An Important Role for N-Acylethanolamine Acid Amidase in the Complete Freunds Adjuvant Rat Model of Arthritis

F T Bonezzi,F Bertozzi,O Sasso,N Realini,S Pontis,S Ponzano,E Romeo,D Piomelli,A Fiasella,A Nuzzi

doi:10.1124/jpet.115.230516

Abstract

The endogenous lipid amides, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), exert marked antinociceptive and anti-inflammatory effects in animal models by engaging nuclear peroxisome proliferator-activated receptor-α. PEA and OEA are produced by macrophages and other host-defense cells and are deactivated by the cysteine amidase, N-acylethanolamine acid amidase (NAAA), which is highly expressed in macrophages and B-lymphocytes. In the present study, we examined whether a) NAAA might be involved in the inflammatory reaction triggered by injection of complete Freund's adjuvant (CFA) into the rat paw and b) administration of 4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]-carbamate (ARN726), a novel systemically active NAAA inhibitor, attenuates such reaction. Injection of CFA into the paw produced local edema and heat hyperalgesia, which were accompanied by decreased PEA and OEA content (assessed by liquid chromatography/mass spectrometry) and increased NAAA levels (assessed by Western blot and ex vivo enzyme activity measurements) in paw tissue. Administration of undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl] carbamate (ARN14686), a NAAA-preferring activity-based probe, revealed that NAAA was catalytically active in CFA-treated paws. Administration of ARN726 reduced NAAA activity and restored PEA and OEA levels in inflamed tissues, and significantly decreased CFA-induced inflammatory symptoms, including pus production and myeloperoxidase activity. The results confirm the usefulness of ARN726 as a probe to investigate the functions of NAAA in health and disease and suggest that this enzyme may provide a new molecular target for the treatment of arthritis.

Highlights

Macrophages and other cellular components of the body’s host-defense system produce a variety of lipid-derived mediators that are involved in the promotion of inflammation
As a direct test of this idea, in the present study we examined whether NAAA might be involved in the inflammatory reaction elicited by complete Freund’s adjuvant (CFA) in ABBREVIATIONS: ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl] carbamate; ARN726, 4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate; CFA, complete Freund’s adjuvant; DRG, dorsal root ganglia; fatty acid ethanolamides (FAE), fatty acid ethanolamide; H&E, hematoxylin and eosin; LPS, lipopolysaccharide; MPO, myeloperoxidase; NAAA, N-acylethanolamine acid amidase; OEA, oleoylethanolamide; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; PEA, palmitoylethanolamide; PPAR-a, peroxisome proliferator-activated receptor-a; (S)-OOPP, N-[(3S)-2-oxo-3oxetanyl]-3-phenylpropanamide; Trisbuffered saline (TBS), Tris-buffered saline
We found that administration of CFA into the paw of rats causes a substantial decrement in the tissue content of PEA and OEA, as previously shown in other animal models of inflammation (Capasso et al, 2001; De Filippis et al, 2009; Solorzano et al, 2009) and in patients suffering from rheumatoid arthritis (Richardson et al, 2008)

Summary

Introduction

Macrophages and other cellular components of the body’s host-defense system produce a variety of lipid-derived mediators that are involved in the promotion of inflammation. Recent experiments have shown that the compound 4-cyclohexylbutylN-[(S)-2-oxoazetidin-3-yl]-carbamate) (ARN726)—a systemically active b-lactam-based NAAA inhibitor—suppresses both lung inflammation in mice, via a mechanism that requires PPAR-a activation, and endotoxin-induced responses in human macrophages (Ribeiro et al, 2015). These results suggest that NAAA inhibition might offer a novel mechanistic approach to treat human inflammatory conditions, such as rheumatoid arthritis and osteoarthritis, in which the levels of PEA and OEA are markedly reduced (Richardson et al, 2008)

Methods

Results

Conclusion