PROJECT SUMMARY/ABSTRACT Beryllium (Be) is used in several industries and the US is the largest exporter of Be in the world. Despite mitigation strategies, Be exposure at the workplace results in Be sensitization (BeS) and Chronic Beryllium Disease (CBD). CBD is an important but understudied organ-specific immune-mediated disease characterized by granulomatous lung inflammation, fibrosis, and death. Hence, CBD is a public health concern resulting in the promulgation of new exposure standards recently. BeS develops in up to 20% of Be-exposed individuals and progresses to CBD in 50-100% of these at-risk individuals. The goal of this study is to determine if complement activation in nasal and bronchial epithelial cells perpetuates the immune response contributing to the exposure-mediated immune disease, CBD. This project hypothesizes that nasal and bronchial epithelial cells share common CBD-related and complement-related gene expression and activation, leading to enhanced immune responses to Be-exposure in BeS and CBD. In Aim 1, the study will determine gene expression profiling from paired nasal and bronchial brushings cells in CBD and BeS as compared to healthy controls, primarily focused on complement genes and pathways. We will determine Be-related airway epithelial cells transcription of paired nasal and bronchial brushings cells in BeS (n=16), CBD (n=16), and controls (n=10). We aim to 1) investigate if gene expression profiling of nasal brushings is similar to that of bronchial brushings, and to determine whether nasal brushing can be used as a non-invasive biomarker of the lower airways in the study of CBD. 2) analyze gene expression levels of complement pathway gene expression. In Aim 2, the study will validate the complement genes identified as key drivers of immune response to Be in epithelial cells. We will use patient-autologous CD4+ T cells and bronchial epithelial cells grown at air-liquid interface (ALI) co-culture models. Specifically, we will use the CRISPR-dCas9 system to target the expression of candidate complement genes (knockdown and overexpression) in airway epithelial cells and determine the impact of complement activation on CD4+ T cell Th1 responses to Be. At the completion of this project, the proposal will enhance our understanding of the identificationcation of novel genes and regulatory pathways associated with Be lung disease, launching an investigation of novel pathogenic mechanisms and setting the stage to determining more rapid and non-invasive detection of CBD and BeS in future studies. Finally, this study will provide a framework to investigate other granulomatous or exposure-related lung diseases, such as hypersensitivity pneumonitis and sarcoidosis.