Single-Cell Analysis Reveals Novel Immune Perturbations in Fibrotic Hypersensitivity Pneumonitis

Rationale: Fibrotic hypersensitivity pneumonitis (FHP) is a debilitating interstitial lung disease driven by incompletely understood immune mechanisms. Objectives: To elucidate immune aberrations in FHP in single-cell resolution. Methods: Single-cell 5' RNA sequencing was conducted on peripheral blood mononuclear cells and BAL cells obtained from 45 patients with FHP, 63 patients with idiopathic pulmonary fibrosis (IPF), 4 patients with nonfibrotic hypersensitivity pneumonitis, and 36 healthy control subjects in the United States and Mexico. Analyses included differential gene expression (Seurat), TF (transcription factor) activity imputation (DoRothEA-VIPER), and trajectory analyses (Monocle3 and Velocyto-scVelo-CellRank). Measurements and Main Results: Overall, 501,534 peripheral blood mononuclear cells from 110 patients and control subjects and 88,336 BAL cells from 19 patients were profiled. Compared with control samples, FHP has elevated classical-monocytes (adjusted-P< 2.5x10(-3)) and is enriched in CCL3(hi)/CCL4(hi) and S100A(hi) classical monocytes (adjusted-P, 2.2x10(-16)). Trajectory analyses demonstrate that S100Ahi classical monocytes differentiate into SPP1(hi) lung macrophages associated with fibrosis. Compared with both control subjects and IPF, cells frompatients with FHP are significantly enriched in GZM(hi) cytotoxic T cells. These cells exhibit TF activities indicative of TGF beta and TNF alpha and NF-kappa B pathways. These results are publicly available at http://ildimmunecellatlas.com. Conclusions: Single-cell transcriptomics of patients with FHP uncovered novel immune perturbations, including previously undescribed increases in GZM(hi) cytotoxic CD4(+) and CD8(+) T cells-reflecting this disease's unique inflammatory T cell-driven nature-as well as increased S100A(hi) and CCL3(hi)/CCL4(hi) classical monocytes also observed in IPF. Both cell populations may guide the development of new biomarkers and therapeutic interventions.