOVA. n = 5 mice/group. Data represent the mean ± SEM.

Figure 8. IL-10 contributes to the resolution of inflammatory pain by MΦs.

(A) Experimental diagram showing the timeline of drug treatments and behavioral tests. (B) IL-10–neutralizing antibody (i.pl., 10 μg, twice, 0.5 h before and 1 day after the zymosan injection) delayed the resolution of heat hyperalgesia and mechanical allodynia. *P < 0.05 versus control IgG; 2-way ANOVA. n = 6 mice/group. (C) Experimental diagram showing the timeline of zymosan injection, adoptive transfer of MΦs with IL-10–neutralizing antibody injection, and behavioral tests shown in D. (D) IL-10–neutralizing antibody (i.pl., 10 μg, 1 day after zymosan injection) impaired the proresolution effects on heat hyperalgesia and mechanical allodynia induced by adoptive transfer of WT MΦs (i.pl., 50,000 cells) into Gpr37^–/– mice. *P < 0.05, WT versus Gpr37^–/–; 2-way ANOVA. n = 6 mice/group. Data represent the mean ± SEM.