A SPLUNC1 Peptidomimetic Inhibits Orai1 and Reduces Inflammation in a Murine Allergic Asthma Model

-Orai1 is a plasma membrane Ca2+ channel that mediates store-operated Ca2+ entry (SOCE) and regulates inflammation. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is an asthma gene modifier that inhibits Orai1 and SOCE via its C-terminal alpha 6 region. SPLUNC1 levels are diminished in asthma patient airways. Thus, we hypothesized that inhaled alpha 6 peptidomimetics could inhibit Orai1 and reduce airway inflammation in a murine asthma model. To evaluate alpha 6-Orai1 interactions, we used fluorescent assays to measure Ca2+ signaling, Forster resonance energy transfer, fluorescent recovery after photobleaching, immunostaining, total internal reflection microscopy, and Western blotting. To test whether alpha 6 peptidomimetics inhibited SOCE and decreased inflammation in vivo, wild-type and SPLUNC1(-/-) mice were exposed to house dust mite (HDM) extract with or without alpha 6 peptide. We also performed nebulization, jet milling, and scanning electron microscopy to evaluate a6 for inhalation. SPLUNC1(-/-) mice had an exaggerated response to HDM. In BAL-derived immune cells, Orai1 levels increased after HDM exposure in SPLUNC1(-/-) but not wild-type mice. Inhaled alpha 6 reduced Orai1 levels in mice regardless of genotype. In HDM-exposed mice, alpha 6 dose-dependently reduced eosinophilia and neutrophilia. In vitro, alpha 6 inhibited SOCE in multiple immune cell types, and alpha 6 could be nebulized or jet milled without loss of function. These data suggest that alpha 6 peptidomimetics may be a novel, effective antiinflammatory therapy for patients with asthma.