James Buckley

EP4 receptor as a new target for bronchodilator therapy

Background Asthma and chronic obstructive pulmonary disease are airway inflammatory diseases characterised by airflow obstruction. Currently approved bronchodilators such as long-acting β2 adrenoceptor agonists are the mainstay treatments but often fail to relieve symptoms of chronic obstructive pulmonary disease and severe asthma and safety concerns have been raised over long-term use. The aim of the study was to identify the receptor involved in prostaglandin E2 (PGE2)-induced relaxation in guinea pig, murine, monkey, rat and human airways in vitro. Methods Using an extensive range of pharmacological tools, the relaxant potential of PGE2 and selective agonists for the EP1–4 receptors in the presence and absence of selective antagonists in guinea pig, murine, monkey, rat and human isolated airways was investigated. Results In agreement with previous studies, it was found that the EP2 receptor mediates PGE2-induced relaxation of guinea pig, murine and monkey trachea and that the EP4 receptor mediates PGE2-induced relaxation of the rat trachea. These data have been confirmed in murine airways from EP2 receptor-deficient mice (Ptger2). In contrast to previous publications, a role for the EP4 receptor in relaxant responses in human airways in vitro was found. Relaxant activity of AH13205 (EP2 agonist) was also demonstrated in guinea pig but not human airway tissue, which may explain its failure in clinical studies. Conclusion Identification of the receptor mediating PGE2-induced relaxation represents a key step in developing a novel bronchodilator therapy. These data explain the lack of bronchodilator activity observed with selective EP2 receptor agonists in clinical studies.

Pulmonary ORMDL3 is critical for induction of Alternaria-induced allergic airways disease

Background: Genome‐wide association studies have identified the ORM (yeast)‐like protein isoform 3 (ORMDL3) gene locus on human chromosome 17q to be a highly significant risk factor for childhood‐onset asthma. Objective: We sought to investigate in vivo the functional role of ORMDL3 in disease inception. Methods: An Ormdl3‐deficient mouse was generated and the role of ORMDL3 in the generation of allergic airways disease to the fungal aeroallergen Alternaria alternata was determined. An adeno‐associated viral vector was also used to reconstitute ORMDL3 expression in airway epithelial cells of Ormdl3 knockout mice. Results: Ormdl3 knockout mice were found to be protected from developing allergic airways disease and showed a marked decrease in pathophysiology, including lung function and airway eosinophilia induced by Alternaria. Alternaria is a potent inducer of cellular stress and the unfolded protein response, and ORMDL3 was found to play a critical role in driving the activating transcription factor 6–mediated arm of this response through Xbp1 and downstream activation of the endoplasmic reticulum–associated degradation pathway. In addition, ORMDL3 mediated uric acid release, another marker of cellular stress. In the knockout mice, reconstitution of Ormdl3 transcript levels specifically in the bronchial epithelium resulted in reinstatement of susceptibility to fungal allergen–induced allergic airways disease. Conclusions: This study demonstrates that ORMDL3, an asthma susceptibility gene identified by genome‐wide association studies, contributes to key pathways that promote changes in airway physiology during allergic immune responses.

Selectivity profiling of the novel EP2 receptor antagonist, PF-04418948, in functional bioassay systems: atypical affinity at the guinea pig EP2 receptor

Understanding the role of the EP2 receptor has been hampered by the lack of a selective antagonist. Recently, a selective EP2 receptor antagonist, PF‐04418948, has been discovered. The aim of this study was to demonstrate the selectivity profile of PF‐04418948 for the EP2 receptor over other EP receptors using a range of isolated tissue systems.

Perinatal paracetamol exposure in mice does not affect the development of allergic airways disease in early life

Background Current data concerning maternal paracetamol intake during pregnancy, or intake during infancy and risk of wheezing or asthma in childhood is inconclusive based on epidemiological studies. We have investigated whether there is a causal link between maternal paracetamol intake during pregnancy and lactation and the development of house dust mite (HDM) induced allergic airways disease (AAD) in offspring using a neonatal mouse model. Methods Pregnant mice were administered paracetamol or saline by oral gavage from the day of mating throughout pregnancy and/or lactation. Subsequently, their pups were exposed to intranasal HDM or saline from day 3 of life for up to 6 weeks. Assessments of airway hyper-responsiveness, inflammation and remodelling were made at weaning (3 weeks) and 6 weeks of age. Results Maternal paracetamol exposure either during pregnancy and/or lactation did not affect development of AAD in offspring at weaning or at 6 weeks. There were no effects of maternal paracetamol at any time point on airway remodelling or IgE levels. Conclusions Maternal paracetamol did not enhance HDM induced AAD in offspring. Our mechanistic data do not support the hypothesis that prenatal paracetamol exposure increases the risk of childhood asthma.

The role of adenylyl cyclase isoform 6 in β-adrenoceptor signalling in murine airways.

Adenylyl cyclase (AC) is a key signalling enzyme for many GPCRs and catalyses the conversion of ATP to cAMP which, in turn, is a crucial determinant of many biological responses. β‐Adrenoceptor agonists are prescribed as bronchodilators for asthma and chronic obstructive pulmonary disease, and it is commonly assumed that they elicit their actions via AC‐dependent production of cAMP. However, empirical evidence in support of this is lacking and the exact mechanism by which these drugs acts remains elusive. This is partly due to the existence of at least 10 different isoforms of AC and the absence of any truly selective pharmacological inhibitors. Here, we have used genetically modified mice and model systems to establish the role of AC isoforms in the airway responses to β‐adrenoceptor agonists.

Inception of early-life allergen–induced airway hyperresponsiveness is reliant on IL-13+CD4+ T cells

The lymphoid cellular source of IL-13 driving AHR is age-dependent. Age matters in allergy Development of allergy is driven by type 2 cytokines, IL-4, IL-5, and IL-13. With the discovery that type 2 innate lymphoid cells (ILC2s) and T cells can produce these cytokines, understanding the contributions of T cells and ILC2s in allergic responses has become important. Using mouse models of allergic airway inflammation, Saglani et al. report the contributions of T cells and ILC2s to be dependent on age. They found T cells to be the predominant source of IL-13 in neonatal mice as compared with ILC2s in adult mice. Given that neonates have fewer T cells as compared with adults, the results are contrary to expectations and bring to the fore an unappreciated role of neonatal T cells in this context. Airway hyperresponsiveness (AHR) is a critical feature of wheezing and asthma in children, but the initiating immune mechanisms remain unconfirmed. We demonstrate that both recombinant interleukin-33 (rIL-33) and allergen [house dust mite (HDM) or Alternaria alternata] exposure from day 3 of life resulted in significantly increased pulmonary IL-13+CD4+ T cells, which were indispensable for the development of AHR. In contrast, adult mice had a predominance of pulmonary LinnegCD45+CD90+IL-13+ type 2 innate lymphoid cells (ILC2s) after administration of rIL-33. HDM exposure of neonatal IL-33 knockout (KO) mice still resulted in AHR. However, neonatal CD4creIL-13 KO mice (lacking IL-13+CD4+ T cells) exposed to allergen from day 3 of life were protected from AHR despite persistent pulmonary eosinophilia, elevated IL-33 levels, and IL-13+ ILCs. Moreover, neonatal mice were protected from AHR when inhaled Acinetobacter lwoffii (an environmental bacterial isolate found in cattle farms, which is known to protect from childhood asthma) was administered concurrent with HDM. A. lwoffii blocked the expansion of pulmonary IL-13+CD4+ T cells, whereas IL-13+ ILCs and IL-33 remained elevated. Administration of A. lwoffii mirrored the findings from the CD4creIL-13 KO mice, providing a translational approach for disease protection in early life. These data demonstrate that IL-13+CD4+ T cells, rather than IL-13+ ILCs or IL-33, are critical for inception of allergic AHR in early life.