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Antagonizing cholecystokinin and its receptor CCKAR in the lung abolishes obesity-induced airway hyperresponsiveness

Ronald Allan M. Panganiban¹, Maoyun Sun¹, Chan-Young Park¹, Alvin Kho², David Kasahara¹, Elliot Israel³, Marc B. Hershenson⁴, Scott T. Weiss², Jeffrey Fredberg¹, Kelan Tantisira², Stephanie A. Shore¹, and Quan Lu¹

¹Program in Molecular and Integrative Physiological Sciences, Harvard T.H. Chan School of Public Health, Boston, MA 02115

²Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115

³Asthma Research Center, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115

⁴Division of Pediatric Pulmonology, University of Michigan Medical School, MI 48109

ABSTRACT

Obesity is a significant co-morbidity factor that associates with increased prevalence and severity of asthma; however, the mechanisms underlying the obesity-asthma association remain largely unknown, and furthermore, there is no effective medication to treat obese asthmatics. Here, we show that cholecystokinin (CCK)—a hormone best known for its roles in satiety regulation and fat metabolism--is increased in the lungs of obese mice and that pharmacological blockade of CCK and its receptor signaling in the lung abolishes obesity-associated airway hyperresponsiveness—a hallmark of asthma. By mining existing RNA-seq transcriptomic data, we first identified CCKAR—a receptor for CCK--as a highly expressed G-protein coupled receptor in primary human airway smooth muscle (ASM) cells. CCK is also expressed in ASM cells and is induced by free fatty acids. Activation of CCKAR by CCK induces ASM stiffening and contraction; the ASM contraction is abolished by CRISPR-mediated CCKAR inactivation or CCKAR antagonists. In vivo, CCK levels are elevated in the lung of both genetically (db/db) and diet-induced obese mice. Importantly, intranasal administration of highly potent CCKAR antagonists (proglumide and devazepide) abolished airway hyperresponsiveness in both genetically and diet-induced obese mice. Together, our results identify an unexpected role for CCK/CCKAR in obesity-associated asthma and provide critical pre-clinical data that support the repurposing of CCKAR antagonists as a novel therapy for obese asthmatics

Upregulation and Direct Cleavage of GSDMB by Asthma Triggers

Ronald Allan M. Panganiban¹, Michael O’Sullivan¹, Mengyuan Kan², Blanca Himes², Jin-ah Park¹, and Quan Lu¹

¹Program in Molecular and Integrative Physiological Sciences, Harvard T.H. Chan School of Public Health, Boston, MA 02115

²Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania

Asthma is a complex airway disease caused by a poorly understood interplay between environmental and genetic factors. Genome-wide association studies (GWAS) have identified numerous asthma-associated genes including GSDMB, a gasdermin family member that resides in 17q21 locus, the strongest and most replicated asthma GWAS signal. However, the functional role of GSDMB in asthma remains incompletely understood. Our previous work demonstrated that 1) caspase-1 cleaves GSDMB to liberate the N-terminal fragment which induces pyroptosis, a recently characterized mode of pro-inflammatory cell death; 2) a functional splice variant of GSDMB (rs11078928) associated with lower asthma risk abolishes GSDMB’s ability to induce pyroptosis; and 3) GSDMB is highly expressed in the airway epithelium, the airway’s first line of defense against asthma triggers. While these findings strongly implicate GSDMB as a bona fide asthma gene that mediates airway epithelial pyroptosis, it remains unclear how and whether asthma triggers affect GSDMB expression and function. Using in vitro cleavage assay, we show that GSDMB is cleaved directly by house dust mite (HDM), mold, and cockroach extracts. We also show that the GSDMB cleavage site for HDM is different from the cleavage site for caspase-1, which we previously demonstrated to be at Asp236. On the other hand, treatment of human bronchial epithelial cells cultured at the air-liquid interface (HBE-ALI) with rhinovirus A (RVA), but neither mechanical compression nor HDM, induces ~2 fold increase in GSDMB expression compared to untreated control. GSDMB promoter analysis by ContraV3 identifies a GATA-2 consensus binding site within the 1.0 kb promoter region upstream of GSDMB transcription start site suggesting that RVA possibly induces the transcription factor GATA-2 to promote GSDMB expression. Our data provides clues to the effect of different asthma triggers on GSDMB and reveals insights into the possible mechanistic link between asthma triggers and airway epithelial pyroptosis. Future studies are aimed at identifying the specific GSDMB cleavage sites for the asthma triggers, identifying the specific proteases in the asthma trigger extracts capable of cleaving GSDMB, as well as investigating the effects of multiple exposures to asthma triggers (e.g. RVA and HDM extracts) on GSDMB and pyroptosis.