Maresin 1 activates LGR6 receptor promoting phagocyte immunoresolvent functions
Nan Chiang, … , Xavier de la Rosa, Charles N. Serhan
Nan Chiang, … , Xavier de la Rosa, Charles N. Serhan
Published October 28, 2019
Citation Information: J Clin Invest. 2019;129(12):5294-5311. https://doi.org/10.1172/JCI129448.
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Research Article Inflammation

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.

Authors

Nan Chiang, Stephania Libreros, Paul C. Norris, Xavier de la Rosa, Charles N. Serhan

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Figure 1

MaR1 candidate receptors.

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MaR1 candidate receptors. (A) A panel of orphan GPCRs was screened in th...
(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.