PROJECT SUMMARY The processes that regulate airway inflammation in asthma are not fully understood and patients with asthma often have persistent airway inflammation and poor disease control despite current therapies, highlighting the need to identify novel pathways and develop new therapies for patients with uncontrolled disease. Recent studies have implicated the airway epithelium in asthma pathogenesis, particularly interactions between the epithelium and innate immune cells that serve to regulate the immune response to inhaled allergens and other triggers for asthma. We recently identified increased numbers of eosinophils (EOS) embedded within the airway wall of subjects with asthma and identified key associations between EOS infiltrating the epithelium (“intraepithelial EOS”) and endogenous airway hyperresponsiveness (AHR), a specific feature of asthma, as well as markers of airway inflammation. RNA-sequencing (RNA-seq) analysis of epithelial brushings, which capture both epithelial cells and leukocytes infiltrating the epithelium, obtained from this same cohort of patients revealed increased expression of genes encoding interferon (IFN)- (IFNG) and interleukin (IL)-18 receptor 1 (IL18R1) amongst subjects with endogenous AHR and intraepithelial EOS. Previous studies have implicated IL-18, IL-18R1, and IFN- in AHR and airway inflammation in asthma and IL-18 is known to induce both IFN- and type 2 (T2) cytokines in multiple cell types but has not been studied in EOS. Based on these associations between intraepithelial EOS and AHR in humans with asthma, we hypothesize that IL-18 induces EOS to express and produce IFN- and T2 cytokines via IL-18R1, which enhances AHR and promotes airway inflammation. The major goal of this research is to develop a murine model system and human airway epithelium-EOS coculture model in order to examine the role of the IL-18-IL-18R1-IFN--T2 cytokine axis in EOS and explore the function of this axis using in vivo and ex vivo models of asthma. These studies will generate critical data that will serve as the foundation for a mentored career development award that further explores this pathway. In Aim 1, we focus on the role of IL-18R1 in driving AHR and airway inflammation using a murine model of asthma and specifically examine the role of IL-18R1 in murine lung EOS. In Aim 2, we characterize the IL-18-IL-18R1-IFN--T2 cytokine axis in human peripheral blood EOS and examine the role of this inflammatory cascade in the crosstalk between EOS and the human airway epithelium. Completion of these studies will provide a better understanding of the IL-18-IL-18R1-IFN--T2 cytokine EOS axis and its contribution to AHR and airway inflammation in asthma as well as provide compelling preliminary data for a mentored career development award on this topic.