PROJECT SUMMARY Cystic fibrosis (CF) is the most common life-shortening genetic disease in the United States. The primary cause of mortality in CF is lung disease, characterized by airway inflammation and lung function decline. CF acute exacerbations (AE) are episodes of rapid clinical deterioration, often associated with worsening airway inflammation and lung function. Limited data link sputum biomarkers and AE, but these markers are not predictive of future events. There are even fewer data analyzing sputum cellular components to diagnose or characterize AE. The lack of readily available noninvasive markers of AE is a critical unmet need, as they could guide therapeutic interventions to minimize morbidity, and facilitate patient selection for clinical trials in an era of accelerated therapeutic developments in CF. We previously showed that airway concentrations of host defense protein Short Palate Lung epithelium Clone 1 (SPLUNC1) are tightly regulated by airway inflammation and respiratory pathogens. Continuing this work on the soluble fraction of sputum, we show in preliminary data that SPLUNC1 is acutely decreased in AE and that stable CF subjects with low basal SPLUNC1 levels have a 6-fold increase in AE likelihood within 60 days. We are now integrating those studies with data from the cellular component of sputum, where preliminary data using single-cell RNA sequencing (scRNAseq) show that CF neutrophils and macrophages exhibit distinct transcriptomic profiles that separate AE from stable states. Our overall hypothesis is that a sputum panel combining soluble (SPLUNC1) and cellular (single- cell transcriptome profile) markers can robustly diagnose and predict early airway inflammation during AE in CF. We will test this using the following specific aims: Aim 1: Establish SPLUNC1 as a diagnostic tool and predictor of AE in CF; Aim 2: Define the AE-associated sputum single-cell transcriptome profile and its ability to predict AE; and Aim 3: Determine if a combined SPLUNC1-scRNAseq panel is a better predictor of AE than FEV1. This proposal is a translational application of mechanistic data generated during my K01 award. Our K01 work uncovered the immunomodulatory role of SPLUNC1 in lung host-pathogen interactions. The goal of this project is to investigate the link between SPLUNC1's immune properties and the sputum cell transcriptomic changes that occur during immune activation in AE. The R03 Award will support complementary studies for my NHLBI-K01-funded work as I apply for my first R01, focused on SPLUNC1-controlled mechanisms of acute lung injury during respiratory infections. The proposed work will translate our findings into clinically relevant tools that inform patient care and minimize the impact of AE for patients with CF and other airway diseases.