Ultramicronized PEA (powder particle size <10 μm, with the following distribution: <6 μm, 99.9%; <2 μm, 59.6%; <1 μm, 14.7%; <0.6 μm, 2%, as described in patent EP2475352 A1, with text from patent WO2011027373A1) was kindly provided by Epitech Group (Saccolongo, Italy). All other reagents and fine chemicals were obtained from Sigma–Aldrich (Milan, Italy).

The animal studies are reported in accordance with the ARRIVE guidelines for reporting animal research (Kilkenny et al., 2010). Female BALB/c mice (18 ± 2 g body weight, Charles River, Calco, Italy) were housed in a controlled environment (21 ± 2°C) and provided with standard rodent chow and water. All animals were allowed to acclimate for 4 days prior to experiments and were subjected to 12 h light – 12 h dark schedule. Experiments were conducted during the light phase. The experimental procedures, according to Italian (DL 26/2014) and European (n. 63/2010/UE) regulations on the protection of animals used for experimental and other scientific purposes, were approved by the Italian Ministry (Number 1092/2015).

Mice were injected subcutaneously (s.c.). with 0.4 mL of a suspension containing 100 μg of OVA absorbed to 3.3 mg of aluminium hydroxide gel on days 1 and 8 (OVA-sensitized mice), while control animals received an equal volume of vehicle (Figure 1A) (Roviezzo et al., 2007, 2015; Sullo et al., 2013). Mice were sacrificed at 15 days after OVA administration to take the bronchi and blood to evaluate PEA levels and metabolism.

In another set of experiments PEA (10 mg/kg; OVA+PEA) (Impellizzeri et al., 2013; Capasso et al., 2014) or vehicle (saline, ETOH and Tween-20, 8:1:1, by volume; OVA) were administered intraperitoneally (i.p.) 15 min before each OVA administration. Mice were euthanized 15 (Figure 5A) and 22 (Figure 3A) days after OVA sensitization and main bronchi and lungs were collected and processed as described below.

Main bronchi and blood were harvested from mice, 15 days after OVA or vehicle administration, and immediately immersed into liquid nitrogen and stored at -80°C until extraction of PEA. PEA was extracted from 100 μL of plasma by liquid–liquid extraction and separated by liquid chromatography (Ultimate 3000 RS, Dionex, CA, United States). Quantitative analyses were performed on a 5500 QTrap^® triple quadrupole/linear ion trap (QqQLIT) mass spectrometer equipped with a TurboIon-Spray TM interface (AB Sciex, Concord, ON, Canada).

Tissues were extracted with chloroform/methanol (2:1, by volume) containing 10 pmol of d4-PEA (provided by Cayman Chemicals, United States). The lipid extracts were purified by silica column chromatography and the fractions containing PEA were analyzed by isotope dilution liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (LC-MS). Results were expressed as picomoles per milligram of tissue (Borrelli et al., 2015).

The bronchi from mice treated with vehicle (control group) or OVA were removed (15 days after the administration of OVA or vehicle), collected in RNA later (Invitrogen, Carlsbad, CA, United States) and homogenized by a rotorstator homogenizer in 1.5 mL of Trizol^® (Invitrogen). Total RNA was purified, quantified, characterized and retrotranscribed as previously described (Grimaldi et al., 2009). For all samples tested, the RNA integrity number (Bioanalyzer 2100, Agilent) was greater than eight relative to a 0–10 scale. Quantitative real-time PCR was performed by an iCycleriQ5^® (Bio-Rad, Milan, Italy) in a 20 μL reaction mixture as described. Assays were performed in quadruplicate (maximum ΔCt of replicate samples <0.5), and a standard curve from consecutive fivefold dilutions (100–0.16 ng) of acDNA pool representative of all samples was included for PCR efficiency determination. Optimized primers for SYBR Green analysis and optimum annealing temperatures were designed by the Allele-Id software version 7.0 (Biosoft International, Palo Alto, CA, United States) and were synthesized (HPLC purification grade) by MWG-Biotech (Ebersberg, Germany). For each target, all mRNA sequences at http://www.ncbi.nlm.nih.gov/gene/ were aligned and common primers were designed. Relative expression calculation, correct for PCR efficiency and normalized with respect to reference genes β-actin and hypoxanthine-guanine phosphoribosyltransferase, was performed by the iQ5 software. Results are expressed as fold expression (Romano et al., 2016). Statistical significance was evaluated by the by the REST 2009 software (Pfaffl et al., 2002).

Main bronchi, collected from mice 22 days after sensitization, were rapidly dissected and cleaned from fat and connective tissue. Rings of 1–2 mm length were cut and mounted in 2.5 mL isolated organ baths containing Krebs solution, at 37°C, oxygenated (95% O₂ and 5% CO₂), and connected to an isometric force transducer (type 7006, Ugo Basile, Comerio, Italy) associated to a Powerlab800 (AD Instruments). Rings were initially stretched until a resting tension of 0.5 g was reached and allowed to equilibrate for at least 30 min during which tension was adjusted, when necessary, to a 0.5 g and bathing solution was periodically changed. In each experiment bronchial rings were previously challenged with acetylcholine (10^-6 M) until a reproducible response was obtained. Subsequently, after tissue washing, a cumulative concentration response curve to carbachol (10^-9 – 3 × 10^-6 M) was performed. Carbachol–induced contractions were also measured in presence of PEA 10^-5 M (Romano and Lograno, 2012), administered in the organ bath 30 min before carbachol. Results were expressed as dine per mg tissue.

In another set of experiments OVA-sensitized mice were sacrificed at 15 days to take pulmonary tissues and blood for biochemical studies and IgE evaluation, respectively. Plasma IgE levels were measured by means of ELISA using matched antibody pairs (BD Pharmingen, Franklin Lakes, NJ, United States). Each lung was divided into two parts. One part was frozen at -80°C and subsequently homogenized for cytokine and LTC₄ measurements by ELISA, and the other was fixed in 10% neutralized buffered formalin for histological analysis. Levels of cytokines and LTC₄ were expressed as pg/mg of tissue.

Lung sections were cut (7 μm thick) and stained with H&E for morphological analysis. Mast cell degranulation was evaluated by toluidine staining, following the method described by Iuvone et al. (1999) with some modifications. In brief, it was calculated the percentage of red purple/violet stained cells, i.e., degranulated mast cells, on the total number of mast cells, per mm². Non-degranulated mast cells appeared dark blue stained. Sections were analyzed by blinded operators using a standard light microscope (20× magnification, for H&E staining, and 40 and 100× magnification, for toluidine blue staining) and photographed under low power. Images were taken by a Leica DFC320 video-camera (Leica, Milan, Italy) connected to a Leica DM RB microscope using the Leica Application Suite software V.4.1.0.

Mice, sensitized as described above, received on days 9 and 12 on the shaved dorsal surface, 2.5 mL s.c. of air to initiate the development of the air-pouches as described previously (Das et al., 1997) (Figure 4A). On day 15 (6 days after the first air injection) animals were challenged by injection into the air-pouch with 0.4 mL of sterile saline alone (control group) or containing 10 μg OVA. At different time-points (2 h or 24 h) after OVA or saline injection into the air-pouch, mice were sacrificed by exposition to CO₂. Air-pouches were washed with 1 mL phosphate-buffered saline (pH = 7.4). Lavage fluids were centrifuged at 300 × g for 10 min at 4°C. Supernatants were then collected and stored at -80°C until assayed for IL-4 and IL-13 evaluation by ELISA kits according to manufacturer’s instructions. Levels were expressed as pg/mL. Cell pellets were suspended in phosphate-buffered saline and total cell counts were performed by optical microscopy following Trypan blue staining.

Data are expressed as mean ± SEM of n observations, were n represents the number of animals. Statistical analysis has been performed by using one- or two-tailed Student T-test, one- or two-way ANOVA for multiple comparisons followed by Bonferroni’s post test (GraphPad Prism 5.0 software; San Diego, CA, United States). Data were considered statistically significant when a value of at least p < 0.05 was achieved.

Increasing evidence suggest that mast cells critically contribute to sensitization mechanisms, but very little is known about endogenous molecules and mechanisms capable of modulating mast cell activation. We have already established that mast cell activation during allergen sensitization is responsible for significant changes in bronchial reactivity (Roviezzo et al., 2015). Although it has been demonstrated that endogenous PEA levels are reduced an inflammatory environment (Capasso et al., 2001; De Filippis et al., 2010; Bandiera et al., 2014), the level of reduction has not been formally tested in airways. To demonstrate the extent to which allergic sensitization decreases PEA levels, BALB/c mice were sensitized and PEA levels were measured in bronchi harvested from vehicle- (control) and OVA-treated mice (Figure 1A). In bronchi harvested from control mice the amount of PEA was about 6 pmol/mg of wet weight tissue. Following OVA sensitization a significant reduction of PEA levels occurred in the bronchi (Figure 1B). Conversely PEA plasma levels remained unaltered by OVA sensitization (Figure 1C).

Direct or indirect (via effects on endogenous ligand levels or receptor expression) molecular targets for PEA include PPAR-α, CB₁ and CB₂ receptors, and GPR55 (De Petrocellis et al., 2004; LoVerme et al., 2005; Ryberg et al., 2007; Ho et al., 2008; Pertwee et al., 2010). RT-PCR analysis performed on bronchi harvested from both control and OVA-treated mice showed that CB₂ (Figure 2A) and GPR55 (Figure 2B) were up-regulated by OVA sensitization, while CB₁ (Figure 2C) and PPAR-α (Figure 2D) were unchanged.

Palmitoylethanolamide is biosynthesized “on demand” from membrane phospholipids (Cadas et al., 1996; Cravatt et al., 1996; Ueda et al., 2013) and has been proposed to behave as a local autacoid mediator able to down-regulate mast cell activation and inflammation. In order to correlate down-regulation of bronchial PEA to an altered reactivity, we performed functional experiments on bronchi obtained from sensitized mice treated with PEA (10 mg/kg) or with the vehicle (saline, ETOH and Tween-20, 8:1:1,v/v) 15 min before each OVA administration (Figure 3A). Bronchi excised from OVA-sensitized mice showed a significant increased reactivity to carbachol, compared to control group and to PEA–treated group (Figure 3B).

In another set of experiments, we added PEA in the organ bath and evaluated its effect on carbachol-induced contractions of bronchi harvested from both control and sensitized-mice. In this experimental set up PEA (10 μM) did not exert any effect on bronchial reactivity to carbachol (Figure 3C). Thus, PEA effect on bronchial tone is not mediated by stromal cells but its targets might be expressed by infiltrating immune cells.

It is a commonly held view that the disordered airway physiology and airway remodeling characteristics of asthma are consequences of airway inflammation and cell infiltration that is typically eosinophilic. In order to assess the role of PEA on eosinophil infiltration we chose the model of allergen-induced eosinophil extravasation into mouse air-pouches (Figure 4A) (Das et al., 1997; Rossi et al., 2016). Specifically, air pouch provides a convenient cavity from which cells and mediators can be easily harvested. Injection of OVA into the air pouch of sensitized mice, as described above, provoked an intense allergen-dependent leukocyte infiltration, mainly eosinophilic, as early as 6 h after OVA challenge, with a peak at 24 h (Das et al., 1997). A significant inhibition of cell recruitment triggered by allergen challenge was observed followed pre-treatment with PEA (10 mg/kg; 15 min before OVA administration) (Figure 4B). This effect was coupled to PEA ability to prevent OVA-induced increase of both IL-4 and IL-13 cytokine levels (Figures 4C,D).

Since bronchial hyperreactivity is closely related to airway inflammation and PEA displayed a significant inhibition of allergic inflammation, we also evaluated the effect of PEA (10 mg/kg, i.p.; 15 min prior each OVA injection) on the development of inflammation in the lung of allergen sensitized mice. For this reason, mice were sacrificed 15 days following sensitization and pulmonary sections underwent morphological and biochemical analysis (Figure 5A). The data obtained demonstrate that PEA prevented pulmonary inflammation; indeed, H&E staining showed reduced peribronchial and alveolar septal inflammatory cell infiltration in the lung of sensitized mice pre-treated with PEA compared to lung sections obtained from OVA-sensitized mice (Figure 5B). In order to assess if the beneficial actions of PEA were ascribed to an inhibitory effect on sensitization mechanisms, plasma IgE levels were quantified. The data obtained evidenced that treatment with PEA during sensitization period did not cause any change in plasma IgE levels (Figure 5C), but prevented the increase in IL-13 and IL-4 pulmonary levels following sensitization (Figures 5D,E).

Another key process central in asthma is mast cell recruitment and their localization to different structural components of the airway wall (Galli et al., 2005; Galli and Tsai, 2012). Normal airway epithelium exerts an inhibitory effect on the activity of mast cells, which is subsequently lost in asthma when the epithelium is damaged. As highlighted by toluidine staining (Figure 6), OVA sensitization significantly increased mast cell recruitment into the lung, as well as mast cell degranulation (Figures 7A,B), effects that well correlate with reduced PEA availability (Figure 1B). Conversely, PEA administration (10 mg/kg, i.p.; 15 min prior each OVA injection) prevented both mast cell recruitment and degranulation (Figures 6, 7A,B). LTC₄ is recognized as the main mediator released by mast cells in allergic asthma; we found that PEA also prevented the increase in LTC₄ pulmonary levels observed in sensitized mice (Figure 7C).