Abstract Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness (AHR), and airway remodeling. Epidemiologic data has revealed that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society. Airway epithelial cells, once considered a static physical barrier between the body and the external world, are now widely recognized as immunologically active cells that can initiate, maintain, and restrain inflammatory responses, such as those that mediate allergic disease. Airway epithelial cells can sense allergens via myriad expression of Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs). We sought to determine whether the innate immune response stimulated by a combination of Pam2CSK4 (“Pam2”, TLR2/6 ligand) and a class C oligodeoxynucleotide ODN362 (“ODN”, TLR9 ligand) when delivered together by aerosol (“Pam2ODN”), can modulate the allergic immune response to allergens. Treatment with Pam2ODN 7 days before sensitization to House Dust Mite (HDM) extract resulted in a strong reduction in eosinophilic and lymphocytic inflammation. This Pam2ODN immunomodulatory effect was also seen using Ovalbumin (OVA) and A. oryzae (Ao) mouse models. The immunomodulatory effect was observed as much as 30 days before sensitization to HDM, but ineffective just 2 days after sensitization, suggesting that Pam2ODN immunomodulation lowers the allergic responsiveness of airway epithelial cells. Furthermore, Pam2 and ODN cooperated synergistically suggesting that this treatment is superior to any single agonist in the setting of allergen immunotherapy. One Sentence Summary A synergistic combination of Toll-like Receptor agonists, delivered directly into the lung mucosa, can attenuate allergic responsiveness of airway epithelial cells and prevent host sensitization to aeroallergens. What is already known Allergic sensitization has increased in the 20 th century due to reduced contact with microbial organisms in industrialized society (ie. hygiene hypothesis) We have previously identified a pharmacological means to stimulate innate immunity of lung epithelial cells. What this study adds Activation of innate immunity in lung epithelial cells attenuates the allergic responsiveness of mice. Synergistic cooperation of pattern recognition receptors induces stronger immunomodulatory responses What is the clinical significance Aerosolized Toll-like Receptor agonists have been demonstrated as safe in human clinical trials This study provides proof-of-principle that aerosolized toll-like receptor agonists could have clinical efficacy in the setting of the allergen immunotherapy

Epithelial Immunomodulation by Aerosolized Toll-like Receptor Agonists Attenuates Allergic Responsiveness in Mice