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 COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, creates an urgent need for identifying molecular mechanisms that mediate viral entry, propagation, and tissue pathology. Cell membrane bound angiotensin-converting enzyme 2 (ACE2) and associated proteases, transmembrane protease serine 2 (TMPRSS2) and Cathepsin L (CTSL), were previously identified as mediators of SARS-CoV2 cellular entry. Here, we assess the cell type-specific RNA expression of ACE2, TMPRSS2, and CTSL through an integrated analysis of 107 single-cell and single-nucleus RNA-Seq studies, including 22 lung and airways datasets (16 unpublished), and 85 datasets from other diverse organs. Joint expression of ACE2 and the accessory proteases identifies specific subsets of respiratory epithelial cells as putative targets of viral infection in the nasal passages, airways, and alveoli. Cells that co-express ACE2 and proteases are also identified in cells from other organs, some of which have been associated with COVID-19 transmission or pathology, including gut enterocytes, corneal epithelial cells, cardiomyocytes, heart pericytes, olfactory sustentacular cells, and renal epithelial cells. Performing the first meta-analyses of scRNA-seq studies, we analyzed 1,176,683 cells from 282 nasal, airway, and lung parenchyma samples from 164 donors spanning fetal, childhood, adult, and elderly age groups, associate increased levels of ACE2, TMPRSS2, and CTSL in specific cell types with increasing age, male gender, and smoking, all of which are epidemiologically linked to COVID-19 susceptibility and outcomes. Notably, there was a particularly low expression of ACE2 in the few young pediatric samples in the analysis. Further analysis reveals a gene expression program shared by ACE2 + TMPRSS2 + cells in nasal, lung and gut tissues, including genes that may mediate viral entry, subtend key immune functions, and mediate epithelial-macrophage cross-talk. Amongst these are IL6, its receptor and co-receptor, IL1R, TNF response pathways, and complement genes. Cell type specificity in the lung and airways and smoking effects were conserved in mice. Our analyses suggest that differences in the cell type-specific expression of mediators of SARS-CoV-2 viral entry may be responsible for aspects of COVID-19 epidemiology and clinical course, and point to putative molecular pathways involved in disease susceptibility and pathogenesis.

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2 + TMPRSS2 + cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

Background: The cellular diversity of the lung endothelium has not been systematically characterized in humans. Here, we provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. Methods: We reprocessed human control single cell RNA sequencing (scRNAseq) data from six datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in-vitro was performed. The signaling network between different lung cell types was studied. For cross species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. Results: Six lung scRNAseq datasets were reanalyzed and annotated to identify over 15,000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including pan-endothelial, pan-vascular and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial and venous ECs, we found previously indistinguishable subpopulations: among venous EC, we identified two previously indistinguishable populations, pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary EC, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1 and TBX2 and general capillary EC. We confirmed that all six endothelial cell types, including the systemic-venous EC and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that the endothelial diversity is maintained in pulmonary hypertension. Our manuscript is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). Conclusions: Our integrated analysis provides the comprehensive and well-crafted reference atlas of lung endothelial cells in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages – wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.

No abstract

Although the lung is the most common metastatic site for cancer cells, biological mechanisms regulating lung metastasis are not fully understood. Using heterotopic and intravenous injection models of lung metastasis in mice, we found that IL-5, a cytokine involved in allergic and infectious diseases, facilitates metastatic colonization through recruitment of sentinel eosinophils and regulation of other inflammatory/immune cells in the microenvironment of the distal lung. Genetic IL-5 deficiency offered marked protection of the lungs from metastasis of different types of tumor cells, including lung cancer, melanoma and colon cancer. IL-5 neutralization protected subjects from metastasis, whereas IL-5 reconstitution or adoptive transfer of eosinophils into IL-5 deficient mice exerted pro-metastatic effects. However, IL-5 deficiency did not affect the growth of the primary tumor or the size of metastatic lesions. Mechanistic investigations revealed that eosinophils produce CCL22, which recruits regulatory T cells (Treg) to the lungs. During early stages of metastasis Treg created a pro-tumorigenic microenvironment, potentially by suppressing IFNγ-producing natural killer cells and M1-polarized macrophages. Together, our results establish a network of allergic inflammatory circuitry that can be co-opted by metastatic cancer cells to facilitate lung colonization, suggesting interventions to target this pathway may offer therapeutic benefits to prevent or treat lung metastasis.

Pulmonary fibrosis is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix. In order to comprehensively define the cell types, mechanisms and mediators driving fibrotic remodeling in lungs with pulmonary fibrosis, we performed single-cell RNAsequencing of single-cell suspensions from 10 non-fibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell types. We report 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. One Sentence SummarySingle-cell RNA-sequencing provides new insights into pathologic epithelial and mesenchymal remodeling in the human lung.