Pulmonary fibrosis (PF) is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix (ECM). To comprehensively define the cell types, mechanisms, and mediators driving fibrotic remodeling in lungs with PF, we performed single-cell RNA sequencing of single-cell suspensions from 10 nonfibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell subsets/states. We report that a remarkable shift in epithelial cell phenotypes occurs in the peripheral lung in PF and identify several previously unrecognized epithelial cell phenotypes, including a KRT5−/KRT17+ pathologic, ECM-producing epithelial cell population that was highly enriched in PF lungs. Multiple fibroblast subtypes were observed to contribute to ECM expansion in a spatially discrete manner. Together, these data provide high-resolution insights into the complexity and plasticity of the distal lung epithelium in human disease and indicate a diversity of epithelial and mesenchymal cells contribute to pathologic lung fibrosis.

The immunological role of exosomes in allograft rejection remains unknown. We sought to determine whether exosomes are induced during lung allograft rejection and to define the antigenic compositions of human leukocyte antigen (HLA), lung associated self-antigens (SAgs), and microRNAs (miRNAs). Exosomes were isolated from sera and bronchoalveolar lavage fluid from 30 lung transplant recipients (LTxR) who were stable or had acute rejection (AR) or bronchiolitis obliterans syndrome (BOS). Exosomes were defined by flow cytometry for CD63 and Western blotting for Annexin V. SAgs, Collagen-V (Col-V), and Kα1 Tubulin were examined by electron microscopy; miRNAs were profiled by Affymetrix miRNA array. Donor HLA and SAgs were detected on exosomes from LTxRs with AR and BOS, but not from stable LTxRs. Exosomes expressing Col-V were isolated from LTxR sera 3 months before AR and 6 months before BOS diagnosis, suggesting that exosomes with SAgs may be a noninvasive rejection biomarker. Exosomes isolated from LTxRs with AR or BOS also contained immunoregulatory miRNAs. We conclude that exosomes expressing donor HLA, SAgs, and immunoregulatory miRNAs are present in the circulation and local site after human lung transplantation, playing an important role in the immune-pathogenesis of acute allograft rejection and BOS.

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