PROJECT SUMMARY Although there has been substantial progress over the past decade in deciphering the pathobiology of progressive pulmonary fibrosis (PF), a large knowledge gap exists regarding the pre-clinical stages of the disease. To address this need, we propose to leverage our ongoing cohort study of asymptomatic relatives of patients with familial PF (FPF) in order to define critical mechanisms driving early disease progression and develop better biomarkers of pre-clinical PF. In this At-Risk for FPF cohort study, 22% of subjects have detectable interstitial lung abnormalities (ILA) on high resolution CT scanning (HRCT) at study enrollment and many of these individuals with ILA have evidence of progression during follow-up. In addition to extensive genotype and phenotype information collected on subjects enrolled in this cohort, we recently performed single-cell RNA-sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) from 49 subjects in this cohort and found that transcriptional profiles of several subsets of immune cells, including monocytes and lymphocytes (T and B cell subsets), differ based on the presence or absence of ILA on HRCT. For example, we identified a subset of CD14+ monocytes that was over-represented in subjects with ILA on HRCT and demonstrated gene expression profile enrichment for growth factor and cytokine signaling pathways. We then determined that PBMCs from subjects with clinical PF undergoing scRNA-seq contained a prominent CD14+ monocyte sub-cluster with a similar transcriptomic profile, thus supporting the potential biological relevance of this approach. In addition, we measured DNA methylation patterns in PBMCs using the DNAge platform and found that At-Risk subjects had increased DNAge compared to chronological age, which was highest in individuals with ILA on HRCT. Overall, we are poised to transform our current understanding of pre-clinical PF by continuing to study this unique cohort. Proposed studies will test the hypothesis that integration of transcriptomic profiling, genetic/epigenetic phenotyping, and HRCT imaging will improve understanding of early disease mechanisms and enable prediction of pre-clinical progression of PF. Specific aims are to: 1) detect transcriptomic biomarkers of pre-clinical disease in a cohort of subjects at-risk for FPF, 2) identify cellular signatures of pre-clinical disease in the lungs of subjects at-risk for FPF, and 3) integrate transcriptomic profiling with measures of “epigenetic age”, genetic susceptibility, and telomere length to identify critical features of early disease activity in subjects at-risk for FPF. By integrating data obtained in this Project with information generated across the Program, we will determine mechanisms of pre-clinical FPF and optimize biomarkers for predicting early disease progression.