RGS4 promotes allergen- and aspirin-associated airway hyper-responsiveness by inhibiting PGE2 biosynthesis

Abstract

BACKGROUND:

Allergens elicit host production of mediators acting on G-protein coupled receptors (GPCRs) to regulate airway tone. Among these is prostaglandin E2 (PGE2), which, in addition to its role as a bronchodilator, has anti-inflammatory actions. Some patients with asthma develop bronchospasm following ingestion of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs), a disorder termed aspirin-exacerbated respiratory disease (AERD). This condition may result in part from abnormal dependence on the bronchoprotective actions of prostaglandin E2 (PGE2).

OBJECTIVE:

We sought to understand the functions of Regulator of G Protein Signaling 4 (RGS4), a cytoplasmic protein expressed in airway smooth muscle (ASM) and bronchial epithelium that regulates activity of GPCRs, in asthma.

METHODS:

We examined RGS4 expression in human lung biopsies by immunohistochemistry. We assessed airways hyper-responsiveness (AHR) and lung inflammation in germline and ASM-specific Rgs4^−/− mice and in mice treated with an RGS4 antagonist following challenge with Aspergillus fumigatus. We examined the role of RGS4 in NSAID-associated bronchoconstriction by challenging AERD-like (ptges1−/−) mice with aspirin.

RESULTS:

RGS4 expression in respiratory epithelium is increased in subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in Rgs4^−/− mice, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation. The RGS4 antagonist CCG203769 attenuated AHR induced by allergen or aspirin challenge of wild type (WT) or ptges1−/− mice, respectively, in association with increased airway PGE2 levels.

CONCLUSIONS:

RGS4 may contribute to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma.

Graphical Abstract

CAPSULE SUMMARY

RGS4 promotes allergen-associated airways hyper-responsiveness by inhibiting production of prostaglandin E2 in lungs and thus may represent a novel therapeutic target in severe asthma.

INTRODUCTION

Asthma is a heterogeneous syndrome that afflicts more than 300 million people worldwide (1). The pathological hallmarks of asthma manifest as lung inflammation, excessive mucus production, airway remodeling (epithelial sloughing, subepithelial collagen deposition, airway smooth muscle hypertrophy), and bronchoconstriction, which induces reversible airflow obstruction, wheezing, cough, and in severe cases, hypoxemia and respiratory failure (2, 3). In more than half of patients with asthma, evidence of exuberant type 2 adaptive and innate immune responses are observed in the lungs, including increased eosinophils and elevated levels of type 2 cytokines (e.g. IL-4, IL-5, and IL-13) (4). Allergens and environmental irritants may also directly activate mast cells and respiratory epithelial cells, which release cytokines including IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), which in turn stimulate lung resident innate lymphoid type 2 cells (ILC2s) (5). Other proinflammatory mediators including bradykinin, histamine, and leukotrienes may also affect bronchial tone (6–8).

Many of these factors induce airway smooth muscle (ASM) shortening by acting on GPCRs bound to Gαq, a cytoplasmic protein that interacts with guanosine diphosphate (GDP) in its quiescent state (9). Ligand binding to the GPCR promotes exchange of guanosine triphosphate (GTP) for GDP on Gαq, a response that in turn evokes a downstream signaling cascade in which cytosolic Ca²⁺ flux promotes actin-myosin interactions and force generation (10). A highly selective antagonist of Gαq elicits profound bronchorelaxation in several species without a substantial impact on allergen-induced inflammation and airway remodeling (11).

Endogenous mechanisms also limit the magnitude of GPCR-mediated responses and modulate bronchial tone. Regulators of G protein Signaling (RGS) proteins restrict heterotrimeric G protein activity by accelerating hydrolysis of GTP by Gα subunits (“GAP” activity) and thereby facilitate Gα inactivation (12). Previously we detected RGS4 immunoreactivity sporadically in cells within the bronchial smooth muscle bundle in lung sections from subjects with asthma and demonstrated that RGS4 knockdown enhanced Ca²⁺ responses of ASM cells from healthy human subjects to histamine ex vivo (13). Here we studied the role of RGS4 in AHR and allergen-associated airway inflammation in mice. We found that Rgs4^−/− mice were protected from developing AHR following allergen or allergen plus aspirin challenge, both findings that could be explained primarily by modification of airway levels of PGE2.

METHODS

Patients

Human lung specimens and clinical parameters were obtained by the Respiratory Health Network Tissue Bank of the Fonds de Recherché Québécois en Santé, IUCPQ site (www.tissuebank.ca). The IUCPQ Ethics Committee approved the collection of tissue samples by the tissue bank and their use for these studies. All subjects gave informed consent for the tissue sampling and future use of samples for research purposes. Subjects with asthma were classified as having mild to moderate or severe asthma based on criteria established by the American Thoracic Society (ATS) (14). All clinical studies and medication dosages were evaluated on the same day or within less than a week from the date of the biopsy.

Allergic airway inflammation model in mice

6–12-week-old male and female mice were sensitized with a mixture of Aspergillus fumigatus (Af) crude extract (20 μg, Hollister-Stier Allergy) mixed with Imject Alum (20 mg, Pierce) via intraperitoneal injection on day 0 and 14. All mice were then challenged intranasally with either PBS (naïve) or Af extract (25 μg) one week after the last immunization. In certain experiments RGS4 inhibitor (6 ng/kg intratracheally) or indomethacin (10 mg/kg i.p.) or an equivalent volume of diluent was administered together with or just prior to allergen. Studies of respiratory function and organ harvest were conducted 24 hours after the last challenge. Lungs were fixed in 10% neutral buffered formalin. Paraffin-embedded sections were analyzed by staining with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). Images were obtained at 10X original magnification using a Leica DMI400 light microscope equipped with an Infinity3 CCD camera (Teledyne Lumenera). PAS staining in the epithelium was determined using the Color Deconvolution plugin of ImageJ and presented as the area of the stained pixels divided by area of each airway. For assessment of leukocyte counts, tracheas were injected with ice cold HBSS and BALF was collected. Red blood cells were hemolyzed using ACK lysis buffer, followed by an additional wash with PBS. Total cell numbers were determined by hemocytometry, and composition of the cells was ascertained by Diff-Quick stain and microscopy (>250 total cells/sample). Cytokines in BALF supernatants were analyzed using a customized MultiPlex bead array (BioRad) in duplicate per the manufacturer’s instructions.

Analysis of lung function in anesthetized mice

Mice were anesthetized with a ketamine (100 mg/kg) and xylazine (20 mg/kg) mixture via i.p. injection followed by transtracheal intubation with a 20-gauge SURFLO Teflon intravenous catheter (Santa Cruz Animal Health). Mice were paralyzed by i.p. injection of vecuronium bromide (1 mg/kg) and mechanically ventilated using the FlexiVent FX1 respirator (SCIREQ). Resistance of the conducting airways (R_n) was measured after inhalation of PBS and increasing concentrations of MCh via nebulizer.

Methods for this paper can be found in the Online Repository.

RESULTS

RGS4 expression in airway epithelium correlates with asthma severity and respiratory dysfunction

To explore the patterns of RGS4 expression in human lung, we examined RGS4 immunoreactivity in lung biopsies from a cohort of subjects diagnosed with asthma of varying severities. The patient characteristics are described in Table E2. RGS4 immunoreactivity was much more extensive in the bronchial epithelium of lungs from subjects with asthma compared to healthy controls, and the extent of coverage increased proportionally with disease severity (Figs. 1A–B). No immunostaining was observed in lung sections probed with rabbit IgG or anti-rabbit IgG controls (Fig. E1). RGS4 immunostaining correlated inversely with lung function (FEV₁) (Fig. 1C) and was also higher in patients treated with inhaled corticosteroids than in those treated with short-acting β-adrenergic agonists (SABA) alone (Fig. 1D). Among those patients treated with steroids, RGS4 immunostaining correlated with dosage requirements for symptom management (Fig. 1E), suggesting a linkage between corticosteroids and RGS4 expression. In a previous study, corticosteroid treatment (dexamethasone) increased RGS2 expression in ASM (15). To examine the impact of dexamethasone on RGS4 quantities in airway epithelial cells (AECs), we applied dexamethasone at concentrations equivalent to those detected in the airways of patients and likewise previously shown to induce RGS2 expression (15). As shown in Fig. 1F, addition of dexamethasone had no impact on RGS4 expression in AECs. This finding suggests that corticosteroids are unlikely to promote expression of RGS4 in the bronchial epithelium. There was also a significant correlation between age of the subjects and RGS4 expression (Fig. E2A). However, as shown in Fig. 1B, RGS4 expression was elevated in subjects with severe asthma compared to those with mild/moderate disease in the absence of any age discrepancies between these two groups. These results indicate that age alone cannot account for the increased RGS4 immunoreactivity detected in respiratory epithelium in severe asthma.

Figure 1. Epithelial RGS4 expression is increased in lungs of patients with asthma.

(A–B) Immunohistochemistry of RGS4 in lung epithelium; *p=0.02, **p=0.008, ****p<0.0001, Kruskal Wallis, Dunn’s multiple comparisons. (C) Negative correlation between RGS4 expression and forced expiratory volume in 1 second (FEV₁). (D) RGS4 expression in patients treated with either inhaled corticosteroids (CCS) or SABA alone; **p=0.003, Mann-Whitney u test. (E) Correlation between RGS4 immunoreactivity and daily dose of inhaled fluticasone or equivalent. (F) RGS4 expression in AECs treated with dexamethasone (1 μM) for the indicated times assessed by immunoblotting. Each symbol represents results of a separate experiment. Non-parametric Pearson correlation coefficients were calculated in C, E.

RGS4 immunoreactivity was similar in patients with allergic and non-allergic asthma (Fig. E2B). To further examine the relationship of RGS4 to allergic inflammation, we assessed expression of MUC5AC, a critical glycoprotein component of airway mucin that is increased in human airways following allergen challenge and required for AHR in mice (16, 17). MUC5AC immunoreactivity in lung epithelium was significantly higher in the group with severe asthma than in subjects with mild/moderate disease or healthy controls (Figs. E2C–D) and correlated strongly with RGS4 expression (Fig. E2E). From these findings we conclude that RGS4 immunostaining in the respiratory epithelium may be a biomarker of asthma severity.

Rgs4^−/− mice have diminished allergen-induced bronchospasm

We localized RGS4 protein expression in the mouse lower respiratory tract by evaluating β-galactosidase (β-gal) activity in lung sections from mice harboring alleles in which Rgs4 was replaced by an in-frame LacZ insertion. As expected, we detected no β-gal activity in naïve wild type (WT) mice; however, we detected prominent β-gal staining in ciliated respiratory epithelial cells and intermittent staining of airway smooth muscle (ASM) cells in lung sections from naïve Rgs4^LacZ/LacZ mice (hereafter referred to as Rgs4^−/−) (Fig. 2A). To determine the mode of action of RGS4 in murine airway inflammation, we subjected wild-type (WT) and Rgs4^−/− mice to sensitization and challenge with extracts derived from the ubiquitous mold Aspergillus fumigatus (Af), a respiratory allergen linked to severe asthma in humans (18). Although total β-gal activity in lungs of Af-challenged mice was similar to that of naïve controls, we detected less activity within the epithelium but increased numbers of β-gal⁺ cells in ASM bundles of allergen-challenged mice (Fig. E3). We next assessed airway tone by invasive measurement of airway resistance in response to the bronchoconstrictor methacholine (MCh). While Rgs4 deletion had no effect on airway resistance PBS-treated mice, Af-challenged Rgs4^−/− mice displayed markedly reduced responses to MCh when compared with responses of Af-challenged WT mice (Fig. 2B).

Figure 2. Diminished allergen-induced airway hyper-responsiveness of Rgs4^−/− mice.

(A) β-galactosidase activity in lungs of naïve WT or Rgs4^LacZ/LacZ mice. Epithelium (black arrowheads) and ASM cells (white arrows) are marked. (B–J) Analysis of lungs from WT and Rgs4^−/− mice as follows: (B) airway resistance (mean ± s.e.m. of 6–12 mice/group analyzed in 2 independent experiments; ****p<0.0001, 2-way ANOVA; (C) airway inflammation (H&E staining); (D) BALF total leukocyte counts and (E) leukocyte composition; (F) BALF type 2 cytokines IL-5 and IL-13 (mean ± s.e.m., ****p<0.0001, 1-way ANOVA, Tukey multiple comparisons); (G–H) airway mucous (PAS staining) (mean ± s.e.m. of 7–8 mice/group, ****p<0.00001, 2-way ANOVA, Sidak multiple comparisons); relative expression of Muc5ac (I) or Il33 (J) (mean ± s.e.m., ***p=0.0001, ****p<0.0001, 2-way ANOVA, Sidak multiple comparisons). (K) Percentages of cytokine⁺ ILC2s (CD45⁺Lin⁻IL-7R⁺GATA3⁺) in lung cells determined by flow cytometry (mean ± s.e.m., of 4 mice/group; ***p<0.0008, ****p<0.00001, 1-way ANOVA, Tukey multiple comparisons).

Lungs of WT and Rgs4^−/− mice had a similar appearance at homeostasis and likewise following Af allergen challenge (Fig. 2C). Total cells and leukocyte differentials in bronchoalveolar lavage fluid (BALF) in WT and Rgs4^−/− mice were also indistinguishable from one another (Figs. 2D–E). By contrast, type 2 cytokines (IL-5, IL-13) were significantly increased in BALF from Rgs4^−/− mice compared to WT controls (Fig. 2F). Other asthma-associated cytokines (IL-4, IL-9, IL-10, and IL-17A) were similar in BALF of mice of either genotype (Fig. E4). We also observed significantly more mucin in the airways of allergen-challenged mice compared to PBS controls (Fig. 2G), but the magnitude of increase was equivalent in WT and Rgs4^−/− mice (Fig. 2H). Surprisingly, we detected more Muc5ac and Il33 expression in lungs of allergen-challenged Rgs4^−/− mice relative to those from WT counterparts (Figs. 2I–J).

To determine the source of allergic cytokines in this model, we immunophenotyped lung cells by flow cytometry. Percentages of lung CD4⁺ and ILC2 cells were equivalent in naïve and Af-challenged mice, and likewise did not differ between WT and Rgs4^−/− mice (Fig. E5A–B). IL-5⁺ and IL-13⁺ ILC2 cells were detected with greater frequency in allergen-challenged mice compared to PBS-treated controls and were increased in Rgs4^−/− mice compared to WT regardless of allergen exposure (Figs. 2K, E5C–D). By contrast, CD4⁺IL-5⁺ or CD4⁺IL-13⁺ cells were nearly undetectable in PBS- or Af-challenged mice of either genotype (Fig. E5E). Finally, expression of airway remodeling-associated genes (Cola1, Cola3, Il25, Pstn) was similar in Af-challenged WT and Rgs4^−/− mice (Fig. E6). These findings favor the interpretation that absence of RGS4 reduces susceptibility to bronchospasm through a mechanism that is independent of allergic inflammation and airway remodeling.

Depletion of RGS4 in ASM recapitulates the phenotype of Rgs4^−/− mice

To investigate further how RGS4 controls AHR, we evaluated mice in which RGS4 was specifically eliminated in ASM (Rgs4^fl/fl x smooth muscle α actin [SMAA]-Cre) (19). RGS4 was virtually undetectable in lysates of ASM cells from Rgs4^Cre/fl mice compared to those from Rgs4^fl/fl mice whereas RGS4 expression in brain tissue of Rgs4^Cre/fl mice was comparable to controls (Fig. 3A). Although this genetic manipulation had no impact on airway resistance in PBS-challenged mice, Rgs4^Cre/fl mice had significantly reduced AHR following Af challenge compared to fl/fl controls (Fig. 3B). Af induced-inflammation was comparable in lungs of Rgs4^Cre/fl and Rgs4^fl/fl mice including peri- bronchial leukocyte infiltration (Fig. 3C), leukocyte counts and composition of BALF (Figs. 3D–E), and mucin staining within the bronchial epithelium (Fig. 3F). Although IL-5 and IL-13 were significantly increased in BALF from allergen-challenged mice of both strains compared with PBS-challenged controls, levels were lower in allergen-challenged Rgs4^Cre/fl mice than in Rgs4^fl/fl mice (Fig. 3G). Lung Il33 expression was also significantly lower in allergen challenged Rgs4^Cre/fl mice than in fl/fl controls (Fig. 3H). These findings suggest that RGS4 expressed in ASM regulates bronchial responsiveness through mechanism(s) unrelated to leukocyte recruitment and mucous but possibly related to decreased type 2 cytokine levels.

Figure 3. ASM-specific deletion of Rgs4 phenocopies global RGS4 deficiency.

(A) RGS4 expression in brain tissue or ASM cells from Rgs4^fl/fl or Rgs4^Cre/fl mice (ASM cells pooled from 4–5 mice/group or brains from 2 mice). (B) Airway resistance (mean ± s.e.m. of 6–12 mice/group analyzed in 2 separate experiments; ****p<0.0001, 2-way ANOVA). (C–G): airway inflammation (H&E staining) (C), total leukocyte counts (D) and leukocyte composition (E) in BALF; airway mucous assessed by PAS staining (F); type 2 cytokines IL-5 and IL-13 in BALF (G) (mean ± s.e.m.; *p=0.02, **p=0.002, 1-way ANOVA, Tukey multiple comparisons) in Rgs4^fl/fl or Rgs4^Cre/fl mice. Images are representative of 6–9 mice/group. (H) Lung Il33 expression (mean ± s.e.m., *p=0.01, **p=0.002, 1-way ANOVA, Tukey multiple comparisons).

RGS4 deficiency enhances Ca²⁺ signaling but not airway contraction ex vivo

RGS4 homologues (RGS2 or RGS5) inhibit AHR in mice by impairing pro-contractile Ca²⁺ signaling in ASM (20–22). To evaluate how RGS4 regulates ASM contraction-related signaling pathways, we measured Ca²⁺ mobilization in tracheal ASM. Cells from Rgs4^−/− mice had increased peak Ca²⁺ responses and prolonged recovery following treatment with acetylcholine (Fig. 4A) or thrombin (Fig. 4B), but not bradykinin (Fig. 4C), compared to cells from WT mice. These results indicate that RGS4 regulates specific excitation-contraction signaling in ASM. To assess airway contraction directly, we studied precision-cut lung slices (PCLS) from WT and RGS4-deficient mice treated with the bronchoconstrictor carbachol (CCh). Surprisingly, airway contraction in PCLS from PBS- or Af-challenged Rgs4^Cre/fl mice was comparable to that of Rgs4^fl/fl controls (Figs. 4D–E).

Figure 4. RGS4 enhances contraction signaling in ASM.

(A-C) Intracellular Ca²⁺ in ASM cells treated with acetylcholine (Ach, 1 mM) (A), thrombin (3 units/ml) (B), or bradykinin (500 μM) (C). Results compiled from at least 60 cells/condition analyzed in 2–3 independent experiments each using cells pooled from 4–5 mice/group; time of agonist addition is noted by the arrow (mean ± s.e.m., AU=arbitrary units, ****p<0.0001, t test). (I) Airway contraction in PCLS from PBS- or allergen-challenged Rgs4^fl/fl or Rgs4^Cre/fl mice treated with the indicated doses of carbachol (CCh) (mean ± s.e.m. of 8–9 airways from 2–3 mice/group analyzed in 2 independent experiments).

RGS4 impairs secretion of PGE2 in the airways of allergen challenged mice

Given these findings, we hypothesized that mediators present in the airways of Rgs4^−/− mice might reduce susceptibility to AHR by mitigating contraction signaling pathways in ASM. Among many candidate bronchorelaxant factors, we focused on PGE2, a lipid mediator generated through metabolism of arachidonic acid (AA) by cyclooxygenases (COX1–2) and PGE2 synthases (23). As shown in Figs. 5A–B, PGE2 was detected at significantly higher levels in BALF from Af-challenged mice compared to PBS controls, and likewise at significantly higher levels in airways of Af-challenged Rgs4^−/− mice and ASM-specific Rgs4 knockout mice compared to respective controls. To determine the importance of PGE2 for the attenuated AHR observed in Rgs4^−/− mice, we performed Af-challenge together with indomethacin, a non-selective COX1–2 inhibitor (Fig. 5C). Indomethacin pretreatment of Rgs4^−/− mice significantly increased airway resistance following Af challenge compared to diluent alone, comparable to levels seen in Af-challenged WT mice (Fig. 5D). Collectively, these findings suggest that Rgs4^−/− mice do not develop AHR following allergen challenge due to an associated increase in PGE2 in the airways.

Figure 5. RGS4 regulates AHR through prostaglandin-dependent mechanisms.

(A–B) PGE2 levels in BALF from WT and Rgs4^−/− (A) or Rgs4^fl/fl and Rgs4^Cre/fl mice (B) (mean ± s.e.m, **p=0.002, ***p=0.0002, Holm-Sidak corrected t tests, PBS v. Af; *p=0.01 Rgs4^fl/fl v. Rgs4^Cre/fl; **p=0.002, WT v. Rgs4^−/− 2-way ANOVA, Sidak multiple comparisons). (B) Schematic of inoculation strategy for indomethacin experiments (i.p.=intraperitoneal; i.n.= intranasal). (C) Lung resistance in WT or Rgs4^−/− mice challenged with Af together with diluent or indomethacin (mean ± s.e.m. of 7–17 mice/group analyzed in 4 independent experiments; ***p=0.001, 2-way ANOVA, Tukey multiple comparisons). (D) PGE2 levels in supernatants from human AECs cells incubated as indicated [NT=not treated, Aa (200 μg/ml), Alp1 (10 μg/ml), SLIGKV (300 μM)] for 6 hours (mean ± s.e.m. of 4–7 independent experiments performed in duplicate; *p<0.03, **p<0.004, ***p=0.0003, ****p<0.0001, 1-way ANOVA, Holm-Sidak multiple comparisons). (F) PGE2 levels (graph) in AEC supernatants treated as indicated; blot shows RGS4 expression in siRNA-treated AECs (mean ± s.e.m. of 4–5 independent experiments performed in duplicate, **p<0.006, 2-way ANOVA, Sidak multiple comparisons). (G) LTE4 levels in BALF of Af-challenged WT or Rgs4^−/− mice. (H) Bronchodilation of PCLS airways pre-treated with RGS4 inhibitor (CCG) or diluent (DMSO) followed by sequential treatment with CCh and PAR2 peptide SLIGRL (100 μM) (mean ± s.e.m. of 12–14 airways/group analyzed in 2–4 independent experiments; *p=0.04, Mann-Whitney).

RGS4 inhibits an epithelial response to allergens

The increased airway PGE2 levels in Rgs4^−/− mice led us to explore pathways leading to PGE2 secretion that might be targeted by RGS4. Af and other allergenic fungi [e.g. Penicillium species and Alternaria alternata (Aa)] linked to severe asthma (24) secrete proteases that can elicit release of proinflammatory mediators including IL-6, IL-8, and TNFα (25–27). We hypothesized that activation of proteinase-activated receptors (PARs) on AECs by allergens may be a critical step in the generation of PGE2. Allergen extracts of Aa, a purified serine protease derived from Af that we have previously linked to AHR (alkaline protease 1, Alp1) (28, 29) or the PAR2 agonist peptide (SLIGKV) elicited a significant increase in PGE2 secretion in AECs compared to cells treated with medium alone (Fig. 5E). Pre-incubation with a PAR2-specific antagonist (ENMD-1068) significantly reduced Aa- and SLIGKV-induced PGE2 secretion but had no effect on Alp1-mediated responses. Finally, RGS4 knockdown by siRNA enhanced Aa- or SLIGKV-induced PGE2 secretion by AECs compared to a control siRNA (Fig. 5F). These findings indicate that fungal allergens induce PGE2 secretion by AECs, a response that is negatively regulated by RGS4.

RGS4 inhibits prostaglandin-dependent airway relaxation

PGE2 has important anti-inflammatory actions on mast cell, lymphocyte, and ILC2 activities (30). To explore the mechanisms by which PGE2 reduces AHR in Rgs4−/− mice, we first assessed mast cell activation by measuring leukotriene E4 (LTE4), a stable mast cell-derived cysteinyl leukotriene (cysLT) metabolite, in BALF (31). LTE4 levels in BALF from allergen-challenged WT and Rgs4−/− mice were indistinguishable from one another (Fig. 5G). This result and the observed increase in ILC2-derived type 2 cytokines in Rgs4−/− mice (Fig. 2K) suggest that PGE2 decreases AHR through mechanisms that are in part independent of allergic inflammation.

To examine the effects of RGS4 on PGE2-mediated bronchial tone via direct means, we used a recently-developed small molecule (CCG203769, hereafter referred to as CCG), which inhibits RGS4-Gα interactions through covalent modification of Cys residues in the RGS domain and has high selectivity for RGS4 over close homologues such as RGS16 or RGS19 (IC₅₀ = 140 nM for RGS4 v. 6 μM for RGS16/19) (32). We applied CCG or diluent alone (DMSO) to PCLS of naïve mice, followed by sequential measurements of CCh-induced bronchoconstriction and responses to the mouse PAR2 agonist SLIGRL, a hexapeptide that induces prostaglandin-dependent bronchorelaxation ex vivo (33–36). Similar to the results of our studies featuring RGS4-deficient mice (Fig. 5E), CCG had no effect on Balb/c WT PCLS contraction in response to CCh (Fig. E7). Administration of SLIGRL resulted in significant dilation of pre-contracted airways (12.2 ± 1.3%, p<0.0001); relaxation of airways in CCG-pre-treated PCLS was nearly double that of controls (20.1 ± 3.3%, p=0.01; Fig. 5H). These results support the conclusion that PAR2 mediates bronchodilation through an RGS4-regulated pathway in mice.

RGS4 modulates GPCR signaling in epithelial cells

To understand the mechanism(s) by which RGS4 modulates PGE2 secretion, we evaluated PAR2-mediated signaling in AECs. PAR2 couples to Gαq, which induces intracellular Ca²⁺ mobilization, Erk phosphorylation, and generation of AA through the actions of cytosolic phospholipase A2 (cPLA2) (37). Since RGS4 is a GAP for Gαq (38), we tested its effects on this pathway by measuring intracellular Ca²⁺ flux in AECs transfected with RGS4 siRNA or treated with CCG. Either manipulation enhanced the peak Ca²⁺ response to SLIGKV or to the GPCR agonist, bradykinin (Figs. 6A–B and E8A). In contrast, neither treatment had an effect on Ca²⁺ flux induced by a GPCR-independent stimulus (Ca²⁺ ionophore) (Figs. 6C, E8B). In a separate set of experiments, overexpression of WT RGS4, but not a mutant lacking GAP activity (N128A)(39) in AECs (BEAS2B) (Fig. 6D) inhibited SLIGKV- and bradykinin-mediated Ca²⁺ flux but had no effect on ionomycin-induced responses (Figs. 6E–G). Taken together, these results indicate that RGS4 inhibits PGE2 secretion primarily by limiting GPCR-induced G protein activation, upstream of PGE2 biosynthesis.

Figure 6. RGS4 inhibits GPCR-mediated Ca²⁺ signaling in airway epithelial cells.

Intracellular Ca²⁺ in AECs transfected with the indicated siRNAs (A–C) or BEAS2B cells transfected with the indicated plasmids (E–G) and stimulated with SLIGKV (300 μM), bradykinin (1 μM), or ionomycin (1 μM); arrows denote time of agonist addition. Bar graphs show peak Ca²⁺ response (mean ± s.e.m. of 4–6 biological replicates evaluated in 2–3 separate experiments; *p<0.03, ***p=0.0003, 1-way ANOVA, Tukey multiple comparisons. RFU=relative fluorescence units. (D) Expression of HA-RGS4 in BEAS2B cells evaluated by immunoblotting (vec=empty control vector).

Pharmacological RGS4 inhibition mimics genetic RGS4 deficiency

The lower predisposition of Rgs4^−/− mice to develop a severe asthma-like phenotype prompted us to investigate pharmacological inhibition of RGS4 activity in this disease model. We sensitized mice with Af as previously and then challenged intranasally with Af together with CCG or diluent (DMSO) alone (schematic shown in Fig. E9A). While CCG had virtually no effect on airway resistance in PBS-challenged WT mice, allergen-challenged mice treated with CCG had significantly reduced MCh-induced bronchospasm compared to mice treated with diluent alone (Fig. 7A). CCG had no effect on AHR in Rgs4^−/− mice regardless of allergen exposure (Fig. E9B), confirming its specificity. CCG had no impact on lung inflammation, airway mucous, or type 2 cytokines (Figs. 7B–F). However, CCG reduced lung Il33 expression (Fig. 7G) and significantly increased airway PGE2 levels (Fig. 7H) in Af-challenged mice compared to controls. These findings suggest that allergen induced AHR is modulated by the actions of RGS4 on a PGE2-dependent pathway.

Figure 7. Pharmacological RGS4 inhibition reduces development of allergen-induced bronchospasm in mice.

(A–G) Airway resistance (A) (****p<0.0001, 2-way ANOVA); airway inflammation (H&E staining) (B); total leukocyte counts (C) and leukocyte composition (D) in BALF; airway mucous (PAS staining) (E); IL-5 and IL-13 in BALF (F); lung Il33 expression (*p=0.01; **p=0.001, 1-way ANOVA, Tukey multiple comparisons) (G) in Balb/c mice challenged with Af together with RGS4 inhibitor CCG203769 (CCG) or diluent (DMSO) alone. Images are representative of 18 mice/group analyzed in 4 independent experiments. (G) PGE2 levels in BALF of mice challenged with Af together with diluent (DMSO) or RGS4 inhibitor (CCG) (mean ± s.e.m. of 8–10 mice/group analyzed in 2 independent experiments, *p=0.04, Mann-Whitney).

Potential role of RGS4 in aspirin-associated bronchospasm

Given our findings on the relationship between RGS4, PGE2, and allergen induced AHR, we speculated that RGS4 regulates bronchospasm related to aspirin (ASA) intake. RGS4 expression in bronchial epithelium of biopsies from patients with severe asthma and a history of ASA-induced bronchospasm was lower than that detected in subjects with asthma of comparable severity but lacking responses to ASA (Fig. 8A). However, RGS4 was transiently upregulated in AECs after acute treatment with the water soluble NSAID ketorolac (Fig. 8B), suggesting potential relevance to ASA-induced respiratory reactions. To investigate this relationship further, we used a mouse model of AERD that features mice lacking the gene encoding microsomal PGE2 synthase 1 (mPGES1; ptges1^−/− mice). ptges1^−/− mice have reduced (but detectable) PGE2 levels in lungs at homeostasis (40) and develop bronchoconstriction following ASA challenge in the setting of existing allergic inflammation (house dust mite (HDM) allergen challenge) (41).

Figure 8. Potential role of RGS4 in aspirin-associated bronchospasm.

(A) Immunohistochemistry of RGS4 in lung epithelium in subjects with severe asthma (boxed data also presented in Fig. 1B) with or without a history of NSAID-associated bronchospasm; **p=0.004, Mann-Whitney. (B) RGS4 expression in AECs left untreated (NT=not treated) or treated with ketorolac (200 μg/ml) for the indicated times (mean ± s.e.m. of 4 independent experiments using cells from 2 different individual donors, *p=0.03, **p=0.005, 1-way ANOVA, Holm-Sidak multiple comparisons). (C) ptges1^−/− mice were challenged with HDM together with diluent (DMSO) or RGS4 inhibitor (CCG) followed by a single intranasal challenge with diluent or lys-ASA. (D) Rgs4 transcript expression in HDM-challenged ptges1^−/− mice (mean ± s.e.m., relative to a calibrator value, set as ‘1’; *p=0.02, unpaired t test). (E–J) Analysis of HDM-challenged ptges1^−/− mice as follows: H&E staining (E); BALF leukocyte counts (F); BALF leukocyte composition (G); and BALF PGE2 levels (H) (mean ± s.e.m. of 5–13 mice/group analyzed in 2–3 independent experiments, *p=0.01, Mann-Whitney); airway resistance following challenge with lys-ASA starting at t= ~8 minutes post-challenge (I); endpoint resistance at 50 minutes post-challenge (mean ± s.e.m. of 4–5 mice/group analyzed in 2 independent experiments; *p<0.04, **p<0.005, 2-way ANOVA, Sidak multiple comparisons) (J).

To determine whether RGS4 inhibition mitigates the AERD-like phenotype of ptges1^−/− mice, we applied HDM repeatedly to the lower airway mucosa in the presence or absence of CCG, followed by an additional treatment with diluent alone (DMSO) or CCG and intranasal inhalation of lysine-ASA two hours later (Fig. 8C). Paralleling the human studies, we detected increased Rgs4 expression in lungs from HDM-challenged ptges1^−/− mice after ASA challenge compared to diluent-treated mice (Fig. 8D). CCG did not significantly affect airway inflammation including lung histology (Fig. 8E), inflammatory cell counts, or airway eosinophils (Figs. 8F–G). By contrast, although levels of PGE2 in BALF from both groups were lower overall than in allergen-challenged WT mice, they were significantly increased in CCG-treated mice compared to controls (Fig. 8H). Consistent with published studies, these mice developed increased lung resistance over a period of 50 minutes following ASA inhalation (basal: 0.46 ± 0.03 v. endpoint: 0.57 ± 0.04; p=0.03, paired t test) (Fig. 8I). Pre-treatment with CCG significantly reduced bronchospasm induced by ASA challenge (Figs. 8I–J). Collectively, our findings suggest that acute upregulation of RGS4 may contribute to ASA-induced bronchospasm by exacerbating PGE2 deficiency in the airways.

DISCUSSION

Here we have identified a lung epithelial response to allergens that is in part mediated by activation of PAR2; the response includes production of PGE2 and is inhibited by RGS4, a protein upregulated in the respiratory epithelium in subjects with asthma. In patients without asthma, RGS4 is expressed at lower levels, facilitating PGE2 secretion following allergen exposure and its resultant protective effects on allergic inflammation and bronchial tone. As such, RGS4 may be a critical target in specific asthma endotypes such as AERD or fungal asthma.

The beneficial effects of RGS4 loss of function were quite unexpected given the results of previous studies of RGS family members in asthma. Rgs2^−/− or Rgs5^−/− mice exhibit spontaneous AHR even in the absence of allergen challenge and manifest allergic inflammation essentially equivalent to respective controls (21, 42). Our previous work on RGS4 demonstrated that pre-treatment of PCLS with platelet-derived growth factor (PDGF) increased RGS4 expression and diminished carbachol (CCh)-induced contraction (13). In a separate study, transgenic mice expressing extra copies of human RGS4 under the control of its native promoter were less susceptible to allergen-induced AHR primarily due to reduced ASM contraction and with equivalent inflammatory responses (43). Here we analyzed loss of RGS4 expression and function and its impact on the airways in experiments performed with Rgs4−/− mice or with an RGS4 inhibitor, respectively. As such, it is not possible to compare the results of our current work with the findings presented previously.

Because it principally targets heterotrimeric G proteins, RGS4 may exert pleiotropic effects on inflammation and AHR by simultaneously regulating several GPCR pathways. In support of this hypothesis are findings that document that the loss of RGS4 expression: 1) enhances PAR2-mediated PGE2 secretion in AECs (Fig. 6E); 2) increases expression of Il33, which encodes an epithelial-derived proinflammatory cytokine (Fig. 3H); and 3) promotes excitation-contraction signaling in ASM cells (Figs. 5A–B). Increased Il33 expression detected in Af-challenged Rgs4^−/− mice suggests that in addition to its effects on ASM cells, RGS4 regulates epithelial activation, which in turn may affect cytokine production by activated ILC2s (44). Since previous work has demonstrated that PGE2 inhibits human ILC2 functions including production of IL-5 and IL-13 (45), it is critical to consider the possibility that the anti-inflammatory actions of PGE2 might counteract the actions of allergic cytokines. Specifically, in contrast to germline Rgs4^−/− mice, we detected less Il33 expression in both ASM-specific RGS4 knockout mice and RGS4 inhibitor-treated mice than in respective controls, suggesting that increased PGE2 in these mice may inhibit ILC2-mediated cytokine production. PGE2 is also known to inhibit eosinophil migration in response to eotaxin and other chemoattractants (46), which may account for the discrepancy between IL-5 levels and the degree of eosinophil infiltration observed.

Our studies suggest that RGS4 promotes AHR by affecting PGE2-dependent bronchorelaxation. PGE2 is a potent bronchodilator of both human and mouse airways ex vivo (47, 48). However, while PGE2 inhalation protects against the development of immediate bronchoconstriction in patients with AERD following ingestion of NSAIDs (30) and in subjects with asthma following allergen challenge (49), its role in regulating airway tone in chronic allergic asthma remains uncertain (50). In addition to PGE2 deficiency, additional mechanisms may contribute to bronchospasm in AERD, including reduced expression of and signaling through the EP2 receptor expressed on ASM (51, 52). PGE2 also inhibits 5-lipoxygenase, an enzyme required for synthesis of bronchoconstrictive CysLTs, and PGE2 deficiency in AERD thereby leads to increased lung CysLT levels (30). We did not detect significantly altered levels of mast cell-secreted LTE4 in lungs of PBS or Af-challenged Rgs4^−/− mice compared to WT (Fig. 6G), nor did we observe changes in expression of Cox1/2, ptges1, or ptger1–4 (encoding PGE2 receptors EP1–4) by RNA sequencing (data not shown). Thus, RGS4 may promote AHR principally by inhibition of PGE2 biosynthesis by activated epithelial cells upstream of enzymatic steps and independent of transcription. Overall, our findings are consistent with those identified in related mouse models, including those in mice lacking 15-hydroxyprostaglandin dehydrogenase (HPGD), the major catabolic enzyme of PGE2. Hpgd^−/− mice have elevated airway PGE2 levels at homeostasis and reduced AHR (53).

RGS4 expression in bronchial epithelium of biopsies from patients with severe asthma and a history of ASA-induced bronchospasm was surprisingly lower than that detected in subjects with asthma of comparable severity but lacking responses to ASA (Fig. 9A), and administration of ASA induced upregulation of Rgs4 in AECs (Fig. 9B) and in the ptges1^−/− mouse AERD model (Fig. 9D). Although these results suggest the possibility of dysregulated RGS4 expression in AERD, more extensive studies of RGS4 expression in cells and tissue from patients with AERD will be required to clarify the role of RGS4 in disease pathogenesis. Most important, pharmacological RGS4 inhibition sustainably reduced bronchospasm and increased PGE2 levels in airways in models of severe and ASA-associated asthma, suggesting a tractable therapeutic approach meriting further investigation.

KEY MESSAGES.

• RGS4 expression is increased in respiratory epithelium in severe asthma, a finding which correlates with functional lung impairment.

• RGS4 promotes AHR in a mouse model of allergic lung inflammation and inhibits PGE2 synthesis in the airways.

• A small molecule RGS4 antagonist inhibits allergen- and NSAID-induced AHR in mice, suggesting a new therapeutic approach.

Abbreviations:

AERD

aspirin-exacerbated respiratory disease

AECs

airway epithelial cells

AHR

airways hyper-responsiveness

RGS4

Regulator of G Protein Signaling 4

PGE2

prostaglandin E2

ASM

airway smooth muscle

PCLS

precision cut lung slices

PAR2

proteinase activated receptor 2

NSAIDs

non-steroidal anti-inflammatory drugs

CysLTs

cysteinyl leukotrienes

COX1–2

cyclooxygenase 1–2

CCh

carbachol

MCh

methacholine

Ach

acetylcholine