Effects of the monoacylglycerol lipase inhibitor JZL184 on chickens infected with avian pathogenic Escherichia coli O78: A preliminary pharmacokinetic and infection study

Abstract

The endocannabinoid (eCB) system modulates the degree of injury caused by inflammation, while enhancing the activity of phagocytes that promote resolution of inflammation and tissue repair. In-vitro studies with the monoacylglycerol lipase (MAGL) inhibitor JZL184 have suggested that increased eCB signaling might enhance the ability of the host immune system to clear invading pathogens. Although the neurochemical effects of JZL184 on the eCB system in rodents are well-known, its immuneregulating effects are less clear, especially in chickens. The primary objective of this study was to explore whether modulating the eCB system affects immune responses in chickens. To do this, we administered JZL184 [10 and 40 mg/kg body weight (BW), intraperitoneal injection] into chickens prior to a challenge with avian pathogenic Escherichia coli (APEC) O78. Bacteria were isolated from livers, blood, air sacs, and hearts at 8, 28, and 56 h post-infection and the gross lesions in air sacs, livers, and hearts were also examined. Serum levels of JZL184 were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which indicated that the drug was distributed systemically. The number of birds positive for airsacculitis after APEC O78 challenge was marginally higher in groups treated with JZL184 than in the control group (P = 0.064). Rather than augmenting host defense and enhancing pathogen clearance, these results suggested that JZL184 might have immunosuppressive effects that exacerbated APEC O78 infection in chickens.

Figure 1

A — Schematic of the study design. The experiment started with injection of 0, 10, or 40 mg/kg body weight (BW) of JZL184 to 5-week-old chickens intraperitoneally. Intratracheal E. coli challenge of 1 × 10⁸ CFU APEC O78 was administered to the various groups 4 h after JZL184 injection. Necropsies and sample collections were done at 3 different time points (8, 28, and 56 h) post-infection. B — Serum JZL184 levels in chickens after 10 mg/kg BW and 40 mg/kg BW intraperitoneal injections. Chicken blood serum was collected at 8, 28, and 56 hpi and was analyzed for JZL184 by LC-MS/MS, as described in the materials and methods section. Area under the curve (AUC) values for the low- and high-dose treatments were 224.4 nM · h and 744.7 nM · h, respectively. Symbols represent the mean ± SD (n = 6 biological replicates).

Figure 2

A — Activity-based protein profiling (ABPP) gel analysis of chicken serum. Increasing amounts of serum (0 to 10 μL) were treated with fluorophosphonate (FP)-biotin for 30 min at room temperature before stopping the reaction. The quenched reaction products were separated by PAGE and the blotted proteins were probed with avidin-horseradish peroxidase (HRP). The band detected at 60 kDa in each sample represents an active serum carboxylesterase (CES) enzyme that is covalently modified by the FP-biotin probe (as shown). The diffuse nature of the CES band on the gel is likely due to the heterogeneity caused by glycosylation of this soluble enzyme. B — Potency of JZL184-mediated inhibition of CES activity in naive chicken serum (chemical structure of JZL184 is shown). JZL184 at final concentrations of 0, 0.1, 1, and 10 μM was preincubated with 5 μL of undiluted chicken serum for 30 min at 37°C. The residual CES enzyme activity was then measured at 405 nm for 5 min after addition of para-nitrophenyl valerate (p-NPV), as described in the materials and methods section. Dimethyl sulfoxide (DMSO) was the solvent vehicle used for JZL184. Data represents the mean ± SD of at least 3 technical replicates. The IC₅₀ value was determined by fitting the data to a hyperbola function (r² = 0.99). C — CES activities in the 8-hour and 28-hpi serum of the different groups described in Figure 1A were determined using the substrate p-NPV. Data represents the mean ± SD of n = 6 biological replicates, each carried out with at least 2 technical replicates. No significant differences were noted when the data was assessed by 1-way ANOVA.

Figure 3

Occurrence of airsacculitis induced with 10⁸ CFU E. coli O78. Chickens from each group were euthanized at 8, 28, and 56 h post-infection (hpi). The percentage of birds in each group with gross lesions in air sacs (airsacculitis) is indicated at each time point. Symbols represent the proportion of birds exhibiting gross lesions in air sacs (n = 5 biological replicates, vehicle control group, 56 hpi; n = 6 biological replicates, vehicle control group, 8 and 28 hpi; n = 6 biological replicates, JZL184 treatment groups, all time points; n = 3 biological replicates, negative control group, all time points). Data were analyzed using Fisher’s exact test.

Figure 4

Concentrations of interleukin-1 beta (IL-1β) in chicken serum were measured using an ELISA assay. Levels of IL-1β in serum from the vehicle control, JZL184 treatment [10 mg/kg body weight (BW)], JZL184 treatment (40 mg/kg BW), and negative control groups are given at 8, 28, and 56 h post-infection (hpi). Symbols represent the mean ± SD (n = 5 to 6 biological replicates). *indicates a significant difference (P < 0.05) when the vehicle- and 10-mg/kg BW JZL184-treated groups were compared (1-way ANOVA).