Novel anti-inflammatory--pro-resolving mediators and their receptors

Abstract

Resolution of inflammation, an actively coordinated program, is essential to maintain host health. It involves effective removal of inflammatory stimuli and the spatio-temporal control of leukocyte trafficking as well as chemical mediator generation. During the active resolution process, new classes of small, local acting endogenous autacoids, namely the lipoxins, D and E series resolvins, (neuro)protectins, and maresins have been identified. These specialized pro-resolving lipid mediators (SPM) prevent excessive inflammation and promote removal of microbes and apoptotic cells, thereby expediting resolution and return to tissue homeostasis. As part of their molecular mechanism, SPM exert their potent actions via activating specific pro-resolving G-protein coupled receptors. Together these SPM and their receptors provide new concepts and opportunities for therapeutics, namely promoting active resolution as opposed to the conventionally used enzyme inhibitors and receptor antagonists. This approach may offer new targets suitable for drug design for treating inflammation related diseases, for the new terrain of resolution pharmacology.

Figure 1

Structures of LXA₄, ATL, ATLa (A), RvE1, RvD1, PD1 (B) and their tritiated radioligands.

Figure 2

Identification of RvE1 receptor. (A) Functional screening for RvE1 receptors. HEK293 cells cotransfected with pNF-κB-luciferase and pcDNA3-GPCRs were exposed to RvE1 (10 nM) and TNF-α (inset) Phylogenetic tree representing amino acid sequence similarities between the human LXA₄ receptor (ALX), leukotriene B4 receptor (BLT1) and related GPCRs. (B) Receptor dependence. RvE1 inhibits luciferase activity in a concentration-dependent manner on cells transfected with ChemR23 but not mock transfected cells. (C) Ligand specificity. Cells transfected with pcDNA3-ChemR23 were exposed to 100 nM of each compound. RvE1 showed ~60%, while EPA and 18-HEPE do not have inhibition of NF-κB luciferase activity,

Figure 3

[³H]-RvE1 binding to ChemR23. Saturation binding. Human ChemR23–transfected CHO cells (10⁶ cells) were incubated with indicated concentrations of [³H]-RvE1 in the presence or absence of 10 μM of unlabeled RvE1. (Inset) Scatchard plot.

Figure 4

(A) Tissue distribution of human ChemR23 determined by dot blot hybridization (B) Mechanisms of action for RvE1. In a ChemR3-dependent manner, RvE1 activates MAPK in monocytes, rS6 phosphorylation in macrophages, and reduces IL-12 production and migration in dendritic cells. RvE1 also induces expression of an anti-adhesive molecule CD55 on the apical surface of mucosal epithelium, promoting clearance of PMN across mucosal surface. In addition, RvE1 directly interact with BLT1 on human PMN, inhibiting calcium mobilization, NF-κB activation in vitro and PMN infiltration in vivo. Therefore RvE1 gave multi-level and cell-type specific actions, serving as an agonist for ChemR23 on mononuclear and dendritic cells as well as an antagonist for BLT1 signals on PMN.

Figure 5

[³H]-RvD1 binding to human PMN. (A) Saturation binding. Human PMN (5 × 10⁶ cells) were incubated with indicated concentrations of [³H]-RvD1 in the presence or absence of 10 μM of unlabeled RvD1. (Inset) Scatchard plot. (B) Competition binding. Human PMN were incubated with 10 nM of [³H]-RvD1 in the presence of 10 μM of LXA₄, or Ac2-12 peptide. Results are expressed as percent competition of [³H]-RvD1–specific binding.

Figure 6

(A) Ligand-dependent receptor activation for monitoring receptor ligand interactions. This system is engineered by stably co-expressing the target GPCR (ALX or GPR32) tagged with the β-gal Pro-Link peptide with β-arrestin linked to the β-gal EA fragment. In the presence of ligand, activated GPCR interacts with β-arrestin, bringing to proximity the EA and Pro-Link fragments, forming a functional enzyme, whose activity can be measured by adding the substrate and generating a chemiluminescent signal. (B) Dose response activation curves of LXA₄ and compound 43 with β-arrestin cells stably overexpressing GPR32. Results are mean ± SEM (n = 4 – 8). (C) Dose response activation curves of LXA₄ and compound 43 with β-arrestin cells stably overexpressing ALX. Results are mean ± SEM (n = 4 – 7). (RLU – Relative Luminiscence Unit).