Endocannabinoid hydrolases in avian HD11 macrophages identified by chemoproteomics: inactivation by small molecule inhibitors and pathogen-induced downregulation of their activity

Authors: ROSS, MATTHEW - Mississippi State University; LEE, J - Mississippi State University; HOU, X - Jiangxi Academy Of Agricultural Sciences; KUMMARI, E - Mississippi State University; BORAZJANI, A - Mississippi State University; EDELMANN, M - University Of Florida

Submitted to: Molecular and Cellular Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/23/2017
Publication Date: 1/1/2017
Citation: Ross, M.K., Lee, J.H., Hou, X., Kummari, E., Borazjani, A., Edelmann, M.J. 2017. Endocannabinoid hydrolases in avian HD11 macrophages identified by chemoproteomics: inactivation by small molecule inhibitors and pathogen-induced downregulation of their activity. Molecular and Cellular Biochemistry. http://doi.org/10.1007/s11010-017-3237-0.
DOI: https://doi.org/10.1007/s11010-017-3237-0

Interpretive Summary: Macrophages are immune cells that activate the biosynthesis of toxic molecules, including cytokines and lipids, for the purpose of killing foreign organisms that invade the host. The endocannabinoid (eCB) system influences host-pathogen interactions. The eCB system is comprised of several components including two G-protein coupled receptors (CB1 and CB2); arachidonoyl-containing ligands, 2-arachidonoylglycerol (2-AG) and anandamide (AEA); and enzymes responsible for their biosynthesis and inactivation. eCBs regulate host defense by stimulating innate immune cells to fight bacteria and viruses. Strategies that increase eCB levels might significantly improve host defense against pathogens. 2-AG levels in an organism are determined by the balance of synthesis and degradation rates, which are regulated by specific enzymes. Our hypothesis was that the eCB system has an important role in strengthening innate immunity. The objective of this study was to identify serine hydrolases (enzymes) in macrophages that can be targeted (i.e., inhibited) by small molecules for the purpose of increasing eCB levels during microbial infection, and to establish whether the microbicidal activity of macrophages is concomitantly enhanced. Our results showed that two serine hydrolases (ABHD6 and FAAH) in chicken macrophages are involved in 2-AG degradation and could be inactivated by irreverisible inhibitors (e.g., JZL184). Moreover, 2-AG levels were increased following treatment of cultured macrophages with JZL184 and that immune functions of the macrophages were augmented by pre-treatment with the inhibitor. These results have implications for host-pathogen interactions research.

Technical Abstract: The endocannabinoids (eCBs) are endogenous arachidonoyl-containing lipid mediators with important roles in host defense. Macrophages are first-line defenders of the innate immune system and biosynthesize large amounts of eCBs when activated. The cellular levels of eCBs are controlled by the activities of their biosynthetic enzymes and catabolic enzymes, which include members of the serine hydrolase (SH) superfamily. The physiologic activity of SHs can be assessed in a class-specific way using chemoproteomic activity-based protein profiling (ABPP) methods. Here, we have examined avian (chicken) HD11 macrophages, a widely used cell line in host–pathogen research, using gel-based ABPP and ABPP-multidimensional protein identification technology (MudPIT) to profile the changes in SH activities under baseline, chemical-inhibitor-treated, and pathogen-challenged conditions. We identified a/ß-hydrolase domain 6 (ABHD6) and fatty acid amide hydrolase (FAAH) as the principal SHs responsible for 2-arachidonoylglycerol (2AG) hydrolysis, thereby regulating the concentration of this lipid in HD11 cells. We further discovered that infection of HD11 macrophages by Salmonella Typhimurium caused the activities of these 2AG hydrolases to be downregulated in the host cells. ABHD6 and FAAH were potently inhibited by a variety of small-molecule inhibitors in intact live cells, and thus these compounds might be useful host-directed adjuvants to combat antimicrobial resistance in agriculture. 2AG was further shown to augment the phagocytic function of HD11 macrophages, which suggests that pathogen-induced downregulation of enzymes controlling 2AG hydrolytic activity might be a physiological mechanism to increase 2AG levels, thus enhancing phagocytosis. Together these results define ABHD6 and FAAH as 2AG hydrolases in avian macrophages that can be inactivated pharmacologically and decreased in activity during Salmonella Typhimurium infection.