Rationale: CC16 (club cell secretory protein-16), a member of the secretoglobin family, is one of the most abundant proteins in normal airway secretions and has been described as a serum biomarker for obstructive lung diseases. Objectives: To determine whether low CC16 is a marker for airway pathology or is implicated in the pathophysiology of progressive airway damage in these conditions. Methods: Using human data from the birth cohort of the Tucson Children's Respiratory Study, we examined the relation of circulating CC16 levels with pulmonary function and responses to bronchial methacholine challenge from childhood up to age 32 years. In wild-type and CC16 2/2 mice, we set out to comprehensively examine pulmonary physiology, inflammation, and remodeling in the naive airway. Measurements and Main Results: We observed that Tucson Children's Respiratory Study participants in the lowest tertile of serum CC16 had significant deficits in their lung function and enhanced airway hyperresponsiveness to methacholine challenge from 11 years throughout young adult life. Similarly, CC16 2/2 mice had significant deficits in lung function and enhanced airway hyperresponsiveness to methacholine as compared with wild-type mice, which were independent of inflammation and mucin production. As compared with wild-type mice, CC16 2/2 mice had significantly elevated gene expression of procollagen type I, procollagen type III, and a-smooth muscle actin, areas of pronounced collagen deposition and significantly enhanced smooth muscle thickness. Conclusions: Our findings support clinical observations by providing evidence that lack of CC16 in the lung results in dramatically altered pulmonary function and structural alterations consistent with enhanced remodeling.

SP-A functions as an important mediator in resolving tissue and lavage fluid eosinophilia in allergic mouse models. Decreased levels of SP-A in OAs, which could be due to increased local TNF-α levels, might lead to impaired eosinophil resolution and could contribute to the eosinophilic asthma phenotype.

Studies have shown that club cell secretory protein (CC16) plays important protective roles in the lungs, yet its complete biological functions are unclear. We devised a translational mouse model in order to investigate the impact of early life infections, in the context of CC16 deficiency, on lung function in adult mice. CC16 sufficient (WT) and deficient (CC16 -/- ) mice were infected with Mycoplasma pneumoniae (Mp) as weanlings and assessed as adults ( e arly l ife i nfection m odel; ELIM) and compared to adult mice infected for only three days ( a dult i nfection m odel; AIM). CC16 -/- Mp-infected mice had significantly increased airway hyperresponsiveness (AHR) in both models compared to WT mice. However, CC16 -/- mice infected in early life (ELIM) displayed significantly increased AHR compared to CC16 -/- mice infected in adulthood (AIM). In stark contrast, lung function in ELIM WT mice returned to levels similar to saline-treated controls. While WT mice cleared Mp infection in the ELIM, CC16 -/- mice remained colonized with Mp throughout the model, which likely contributed to increased airway remodeling and persistence of Muc5ac expression. When CC16 -/- mouse tracheal epithelial cells (MTECs) were infected with Mp, increased Mp colonization and collagen gene expression were also detected compared to WT cells, suggesting that CC16 plays a protective role during Mp infection, in part through epithelial-driven host defense mechanisms.

Background and Purpose Asthma is a heterogenous disease strongly associated with inflammation that has many different causes and triggers. Current asthma treatments target symptoms such as bronchoconstriction and airway inflammation. Despite recent advances in biological therapies, there remains a need for new classes of therapeutic agents with novel, upstream targets. The proteinase‐activated receptor‐2 (PAR2) has long been implicated in allergic airway inflammation and asthma and it remains an intriguing target for novel therapies. Here, we describe the actions of C781, a newly developed low MW PAR2 biased antagonist, in vitro and in vivo in the context of acute allergen exposure. Experimental Approach A human bronchial epithelial cell line expressing PAR2 (16HBE14o‐ cells) was used to evaluate the modulation in vitro, by C781, of physiological responses to PAR2 activation and downstream β‐arrestin/MAPK and Gq/Ca2+ signalling. Acute Alternaria alternata sensitized and challenged mice were used to evaluate C781 as a prophylactically administered modulator of airway hyperresponsiveness, inflammation and mucus overproduction in vivo. Key Results C781 reduced in vitro physiological signalling in response to ligand and proteinase activation. C781 effectively antagonized β‐arrestin/MAPK signalling without significant effect on Gq/Ca2+ signalling in vitro. Given prophylactically, C781 modulated airway hyperresponsiveness, airway inflammation and mucus overproduction of the small airways in an acute allergen‐challenged mouse model. Conclusion and Implications Our work demonstrates the first biased PAR2 antagonist for β‐arrestin/MAPK signalling. C781 is efficacious as a prophylactic treatment for allergen‐induced airway hyperresponsiveness and inflammation in mice. It exemplifies a key pharmacophore for PAR2 that can be optimized for clinical development.

Menopause-associated asthma impacts a subset of women, tends to be more severe, and is less responsive to current treatments. We recently developed a model of menopause-associated asthma using 4-Vinylcyclohexene Diepoxide (VCD) and house dust mites (HDM). The goal of this study was to uncover potential biomarkers and drivers of menopause-onset asthma by assessing serum and bronchoalveolar lavage fluid (BALF) samples from mice with and without menopause and HDM challenge by large-scale targeted metabolomics. Female mice were treated with VCD/HDM to model menopause-associated asthma, and serum and BALF samples were processed for large-scale targeted metabolomic assessment. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was used to examine metabolites of potential biological significance. We identified over 50 individual metabolites, impacting 46 metabolic pathways, in the serum and BALF that were significantly different across the four study groups. In particular, glutamate, GABA, phosphocreatine, and pyroglutamic acid, which are involved in glutamate/glutamine, glutathione, and arginine and proline metabolisms, were significantly impacted in the menopausal HDM-challenged mice. Additionally, several metabolites had significant correlations with total airway resistance including glutamic acid, histamine, uridine, cytosine, cytidine, and acetamide. Using metabolic profiling, we identified metabolites and metabolic pathways that may aid in discriminating potential biomarkers for and drivers of menopause-associated asthma.

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Asthma is a complex disease characterized by airway hyperresponsiveness (AHR) inflammation, and mucus overproduction, and has differential presentation in males and females. There exists strong demand to improve upon current asthma treatments by targeting upstream signaling pathways, including the G-protein coupled receptor protease-activated receptor-2 (PAR2). We have recently shown effectiveness of full (C391) and ß-arrestin biased (C781) PAR2 antagonism in limiting allergen-induced asthma indicators in mouse models (C57Bl/6 and Balb/C). To better test PAR2 antagonists as asthma drugs, we have developed a transgenic mouse model that expresses human PAR2 without mouse PAR2 expression in a C57Bl/6 background (htgPAR2). The htgPAR2 mice have heightened responses to asthma allergens [house dust mite (HDM) or Alternaria alternata] following acute exposure. Limited sex differences were observed in the HDM challenge model. However, when challenged with A. alternata there was significantly higher AHR in the male mice and significantly higher inflammatory and mucus responses in the female mice. The full and ß-arrestin biased PAR2 antagonists also displayed differential effects with C781 providing broader control of allergen-induced indicators (AHR, inflammation and mucus overproduction). We conclude that the htgPAR2 mouse model is a promising tool for preclinical in vivo screening of potential asthma treatments targeting PAR2. Grants from the National Institute of Health (NS098826, AI140257, HL16024, HL152942) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Declines in lung function worsen quality of life and increase the risk of mortality. Obesity and non-alcoholic fatty liver disease are associated with worsened lung function. To investigate this association, we assessed lung function in lean and diet-induced obese conscious mice using our newly developed leak-free head-out plethysmography system. Obesity was associated with increased volume (P<0.0001), minute ventilation (volume per minute; P<0.0001), mid-expiratory flow (flow rate at 50% expiratory volume; P<0.0001), end-inspiratory pause (pause at end of inspiration; P<0.0001) and decreased expiratory time (P<0.0001). We next compared the response to methacholine (0, 25, 50, 100 mg/ml in PBS flow 0.2ml/30sec) measured using our head-out plethysmography system with forced oscillation technique (using the standard flexiVent system) measures taken in the same mice. Many of the measures gathered using head-out plethysmography were associated with measures collected using the forced oscillation technique. Minute ventilation was most significantly associated with maximal airway resistance, maximal airway elastance, tissue damping, and tissue elastance (r=-0.59 P<0.0001; r=-0.54 P<0.005; r=-0.48 P<0.005; r=-0.40 P<0.005 respectively). Volume, corrected for energy expenditure, was most significantly associated with maximal resistance of the conducting airways (r=-0.57 P<0.0001). Although fatty liver is associated with changes in lung function, we found neither hepatic vagotomy nor knocking down obesity-induced hepatic GABA production improved lung function in obese mice. Still, our head-out plethysmography system is ideal for assessing the response to interventions aimed at improving obesity-associated declines in lung function.