Maresin 1 activates LGR6 receptor promoting phagocyte immunoresolvent functions

Abstract

Resolution of acute inflammation is an active process orchestrated by endogenous mediators and mechanisms pivotal in host defense and homeostasis. The macrophage mediator in resolving inflammation, maresin 1 (MaR1), is a potent immunoresolvent, stimulating resolution of acute inflammation and organ protection. Using an unbiased screening of greater than 200 GPCRs, we identified MaR1 as a stereoselective activator for human leucine-rich repeat containing G protein-coupled receptor 6 (LGR6), expressed in phagocytes. MaR1 specificity for recombinant human LGR6 activation was established using reporter cells expressing LGR6 and functional impedance sensing. MaR1-specific binding to LGR6 was confirmed using 3H-labeled MaR1. With human and mouse phagocytes, MaR1 (0.01-10 nM) enhanced phagocytosis, efferocytosis, and phosphorylation of a panel of proteins including the ERK and cAMP response element-binding protein. These MaR1 actions were significantly amplified with LGR6 overexpression and diminished by gene silencing in phagocytes. Thus, we provide evidence for MaR1 as an endogenous activator of human LGR6 and a novel role of LGR6 in stimulating MaR1's key proresolving functions of phagocytes.

Keywords: Inflammation; Macrophages; Neutrophils.

Figure 1. MaR1 candidate receptors.

(A) A panel of orphan GPCRs was screened in the presence of 10-nM MaR1 or vehicle (0.1% ethanol) using the β-arrestin PathHunter GPCR system. The % activity = 100% × (mean RLU of test sample – mean RLU of vehicle control)/(mean RLU of vehicle control). (B–E) Ligand (MaR1)-receptor interaction was monitored using the CHO-β-arrestin system overexpressing LGR6 or GPR148. Results are mean ± SEM from 3 independent experiments. (B) LGR6 or GPR148 cells with MaR1. ^#P < 0.05, ^##P < 0.01. MaR1 versus vehicle with LGR6 cells. *P < 0.05, ***P < 0.001. LGR6 versus GPR148. (C) LGR6 cells with MaR1 or MaR1 ME. *P < 0.05, **P < 0.01; ***P < 0.001 versus vehicle. (D) LGR6 cells with MaR1, MCTR1, MCTR2, or MCTR3. *P < 0.05, **P < 0.01; ***P < 0.001 versus vehicle. ^#P < 0.05, ^##P < 0.01; ^###P < 0.001 versus MaR1. (E) LGR6 cells with MaR1, Rspo-2, or Rspo-2+MaR1. *P < 0.05, **P < 0.01; ***P < 0.001 versus vehicle. ^#P < 0.05, ^##P < 0.01; ^###P < 0.001 versus MaR1. For D and E, the 6 groups (MaR1, MCTR1, MCTR2, MCTR3, Rspo-2, Rspo-2+MaR1) were carried out in the same experiments (n = 3). For clarity, the results were separated into D and E. The same MaR1 response curve is presented in both panels for direct comparisons. The statistical analysis (2-way ANOVA with Tukey’s multiple comparisons test) was carried out with all 6 groups. (F) MaR1 (0.1–10 nM) was incubated with CHO-β-arrestin-LGR6 at 4°C, 25°C, 37°C, or 40°C. Results are mean ± SEM from 3 independent experiments. ^#P < 0.05, versus 4°C; **P < 0.01, versus 4°C and 25°C. (G) Intracellular cAMP. HEK cells transfected with human LGR6 or mock plasmids were incubated with 1- to 100-nM MaR1 for 15 minutes, and cAMP levels were determined. Results are mean ± SEM from 4 independent experiments. ***P < 0.001, versus HEK-mock cells; ^###P < 0.001 versus vehicle control. (B–G) Statistical analysis was carried out using 2-way ANOVA with Tukey’s multiple comparisons test. (H) Maresin biosynthesis pathways.

Figure 2. Human recombinant LGR6 receptor specificity.

CHO-LGR6 cells were plated onto 8-well ECIS arrays (8W10E+), incubated with (A and B) MaR1 (0.01-100 nM), (C and D) MaR1, PD1, LTB₄ (100 nM) or vehicle alone (control), and impedance changes across CHO cell monolayers were continuously recorded every 4 seconds for 10 minutes using ECIS. Results are mean (A and C) or mean ± SEM (B and D) (n = 3–4). **P < 0.01, versus vehicle. ^##P < 0.01; ^###P < 0.001, versus MaR1. One-way ANOVA with Tukey’s multiple comparisons test. (E) Space-filling 3D molecular models of MaR1, PD1, and LTB₄ with energy minimization. R and S denote the stereochemistry of the hydroxyl groups; E and Z denote the double-bond geometry. Area within the blue dashed lines denotes the dihydroxyl and triene structures oriented in the E, E, Z configuration of these mediators.

Figure 3. [³H]-MaR1 preparation and specific binding with human recombinant LGR6.

(A) Synthetic [12,13]-acetylenic-MaR1 ME was converted to [12,13-³H]-MaR1-ME via catalytic hydrogenation using tritium gas (³H₂). See Methods. (B) Chromatographic (green line) and radioactive (gray bars) tracing of [³H]-MaR1-ME. Results are mean ± SEM (n = 3). (Inset) Online UV spectra of [³H]-MaR1-ME; λ_max 271 nm; representative of 3 separate experiments. (C and D) Competition binding. CHO cells were transfected with a human LGR6 plasmid. Transfected CHO cells (0.5 × 10⁶ cells in 100-μl DPBS⁺⁺) were incubated with 2 nM of [³H]-MaR1-ME in the absence or presence of (C) increasing concentrations of unlabeled MaR1-ME (10^–10–10^–5 M) or (D) unlabeled MaR1-ME (taken as 100% competition), MaR1, MCTR1, MCTR2 or MCTR3 (10^–6 M) for 60 minutes at 4°C. Results are mean ± SEM (n = 3). *P < 0.05; ***P < 0.001, versus [³H]-MaR1-ME plus vehicle. One-way ANOVA with Tukey’s multiple comparisons test.

Figure 4. Human LGR6-mediated MaR1 actions on human macrophage phagocytosis: overexpression and knockdown of LGR6.

(A–C) Human MΦ were transfected with human LGR6 or mock plasmids. Seventy-two hours later, MΦ were plated onto chamber slides (0.1 × 10⁶ cells/well), incubated with 1-nM MaR1 or vehicle for 15 minutes at 37°C, followed by addition of BacLight Green-labeled E. coli to initiate phagocytosis. Fluorescent images were recorded every 10 minutes. Four separate experiments with separate donors were carried out. In each experiment, 4 fields (×20) per condition (per well) were recorded. (A) (Upper left) LGR6 expression monitored by flow cytometry. (Lower left) Representative fluorescence images. Arrows denote MΦ with ingested fluorescent E. coli. Scale bars: 20 μm. (Right) mean fluorescence intensity (MFI)/cell from 1 representative experiment. (B) Kinetics of phagocytosis. Rate (MFI/min) = (MFI_60min – MFI_{20 min})/40 min obtained from the same experiment as in (A). (See Supplemental Figure 6 for results obtained from additional 3 donors.) (C) Percent increases of phagocytosis by MaR1. Results are mean ± SEM (n = 4). *P < 0.05, LGR6 versus mock transfection. (D) Human MΦ were transfected with human LGR6 or mock plasmids. MΦ were incubated with MaR1 (10^–13 to 10^–8 M) or vehicle control for 15 minutes, followed by addition of BacLight Green-labeled E. coli, CFDA-labeled apoptotic PMN, or FITC-labeled STZ. Results are percent increases of phagocytosis above vehicle. Results are mean ± SEM from 3 independent experiments with separate donors and triplicates in each experiment. *P < 0.05; **P < 0.01, versus mock transfection. ^#P < 0.05, ^##P < 0.01; ^###P < 0.001, versus vehicle. (E and F) Human MΦ were transfected with scramble control (SC)-shRNA or human LGR6-shRNA plasmids and phagocytosis carried out as in A. (E) MFI/cell from 1 representative experiment. (F) Percent increases of phagocytosis by MaR1. Mean ± SEM (n = 4). **P < 0.01, LGR6-shRNA versus SC-shRNA transfections. (C, D, and F) Two-way ANOVA with Bonferroni’s multiple comparisons test.

Figure 5. MaR1-LGR6–dependent binding, signal, and phagocytosis: knockdown of human LGR6.

(A and B) LGR6 knockdown in THP-1 cells. (A) Gating strategy: Cells were gated on FSC-SSC dot plots (left), GFP⁺ populations were selected on the histograms (middle), then LGR6 expression was determined within the GFP⁺ population (right). (B) LGR6 expression (MFI) in GFP⁺ THP-1 cells. Results are mean ± SEM (n = 4). ****P < 0.001 versus control shRNA (Ren 713) and mock vector. (C) [³H]-MaR1 binding. THP-1 cells were incubated with 2-nM [³H]-MaR1-ME in the presence or absence of unlabeled 1-μM MaR1-ME for 60 minutes at 4°C. Results are specific binding (CPM), calculated as total CPM ([³H]-MaR1 plus vehicle) – nonspecific CPM ([³H]-MaR1 plus unlabeled MaR1-ME). Results are mean ± SEM (n = 3). *P < 0.05, versus shRNA LGR6. Two-tailed paired Student’s t test. (D) cAMP. THP-1 cells were incubated with MaR1 (1–100 nM) for 15 minutes and cAMP levels determined. Results are mean ± SEM (n = 3). **P < 0.01, versus shRNA LGR6; ^##P < 0.01, versus 1 nM (mock vector). (E–H) Phagocytosis. THP-1 cells were incubated with 10-nM MaR1 or vehicle (0.01% ethanol) for 15 minutes prior to addition of BacLight Red-labeled (PE-Texas Red) E. coli (1:50 THP-1:E.coli) for 45 minutes at 37°C. Flow cytometry was carried out. (E) Representative histograms for GFP (top panels), GFP⁺ BacLight Red E. coli+ (middle panels) and GFP- BacLight Red E. coli+ (bottom panels). (F) Quantification of BacLight Red E. coli (MFI) in GFP⁺ cells (top) and GFP- cells (bottom). Results are mean ± SEM (n = 3). *P < 0.05; **P < 0.01, MaR1 versus vehicle. (G) Structures of MaR1 and 12E-MaR1 (left) and representative histograms (right) of BacLight Red E. coli in GFP⁺ cells. (H) Percent increase of phagocytosis. ****P < 0.0001, versus MaR1-treated mock vector transfected cells. (B and H) One-way ANOVA or (D and F) 2-way ANOVA with Tukey’s multiple comparisons test.

Figure 6. MaR1-LGR6–dependent phosphorylation signals.

(A) MaR1-dependent protein phosphorylation in human macrophages. Heat maps of phosphorylated signaling molecules at 0, 1, 2, 5, and 15 minutes after exposure of 10-nM MaR1 in M1 and M2 human macrophages was obtained using CyTOF (see Methods). (B, C) THP-1 cells transfected with either a mock vector or LGR6-specific shRNA were incubated with 10-nM MaR1 for 0 to 5 minutes. pCREB and pERK levels in GFP⁺ cells were determined using flow cytometry. Results are (B) representative histograms and (C) heat maps from n = 4. (D and E) Comparisons of MaR1 and its 12E isomer. THP-1 cells transfected with either a mock vector or LGR6-specific shRNA were incubated with MaR1 or 10-nM 12E-MaR1 for 1 (for pERK) or 2 minutes (for pCREB). pCREB and pERK levels were determined using flow cytometry. Results are (D) representative histograms and (E) mean ± SEM from 4 independent experiments. **P < 0.01; ****P < 0.0001 versus MaR1-treated mock vector transfected cells. One-way ANOVA with Tukey’s multiple comparisons test.

Figure 7. In vivo knockdown of mouse LGR6 reduces MaR1 actions in limiting PMN and stimulating macrophage phagocytosis.

Mice were injected i.p. with siRNA for mouse LGR6 (10 μg/mouse) or control non-target siRNA. (A–C) Three days after siRNA injection, 1-mg zymosan was injected i.p. (time 0) to initiate peritonitis. At 12 hours, MaR1 (100 ng/mouse) was injected i.p., and peritoneal exudates were collected at 24 hours. (A) Flow cytometry gating strategy and histograms for LGR6 expression in specific leukocytes. Live cells were first selected from FSC and SSC dot plots, within which leukocytes (CD45⁺) were further selected to identify PMN (CD11b⁺Ly6G⁺Ly6C^–), monocytes (CD11b⁺Ly6G^–Ly6C⁺), and macrophages (CD11b⁺F4/80⁺). (B) Quantification of LGR6 expression. **P < 0.01. LGR6 siRNA versus nontarget siRNA. Two-tailed unpaired Student’s t test. (C) Exudate PMN numbers. *P < 0.05. Vehicle versus MaR1. Two-tailed unpaired Student’s t test. (D–F) Three days after siRNA injection, peritoneal macrophages were collected. (D) Flow cytometry gating strategy for macrophages (CD45⁺F4/80⁺Ly6C^–), representative histograms, and quantification of LGR6 expression. **P < 0.01. LGR6 siRNA versus non-target siRNA. Two-tailed unpaired Student’s t test. (E) Phagocytosis of BacLight Green-labeled E. coli carried out using a real-time imaging microscope as in Figure 4. Results are MFI/cell from 4 fields/condition in 1 representative experiment with macrophages collected from nontarget siRNA (left) or LGR6 siRNA (right) injected mice. 1-nM MaR1. (F) Percent increases of phagocytosis by 1- or 10-nM MaR1 with macrophages collected from LGR6 siRNA or nontarget siRNA injected mice. Results are mean ± SEM (n = 3). *P < 0.05. LGR6 siRNA versus nontarget siRNA. Two-tailed paired Student’s t test.

Figure 8. In vivo knockdown of mouse LGR6 diminishes MaR1 phosphorylation signals on peripheral blood PMN and monocytes.

Mice were injected i.p. with siRNA for mouse LGR6 (10 μg/mouse) or control nontarget siRNA. Three days after siRNA injection, peripheral blood was collected and incubated with 10-nM MaR1 for 0 to 2 minutes. RBC was lysed and pCREB and pERK levels were determined using flow cytometry. (A) (Top panels) Flow cytometry gating strategy for PMN (CD11b⁺Ly6G⁺Ly6C^–) and monocytes (CD11b⁺Ly6G^–Ly6C⁺). (Bottom panels) representative histograms and quantification of LGR6 expression. *P < 0.05, **P < 0.01. LGR6 siRNA versus nontarget siRNA. Two-tailed unpaired Student’s t test. (B–D) pCREB and pERK levels in PMN. (B) Representative histograms (C), heat maps, and (D) quantification. Results are mean ± SEM (n = 5). (E–G) pCREB and pERK levels in monocytes. (E) Representative histograms (F), heat maps, and (G) quantification. Results are mean ± SEM (n = 5). (D and G) *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, versus time 0. #P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001. LGR6 siRNA versus nontarget siRNA at the same time points. Two-way ANOVA with Bonferroni’s multiple comparisons test.