Project Summary In addition to their role as subunits of nucleic acids, extracellular nucleotides (ATP, ADP, UTP, UDP) serve as bioactive mediators by activating receptors localized in the plasma membrane of target cells. However, little is understood about their role in many biologic processes and disease states due to a lack of conventional assay to detect them. We recently developed a completely novel high-sensitivity bioassay system for the detection of extracellular nucleotides in biological fluids. Using this system, we demonstrated that UDP release in lungs facilitated a protective innate immune response against experimental asthma in mice through alveolar macrophage/IL-12/NK cell/IFN-γ axis. The long-term goal is to validate each nucleotide profile as a useful diagnostic or predictive allergic/inflammatory state biomarker. In pursuit of this goal, we will demonstrate the effectiveness of this assay to quantify the nucleotides in biological fluids obtained from human subjects with severe asthma and aspirin-exacerbated respiratory disease (AERD) and corresponding animal models and identify the responsive cell-type. We also seek to expand the scale of our bioassay to provide a broadly applicable platform through which other investigators can detect and monitor extracellular nucleotides in biological fluids at a high throughput. The central hypothesis will be tested by way of two specific aims: 1) To determine the relevance of the nucleotide profile in biological fluids from humans and animals with allergic inflammatory pulmonary diseases to clinical characteristics and severity; 2) To identify cell-intrinsic nucleotide release profiles. In Aim 1, we hypothesize that each extracellular nucleotide is pathogenetically relevant and can be a useful biomarker in humans with allergic/inflammatory pulmonary diseases. We will quantify and profile extracellular nucleotide secretion of bronchoalveolar lavage (BAL) fluids from subjects with severe asthma and nasal surface fluids from patients with AERD, and determine the relevance of each nucleotide to disease severity. In Aim 2, to our knowledge, there is no comprehensive characterization of the cell type- or stimulus-specific profile of nucleotide release. We will characterize nucleotides released by immune (Macrophage) and structural airway cells (Epithelial cells) in response to pathogenetically relevant stimuli. Together, these anticipated results and new approaches are expected to validate each nucleotide level (and nucleotide profile) as a useful diagnostic or predictive allergic/inflammatory state biomarker. Results also promise to deepen the understanding of mechanisms of nucleotides release and asthmatic patient variants, and address the limitation on developing extracellular nucleotide-based therapeutic strategies.