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.

Introduction

Due to the overuse of antimicrobial agents (AMAs) and the resulting antibiotic resistance in animals and humans, there is an urgent need for alternative AMA therapeutics and growth promoters to maintain livestock health and production in the poultry industry (1). Chickens in the poultry broiler industry are reared under intensive conditions where they are often exposed to opportunistic pathogens, such as Escherichia coli O78, which is responsible for about half the cases of avian colisepticemia. In this condition, the virulent strain of the bacteria adheres to epithelial tissues in the airway and invades the trachea (2).

In general, livestock with clinical and subclinical infections eat less, grow poorly, and have higher feed conversion ratios (3). Several vaccines, including live-attenuated vaccines, have been developed for chickens that provide long-term immunity against infectious diseases (4). However, the rise of multiple virulent pathogen strains still poses a serious threat to animal and human health (5,6). Although reducing the use of antibiotics in poultry husbandry tackles the resistance issue, this in turn can negatively affect poultry production and profitability (7). Restrictions on the use of AMAs in the United States have therefore prompted the food animal industry to develop innovative yet safe therapeutic measures to protect livestock health and increase production.

The discovery that the endogenous endocannabinoid (eCB) system is an important regulator of physiological homeostasis has spawned the development of novel therapies to treat inflammatory diseases (8–10). The eCB system plays an important role in regulating immune responses in animals (11). The cannabinoid receptors, types 1 and 2 (CB1 and CB2), are G-protein coupled receptors that engage the endogenous-signaling lipids, 2-arachidonoylglycerol (2-AG) and anandamide (AEA), thereby activating an extensive range of physiological processes and immune cell functions (12). As in mammalian species, chickens also express CB1 and CB2 receptors (13). 2-AG and AEA are lipid mediators that are produced on demand from membrane phospholipid precursors in an activity-dependent manner, i.e., they are not preformed and stored in vesicles, then released when stimulated.

Due to their hydrolytic lability, both 2-AG and AEA are thought to exhibit short-range autocrine and paracrine activities on cells rather than long-range endocrine functions. Furthemore, 2-AG was shown to be a full agonist of the CB2 receptor, which is expressed abundantly in leukocytes, such as lymphocytes and monocytes/macrophages, whereas AEA is only considered to be a partial agonist. Thus 2-AG is considered to be the most important endogenous ligand for the CB2 receptor (14), which is largely responsible for transducing the immunomodulatory effects that eCBs exert on immune cells (15). These include enhanced chemotaxis, phagocytosis, and antimicrobial peptide release by neutrophils and monocytes (14,16).

Inflammatory stimuli have been shown to upregulate the expression of biosynthetic enzymes that produce endocannabinoids and to increase the levels of cannabinoid receptors that interact with them (16). Interestingly, mice treated with bacterial lipopolysaccharide (LPS) exhibited downregulated Ces2g and Ces1d activities in spleen and lung, respectively [(17); unpublished results]. Both these enzymes are capable of catabolizing 2-AG (17,18). These effects suggest that one compensatory homeostatic response to inflammation is to increase the endocannabinoid tone. A therapeutic strategy to augment the homeostatic effects of the eCB system during inflammation is to use small-molecule inhibitors to inactivate the serine hydrolases known to catabolize eCBs. For example, compounds that target catabolic enzymes that degrade 2-AG, such as monoacylglycerol lipase (MAGL), alpha/beta hydrolase domain 6 (ABHD6), ABHD12, and carboxylesterases (CESs), have been developed to increase the local level of eCBs in cells and tissues (19–22). These enzymes hydrolyze 2-AG, producing arachidonic acid and glycerol as products. Among small-molecule inhibitors that inactivate eCB degradative enzymes, JZL184 is one of the most potent and selective inhibitors of MAGL, which is the enzyme primarily responsible for hydrolyzing 2-AG (20). Although several studies using rodent models have unveiled both anti- and pro-inflammatory effects for JZL184 that correlate with increased levels of endogenous 2-AG (23–25), few have examined the therapeutic potential of this drug in chickens in the context of inflammatory disease or host-pathogen response.

In-vitro studies have suggested that the eCB system can modulate innate immune cell functions in avian and mammalian models. For example, exogenous 2-AG was shown to augment the phagocytic activity of cultured chicken macrophages when challenged with heat-killed Staphylococcus aureus (26). In addition, the inactivation of diacylglycerol lipase β (a biosynthetic enzyme responsible for 2-AG biosynthesis) with small-molecule inhibitors was shown to attenuate LPS-induced interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels in mouse macrophages (27). Interleuken-1 beta and TNF-α are important proinflammatory cytokines secreted by macrophages in response to activation of toll-like receptors at sites of local pathogen invasion, thus inducing an inflammatory response (28). These cytokines act by stimulating macrophage and T-lymphocyte functions, resulting in local tissue damage, fever, and loss of function. These findings suggest that the eCB and immune systems are linked together and that an increased eCB tone helps fine-tune innate immune activity, thereby reducing the degree of host tissue injury caused by inflammation, while enhancing the activity of phagocytes that promote resolution of inflammation.

In this study, we hypothesized that systemic treatment of chickens with JZL184 would block the activity of MAGL and lead to localized increases in 2-AG levels, which would in turn reduce the extent of collateral damage in healthy tissue caused by pathogen infection. The specific objective of this study was to investigate whether JZL184-mediated MAGL inhibition would reduce the pathology in air sacs caused by an intratracheal challenge of avian pathogenic E. coli (APEC) O78. In addition, we assessed the bioavailability of JZL184 in chickens after intraperitoneal administration of the drug by assessing its serum pharmacokinetic behavior, as there are no data on the distribution of this drug in chickens. At a more fundamental level, we wanted to know whether JZL184 could be used to activate the eCB system and trigger immunomodulatory effects that augment the innate immune response in an in-vivo inflammation model.

Materials and methods

Animals

Chickens used in this study were hatched from fertile, specific-pathogen-free Leghorn eggs from Charles River Laboratories (Storrs, Connecticut, USA). A total of 63 chickens was reared in negative-pressure isolators in the Biomedical Research building at the College of Veterinary Medicine at Mississippi State University. Animals were under veterinary care with feed and water available ad libitum. Animal experiments were reviewed and approved by the Mississippi State University Institutional Animal Care and Use Committee (IACUC 16-452).

Study design and infection of chickens

The drug JZL184, (4-nitrophenyl) 4-[bis(1,3-benzodioxol-5-yl)-hydroxymethyl]piperidine-1-carboxylate, was purchased from Cayman Chemicals (Ann Arbor, Michigan, USA). Sixty-three White Leghorn chickens were divided into 4 groups [18/treatment group and 9/negative control group; 3 treatment groups and 3 time points (n = 6 chickens/treatment group/time point)]. See Figure 1A for the experimental design. JZL184 was dissolved overnight in a vehicle: polyethylene glycol (PEG) 300:Tween 80 (4:1 v/v), which was described by Long et al (20). Chickens (5 wk old) received low-dose JZL184 [10 mg/kg body weight (BW)], high-dose JZL184 (40 mg/kg BW), or vehicle alone (n = 6 chickens/group). The birds were injected intraperitoneally with drug 4 h before the E. coli challenge. APEC O78 is a field virulent strain isolated from chickens with airsacculitis. The bacteria were freshly cultured overnight in brain-heart infusion (BHI) broth, harvested by centrifugation, washed in phosphate-buffered saline (PBS), and resuspended in PBS before inoculation. The virulence of the APEC O78 strain was tested in a systemic chicken infection model, as described by La Ragione et al (4). Except for those in the negative control group, each chicken was challenged intratracheally with 1 mL of PBS containing 10⁸ colony-forming units (CFUs) of APEC O78 4 h after JZL184 was administered.

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).

LC-MS/MS analysis of JZL184 in chicken serum

Blood was obtained at 8, 28, and 56 hpi from each treatment group, allowed to clot on ice, and centrifuged to obtain serum. A 50-μL aliquot of serum from each time point was added to 100 μL of ice-cold acetonitrile and vortexed. After chilling for 15 min at −20°C, each sample was centrifuged (16 000 × g, 5 min, 4°C) and the supernatant transferred to an LC vial containing a volume-reducing insert for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of JZL184. Ten μL of each sample was injected onto an Acquity UPLC System (Waters, Milford, Massachusetts, USA), coupled to a TSQ Quantum Tandem Mass Spectrometer equipped with an electrospray ionization source (ThermoFisher Scientific, San Jose, California, USA). Chromatographic separation was carried out using an Acquity UPLC BEH C18 Column (2.1 mm × 100 mm, 1.7 μm) (Waters) equipped with a precolumn (2.1 mm × 5 mm, 1.7 μm) at 40°C.

The mobile phases used were water with 0.1% acetic acid (mobile phase A) and acetonitrile with 0.1% acetic acid (mobile phase B). Mobile phase gradient conditions for each run were as follows: after injection, hold at 85% A and 15% B for 0.5 min, linear increase of B to 90% over 20 min, hold at 90% B for 2 min, then decrease B to 15% over 3 min and re-equilibrate at the initial conditions for 5 min. The overall run time and flow rate were 30 min and 0.2 mL/min, respectively. JZL184 was detected in positive-ion mode by selected reaction monitoring (SRM; m/z 503.2 > m/z 199.2). Optimum collision energy and tube-lens conditions were determined by postcolumn infusion of the compound into a 50% A/50% B blend of the mobile phase pumped at a flow rate of 0.2 mL/min and using the autotune software. Xcalibur software was used for data acquisition and processing. Levels of JZL184 in the chicken serum were quantified by comparing its peak area with that of a calibration standard prepared in blank chicken serum.

Preparation of tissue proteomes and protein quantification

Livers and spleens were harvested and stored at −80°C until used. At time of processing, organs were thawed and ~200 mg of tissue was removed and rinsed with 4% w/v potassium chloride (KCl) solution. Glass Dounce homogenizers were used to grind tissues in 1 mL of sucrose buffer [50 mM Tris-hydrochloride (HCl), 0.32 M sucrose, pH 7.4]. The homogenates were centrifuged at 4°C for 30 min at 10 000 × g and the supernatants (post-mitochondrial fraction) were collected and stored at −80°C until used. Protein concentrations of supernatants were diluted 1:20 v/v in water and incubated with Quick Start Bradford Dye Reagent (Bio-Rad, Hercules, California, USA) for 5 min at room temperature. Absorbance of the diluted solution was measured using a microplate reader (595 nm) and compared against a bovine serum albumin (BSA) standard to determine protein concentrations.

Activity-based protein profiling (ABPP) analysis of chicken serum and tissues

Tissue proteomes (at a concentration of 1 mg protein/mL) or serum (2 to 15 μL/sample) were treated with the serine hydrolase activity probe fluorophosphonate (FP)-biotin (Toronto Chemicals, Toronto, Ontario; 8 μM final concentration) in a total volume of 25 μL of 50 mM Tris-HCl (pH 7.4) for 30 to 60 min (room temperature). These probes assess the activity of serine hydrolases by covalently reacting with the catalytic serine residue in the enzyme-active site (Figure 2A). Reactions were quenched by adding an equal volume of 2x SDS-PAGE loading buffer (reducing), then heating the samples at 95°C for 5 min. The probe-labeled proteins were then separated on polyacrylamide (PAGE) gels (4% to 15%; Bio-Rad) and detected using avidin-horseradish peroxidase (HRP) (Sigma Aldrich, St. Louis, Missouri, USA) and chemiluminescent reagents, as described previously (29).

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.

CES activity assay

The activity and potency of the drug, JZL184, was verified in vitro by its ability to inhibit the activity of a 2-AG catabolic enzyme, termed carboxylesterase (CES), found in chicken blood serum. A stock solution of JZL184 was prepared in dimethyl sulfoxide (DMSO) and added to a reaction mixture containing serum to give final drug concentrations of 0, 0.1, 1, and 10 μM. In a 96-well microtiter plate, JZL184 was preincubated with naive chicken serum (5 μL) diluted in 50 mM Tris–HCl (pH 7.4) buffer (total reaction volume of 160 μL) for 30 min at 37°C. A colorimetric substrate for CESs [para-nitrophenyl valerate (p-NPV)] was then added to give a final concentration of 500 μM and the reactions were monitored continuously in a plate reader at 405 nm for 5 min. The slopes of the activity curves were determined to calculate the activity of the CES enzyme in chicken serum. The enzyme activity data (μmol/min/mL serum) were plotted against each JZL184 concentration to estimate the inhibitor potency of the drug. In additional experiments, the CES activities of serum, liver, and spleen homogenates prepared from the 4 experimental groups (as already described in this article) were also assessed using the p-NPV substrate.

Re-isolation and culture of E. coli

At 8, 28, and 56 h post-infection (hpi), 6 chickens per treatment group and 3 per negative control group were euthanized at each time point by exsanguination and bacteria were isolated from the blood, heart, liver, and air sacs of every bird with sterile cotton swabs and plated on MacConkey agar. MacConkey agar plates were incubated at 37°C for 24 h and examined for bacterial colonies (positive versus negative).

Assay of serum IL-1β by ELISA

Blood samples were collected from chicken wing veins. Serum was obtained after centrifugation of the blood and stored at −20°C until assay of the pro-inflammatory cytokine interleukin-1 beta (IL-1β). For quantitative assay of IL-1β in chicken serum, the concentrations of the cytokine in each sample were determined with an enzyme-linked immunosorbent assay (ELISA) kit (MyBiosource, San Diego, California, USA). The double-antibody sandwich ELISA uses chicken IL-1β monoclonal antibody as the pre-coat and biotin-labeled polyclonal antibody as the detecting antibody.

Statistical analysis

The mean ± SEM or SD was determined for each treatment group. Analysis of chicken serum IL-1β levels and enzymatic activities of serum CES in each experimental group were assessed by 1-way analysis of variance (ANOVA). The measure of JZL184 efficacy in vivo was assessed by the comparative protection from clinical colibacillosis, defined as the presence of lesions typical of colibacillosis in liver, heart, or air sacs. Data for the categorical data (± lesions in air sacs) were assessed using the Fisher’s exact test. Statistical significance was defined at P < 0.05.

Results

LC-MS/MS analysis of serum JZL184 levels in APEC-infected chickens

First, we assessed the bioavailability of JZL184 in 5-week-old White Leghorn chickens after a single intraperitoneal injection of drug (all birds were infected with APEC O78). For this purpose, we analyzed the serum samples using LC-MS/MS. The pharmacokinetic profiles of JZL184 in serum after birds were treated with the various doses of JZL184 are shown in Figure 1B. For the 40 mg/kg BW of JZL184 group, the peak level of drug in serum was observed at 28 hpi (32 h after drug delivery) and was decreased significantly by 56 hpi (60 h after drug delivery). For the 10 mg/kg BW of JZL184 group, the peak level of JZL184 was found at 8 hpi (12 h after drug delivery), then a significant decrease was observed by 28 hpi (32 h after drug delivery). Area-under-the-curve (AUC) values for the low- and high-dose treatments were 224.4 nM · h and 744.7 nM · h, respectively. These data indicated that JZL184 had been absorbed systemically into the intraperitoneal cavity after its injection and that it exhibited good bioavailability in chickens.

Off-target effects of JZL184: Assessment of CES enzyme inhibition by JZL184 in vitro and in vivo

JZL184 covalently inactivates the catalytic serine residue in MAGL, releasing para-nitrophenol valerate (p-NPV) as the leaving group (20). Carboxylesterase (CES) is another member of the serine hydrolase superfamily and is found in the serum of most mammals, with humans being a notable exception. It is an off-target of JZL184 (19). To establish whether CES is present in chicken serum, the serum from naive birds was shown to have a 60-kDa CES enzyme that could be labeled by the serine hydrolase probe FP-biotin (Figure 2A). To assess whether JZL184 will also inhibit this CES enzyme, chicken serum was titrated ex vivo with increasing concentrations of JZL184, followed by assessment of residual CES enzymatic activity (Figure 2B). Analysis of the inhibition data showed that the concentration of JZL184 that inhibited 50% of the CES activity (IC₅₀) was 3.0 μM. It is noteworthy that this IC₅₀ is significantly higher than the peak JZL184 concentrations in chicken serum after in-vivo exposure to the drug (Figure 1). This is important because CESs, like MAGL, are also covalently modified by JZL184, releasing p-NPV as the leaving group in the process, thereby causing the drug to be degraded and potentially minimizing its bioavailability. It is therefore unlikely that JZL184 will interact with chicken CES after doses of 10 mg/kg BW or 40 mg/kg BW, at least in the blood circulation. This notion was corroborated by measuring the serum CES activities of birds dosed with JZL184 in vivo. As indicated in Figure 2C, no evidence of serum CES inhibition was noted at 8 and 28 hpi, which are the time points with peak concentrations of JZL184 after doses of 10 mg/kg BW and 40 mg/kg BW, respectively. Thus, it is unlikely that JZL184 in the circulation will be intercepted by CES and degraded. The compound should therefore be available to interact with MAGL expressed in lung and other tissues.

Pathology and microbiology after exposure to APEC O78

The APEC O78 strain produces a systemic infection in chickens that causes airsacculitis, pericarditis, and perihepatitis. Air sacs, hearts, and livers were examined for gross lesions at 8, 28, and 56 h post-infection. The gross lesions caused by E. coli infection are typically marked with endothelial thickening and development of suppurative exudate in the air sacs, focal liver necrosis, and inflammation of the pericardium and production of excess fluid. As shown in Table I, airsacculitis lesions in the APEC O78-challenged chickens were the most commonly observed lesion and formed as early as 8 hpi, while lesions due to perihepatitis and pericarditis were less apparent and developed more slowly.

Table I.

Pathological findings in chickens challenged with APEC O78 with or without JZL184 treatment.

Hours post-infection	Gross lesions (Number positive/total)
	Airsacculitis			Perihepatitis			Pericarditis			Number of lesions in air sac
	8	28	56	8	28	56	8	28	56	8	28	56
10 mg/kg BW JZL184 + E. coli	3/6	5/6	5/6	0/6	0/6	1/6	0/6	0/6	1/6	3/6	1/6	1/6
40 mg/kg BW JZL184 + E. coli	3/6	6/6	6/6	0/6	2/6	0/6	1/6	1/6	0/6	3/6	0/6	0/6
Vehicle control + E. coli	1/6	4/6	3/5	0/6	0/6	3/5	0/6	1/6	1/5	5/6	2/6	2/5
Negative control	0/3	0/3	0/3	0/3	0/3	0/3	0/3	0/3	0/3	3/3	3/3	3/3

Euthanasia and necropsy were done at 8, 28, and 56 h post-infection (hpi). The number of chickens positive for airsacculitis, perihepatitis, and pericarditis was determined. The rates of E. coli occurrence are presented as the number of positive over the total number of birds tested in each group.

BW — body weight.

Our results showed that JZL184 treatment did not attenuate the number of airsaccultis lesions in APEC O78-challenged chickens (Figure 3), which negates our original hypothesis. Surprisingly, although the results did not reach statistical significance due to a limited number of birds, the JZL184 treatment exhibited a trend that suggested it could exacerbate the APEC O78 infection of air sacs compared to that of vehicle-treated APEC O78-challenged chickens (Figure 3; P = 0.064, Fisher’s exact test). The proportion of birds with gross lesions at 8 hpi was < 20% for the vehicle-treated group, whereas the proportion was 50% in both JZL184-treated groups. By 28 hpi, the proportions of infected birds exhibiting gross air sac lesions in the vehicle, 10-mg/kg BW JZL184, and 40-mg/kg BW JZL184 groups had risen to 60%, 80%, and 100%, respectively, and remained at those levels at 56 hpi. The highest dose (40 mg/kg BW) of JZL184 resulted in 100% occurrence of airsacculitis lesions when compared to all other treatment groups. Throughout the study, only 1 chicken, which was a vehicle-treated bird, was found dead due to systemic infection by APEC O78. It was discovered between 28 and 56 hpi and gross postmortem examination revealed that it had developed airsacculitis lesions. However, because it died before the 56 hpi time point, it was removed from the gross lesion analysis shown in Figure 3.

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.

The number of infected birds that were healthy, i.e., without any lesions, varied among experimental treatment groups (Table I). For example, at 56 hpi, 40%, 16.7%, and 0% of birds were deemed to be healthy in the vehicle, low-dose, and high-dose treatment groups, respectively.

In addition to gross lesions, experiments involving re-isolating of E. coli from air sacs suggested that bacteria were eliminated faster from JZL184-treated birds than vehicle-treated birds (shown in Table II). For example, at 8 hpi, a smaller number of chickens in the vehicle-treated group tested positive for E. coli than either JZL184-treated group. But by 56 hpi, an equal or lower number of birds had tested positive for E. coli in the JZL184-treated groups than in the vehicle-treated group.

Table II.

Escherichia coli re-isolation in chickens challenged with APEC O78 with or without JZL184 treatment.

Hours post-infection	Microbiology (Number positive/total)
	Air sac			Liver			Heart			Blood
	8	28	56	8	28	56	8	28	56	8	28	56
10 mg/kg BW JZL184 + E. coli	6/6	4/6	1/6	3/6	1/6	0/6	3/6	1/6	0/6	1/6	0/6	0/6
40 mg/kg BW JZL184 + E. coli	5/6	4/6	2/6	2/6	1/6	0/6	1/6	0/6	0/6	0/6	0/6	0/6
Vehicle control + E. coli	4/6	3/6	2/6	2/6	1/6	1/6	2/6	0/6	1/6	0/6	0/6	0/6
Negative control	0/3	0/3	0/3	0/3	0/3	0/3	0/3	0/3	0/3	0/3	0/3	0/3

At each time point, chickens were euthanized, and the indicated tissues and blood were swabbed and plated on MacConkey agar and incubated at 37°C for 24 h. The number of chickens positive for E. coli was determined and the rates of E. coli occurrence are presented as the number of positive over the total number of birds tested in each group.

BW — body weight.

Serum IL-1β levels after infection

Interleuken-1 beta (IL-1β) is an immune cytokine responsible for coordinating the host inflammatory response against endotoxins released by pathogenic bacteria. We assessed the levels of IL-1β in serum at 12, 32, and 60 h after injection of JZL184 (or 8, 28, and 56 h post-APEC O78 inoculation). It was noted that serum IL-1β levels in the negative control and APEC O78-infected groups (whether treated with JZL184 or not) were similar at all time points (Figure 4). Thus, it was possible that we had missed the induction phase of pathogen-induced IL-1β in serum, which might have occurred earlier than 8 hpi. Alternatively, it is possible that intratracheal administration of APEC O78 did not induce a strong IL-1β response in chicken serum. In line with this, no evidence of septicemia was noted based on the general absence of E. coli that could be re-isolated from blood (Table II). Nevertheless, analysis of serum IL-1β levels was still useful because JZL184 did not appear to modulate IL-1β cytokine levels at time points that coincided with peak serum concentrations of the drug, i.e., 12 and 32 h after drug was given, or 8 hpi and 28 hpi. At 56 hpi, the serum IL-1β level in the low-dose JZL184 group, but not in the high-dose group, was statistically higher than that in the vehicle group.

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).

Discussion

We used a systemic infection model in 5-week-old White Leghorn chickens to evaluate whether the monoacylglycerol lipase (MAGL) inhibitor JZL184 could modulate the pathological effects of avian pathogenic E. coli APEC O78 infection in air sacs, hearts, and livers. Previous unpublished work had shown that intratracheal inoculation of chickens with 10⁸ CFU of APEC O78 caused systemic infection, with gross lesions of airsacculitis, pericarditis, and perihepatitis. In this study, we observed a trend in which JZL184 pretreatment accelerated the progression and proportion of APEC O78-induced lesions compared to those observed in the vehicle control group (Figure 3, P = 0.064). Specifically, post-sacrifice necropsy showed that birds treated with JZL184 exhibited a larger proportion of airsacculitis than the vehicle-treated birds at 8, 28, and 56 h after APEC O78 challenge.

Although the strategy to inactivate eCB-hydrolyzing enzymes such as MAGL was postulated to reduce airsacculitis lesions in E. coli-challenged chickens, this approach appeared to exacerbate them instead. A possible explanation for this observation is that, instead of augmenting the immune response, JZL184 had suppressed the chickens’ immune system, thereby making the drug-treated birds more susceptible to APEC O78 infection. Previous studies have shown that APEC O78 infection of macrophages caused marked cell death to occur (30–32). It is possible that the activation of resident and elicited macrophages due to JZL184-induced eCB levels, i.e., heightened eCB tone, in the air sacs might have exacerbated APEC O78 virulence and subsequent tissue damage. In line with this, signaling through CB2 receptors has been shown to decrease reactive oxygen species, such as superoxide and hydrogen peroxide in cells, which might reduce the levels of microbicidal products, such as hypochlorous acid (HOCl), to suboptimal amounts.

Numerous studies in mammals have shown that JZL184 is a selective and in-vivo active inhibitor of 2-AG degrading enzymes, thereby indirectly affecting immune responses and cytokine levels (33–35). We hypothesized that administration of JZL184 to chickens would increase local eCB levels, i.e., 2-AG, thereby augmenting the host defense. One of the endpoints that we measured to assess host immune response was serum IL-1β levels. This cytokine is produced by innate immune cells in response to pathogens and initiates inflammation by recruiting myeloid cells and enhancing macrophage retention (36). We assessed IL-1β at 3 time points, 8, 28, and 56 hpi. Surprisingly, systemic levels of IL-1β in the APEC O78-challenged birds were not increased relative to those in the negative controls. Whereas the low-dose JZL184 did not alter serum IL-1β levels compared to those in the vehicle group at 8 and 28 hpi, a significant difference was noted between these groups at 56 hpi, which suggests that JZL184 might modulate this cytokine at later time points.

Tumor necrosis factor alpha (TNF-α), IL-6, and IL-1β are important immune mediators that coordinate inflammatory and immune effector responses and induce the hemodynamic changes and cellular lesions that occur in chickens after exposure to pathogens (37–41). A previous study using APEC O78-challenged chickens tested a Chinese herbal formulation called Xiang-qi-Tang, which is known to contain several anti-inflammatory agents (42). Chickens pretreated with this formulation exhibited significantly lower serum IL-1 levels than those in the vehicle group after subcutaneous inoculation with APEC O78 (42).

In our study, there was no clear dose-dependent association between JZL184 treatment and IL-1β levels, and more surprisingly, there was no apparent increase in serum IL-1β levels caused by E. coli infection. It should be pointed out, however, that in our challenge model, the APEC O78 was given by the intratracheal route, whereas He et al (42) used the subcutaneous route. On the basis of cytokine and pathology data, it is possible that the inflammatory response might have begun to subside by 8 hpi (our earliest timepoint). It might be necessary to use earlier time points in order to assess the levels of proinflammatory cytokines in serum after intratracheal APEC O78 challenge and to determine whether JZL184 can affect those levels. For future studies, we suggest collecting more serum samples at time points earlier than 8 hpi to better monitor the rapid innate immune response after APEC O78 challenge.

Information is limited about the effects of JZL184 in chickens and whether it can affect the eCB system in this species, although we previously showed that the compound can increase eCB levels in cultured chicken macrophages (26). Using a chemoproteomic approach, we also identified other enzyme targets that can interact with JZL184, including fatty acid amide hydrolase (FAAH) (26). Chang et al (43) also showed that JZL184 could cross react with both FAAH and CES enzymes. However, our in-vivo results clearly indicate that CES activity in chicken serum was not inhibited after IP administration of JZL184 (Figure 2B). Although we did not assess the potency (IC₅₀) of JZL184 against MAGL in chickens, it would be interesting in the future to compare it with MAGL in other species.

A better understanding of the different types of drug-solubilizing vehicles would also help establish the effectiveness of this therapeutic approach. The solubilizing agents that are used to deliver JZL184 and other similar inhibitors have been shown to influence their efficacy in vivo (35,43,44). Besides the vehicle (PEG 300:Tween 80, 4:1 v/v) that was used in our study, as well as by Long et al (20), saline-based emulsions, such as saline:cremophor:ethanol (18:1:1 v/v/v) and saline:DMSO:Tween 80:PEG 400 (76.5:15:4.25:4.25 v/v/v/v), have also been used to deliver JZL184 (24,44). It is possible that the vehicle we used might have limited the ability of JZL184 to attenuate the APEC O78-mediated infection. Future studies might be required to compare the solubility and effects of different vehicles in order to find the most suitable formulation for safe delivery of JZL184 into chickens.

In conclusion, we showed that JZL184 (at 10 and 40 mg/kg BW) exhibited good pharmacokinetic behavior in chickens, but did not reduce the severity of lesions produced by an intratracheal challenge with 10⁸ CFU of APEC O78. Surprisingly, on the basis of the increased lesions observed in the airsacs, the drug might have augmented pathogen virulence. Generally, no marked differences were noted in IL-1β levels in serum in the experimental groups at each time point, even when the vehicle-treated APEC O78-challenged birds were compared to the negative controls, which suggests that we had missed the induction peak for this cytokine. This pilot study suggests that modulating the eCB system in chickens might cause immunosuppressive effects that enhance APEC O78 virulence in challenge models, although this needs to be confirmed by assessing endpoints that are more sensitive and quantifiable than mere macroscopic lesions and bacterial re-isolation techniques. Despite the limitations of our current study, it does suggest that caution is required when considering the use of therapeutics that interfere with the eCB system in chickens.