Journal: Science Advances

Article Title: A potent proresolving mediator 17R-resolvin D2 from human macrophages, monocytes, and saliva

doi: 10.1126/sciadv.adq4785

Figure Lengend Snippet: ( A ) Representative time-course of BacLight Green–labeled E. coli phagocytosis by human neutrophils (ratio of 1 neutrophil: 50 E. coli ) pre-incubated for 15 min with 1 and 10 nM 17R-RvD2. ( B ) Percentage increase of E. coli phagocytosis above the vehicle after 45 min in neutrophils incubated with 10 nM 17R-RvD2, RvD2, and GPR18-specific agonist or vehicle. Results are represented as mean ± SEM. n = 3 healthy human donors. Student’s t test compared to vehicle, * P < 0.05 and ** P < 0.01 compared to E.coli plus vehicle. One-way ANOVA with Tukey’s multiple comparisons test was used to compare conditions. ( C ) Percent increase of intracellular ROS in neutrophils pre-incubated with increasing concentrations of 17R-RvD2, RvD2, or vehicle for 15 min and incubated with E. coli for 1 hour. Results are represented as mean ± SEM. n = 3 healthy human donors. Two-way ANOVA with Tukey’s multiple comparisons test, * P < 0.05, *** P < 0.001, and **** P < 0.0001. The EC 50 was estimated using nonlinear regression (dashed blue line) with Rvs versus response (three parameters).

Article Snippet: CHO cells (WT or human GPR18 expressing cells) were incubated with Alexa Fluor 647–labeled rabbit anti-human GPR18 immunoglobulin G (IgG; Novus Biologicals, Centennial, Colorado, NBP2-24918AF647) or Alexa Fluor 647–labeled rabbit IgG isotype control (NBP2-36463AF647, Novus Biologicals) for 30 min at 4°C.

Techniques: Labeling, Incubation

Journal: The Journal of Experimental Medicine

Article Title: Identification of resolvin D2 receptor mediating resolution of infections and organ protection

doi: 10.1084/jem.20150225

Figure Lengend Snippet: Identification of RvD2 receptor candidates. (A) A panel of orphan GPCRs was screened using β-arrestin PathHunter GPCR system in the presence of 10 nM of RvD2 or vehicle control (0.1% ethanol). Results were expressed as a heatmap. The receptors that gave highest chemiluminescence signal in response to RvD2 (see Materials and methods for the screening methodology) were taken as 100% in the heatmap (indicated by arrows). (B) Receptor specificity. Ligand (RvD2)–receptor interaction was monitored using a β-arrestin system overexpressing GPR18 (circle), GPR26 (square), or GPR30 (triangle). Results are mean ± SEM from 3 independent experiments and 4 replicates each experiment. ## , P < 0.01 versus GPR18 (one-way ANOVA with Tukey’s Multiple Comparison test). *, P < 0.05; **, P < 0.01, RvD2 versus vehicle controls (unpaired Student’s t test). RLU, relative luminescence units. (C) Ligand specificity. RvD2 (circle), RvD1, or RvD3 (square) interaction with GPR18 were monitored using β-arrestin system overexpressing GPR18. Results are from 3 (RvD2) or 2 (RVD1 and RvD3) independent experiments and 4 replicates each experiment. *, P < 0.05; **, P < 0.01 RvD2 versus vehicle controls (unpaired Student’s t test). (D) GPR18 expression. Human whole blood (50 µl), isolated PMN (10 6 cells), or GM-CSF–differentiated MΦ (10 6 cells) were incubated with rabbit anti-GPR18 IgG or nonimmune rabbit IgG (1:50 dilutions, 30 min), followed by PE-anti-rabbit IgG (1:200 dilutions, 30 min). GPR18 expression was monitored by flow cytometry. Results are representative of 4 independent experiments using 4 separate healthy donors. Results with whole blood and isolated PMN were obtained from the same donor.

Article Snippet: For antibody incubations, anti-GPR18 Ab (Imgenex) or nonimmune rabbit IgG was incubated with cells in the ECIS chambers at 1:50 dilutions for 30 min before addition of compounds.

Techniques: Control, Comparison, Expressing, Isolation, Incubation, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Identification of resolvin D2 receptor mediating resolution of infections and organ protection

doi: 10.1084/jem.20150225

Figure Lengend Snippet: RvD2-dependent activation of GPR18. (A) Dose response. CHO-GPR18 cells were incubated with RvD2 (1–100 nM) or vehicle alone (control). Impedance changes across CHO cell monolayers were continuously recorded in real-time for 10 min (inset). Representative histograms of GPR18 expression. (B) Ligand specificity. Tracings were CHO-GPR18 cells incubated with RvD2 or NAGly (100 nM each; chemical structures depicted on the right). Time 0 denotes the addition of compounds. (C) CHO-GPR18 cells were incubated with anti-GPR18 Ab (1:50) or nonimmune rabbit IgG for 30 min, followed by addition of 100 nM RvD2. (D) CHO-GPR18 cells were treated with CTX (1 µg/ml, 2 h) or PTX (1 µg/ml, 16 h) followed by addition of 100 nM RvD2. Results are expressed as (A and B) changes in impedance (Ω); mean of 4 separate tracings from 4 independent experiments or (C and D) percentage of changes in impedances. RvD2-initiated impedance changes were taken as 100%; mean ± SEM from 4 separate tracings from 4 independent experiments; **, P < 0.01; ***, P < 0.001 versus RvD2 plus nonimmune IgG (C) or RvD2 alone (D) using one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: For antibody incubations, anti-GPR18 Ab (Imgenex) or nonimmune rabbit IgG was incubated with cells in the ECIS chambers at 1:50 dilutions for 30 min before addition of compounds.

Techniques: Activation Assay, Incubation, Control, Expressing, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Identification of resolvin D2 receptor mediating resolution of infections and organ protection

doi: 10.1084/jem.20150225

Figure Lengend Snippet: Human GPR18-mediated RvD2 actions in MΦ phagocytosis. (A) Human GPR18 was knocked down with GPR18 shRNA in human MΦ. MΦ (0.4 × 10 6 cells) were incubated with indicated concentrations of RvD2 for 2 min (37°C), and cAMP was measured. Results are mean ± SEM of four separate experiments and duplicates in each experiment. *, P < 0.05 obtained with unpaired Student’s t test for GPR18 shRNA (solid red line) versus control scrambled shRNA (dashed blue line) transfected MΦ. (B) Human MΦ were transfected with human GPR18 (circle) or mock (square) plasmids; 72 h later, MΦ were plated onto chamber (0.1 × 10 6 cells/well) incubated with RvD2 at 10 −9 M (blue) or vehicle control (white) for 15 min at 37°C, followed by addition of BacLight Green-labeled E. coli to initiate phagocytosis. Fluorescent images were then recorded every 10 min for 180 min. (top inset) Percent increase in phagocytosis by RvD2 in mock (white) or GPR18 (blue) transfected MΦ. (bottom) Representative fluorescent images at 180 min. Bars, 50 µm. Three separate experiments were performed. In each experiment, 4 fields (20×) per condition (per well) were recorded. Results are mean fluorescence of four fields/well from one representative experiment. (C and D) Human GPR18 was overexpressed (C) or knocked down with shRNA (D) in human MΦ and verified by flow cytometry (insets). MΦ were incubated with RvD2 (10 −13 to 10 −8 M) or vehicle control for 15 min, followed by addition of FITC-zymosan, BacLight Green-labeled E. coli , or CFDA-labeled apoptotic PMN to initiate phagocytosis. Results are percent increases of phagocytosis above vehicle. (C) Mean ± SEM from 5 or (D) mean ± SEM from 3 independent experiments with separate donors and triplicates in each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001, obtained with unpaired Student’s t test for GPR18 overexpression (solid blue lines) versus mock transfection (dashed red lines) in C and GPR18 shRNA (solid red lines) versus control scrambled shRNA (dashed blue lines) in D. (E and F) GPR18, CD206, and CD163 expression. (E) Human MΦ (0.5 × 10 6 cells) or (F) human MΦ overexpressing GPR18 (GPR18-OE) or mock plasmids were incubated with vehicle or RvD2 (0.1, 1, or 10 nM) for 24 h. GPR18, CD206, and CD163 were monitored using flow cytometry. Results are percent increase above vehicle; mean ± SEM from 3 independent experiments with 3 separate donors. # , P < 0.05; ## , P < 0.01 RvD2 versus vehicle; *, P < 0.05, GPR18 overexpression versus mock transfection using unpaired Student’s t test.

Article Snippet: For antibody incubations, anti-GPR18 Ab (Imgenex) or nonimmune rabbit IgG was incubated with cells in the ECIS chambers at 1:50 dilutions for 30 min before addition of compounds.

Techniques: shRNA, Incubation, Control, Transfection, Labeling, Fluorescence, Flow Cytometry, Over Expression, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Identification of resolvin D2 receptor mediating resolution of infections and organ protection

doi: 10.1084/jem.20150225

Figure Lengend Snippet: RvD2 in vivo actions were enhanced by overexpression and reduced by knockdown of GPR18. (A–C) Naive peritoneal MΦ were collected and transfected ex vivo (10 6 cells) with either GPR18 (5 µg) or mock plasmids for 48 h. Zymosan (1 mg) was injected into peritoneum to initiate peritonitis. 12 h later, transfected MΦ (1.5 × 10 5 /mouse) and/or RvD2 (10 ng) was injected i.p. (A) Timeline. (B) PMN numbers (Ly6G + CD11b + ) and (C) efferocytosis (Ly6G + F4/80 + ) were determined using flow cytometry. Results are expressed as mean ± SEM from 2 independent experiments and 6 mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001, versus zymosan (zym) alone. # , P < 0.05, ## , P < 0.01, versus zym+veh+RvD2 (one-way ANOVA with Dunnett’s multiple comparison test). § , P < 0.05, versus zym+MΦ-mock+RvD2 (unpaired Student’s t test). (D–F) Naive peritoneal MΦ (10 6 cells) were transfected ex vivo with either GPR18 shRNA (5 µg) or control-scrambled shRNA. Zymosan (1 mg) was injected to initiate peritonitis. 12 h later, transfected MΦ (2 × 10 5 /mouse) and/or RvD2 (20 ng) was injected i.p. Inflammatory exudates were collected at 24 h. (D) Timeline. (E) PMN numbers (Ly6G + CD11b + ) and (F) efferocytosis (Ly6G + F4/80 + ) were determined using flow cytometry. Results are expressed as mean ± SEM from 2 independent experiments and 6 mice/group. *, P < 0.05; **, P < 0.01, obtained with unpaired Student’s t test for vehicle versus RvD2 in MΦ + control shRNA group.

Article Snippet: For antibody incubations, anti-GPR18 Ab (Imgenex) or nonimmune rabbit IgG was incubated with cells in the ECIS chambers at 1:50 dilutions for 30 min before addition of compounds.

Techniques: In Vivo, Over Expression, Knockdown, Transfection, Ex Vivo, Injection, Flow Cytometry, Comparison, shRNA, Control

Journal: The Journal of Experimental Medicine

Article Title: Identification of resolvin D2 receptor mediating resolution of infections and organ protection

doi: 10.1084/jem.20150225

Figure Lengend Snippet: [ 3 H]-RvD2 isolation and specific binding with human recombinant GPR18. (A and B) [10,11 3 H]-RvD2-ME characterization and isolation. (A) HPLC chromatographs of [10,11 3 H]-RvD2-ME and unlabeled RvD2-ME co-injection. (B) Chromatographic and radioactive tracing; [10,11 3 H]-RvD2-ME (solid line) and radioactivity (dashed line). (insets) Structure and online UV spectra of RvD2-ME and [10,11 3 H]-RvD2-ME. Results are representative of 5 separate experiments. (C–F) CHO cells were transfected with human GPR18. (C) Saturation binding. GPR18-transfected CHO cells (0.5 × 10 6 cells in 100 µl PBS 2+ ) were incubated with [ 3 H]-RvD2-ME at indicated concentrations in the presence or absence of 10 µM unlabeled RvD2-ME for 60 min at 4°C. Bound and unbound radioligands were separated by filtration, and specific binding was determined. Results are representative of 4 independent experiments and 2 replicates in each experiment. (D) Displacement binding . CHO-GPR18 cells (0.5 × 10 6 cells) were incubated with 3 nM of [ 3 H]-RvD2-ME. 1 h later, 1 µM of unlabeled RvD2-ME (square) was added to displace radioligand binding (denoted by an arrow). Radioactivity was determined at indicated time points. Results are representative of 3 independent experiments and 2–3 replicates in each experiment. (E) Competition binding. CHO-GPR18 cells (0.5 × 10 6 cells) were incubated with 3 nM of [ 3 H]-RvD2-ME in the presence or absence of increasing concentrations of unlabeled RvD2-ME (circle) or RvD2 (square) for 60 min at 4°C. (inset) Specific [ 3 H]-RvD2-ME (3 nM) binding on CHO-GPR18 and CHO-WT cells in the absence or presence of 1 µM of RvD2-ME. (F) Ligand specificity. CHO-GPR18 cells (0.5 × 10 6 cells) were incubated with 3 nM of [ 3 H]-RvD2-ME in the absence or presence of 100 nM of RvD2-ME, RvD2, NAGly, RvD1, RvD3, MaR1, or PD1 for 60 min at 4°C. (E and F) Results are mean ± SEM from 4 (RvD2-ME, RvD2, NAGly, RvD1) or 2 (RvD3, MaR1, PD1) independent experiments and 2 replicates in each experiment; *, P < 0.05, compared with incubations with cells and [ 3 H]-RvD2-ME in the absence of competing unlabeled compound (one-way ANOVA with Tukey’s multiple comparison test).

Article Snippet: For antibody incubations, anti-GPR18 Ab (Imgenex) or nonimmune rabbit IgG was incubated with cells in the ECIS chambers at 1:50 dilutions for 30 min before addition of compounds.

Techniques: Isolation, Binding Assay, Recombinant, Injection, Radioactivity, Transfection, Incubation, Filtration, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Identification of resolvin D2 receptor mediating resolution of infections and organ protection

doi: 10.1084/jem.20150225

Figure Lengend Snippet: Targeted deletion of mouse gpr18 delays resolution of E. coli infection. (A) Targeted deletion of mouse gpr18 (NM_182806) was constructed by insertion of bGeo/Puro gene into the coding region of gpr18 (left). Mice tails were collected, genomic DNA was isolated, and PCR was performed using primers specific for KO construct (right). (B–G) GPR18-deficient mice (white) and WT littermates (black) were inoculated with E. coli (10 5 CFU) by i.p. injection, and peritoneal exudates were collected at indicated time points. (B) PMN numbers were determined, and resolution indices were calculated (see Materials and methods). WT (black) and GPR18-KO (white). (C) In vivo efferocytosis (F4/80 + Ly-6G + ), (D) intracellular E. coli levels in PMN (Ly-6G + CD11b + E. coli + ) or monocytes (Ly-6G − CD11b + E. coli + ), and (E) PMN apoptosis (Ly-6G + Annexin V + ) were monitored by flow cytometry. MFI, mean fluorescence intensity. (F) d -series resolvins, protectins, and prostanoids in 24 h infectious exudates. (G) Representative MS/MS spectra of RvD2 and PD1. (B–F) Results are expressed as mean ± SEM from 2 independent experiments and 6–7 mice/group. *, P < 0.05; **, P < 0.01, obtained with unpaired Student’s t test for GPR18-KO versus WT.

Article Snippet: For antibody incubations, anti-GPR18 Ab (Imgenex) or nonimmune rabbit IgG was incubated with cells in the ECIS chambers at 1:50 dilutions for 30 min before addition of compounds.

Techniques: Infection, Construct, Isolation, Injection, In Vivo, Flow Cytometry, Fluorescence, Tandem Mass Spectroscopy

Journal: The Journal of Experimental Medicine

Article Title: Identification of resolvin D2 receptor mediating resolution of infections and organ protection

doi: 10.1084/jem.20150225

Figure Lengend Snippet: RvD2-dependent protection is diminished with GPR18 deficiency in mice. (A–E) E. coli peritonitis; GPR18-deficient mice and WT littermates were inoculated with E. coli (10 5 CFU). 100 ng RvD2 was given by i.p. injection 12 h after E. coli inoculation, and peritoneal exudates collected at indicated time points. (A–C) PMN numbers were determined and resolution indices were calculated. E. coli alone (white), E. coli plus RvD2 (black). Results are expressed as mean ± SEM from 2 independent experiments with 4–5 mice/group (for 4-, 12-, and 48-h time points), or 3 independent experiments with 7–8 mice/group (for 24-h time point). *, P < 0.05; ***, P < 0.001, using unpaired Student’s t test for RvD2 versus vehicle group at 24 h. (D) In vivo efferocytosis (F4/80 + Ly-6G + ) and (E) PMN apoptosis (Ly-6G + Annexin V + ) were monitored by flow cytometry. Results are expressed as mean ± SEM from 2 independent experiments and 5 mice/group; *, P < 0.05; ***, P < 0.001, using unpaired Student’s t test for RvD2 versus vehicle group at 24 h. (F) Mouse peripheral blood was collected from WT (circle) and GPR18 KO (square) mice, incubated with RvD2 (10 −9 –10 −6 M) or vehicle for 15 min, followed by addition of BacLight Green-labeled E. coli for 2 h. RBCs were lysed, and fluorescence associated with phagocytes monitored by flow cytometry. Results are expressed as mean ± SEM from 2 independent experiments, 4 mice/group. *, P < 0.05, using unpaired Student’s t test for WT versus GPR18-KO. (G) Peritoneal MΦ were collected from naive WT (circle) and GPR18 KO (square) mice and incubated with RvD2 (10 −13 – 10 −8 M) or vehicle for 15 min, followed by addition of FITC-zymosan to initiate phagocytosis. Results are mean ± SEM from 2 independent experiments, 4 mice/group and 4 replicates for each experimental condition. *, P < 0.05; **, P < 0.01 using unpaired Student’s t test for WT versus GPR18-KO. (H and I) S. aureus skin infection. Murine dorsal pouches were raised in GPR18-KO mice and WT littermates for 6 d. Live S. aureus (10 5 CFU) was given together with RvD2 (200 ng) or vehicle by intra-pouch injection, and pouch exudates were collected at 4 h. (H) Bacterial counts (CFU/ml) and (I) exudate PMN numbers were determined. Results are expressed as mean ± SEM from 2 independent experiments and 6–8 mice/group. *, P < 0.05, using unpaired Student’s t test for RvD2+ S. aureus versus S. aureus alone.

Article Snippet: For antibody incubations, anti-GPR18 Ab (Imgenex) or nonimmune rabbit IgG was incubated with cells in the ECIS chambers at 1:50 dilutions for 30 min before addition of compounds.

Techniques: Injection, In Vivo, Flow Cytometry, Incubation, Labeling, Fluorescence, Infection

Journal: The Journal of Experimental Medicine

Article Title: Identification of resolvin D2 receptor mediating resolution of infections and organ protection

doi: 10.1084/jem.20150225

Figure Lengend Snippet: I/R injury. RvD2 decreases PMN-mediated lung injury in WT but not GPR18-deficient mice. Mice were subjected to hind limb ischemia (60 min). RvD1, 100 ng RvD2, or vehicle control (0.1% ethanol in saline) was then administered i.v., followed by reperfusion (2 h). Mice were sacrificed and lung was collected. (A) Lung tissue histology. Hematoxylin and eosin (H&E) staining of I/R lungs. Bars, 50 µm. (B) Lung PMN infiltration was quantified by myeloperoxidase (MPO). Results are MPO values (nanogram/milligram lung tissue); mean ± SEM from 2 independent experiments and 5–7 mice/group. *, P < 0.05; **, P < 0.01, treatment (RvD1 or RvD2) versus I/R alone in WT group. #, P < 0.05 versus I/R alone; &, P < 0.05 versus RvD2 in GPR18-KO group using one-way ANOVA with Tukey’s multiple comparison post-test.

Article Snippet: For antibody incubations, anti-GPR18 Ab (Imgenex) or nonimmune rabbit IgG was incubated with cells in the ECIS chambers at 1:50 dilutions for 30 min before addition of compounds.

Techniques: Control, Saline, Staining, Comparison