Maresin 1 activates LGR6 receptor promoting phagocyte immunoresolvent functions
Nan Chiang, Stephania Libreros, Paul C. Norris, Xavier de la Rosa, Charles N. Serhan
Nan Chiang, Stephania Libreros, Paul C. Norris, Xavier de la Rosa, Charles N. Serhan
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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 7

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

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In vivo knockdown of mouse LGR6 reduces MaR1 actions in limiting PMN and...
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.