ABSTRACT
BACKGROUND: Fibroblast-to-myofibroblast conversion is a major driver of tissue remodelling in organ fibrosis. Distinct lineages of fibroblasts support homeostatic tissue niche functions, yet their specific activation states and phenotypic trajectories during injury and repair have remained unclear. METHODS: We combined spatial transcriptomics, multiplexed immunostainings, longitudinal single-cell RNA-sequencing and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. Our findings were validated in idiopathic pulmonary fibrosis patient tissues in situ as well as in cell differentiation and invasion assays using patient lung fibroblasts. Cell differentiation and invasion assays established a function of SFRP1 in regulating human lung fibroblast invasion in response to transforming growth factor (TGF)ß1. MEASUREMENTS AND MAIN RESULTS: We discovered a transitional fibroblast state characterised by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1 + cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage-specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1 + transitional fibroblasts and Cthrc1 + myofibroblasts. TGFß1 downregulated SFRP1 in noninvasive transitional cells and induced their switch to an invasive CTHRC1+ myofibroblast identity. Finally, using loss-of-function studies we showed that SFRP1 modulates TGFß1-induced fibroblast invasion and RHOA pathway activity. CONCLUSIONS: Our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies SFRP1 as a modulator of TGFß1-driven fibroblast phenotypes in fibrogenesis. These findings are relevant in the context of therapeutic interventions that aim at limiting or reversing fibroblast foci formation.
Subject(s)
Idiopathic Pulmonary Fibrosis , Myofibroblasts , Mice , Animals , Humans , Myofibroblasts/metabolism , Fibroblasts/metabolism , Lung/metabolism , Idiopathic Pulmonary Fibrosis/metabolism , Cell Differentiation , Transforming Growth Factor beta1/metabolism , Extracellular Matrix Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolismABSTRACT
INTRODUCTION: Environmental pollutants injure the mucociliary elevator, thereby provoking disease progression in chronic obstructive pulmonary disease (COPD). Epithelial resilience mechanisms to environmental nanoparticles in health and disease are poorly characterised. METHODS: We delineated the impact of prevalent pollutants such as carbon and zinc oxide nanoparticles, on cellular function and progeny in primary human bronchial epithelial cells (pHBECs) from end-stage COPD (COPD-IV, n=4), early disease (COPD-II, n=3) and pulmonary healthy individuals (n=4). After nanoparticle exposure of pHBECs at air-liquid interface, cell cultures were characterised by functional assays, transcriptome and protein analysis, complemented by single-cell analysis in serial samples of pHBEC cultures focusing on basal cell differentiation. RESULTS: COPD-IV was characterised by a prosecretory phenotype (twofold increase in MUC5AC+) at the expense of the multiciliated epithelium (threefold reduction in Ac-Tub+), resulting in an increased resilience towards particle-induced cell damage (fivefold reduction in transepithelial electrical resistance), as exemplified by environmentally abundant doses of zinc oxide nanoparticles. Exposure of COPD-II cultures to cigarette smoke extract provoked the COPD-IV characteristic, prosecretory phenotype. Time-resolved single-cell transcriptomics revealed an underlying COPD-IV unique basal cell state characterised by a twofold increase in KRT5+ (P=0.018) and LAMB3+ (P=0.050) expression, as well as a significant activation of Wnt-specific (P=0.014) and Notch-specific (P=0.021) genes, especially in precursors of suprabasal and secretory cells. CONCLUSION: We identified COPD stage-specific gene alterations in basal cells that affect the cellular composition of the bronchial elevator and may control disease-specific epithelial resilience mechanisms in response to environmental nanoparticles. The identified phenomena likely inform treatment and prevention strategies.
Subject(s)
Epithelial Cells , Pulmonary Disease, Chronic Obstructive , Humans , Pulmonary Disease, Chronic Obstructive/etiology , Epithelial Cells/metabolism , Male , Middle Aged , Cells, Cultured , Bronchi/pathology , Female , Aged , Zinc Oxide , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Cilia , Nanoparticles , Cell DifferentiationABSTRACT
Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.
Subject(s)
Endothelial Protein C Receptor , Fascia , Wound Healing , Animals , Mice , Cell Differentiation , Cell Hypoxia , Cell Lineage , Disease Models, Animal , Endothelial Protein C Receptor/metabolism , Fascia/cytology , Fascia/injuries , Fascia/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Profiling , Inflammation/metabolism , Inflammation/pathology , Myofibroblasts/cytology , Myofibroblasts/metabolism , Signal Transduction , Single-Cell Gene Expression Analysis , Skin/cytology , Skin/injuries , Skin/metabolism , Tretinoin/metabolismABSTRACT
Extravasation of monocytes into tissue and to the site of injury is a fundamental immunological process, which requires rapid responses via post translational modifications (PTM) of proteins. Protein arginine methyltransferase 7 (PRMT7) is an epigenetic factor that has the capacity to mono-methylate histones on arginine residues. Here we show that in chronic obstructive pulmonary disease (COPD) patients, PRMT7 expression is elevated in the lung tissue and localized to the macrophages. In mouse models of COPD, lung fibrosis and skin injury, reduced expression of PRMT7 associates with decreased recruitment of monocytes to the site of injury and hence less severe symptoms. Mechanistically, activation of NF-κB/RelA in monocytes induces PRMT7 transcription and consequential mono-methylation of histones at the regulatory elements of RAP1A, which leads to increased transcription of this gene that is responsible for adhesion and migration of monocytes. Persistent monocyte-derived macrophage accumulation leads to ALOX5 over-expression and accumulation of its metabolite LTB4, which triggers expression of ACSL4 a ferroptosis promoting gene in lung epithelial cells. Conclusively, inhibition of arginine mono-methylation might offer targeted intervention in monocyte-driven inflammatory conditions that lead to extensive tissue damage if left untreated.
Subject(s)
Protein-Arginine N-Methyltransferases , Pulmonary Disease, Chronic Obstructive , Animals , Arginine/metabolism , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Mice , Monocytes/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Pulmonary Disease, Chronic Obstructive/geneticsABSTRACT
The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for 'reverse phenotyping'. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.
Subject(s)
COVID-19/immunology , Gene Expression Profiling/methods , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , T-Lymphocytes/metabolism , Aged , Aged, 80 and over , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , COVID-19/epidemiology , COVID-19/virology , Cells, Cultured , Cohort Studies , Female , Humans , Male , Middle Aged , Pandemics , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , SARS-CoV-2/physiology , T-Lymphocytes/virologyABSTRACT
The correspondence of cell state changes in diseased organs to peripheral protein signatures is currently unknown. Here, we generated and integrated single-cell transcriptomic and proteomic data from multiple large pulmonary fibrosis patient cohorts. Integration of 233,638 single-cell transcriptomes (n = 61) across three independent cohorts enabled us to derive shifts in cell type proportions and a robust core set of genes altered in lung fibrosis for 45 cell types. Mass spectrometry analysis of lung lavage fluid (n = 124) and plasma (n = 141) proteomes identified distinct protein signatures correlated with diagnosis, lung function, and injury status. A novel SSTR2+ pericyte state correlated with disease severity and was reflected in lavage fluid by increased levels of the complement regulatory factor CFHR1. We further discovered CRTAC1 as a biomarker of alveolar type-2 epithelial cell health status in lavage fluid and plasma. Using cross-modal analysis and machine learning, we identified the cellular source of biomarkers and demonstrated that information transfer between modalities correctly predicts disease status, suggesting feasibility of clinical cell state monitoring through longitudinal sampling of body fluid proteomes.
Subject(s)
Proteomics , Pulmonary Fibrosis , Biomarkers , Bronchoalveolar Lavage Fluid , Calcium-Binding Proteins , Humans , Proteome/metabolismABSTRACT
Lymphotoxin ß-receptor (LTßR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures1,2, which are associated with severe chronic inflammatory diseases that span several organ systems3-6. How LTßR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTßR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTßR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTßR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTßR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTßR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFß signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/ß-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTßR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.
Subject(s)
Lung/drug effects , Lung/physiology , Lymphotoxin beta Receptor/antagonists & inhibitors , Regeneration/drug effects , Signal Transduction/drug effects , Wnt Proteins/agonists , Adaptive Immunity , Aging/metabolism , Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/metabolism , Animals , Apoptosis/drug effects , Emphysema/metabolism , Female , Humans , Immunity, Innate , Lung/metabolism , Lymphotoxin beta Receptor/metabolism , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Smoke/adverse effects , Stem Cells/drug effects , Stem Cells/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolismABSTRACT
The cell type specific sequences of transcriptional programs during lung regeneration have remained elusive. Using time-series single cell RNA-seq of the bleomycin lung injury model, we resolved transcriptional dynamics for 28 cell types. Trajectory modeling together with lineage tracing revealed that airway and alveolar stem cells converge on a unique Krt8 + transitional stem cell state during alveolar regeneration. These cells have squamous morphology, feature p53 and NFkB activation and display transcriptional features of cellular senescence. The Krt8+ state appears in several independent models of lung injury and persists in human lung fibrosis, creating a distinct cell-cell communication network with mesenchyme and macrophages during repair. We generated a model of gene regulatory programs leading to Krt8+ transitional cells and their terminal differentiation to alveolar type-1 cells. We propose that in lung fibrosis, perturbed molecular checkpoints on the way to terminal differentiation can cause aberrant persistence of regenerative intermediate stem cell states.
Subject(s)
Alveolar Epithelial Cells/metabolism , Keratin-8/metabolism , Pulmonary Alveoli/physiology , Pulmonary Fibrosis/pathology , Regeneration , Stem Cells/metabolism , Alveolar Epithelial Cells/cytology , Animals , Cell Communication , Disease Models, Animal , Female , Gene Expression Profiling , Humans , Keratin-8/genetics , Lung Injury/chemically induced , Lung Injury/metabolism , Lung Injury/pathology , Mice , Mice, Inbred C57BL , Pulmonary Alveoli/cytology , Pulmonary Fibrosis/metabolism , Single-Cell Analysis , Stem Cells/cytologyABSTRACT
Surgical adhesions are bands of scar tissues that abnormally conjoin organ surfaces. Adhesions are a major cause of post-operative and dialysis-related complications, yet their patho-mechanism remains elusive, and prevention agents in clinical trials have thus far failed to achieve efficacy. Here, we uncover the adhesion initiation mechanism by coating beads with human mesothelial cells that normally line organ surfaces, and viewing them under adhesion stimuli. We document expansive membrane protrusions from mesothelia that tether beads with massive accompanying adherence forces. Membrane protrusions precede matrix deposition, and can transmit adhesion stimuli to healthy surfaces. We identify cytoskeletal effectors and calcium signaling as molecular triggers that initiate surgical adhesions. A single, localized dose targeting these early germinal events completely prevented adhesions in a preclinical mouse model, and in human assays. Our findings classifies the adhesion pathology as originating from mesothelial membrane bridges and offer a radically new therapeutic approach to treat adhesions.
Subject(s)
Calcium/chemistry , Epithelium/metabolism , Tissue Adhesions/metabolism , Animals , Calcium Signaling , Cell Adhesion , Cell Line , Cell Membrane/metabolism , Computational Biology , Cytoskeleton/metabolism , Cytosol/metabolism , Disease Models, Animal , Female , Humans , Imaging, Three-Dimensional , Male , Mice , Mice, Inbred C57BL , Postoperative Complications , Principal Component Analysis , RNA, Small Interfering/metabolism , Single-Cell AnalysisABSTRACT
Inhaled nanoparticles constitute a potential health hazard due to their size-dependent lung deposition and large surface to mass ratio. Exposure to high levels contributes to the risk of developing respiratory and cardiovascular diseases, as well as of lung cancer. Particle-induced acute phase response may be an important mechanism of action of particle-induced cardiovascular disease. Here, the authors review new important scientific evidence showing causal relationships between inhalation of particle and nanomaterials, induction of acute phase response, and risk of cardiovascular disease. Particle-induced acute phase response provides a means for risk assessment of particle-induced cardiovascular disease and underscores cardiovascular disease as an occupational disease.
Subject(s)
Acute-Phase Reaction , Cardiovascular Diseases , Inhalation Exposure , Nanoparticles , Acute-Phase Reaction/chemically induced , Cardiovascular Diseases/chemically induced , Humans , Inhalation Exposure/adverse effects , Lung/drug effects , Nanoparticles/toxicity , Occupational Diseases/chemically induced , Particle Size , Particulate Matter/toxicityABSTRACT
Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.
Subject(s)
Asthma/pathology , Lung/cytology , Adult , Aged , CD4-Positive T-Lymphocytes/physiology , Cell Communication , Epithelial Cells/immunology , Epithelial Cells/physiology , Female , Genome-Wide Association Study , Goblet Cells/metabolism , Humans , Lung/immunology , Lung/pathology , Male , Metaplasia , Middle Aged , Th2 Cells/physiology , TranscriptomeABSTRACT
Lung disease accounts for every sixth death globally. Profiling the molecular state of all lung cell types in health and disease is currently revolutionizing the identification of disease mechanisms and will aid the design of novel diagnostic and personalized therapeutic regimens. Recent progress in high-throughput techniques for single-cell genomic and transcriptomic analyses has opened up new possibilities to study individual cells within a tissue, classify these into cell types, and characterize variations in their molecular profiles as a function of genetics, environment, cell-cell interactions, developmental processes, aging, or disease. Integration of these cell state definitions with spatial information allows the in-depth molecular description of cellular neighborhoods and tissue microenvironments, including the tissue resident structural and immune cells, the tissue matrix, and the microbiome. The Human Cell Atlas consortium aims to characterize all cells in the healthy human body and has prioritized lung tissue as one of the flagship projects. Here, we present the rationale, the approach, and the expected impact of a Human Lung Cell Atlas.
Subject(s)
Lung Diseases/pathology , Lung/pathology , Humans , Lung/metabolism , Transcriptome/geneticsABSTRACT
Alzheimer's disease (AD) is an age-related progressive form of dementia that features neuronal loss, intracellular tau, and extracellular amyloid-ß (Aß) protein deposition. Neurodegeneration is accompanied by neuroinflammation mainly involving microglia, the resident innate immune cell population of the brain. During AD progression, microglia shift their phenotype, and it has been suggested that they express matricellular proteins such as secreted protein acidic and rich in cysteine (SPARC) and Hevin protein, which facilitate the migration of other immune cells, such as blood-derived dendritic cells. We have detected both SPARC and Hevin in postmortem AD brain tissues and confirmed significant alterations in transcript expression using real-time qPCR. We suggest that an infiltration of myeloid-derived immune cells occurs in the areas of diseased tissue. SPARC is highly expressed in AD brain and collocates to Aß protein deposits, thus contributing actively to cerebral inflammation and subsequent tissue repair, and Hevin may be downregulated in the diseased state. However, further research is needed to reveal the exact roles of SPARC and Hevin proteins and associated signaling pathways in AD-related neuroinflammation. Nevertheless, normalizing SPARC/Hevin protein expression such as interdicting heightened SPARC protein expression may confer a novel therapeutic opportunity for modulating AD progression.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Brain Injuries/metabolism , Brain Injuries/pathology , Calcium-Binding Proteins/biosynthesis , Extracellular Matrix Proteins/biosynthesis , Osteonectin/biosynthesis , Adult , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Brain Injuries/genetics , Calcium-Binding Proteins/genetics , Extracellular Matrix Proteins/genetics , Female , Humans , Laser Capture Microdissection/methods , Male , Middle Aged , Osteonectin/geneticsABSTRACT
Aging promotes lung function decline and susceptibility to chronic lung diseases, which are the third leading cause of death worldwide. Here, we use single cell transcriptomics and mass spectrometry-based proteomics to quantify changes in cellular activity states across 30 cell types and chart the lung proteome of young and old mice. We show that aging leads to increased transcriptional noise, indicating deregulated epigenetic control. We observe cell type-specific effects of aging, uncovering increased cholesterol biosynthesis in type-2 pneumocytes and lipofibroblasts and altered relative frequency of airway epithelial cells as hallmarks of lung aging. Proteomic profiling reveals extracellular matrix remodeling in old mice, including increased collagen IV and XVI and decreased Fraser syndrome complex proteins and collagen XIV. Computational integration of the aging proteome with the single cell transcriptomes predicts the cellular source of regulated proteins and creates an unbiased reference map of the aging lung.
Subject(s)
Aging/genetics , Aging/metabolism , Lung/metabolism , Aging/pathology , Animals , Cholesterol/biosynthesis , Collagen/metabolism , Epithelial Cells/metabolism , Extracellular Matrix/metabolism , Gene Expression Profiling , Lung/cytology , Mice , Mice, Inbred C57BL , Proteome/metabolism , Proteomics , Single-Cell AnalysisABSTRACT
RATIONALE: Analyzing the molecular heterogeneity of different forms of organ fibrosis may reveal common and specific factors and thus identify potential future therapeutic targets. OBJECTIVES: We sought to use proteome-wide profiling of human tissue fibrosis to (1) identify common and specific signatures across end-stage interstitial lung disease (ILD) cases, (2) characterize ILD subgroups in an unbiased fashion, and (3) identify common and specific features of lung and skin fibrosis. METHODS: We collected samples of ILD tissue (n = 45) and healthy donor control samples (n = 10), as well as fibrotic skin lesions from localized scleroderma and uninvolved skin (n = 6). Samples were profiled by quantitative label-free mass spectrometry, Western blotting, or confocal imaging. MEASUREMENTS AND MAIN RESULTS: We determined the abundance of more than 7,900 proteins and stratified these proteins according to their detergent solubility profiles. Common protein regulations across all ILD cases, as well as distinct ILD subsets, were observed. Proteomic comparison of lung and skin fibrosis identified a common upregulation of marginal zone B- and B1-cell-specific protein (MZB1), the expression of which identified MZB1+/CD38+/CD138+/CD27+/CD45-/CD20- plasma B cells in fibrotic lung and skin tissue. MZB1 levels correlated positively with tissue IgG and negatively with diffusing capacity of the lung for carbon monoxide. CONCLUSIONS: Despite the presumably high molecular and cellular heterogeneity of ILD, common protein regulations are observed, even across organ boundaries. The surprisingly high prevalence of MZB1-positive plasma B cells in tissue fibrosis warrants future investigations regarding the causative role of antibody-mediated autoimmunity in idiopathic cases of organ fibrosis, such as idiopathic pulmonary fibrosis.