Dos_2026_JDS.Commun_7_284

The endocannabinoid system (ECS) is involved in regulating immune functions in leukocytes. An inflammatory stimulus, specifically LPS, can modulate the activity of endocannabinoids (eCB) receptors, and eCB within the leukocytes can further exert either pro- or anti-inflammatory effects on the immune function of these cells. The effects of exogenous eCB on the cellular inflammatory responses of bovine leukocytes are largely unexplored; therefore, we aimed to evaluate the effects of incubation with the main eCB N-arachidonoylethanolamide (AEA/anandamide) or 2-arachidonyglycerol (2-AG) on gene expression of eCB and inflammatory mediators following an ex vivo LPS challenge in dairy cow leukocytes. To this end, whole blood from mid-lactation dairy cows (n = 6) were subjected to ex vivo incubation with eCB (control [CTL], AEA at 0.29 microM, or 2-AG at 0.26 microM) for 2 h, followed by stimulation with or without LPS (10 ng/mL) for an additional 2 h. Overall, there were 6 treatments for cells from each cow: CTL, AEA, and 2-AG without LPS stimulation, and CTL+LPS, AEA+LPS, and 2-AG+LPS. Subsequently, RNA was extracted from leukocytes and assayed for gene expression levels via real-time quantitative PCR. First, we examined the main effects of LPS stimulation across eCB treatments: LPS decreased expression of the eCB receptors cannabinoid receptor 2 (CNR2), G protein-coupled receptor 55 (GPR55), and peroxisome proliferator-activated receptor gamma (PPARG). Furthermore, LPS increased expression of the eCB enzymes N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD) and monoglyceride lipase (MGLL) while reducing the expression of diacylglycerol lipase B (DAGLB) compared with non-LPS-stimulated groups. Then, we examined the main effects of eCB treatments on gene expression: incubation with AEA increased expression of cannabinoid receptor 1 (CNR1) in leukocytes, whereas 2-AG increased the expression of the CNR2 and tended to increase GPR55. In addition, 2-AG increased the expression of fatty acid amide hydrolase (FAAH) and tended to increase the expression of NAPEPLD. As expected, LPS increased the expression of inflammatory markers; however, incubation with eCB had no discernible effects on these genes. Taken together, ex vivo exposure of dairy cow leukocytes to AEA or 2-AG, with or without stimulation with LPS, resulted in differential effects on the expression of eCB receptors and enzymes, but we did not detect effects of exogenous eCB on the expression of inflammatory genes following an LPS challenge. The findings of the present study provide the first reductionist step in understanding the relationship between the ECS and inflammatory responses in immune cells of dairy cows. The complexity of the regulation of immune function in leukocytes, and its potential interplay with eCB, requires further studies to comprehensively elucidate the cellular mechanisms underlying these responses.