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

Abstract

Background: Allergens elicit host production of mediators acting on G-protein-coupled receptors 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 after the ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, a disorder termed aspirin-exacerbated respiratory disease. This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2.

Objective: We sought to understand the functions of regulator of G protein signaling 4 (RGS4), a cytoplasmic protein expressed in airway smooth muscle and bronchial epithelium that regulates the activity of G-protein-coupled receptors, in asthma.

Methods: We examined RGS4 expression in human lung biopsies by immunohistochemistry. We assessed airways hyperresponsiveness (AHR) and lung inflammation in germline and airway smooth muscle-specific Rgs4^-/- mice and in mice treated with an RGS4 antagonist after challenge with Aspergillus fumigatus. We examined the role of RGS4 in nonsteroidal anti-inflammatory drug-associated bronchoconstriction by challenging aspirin-exacerbated respiratory disease-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 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.

Keywords: Asthma; G proteins; PGE2; aspirin sensitivity; aspirin-exacerbated respiratory disease; regulators of G protein signaling protein.

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.

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).

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).

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).

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).

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).

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).

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).