OGR1-dependent regulation of the allergen-induced asthma phenotype

Chemicals and Reagents

Lorazepam was purchased from Sigma (St. Louis, MO), and sulazepam was purchased from Specs (Delft, The Netherlands). House dust mite (HDM) extract (Dermatophagoides pteronyssinus; Der p) was purchased from Greer Labs (Lenoir, NC). Alexa Fluor-488 conjugated secondary antibodies for immunofluorescence studies were obtained from Life Technologies (Carlsbad, CA). Sircol collagen assay kit was purchased from Biocolor Life Sciences (Carrickfergus, UK). Hematoxylin and eosin (H&E) staining kit was purchased from Vector Laboratories (Burlingame, CA). Periodic acid-Schiff (PAS) staining kit was obtained from Sigma. Acid fuchsin orange G (AFOG) stain was prepared by our laboratory.

Murine Model of Allergic Asthma

All animal procedures were approved by the Institutional Animal Care Committee of Thomas Jefferson University, Philadelphia, PA. All methods were performed in accordance with the guidelines and regulations of the institution.

Male and female wild-type C57BL/6 mice (8–10 wk old) and age-matched OGR1KO mice (C57BL/6 background) developed previously (16) were intranasally challenged 5 days a week for three consecutive weeks with 25 µg of HDM extract in 35 µL of phosphate-buffered saline (PBS; Fig. 1). A subset of mice was also administered with either lorazepam (3 mg/kg), sulazepam (3.2 mg/kg), or vehicle (10% vol/vol DMSO) in 25 µL volume via intranasal route ∼30 min before HDM challenge. Choice of drug dose was based on: 1) drug solubility, 2) our previous cell-based studies (11, 14, 15) that demonstrate micromolar concentrations of lorazepam and sulazepam to maximally activate OGR1 in ASM, and 3) the desire to limit systemic/off-target effect of the drugs. Although administration of both drugs was followed by a brief period of lethargy in mice, we did not observe any mortality, morbidity, or overt signs suggestive of untoward systemic effects. Aerosol challenges in animals were performed under isoflurane (2%–5%) anesthesia. Effects of acute administration of lorazepam or sulazepam in the sensitization model were not tested. Twenty-four hours following the final challenge, lung function measurements were performed, followed by collection of bronchoalveolar lavage (BAL) fluid by perfusing the lungs with 500 µL ice-cold PBS. At the end of the experiment, lungs were filled with 10% formalin and harvested, or fresh lung tissues were frozen in liquid nitrogen for histopathological and biochemical analyses.

Measurement of Lung Mechanics

Lung mechanics were measured using a flexiVent system (Scireq, Montreal, Canada) using methods described previously (8, 17). Animal surgeries were performed under tribromoethanol (Avertin, 250 mg/kg) anesthesia. Briefly, mice were anesthetized with tribromoethanol and the trachea was intubated with a cannula. Mice were ventilated with a tidal volume of 250 μL at 150 breaths/min. A computer-controlled positive end-expiratory pressure (PEEP) of 3 cmH₂O was used for all studies. For data acquisition, mice were subjected to increasing doses of nebulized methacholine (MCh) using a standardized protocol and low-frequency forced oscillation technique. Airway resistance (R) was calculated based on fitting the respiratory mechanical input impedance (Zrs) derived from displacement of ventilator’s piston and the pressure in the cylinder to the constant phase model normalized to mouse body weight.

Assessment of Bronchoalveolar Cellularity

Following lung function measurements, BAL fluid was collected from tracheotomized mice as described earlier. BAL samples were centrifuged at 2,000 rpm for 3 min, and cell pellet was resuspended in 1 mL of Roswell Park Memorial Institute medium (containing 5% fetal bovine serum). Total cell count in BAL samples was determined by hemocytometer and data were expressed as cells/mL. The BAL cells were pelleted onto microscopic slides and subsequently stained with Hema-3 staining kit (Fisher Scientific, Hampton, NH), and differential cell count was determined using a bright-field microscope.

Assessment of BAL Cytokine and Chemokine Profiles

A panel of cytokines and chemokines were measured in BAL fluid by Multiplex LASER Bead Technology (Eve Technologies, Calgary, Canada) using a 31-Plex mouse cytokine/chemokine array (Cat. No. MD31). The concentration of each cytokine was determined by extrapolation from a standard curve and expressed as pg/mL.

Histopathological Evaluation of Lung Tissue

Paraffin-embedded tissues were used for histological evaluation as described previously (17) using 5-µm sections mounted on Superfrost Plus slides. The lung tissues fixed in 10% formalin were embedded in paraffin, cut, and stained with hematoxylin and eosin (H&E), acid fuchsin orange G (AFOG), and periodic acid-Schiff (PAS) using a standard histological protocol. Image acquisition was performed using a bright-field microscope. Airway wall thickness was graded on a scale of 0–3 with 0, no change; 1, mild; 2, moderate; and 3, severe. Scores were reported following blinded and independent evaluation of AFOG-stained tissue sections by two investigators.

Immunofluorescence-based detection of smooth muscle α-actin was conducted using methods described previously (17). Slides were deparaffinized, rehydrated, and subjected to heat-induced epitope retrieval. The sections after blocking were stained with the primary antibody (1:100 anti-sm-α-actin) at 4°C overnight, followed by staining with 1:250 goat anti-mouse Alexa Fluor-conjugated secondary antibody at room temperature for 1 h. The sections were washed in PBS and stained with Draq5 for 15 min. Finally, the sections were washed, mounted with Prolong Antifade (Molecular Probes, Eugene, OR), and fluorescent imaging was performed using an Olympus BX-51 fluorescent microscope. Quantitative estimation of peribronchial sm-α-actin staining was performed using Image J and reported as the ratio of integrated intensity of sm-α-actin staining to peribronchial area.

Preparation of Lung Tissue Protein Lysates

Murine lung lobes were excised, cut into small pieces, and suspended in 250 μL RIPA lysis buffer (Cell Signaling Technology, Danvers, MA) containing protease and phosphatase inhibitors (Bimake, Houston, TX). Lung tissues were homogenized using a handheld homogenizer. The lysate was centrifuged (1,000 g, 10 min) and the supernatant was stored at −80°C for further analysis.

Immunoblotting

Protein concentration of lung tissue lysates was determined using Pierce bicinchoninic acid (BCA) Assay kit (Thermo Fisher Scientific, Rockford, IL) and subjected to immunoblot analysis using standard methodologies. Blots were probed with primary antibodies to smooth muscle myosin heavy chain (sm MHC; 1:1,500; AbCam, Cambridge, MA), smooth muscle alpha actin (sm α-actin; 1:1,000; AbCam), and β-actin (1:100,000; Sigma). IRDye-conjugated anti-mouse and anti-rabbit secondary antibodies (LI-COR Biosciences, Lincoln, NE; 1:15,000) were applied to the blots as secondary antibodies. Immunoblots were scanned and immunoreactive bands were quantified using the Odyssey infrared imaging system and software (LI-COR Biosciences).

Soluble Collagen Assay

Soluble collagen (resulting from active inflammation) was measured in lung lysates obtained from murine lung using Sircol Soluble Collagen Assay (Biocolor, UK) according to the manufacturer’s protocol and as described previously (18). This assay allows for quantitative estimation of acid- and pepsin-soluble collagen species. Lung lysate was mixed with Sircol dye reagent allowing collagen to bind to the dye and precipitate. After acid-salt wash, alkali reagent was used to release collagen-bound dye into solution and absorbance was measured at 550 nm. A standard curve was established using reference standards provided in the assay kit.

Statistical Analysis of Data Sets

All data sets are represented as means ± SE. The statistical significance was determined using one-way or two-way ANOVA with Bonferroni’s post hoc multiple comparisons test, with P < 0.05 being sufficient to establish significant difference between groups. All statistical analyses were performed using Prism8 software (GraphPad, San Diego, CA).

Sulazepam but Not Lorazepam Modulates HDM-Induced Airway Hyperreactivity in Mice in an OGR1-Dependent Manner

Repeated treatment of mice (WT and OGR1KO) with benzodiazepines (lorazepam or sulazepam; PBS treatment serving as a control), in conjunction with PBS or HDM treatment, did not adversely affect their growth and mice exhibited no apparent changes in health or behavior. Following cessation of the dosing schedule after 3 wk, mice were anesthetized to assess changes in airway resistance in response to MCh challenge. Repeated challenge of WT mice with HDM for 3 wk increased the airway resistance in response to increasing doses of MCh (6.125–25 mg/mL) compared with PBS-challenged animals (Fig. 2A). OGR1KO mice undergoing HDM treatment demonstrated a similar increase in airway hyperresponsiveness (AHR; Fig. 2B). Concomitant treatment with lorazepam failed to inhibit the development of AHR in both WT and OGR1KO mice. In contrast, sulazepam treatment significantly (P < 0.05) inhibited the development of AHR in WT mice, but not in OGR1KO mice. These data suggest that sulazepam can effectively inhibit development of increased airway resistance in a murine model of allergen-induced asthma. However, sulazepam treatment did not protect against development of HDM-induced AHR in OGR1KO mice, suggesting the effect of sulazepam is mediated via OGR1.

Lorazepam and Sulazepam Do Not Mitigate HDM-Induced Allergic Airway Inflammation in Mice

Given that allergen-induced AHR was inhibited in mice by treatment with sulazepam in an OGR1-dependent manner, we next sought to determine the mechanism(s) underlying this effect. First, we examined if treatment with benzodiazepines and particularly sulazepam had any effect on HDM-induced airway inflammation. In WT and OGR1KO mice challenged repeatedly with HDM for 3 wk, a comparable increase in BAL cellularity was observed (Fig. 3). In WT mice (Fig. 3A), following treatment with lorazepam and sulazepam, the total cellular influx in airways was not reduced and similar trends were noted for the number of lymphocytes and eosinophils in the BAL fluid. Treatment of OGR1KO mice with either benzodiazepine did not inhibit the total cellular influx in the airways resulting from HDM challenge (Fig. 3B). Somewhat surprisingly, treatment of OGR1KO mice with sulazepam significantly increased HDM-induced accumulation of eosinophils in BAL fluid. Finally, treatment of WT or OGR1KO mice with either benzodiazepine had no significant effect on levels of eotaxin-1, IL-4, IL-6, IP-10, Cxcl1, and leukemia inhibitory factor (LIF) induced by HDM challenge and recovered in the BAL fluid (Fig. 4). For all other cytokines measured using the multiplex system, the levels were below the threshold of detection (data not shown). Collectively, these data indicate benzodiazepines lack any mitigating effect on multiple indices of airway inflammation, suggesting that sulazepam’s ability to inhibit AHR development was not mediated via regulation of airway inflammation.

Effect of Inhalation of Benzodiazepines on Allergen-Induced Lung Tissue Inflammation and Airway Remodeling Features

Next, we sought to examine if sulazepam or lorazepam had any effect on the lung tissue inflammation or histological changes in lung architecture induced by repeated inhalation of HDM. Examination of H&E-stained lung tissue sections from WT and OGR1KO mice that were repeatedly challenged with HDM demonstrated typical histopathological features including pleocellular, peribronchial, and perivascular inflammation (Fig. 5A) and mucus cell metaplasia (Fig. 5B). Consistent with our assessment of lung inflammation evident in BAL fluid, in both WT and OGR1KO mice, treatment with sulazepam and lorazepam did not result in any changes in HDM-induced infiltration of immune cells in the lung tissue (Fig. 5A). Furthermore, treatment with either benzodiazepine failed to inhibit HDM-induced goblet cell metaplasia in either mouse genotype (Fig. 5B).

Next, we examined the effect of lorazepam or sulazepam on features of airway remodeling (AR) induced by HDM challenge (Figs. 6 and 7). Repeated inhalation of HDM resulted in increased collagen staining along the epithelial basement membrane and overall thickening of the airway wall in lungs from both strains of mice (Fig. 6A). However, microscopic examination of histological sections from sulazepam-treated WT mice revealed no changes in accumulation of collagen around the airways. Lorazepam treatment had no effect on increased accumulation of collagen in lung by HDM challenge. In OGR1KO mice, we observed accumulation of collagen around airways upon HDM challenge and no changes with lorazepam and sulazepam treatment. Furthermore, AFOG-stained slides were scored to estimate the airway wall thickness, which increased in mice following challenge with HDM and was unchanged in mice treated with sulazepam or lorazepam (Fig. 6B). To quantify the difference in collagen accumulation between the treatment groups, we performed Sircol Collagen Assay. In WT mice, we observed that treatment with sulazepam (but not lorazepam) significantly reduced the HDM-induced increase in soluble collagen in lungs (Fig. 6C). Baseline collagen levels in PBS-treated OGR1KO lungs were higher but were not significantly increased by HDM challenge. Furthermore, sulazepam and lorazepam treatment did not modulate soluble collagen levels in OGR1KO mice.

We next examined ASM mass by staining for smooth muscle alpha actin (sm α-actin) using an immunofluorescence approach. Sm α-actin increased in animals challenged with HDM and was unchanged in sulazepam- or lorazepam-treated mice (Fig. 7A). Imaging and quantitative analysis revealed significant increase in peribronchial sm α-actin staining in mice repeatedly challenged with HDM, which was unchanged in sulazepam- and lorazepam-treated mice (Fig. 7, A and B). To further quantify changes in ASM mass, we examined the effect of HDM challenge and lorazepam/sulazepam treatment on the expression of smooth muscle markers, sm α-actin and smooth muscle myosin heavy chain (sm MHC), in murine whole lung lysates. Western blotting and subsequent quantitative analysis revealed no significant changes in expression of sm α-actin and sm MHC in lungs of animals challenged with HDM and those concomitantly treated with sulazepam or lorazepam (Fig. 7, C and D).