a purinergic p2y6 receptor agonist prodrug modulates airway inflammation, remodeling, and hyperreactivity in a mouse model of asthma

Anne Chetty,1 Azeem Sharda,1 Rod Warburton,2 Ellen O Weinberg,3 Jinghui Dong,4 Min Fang,5 G Gary Sahagian,5 Tiangmeng Chen,3 Chang Xue,3 John J Castellot,3,6 Philip G Haydon,4 Heber C Nielsen1,6 1Department of Pediatrics, Tufts Medical Center, Boston, MA, USA; 2Department of Medicine, Tufts Medical Center, Boston, MA, USA; 3Department of Integrative Physiology and Pathobiology, Tufts University School of Medicine, Boston, MA, USA; 4Department of Neuroscience, Tufts University School of Medicine, Boston, MA, USA; 5Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, USA; 6Graduate Program in Cell, Molecular and Developmental Biology, Tufts University School of Medicine, Boston, MA, USABackground: Purinergic receptors control cell proliferation, apoptosis, migration, inflammation, and cytokine secretion. Increased expression of specific purinergic receptors is reported in asthma. The role of purinergic P2Y6 receptors (P2Y6R) in asthma is controversial. Hypothesis: P2Y6R activation in asthma improves pulmonary function and reduces inflammation and smooth muscle amount. Methods: Female mice (C57/BL6, age 30 days) were randomly assigned to receive intranasal house dust mite (HDM) antigen (40 or 80 µg) or saline, 5 days/week, for 6 weeks. Randomly selected subgroups received intraperitoneal P2Y6R agonist prodrug (GC021109; 10 or 100 μg/kg weight/dose) simultaneously with HDM. After 6 weeks, lung function was measured. Lung lavage fluid (LLF) was used to measure total cell count, total protein, and cytokines. Immunohistochemistry for alpha smooth muscle actin (α-SMA) was done. Airway wall thickness was measured on micro-computed tomography (micro-CT) images. Results: Pulmonary function testing revealed a HDM dose-dependent airway hyperresponsiveness. Airway resistance was increased 2-fold while compliance was decreased by 50% at the higher HDM dose (P<0.05). GC021109 prevented these changes. HDM-exposed mice had elevated inflammatory cell and total protein levels in LLF which were prevented by GC021109 (P<0.05). HDM mice also had elevated LLF levels of interleukin (IL)-4, IL-5, IL-12, granulocyte colony stimulating factor, chemokine (C-X-C) motif ligand 1, and leukemia inhibitory factor that were reduced by GC021109 with a dose-dependent pattern. HDM mice had increased peribronchial and perivascular inflammatory cell infiltration and increased α-SMA; these changes were absent with GC021109. Airway wall thickness measured on micro-CT images was increased after HDM exposure and significantly reduced by GC021109 treatment. Conclusion: The P2Y6R prodrug GC021109 inhibited allergen-induced changes in pulmonary function, inflammatory responses, and airway and vascular smooth muscle mass. P2Y6R activation may be an effective therapeutic maintenance strategy in asthma. Keywords: airway smooth muscle, pulmonary function, inflammation, cytokines, P2Y6 receptors, GC021109