Targetng the Endocannabinoid System to Enhance Innate Immunity Using Chemoproteomics

摘要

The role of serine hydrolases (SHs) in the endogenous cannabinoid (eCB) system is important in innate immunity because the actvites of these enzymes are responsible for 2-arachidonoylglycerol (2-AG) bio-synthesis and degradaton and can be altered during microbial infecton. To profle alteratons in SH actvites during pathogenic challenge, we analyzed Salmonella Typhimurium-infected chicken HD-11 macrophages using either gel- or liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based actvity-based protein profling (ABPP) approaches. We also determined whether the SHs responsible for endocannabinoid (2-AG) hydrolysis in HD-11 cells can be inhibited by small-molecule inhibitors and whether the elevaton of eCB levels results in enhanced phagocytc actvity. In additon, bioactve lipids (i.e. 2-AG, anadaminde (AEA), prostaglandins and prostaglandin glyceryl esters) in the macrophages were analyzed using LC-MS/MS. A total of 28 SHs were identfed in the HD11 cells using the LC-MS/MS-based ABPP approach; most of the identfed SH actvites appeared to be downregulated afer Salmonella infecton (at 18 h or 24 h) compared with SHs in uninfected controls. Among them, alpha, beta-hydrolasedomain 6 (ABHD6) and faty acid amide hydrolase (FAAH), which are known to catabolize eCBs, were decreased signifcantly at 18 h infecton. A small-molecule inhibitor, JZL184, was discovered to selectvely inhibit ABHD6 and FAAH actvites in intact cells, and the inhibitor increased the level of 2-AG in cells as compared to that in vehicle-treated cells due to inactvaton of 2-AG hydrolysis. In additon, inactivating the metabolism of endocannabinoids with JZL184 augmented the phagocytc actvity of HD11 macrophages and enhanced their microbicidal actvity. Together, the results suggest that inhibiton of endocannabinoid degradaton may augment the innate immune response to Salmonella Typhimurium infecton.