Metabolomic Profiling in Mouse Model of Menopause-Associated Asthma.

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.

ß-Arrestin-biased proteinase-activated receptor-2 antagonist C781 limits allergen-induced airway hyperresponsiveness and inflammation.

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 ß-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 ß-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 ß-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.

CC16 Deficiency in the Context of Early-Life Mycoplasma pneumoniae Infection Results in Augmented Airway Responses in Adult Mice.

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 (early life infection model; ELIM) and compared to adult mice infected for only 3 days (adult infection model; 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.

Genetic variation in surfactant protein-A2 alters responses to ozone.

BACKGROUND: Increased exposure to Ozone (O3) is associated with adverse health effects in individuals afflicted with respiratory diseases. Surfactant protein-A (SP-A), encoded by SP-A1 and SP-A2, is the largest protein component in pulmonary surfactant and is functionally impaired by O3-oxidation. OBJECTIVE: We used humanized SP-A2 transgenic mice with allelic variation corresponding to a glutamine (Q) to lysine (K) amino acid substitution at position 223 in the lectin domain to determine the impact of this genetic variation in regards to O3 exposure. METHODS: Mice were exposed to 2ppm O3 or Filtered Air (FA) for 3 hours and 24 hrs post-challenge pulmonary function tests and other parameters associated with inflammation were assessed in the bronchoalveolar lavage (BAL) fluid and lung tissue. Additionally, mouse tracheal epithelial cells were cultured and TEER measurements recorded for each genotype to determine baseline epithelial integrity. RESULTS: Compared to FA, O3 exposure led to significantly increased sensitivity to methacholine challenge in all groups of mice. SP-A2 223Q variant mice were significantly protected from O3-induced AHR compared to SP-A-/- and SP-A2 223K mice. Neutrophilia was observed in all genotypes of mice post O3-exposure, however, SP-A2 223Q mice had a significantly lower percentage of neutrophils compared to SP-A-/- mice. Albumin levels in BAL were unchanged in O3-exposed SP-A2 223Q mice compared to their FA controls, while levels were significantly increased in all other genotypes of O3-exposed mice. SP-A 223Q MTECS has significant higher TEER values than all other genotypes, and WT MTECS has significantly higher TEER than the SP-A KO and SP-A 223K MTECS. SIGNIFICANCE: Taken together, our study suggests that expression of a glutamine (Q) as position 223 in SP-A2, as opposed to expression of lysine (K), is more protective in acute exposures to ozone and results in attenuated O3-induced AHR, neutrophilia, and vascular permeability.