Regulation of Normal and Asthmatic Lung Function by G-Protein-Coupled Receptors

National Institute Of Allergy And Infectious Diseases

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Abstract

Asthma, a pathological condition of reversible airway obstruction, is comprised of both inflammation of the lung and hyper-contractility of the bronchial smooth muscle. The major naturally occurring substances that induce bronchial smooth muscle contraction are ligands of G-protein-coupled receptors (GPCRs), such as allergen proteases, thrombin, and those contained in allergen-IgE activated mast cell granules (e.g. histamine, cysteinyl leukotrienes (LTD4), endothelin 1, adenosine, and bradykinin). In general, these agonists induce activation of the heterotrimeric G protein G-alpha q, which increases the concentration of intracellular calcium in smooth muscle cells, promoting actin-myosin interactions and muscle fiber shortening. In contrast, ligands acting on G-alpha-s-coupled receptors, such as albuterol, increase intracellular levels of cyclic AMP (cAMP), facilitating ASM relaxation. Although eosinophilic inflammation typifies allergic asthma, it is not a prerequisite for airway hyper-responsiveness (AHR), suggesting that underlying abnormalities in structural cells including ASM contribute to the asthmatic diathesis. Dysregulation of procontractile, GPCR signaling in ASM could mediate enhanced contractility. 10-15% of people with asthma experience severe, life threatening attacks and even death despite aggressive treatment with bronchodilators and corticosteroids. Nearly half of these (10-20 million) are sensitized (i.e. have IgE-mediated allergy) to filamentous fungi (e.g. Aspergillus fumigatus, Af), which has been designated "severe asthma with fungal sensitization" (SAFS). Current therapies for SAFS including antifungals or omalizumab monoclonal antibody (mAb) targeting IgE have not achieved uniform success. Utilizing a model of allergic airway inflammation in mice induced by respiratory Af exposure, we have 2 overarching aims for this project: 1) identify derangements in ASM contraction signaling downstream of inflammatory mediators; 2) examine the functions of allergen protease activity in AHR, particularly in relation to allergen-ASM interactions. Protease activity is a common and important feature of allergens capable of inducing asthma, most notably from ubiquitous fungi such as Af. Whether any allergens affect ASM contraction directly has never been explored. Previously we found a causal link between fungi and asthma occurring independently of allergenicity; in other words, host inflammatory response to the allergen. The secreted Af protease Alkaline protease 1 (Alp1) was detected in the airways of asthmatic subjects but not controls. In mouse and in vitro studies, we found that Alp1 directly promoted AHR by degrading extracellular matrix (ECM) components, leading to dysregulation of ASM contraction. Subsequently, we demonstrated that Alp1 induces AHR in mice devoid of eosinophils or the protease activated receptor 2 (PAR2) and that Alp1 directly increased contractile force of human ASM cells in vitro. In Fiscal Year (FY) 22, we performed a compound screen to identify Alp1 inhibitors in collaboration with Dr. Krishnan through an R21 grant and identified candidate protease inhibitors. A critical therapeutic gap is to identify moieties with specific affinity for fungal Alp1 but without activity on endogenous serine proteases present in the human airway. High throughput screening of existing libraries of commercially available protease inhibitors will be used to identify inhibitors of Alp1 protease. Primary hits can be rank-ordered based on inhibition of Alp1-induced ASM hypercontraction. A prioritized set of efficacious and non-toxic hits can be evaluated for its effects on (i) ASM cells from subjects with fungal asthma; (ii) airways of murine and human precision-cut lung slices (PCLS) treated with Alp1; (iii) murine models of fungal asthma. We expect these studies to identify novel anti-SAFS drug candidates as well as provide novel insight into both ASM-intrinsic and allergen-protease-dependent mechanisms of bronchoconstriction. The second area of emphasis for this project is the study of Regulators of G protein signaling (RGS) proteins in the lung. RGSs bind to the G protein alpha subunits Gi and Gq (but not Gs) through a conserved RGS domain and inactivates them by catalyzing their intrinsic GTPase activity and by blocking downstream effector interactions. Although they are generally considered to act as negative regulators of GPCR signaling pathways, the physiological function of RGS proteins in the lung is mostly unknown. We identified expression of several RGS proteins (RGS4, RGS5) in bronchial smooth muscle of humans and mice. In fiscal year 22, we generated mice with epithelial-specific Rgs4 gene deletion and mice expressing a mutant form of RGS4. The mutation in RGS4 is predicted to eliminate its GAP activity while preserving expression of RGS4 and potentially other functions of the protein. 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 (AERD). This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2. RGS4 expression in respiratory epithelium is increased in human subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in mice with global gene deletion of Rgs4, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen-induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation in mice. 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. These findings suggest that RGS4 contributes to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma. The new mouse strains will be used to determine the relative role of RGS4 GAP activity in bronchial epithelial cells for the asthma phenotype and regulation of airway PGE2 levels.