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Rationale: Changes in peripheral blood cell populations have been observed, but not detailed, at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: We sought to provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with IPF and control subjects were profiled using 10× chromium 5' single-cell RNA sequencing. Flow cytometry was used for validation. Protein concentrations of regulatory T cells (Tregs) and monocyte chemoattractants were measured in plasma and lung homogenates from patients with IPF and control subjects. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched control subjects yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in patients with progressive and stable IPF compared with control subjects (32.1%, 25.2%, and 17.9%, respectively; P  < 0.05). Total lymphocytes were decreased in patients with IPF versus control subjects and in progressive versus stable IPF (52.6% vs. 62.6%, P  = 0.035). Tregs were increased in progressive versus stable IPF (1.8% vs. 1.1% of all PBMCs, P  = 0.007), although not different than controls, and may be associated with decreased survival ( P  = 0.009 in Kaplan-Meier analysis; and P  = 0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of patients with IPF. The fraction of Tregs out of all T cells was also increased in two cohorts of lung single-cell RNA sequencing. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

Rationale: Changes in peripheral blood cell populations have been observed but not detailed at single-cell resolution in idiopathic pulmonary fibrosis (IPF)., Objectives: To provide an atlas of the changes in the peripheral immune system in stable and progressive IPF., Methods: Peripheral blood mononuclear cells (PBMCs) from IPF patients and controls were profiled using 10x Chromium 5' single-cell RNA sequencing (scRNA-seq). Flow cytometry was used for validation. Protein concentrations of Regulatory T-cells (Tregs) and Monocytes chemoattractants were measured in plasma and lung homogenates from patients and controls., Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched controls yielded 149,564 cells that segregated into 23 subpopulations, corresponding to all expected peripheral blood cell populations. Classical monocytes were increased in progressive and stable IPF compared to controls (32.1%, 25.2%, 17.9%, respectively, p<0.05). Total lymphocytes were decreased in IPF vs controls, and in progressive vs stable IPF (52.6% vs 62.6%, p=0.035). Tregs were increased in progressive IPF (1.8% vs 1.1%, p=0.007), and were associated with decreased survival (P=0.009 in Kaplan-Meier analysis). Flow cytometry analysis confirmed this finding in an independent cohort of IPF patients. Tregs were also increased in two cohorts of lung scRNA-seq. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF., Conclusions: The single-cell atlas of the peripheral immune system in IPF, reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs)., Competing Interests: NK is a scientific founder at Thyron, served as a consultant to Biogen Idec, Boehringer Ingelheim, Third Rock, Pliant, Samumed, NuMedii, Theravance, LifeMax, Three Lake Partners, Optikira, Astra Zeneca, RohBar, Veracyte, Augmanity, CSL Behring, Galapagos, Fibrogen, and Thyron over the last 3 years, reports Equity in Pliant and Thyron, and grants from Veracyte, Boehringer Ingelheim, BMS and non-financial support from MiRagen and Astra Zeneca. AU reports receiving research funding from Boehringer Ingelheim, and personal consulting fees or honoraria from Boehringer Ingelheim, Kamada, RemedyCell, Augmanity Nano, Splisense, Veracyte, and 1E Therapeutics in the last 36 months. JCS reports receiving honoraria and travel support from Boehringer Ingelheim. All other authors report no conflict of interest.

Studies comparing single cell RNA-Seq (scRNA-Seq) data between conditions mainly focus on differences in the proportion of cell types or on differentially expressed genes. In many cases these differences are driven by changes in cell interactions which are challenging to infer without spatial information. To determine cell-cell interactions that differ between conditions we developed the Cell Interaction Network Inference (CINS) pipeline. CINS combines Bayesian network analysis with regression-based modeling to identify differential cell type interactions and the proteins that underlie them. We tested CINS on a disease case control and on an aging mouse dataset. In both cases CINS correctly identifies cell type interactions and the ligands involved in these interactions improving on prior methods suggested for cell interaction predictions. We performed additional mouse aging scRNA-Seq experiments which further support the interactions identified by CINS., Competing Interests: The authors have declared that no competing interests exist.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide, however our understanding of cell specific mechanisms underlying COPD pathobiology remains incomplete. Here, we analyze single-cell RNA sequencing profiles of explanted lung tissue from subjects with advanced COPD or control lungs, and we validate findings using single-cell RNA sequencing of lungs from mice exposed to 10 months of cigarette smoke, RNA sequencing of isolated human alveolar epithelial cells, functional in vitro models, and in situ hybridization and immunostaining of human lung tissue samples. We identify a subpopulation of alveolar epithelial type II cells with transcriptional evidence for aberrant cellular metabolism and reduced cellular stress tolerance in COPD. Using transcriptomic network analyses, we predict capillary endothelial cells are inflamed in COPD, particularly through increased CXCL-motif chemokine signaling. Finally, we detect a high-metallothionein expressing macrophage subpopulation enriched in advanced COPD. Collectively, these findings highlight cell-specific mechanisms involved in the pathobiology of advanced COPD., (© 2022. The Author(s).)

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100A hi /HLA-DR lo classical monocytes and activated LAG-3 hi T cells are hallmarks of progressive disease and highlights the abnormal MHC-II/LAG-3 interaction on myeloid and T cells, respectively. We also find skewed T cell receptor repertories in expanded effector CD8 + clones, unmutated IGHG + B cell clones, and mutated B cell clones with stable somatic hypermutation frequency over time. In conclusion, our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19., (© 2022. The Author(s).)

Hypoxia adversely affects the pulmonary circulation of mammals, including vasoconstriction leading to elevated pulmonary arterial pressures. The clinical importance of changes in the structure and function of the large, elastic pulmonary arteries is gaining increased attention, particularly regarding impact in multiple chronic cardiopulmonary conditions. We establish a multi-disciplinary workflow to understand better transcriptional, microstructural, and functional changes of the pulmonary artery in response to sustained hypoxia and how these changes inter-relate. We exposed adult male C57BL/6J mice to normoxic or hypoxic (FiO 2 10%) conditions. Excised pulmonary arteries were profiled transcriptionally using single cell RNA sequencing, imaged with multiphoton microscopy to determine microstructural features under in vivo relevant multiaxial loading, and phenotyped biomechanically to quantify associated changes in material stiffness and vasoactive capacity. Pulmonary arteries of hypoxic mice exhibited an increased material stiffness that was likely due to collagen remodeling rather than excessive deposition (fibrosis), a change in smooth muscle cell phenotype reflected by decreased contractility and altered orientation aligning these cells in the same direction as the remodeled collagen fibers, endothelial proliferation likely representing endothelial-to-mesenchymal transitioning, and a network of cell-type specific transcriptomic changes that drove these changes. These many changes resulted in a system-level increase in pulmonary arterial pulse wave velocity, which may drive a positive feedback loop exacerbating all changes. These findings demonstrate the power of a multi-scale genetic-functional assay. They also highlight the need for systems-level analyses to determine which of the many changes are clinically significant and may be potential therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Manning, Ramachandra, Schupp, Cavinato, Raredon, Bärnthaler, Cosme, Singh, Tellides, Kaminski and Humphrey.)

Background: Cellular diversity of the lung endothelium has not been systematically characterized in humans. 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 6 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 >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, 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 2 previously indistinguishable populations: pulmonary-venous ECs (COL15A1 neg ) localized to the lung parenchyma and systemic-venous ECs (COL15A1 pos ) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB , SOSTDC1 , and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic-venous ECs 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 endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com)., Conclusions: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.