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We present a multiomic cell atlas of human lung development that combines single-cell RNA and ATAC sequencing, high-throughput spatial transcriptomics, and single-cell imaging. Coupling single-cell methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial, and erythrocyte/leukocyte compartments from 5-22 post-conception weeks. We identify previously uncharacterized cell states in all compartments. These include developmental-specific secretory progenitors and a subtype of neuroendocrine cell related to human small cell lung cancer. Our datasets are available through our web interface (https://lungcellatlas.org). To illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signaling and transcription factor hierarchies which we rigorously test using organoid models.
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Feto , Pulmón , Humanos , Diferenciación Celular , Perfilación de la Expresión Génica , Pulmón/citología , Organogénesis , Organoides , Atlas como Asunto , Feto/citologíaRESUMEN
The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the dynamics of early cellular responses to this disease remains limited1. Here in our SARS-CoV-2 human challenge study, we used single-cell multi-omics profiling of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in seronegative individuals challenged with pre-Alpha SARS-CoV-2. Our analyses revealed rapid changes in cell-type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific time points and infection status. We observed that the interferon response in blood preceded the nasopharyngeal response. Moreover, nasopharyngeal immune infiltration occurred early in samples from individuals with only transient infection and later in samples from individuals with sustained infection. High expression of HLA-DQA2 before inoculation was associated with preventing sustained infection. Ciliated cells showed multiple immune responses and were most permissive for viral replication, whereas nasopharyngeal T cells and macrophages were infected non-productively. We resolved 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our new computational pipeline Cell2TCR identifies activated antigen-responding T cells based on a gene expression signature and clusters these into clonotype groups and motifs. Overall, our detailed time series data can serve as a Rosetta stone for epithelial and immune cell responses and reveals early dynamic responses associated with protection against infection.
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COVID-19 , Nasofaringe , SARS-CoV-2 , Análisis de la Célula Individual , Linfocitos T , Humanos , COVID-19/inmunología , COVID-19/virología , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/fisiología , Nasofaringe/virología , Nasofaringe/inmunología , Linfocitos T/inmunología , Linfocitos T/virología , Interferones/inmunología , Interferones/metabolismo , Masculino , Femenino , Macrófagos/inmunología , Macrófagos/virología , Replicación Viral , Células Epiteliales/virología , Células Epiteliales/inmunología , Factores de Tiempo , AdultoRESUMEN
Rationale: Obesity affects 40% of U.S. adults, is associated with a proinflammatory state, and presents a significant risk factor for the development of severe coronavirus disease (COVID-19). To date, there is limited information on how obesity might affect immune cell responses in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Objectives: To determine the impact of obesity on respiratory tract immunity in COVID-19 across the human lifespan. Methods: We analyzed single-cell transcriptomes from BAL in three ventilated adult cohorts with (n = 24) or without (n = 9) COVID-19 from nasal immune cells in children with (n = 14) or without (n = 19) COVID-19, and from peripheral blood mononuclear cells in an independent adult COVID-19 cohort (n = 42), comparing obese and nonobese subjects. Measurements and Main Results: Surprisingly, we found that obese adult subjects had attenuated lung immune or inflammatory responses in SARS-CoV-2 infection, with decreased expression of IFN-α, IFN-γ, and TNF-α (tumor necrosis factor α) response gene signatures in almost all lung epithelial and immune cell subsets, and lower expression of IFNG and TNF in specific lung immune cells. Peripheral blood immune cells in an independent adult cohort showed a similar but less marked reduction in type-I IFN and IFNγ response genes, as well as decreased serum IFNα, in obese patients with SARS-CoV-2. Nasal immune cells from obese children with COVID-19 also showed reduced enrichment of IFN-α and IFN-γ response genes. Conclusions: These findings show blunted tissue immune responses in obese patients with COVID-19, with implications for treatment stratification, supporting the specific application of inhaled recombinant type-I IFNs in this vulnerable subset.
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COVID-19 , Interferón Tipo I , Obesidad Infantil , Adulto , Humanos , Niño , SARS-CoV-2 , Leucocitos Mononucleares , Pulmón/patologíaRESUMEN
The National Heart, Lung, and Blood Institute of the National Institutes of Health, together with the Longfonds BREATH consortium, convened a working group to review the field of lung regeneration and suggest avenues for future research. The meeting took place on May 22, 2019, at the American Thoracic Society 2019 conference in Dallas, Texas, United States, and brought together investigators studying lung development, adult stem-cell biology, induced pluripotent stem cells, biomaterials, and respiratory disease. The purpose of the working group was 1) to examine the present status of basic science approaches to tackling lung disease and promoting lung regeneration in patients and 2) to determine priorities for future research in the field.
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Células Madre Pluripotentes Inducidas , Enfermedades Pulmonares , Pulmón/fisiología , Regeneración , Mucosa Respiratoria/fisiología , Animales , Tratamiento Basado en Trasplante de Células y Tejidos , Congresos como Asunto , Educación , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/terapia , National Heart, Lung, and Blood Institute (U.S.) , Estados UnidosRESUMEN
Mesothelioma is an aggressive cancer that is associated with exposure to asbestos. Although asbestos is banned in several countries, including the UK, an epidemic of mesothelioma is predicted to affect middle-income countries during this century owing to their heavy consumption of asbestos. The prognosis for patients with mesothelioma is poor, reflecting a failure of conventional chemotherapy that has ultimately resulted from an inadequate understanding of its biology. However, recent work has revolutionised the study of mesothelioma, identifying genetic and pathophysiological vulnerabilities, including the loss of tumour suppressors, epigenetic dysregulation and susceptibility to nutrient stress. We discuss how this knowledge, combined with advances in immunotherapy, is enabling the development of novel targeted therapies.
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Amianto/toxicidad , Redes Reguladoras de Genes , Mesotelioma/terapia , Terapia Combinada , Epigénesis Genética , Humanos , Mesotelioma/inducido químicamente , Mesotelioma/genética , Mesotelioma/patología , PronósticoRESUMEN
Recent studies have revealed biologically significant differences between human and mouse lung development, and have reported new in vitro systems that allow experimental manipulation of human lung models. At the same time, emerging clinical data suggest that the origins of some adult lung diseases are found in embryonic development and childhood. The convergence of these research themes has fuelled a resurgence of interest in human lung developmental biology. In this Review, we discuss our current understanding of human lung development, which has been profoundly influenced by studies in mice and, more recently, by experiments using in vitro human lung developmental models and RNA sequencing of human foetal lung tissue. Together, these approaches are helping to shed light on the mechanisms underlying human lung development and disease, and may help pave the way for new therapies.
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Embrión de Mamíferos , Desarrollo Embrionario , Enfermedades Pulmonares/embriología , Pulmón/embriología , Modelos Biológicos , Adulto , Humanos , Pulmón/patología , Enfermedades Pulmonares/patologíaRESUMEN
Insufficient alveolar gas exchange capacity is a major contributor to lung disease. During lung development, a population of distal epithelial progenitors first produce bronchiolar-fated and subsequently alveolar-fated progeny. The mechanisms controlling this bronchiolar-to-alveolar developmental transition remain largely unknown. We developed a novel grafting assay to test if lung epithelial progenitors are intrinsically programmed or if alveolar cell identity is determined by environmental factors. These experiments revealed that embryonic lung epithelial identity is extrinsically determined. We show that both glucocorticoid and STAT3 signalling can control the timing of alveolar initiation, but that neither pathway is absolutely required for alveolar fate specification; rather, glucocorticoid receptor and STAT3 work in parallel to promote alveolar differentiation. Thus, developmental acquisition of lung alveolar fate is a robust process controlled by at least two independent extrinsic signalling inputs. Further elucidation of these pathways might provide therapeutic opportunities for restoring alveolar capacity.
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Células Epiteliales/citología , Células Epiteliales/metabolismo , Glucocorticoides/metabolismo , Pulmón/citología , Pulmón/metabolismo , Células Madre/citología , Células Madre/metabolismo , Adenoviridae/genética , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Glucocorticoides/antagonistas & inhibidores , Humanos , Ratones , Mifepristona/farmacología , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Background: Persistent radiological lung abnormalities are evident in many survivors of acute coronavirus disease 2019 (COVID-19). Consolidation and ground glass opacities are interpreted to indicate subacute inflammation whereas reticulation is thought to reflect fibrosis. We sought to identify differences at molecular and cellular level, in the local immunopathology of post-COVID inflammation and fibrosis. Methods: We compared single-cell transcriptomic profiles and T cell receptor (TCR) repertoires of bronchoalveolar cells obtained from convalescent individuals with each radiological pattern, targeting lung segments affected by the predominant abnormality. Results: CD4 central memory T cells and CD8 effector memory T cells were significantly more abundant in those with inflammatory radiology. Clustering of similar TCRs from multiple donors was a striking feature of both phenotypes, consistent with tissue localised antigen-specific immune responses. There was no enrichment for known SARS-CoV-2-reactive TCRs, raising the possibility of T cell-mediated immunopathology driven by failure in immune self-tolerance. Conclusions: Post-COVID radiological inflammation and fibrosis show evidence of shared antigen-specific T cell responses, suggesting a role for therapies targeting T cells in limiting post-COVID lung damage.
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COVID-19 , SARS-CoV-2 , Análisis de la Célula Individual , Humanos , COVID-19/inmunología , COVID-19/patología , SARS-CoV-2/inmunología , Masculino , Femenino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD4-Positivos/inmunología , Pulmón/inmunología , Pulmón/patología , Pulmón/diagnóstico por imagen , Anciano , Adulto , Inflamación/inmunología , Inflamación/patología , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/citología , Células T de Memoria/inmunología , TranscriptomaRESUMEN
Children infected with SARS-CoV-2 rarely progress to respiratory failure. However, the risk of mortality in infected people over 85 years of age remains high. Here we investigate differences in the cellular landscape and function of paediatric (<12 years), adult (30-50 years) and older adult (>70 years) ex vivo cultured nasal epithelial cells in response to infection with SARS-CoV-2. We show that cell tropism of SARS-CoV-2, and expression of ACE2 and TMPRSS2 in nasal epithelial cell subtypes, differ between age groups. While ciliated cells are viral replication centres across all age groups, a distinct goblet inflammatory subtype emerges in infected paediatric cultures and shows high expression of interferon-stimulated genes and incomplete viral replication. In contrast, older adult cultures infected with SARS-CoV-2 show a proportional increase in basaloid-like cells, which facilitate viral spread and are associated with altered epithelial repair pathways. We confirm age-specific induction of these cell types by integrating data from in vivo COVID-19 studies and validate that our in vitro model recapitulates early epithelial responses to SARS-CoV-2 infection.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Células Epiteliales , Mucosa Nasal , SARS-CoV-2 , Serina Endopeptidasas , Humanos , COVID-19/virología , SARS-CoV-2/fisiología , SARS-CoV-2/patogenicidad , SARS-CoV-2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Adulto , Persona de Mediana Edad , Anciano , Células Epiteliales/virología , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Mucosa Nasal/virología , Niño , Factores de Edad , Replicación Viral , Preescolar , Tropismo Viral , Masculino , Femenino , Anciano de 80 o más Años , Células Cultivadas , Adolescente , LactanteRESUMEN
Common genetic variants across individuals modulate the cellular response to pathogens and are implicated in diverse immune pathologies, yet how they dynamically alter the response upon infection is not well understood. Here, we triggered antiviral responses in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-sequencing. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), a statistical approach designed to identify nonlinear dynamic genetic effects across transcriptional trajectories of cells. This approach identified 1,275 expression quantitative trait loci (local false discovery rate 10%) that manifested during the responses, many of which were colocalized with susceptibility loci identified by genome-wide association studies of infectious and autoimmune diseases, including the OAS1 splicing quantitative trait locus in a COVID-19 susceptibility locus. In summary, our analytical approach provides a unique framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.
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Enfermedades Autoinmunes , COVID-19 , Tetranitrato de Pentaeritritol , Humanos , Estudio de Asociación del Genoma Completo , Inmunidad InnataRESUMEN
Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org ). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new 'gland-associated immune niche' has implications for respiratory health.
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Pulmón , Mucosa Respiratoria , Humanos , Mucosa Respiratoria/metabolismo , Células Epiteliales/metabolismo , Linfocitos B , Inmunoglobulina A/metabolismoRESUMEN
Studies of human lung development have focused on epithelial and mesenchymal cell types and function, but much less is known about the developing lung immune cells, even though the airways are a major site of mucosal immunity after birth. An unanswered question is whether tissue-resident immune cells play a role in shaping the tissue as it develops in utero. Here, we profiled human embryonic and fetal lung immune cells using scRNA-seq, smFISH, and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including innate lymphoid cells, natural killer cells, myeloid cells, and lineage progenitors. By the canalicular stage, we detected naive T lymphocytes expressing high levels of cytotoxicity genes and the presence of mature B lymphocytes, including B-1 cells. Our analysis suggests that fetal lungs provide a niche for full B cell maturation. Given the presence and diversity of immune cells during development, we also investigated their possible effect on epithelial maturation. We found that IL-1ß drives epithelial progenitor exit from self-renewal and differentiation to basal cells in vitro. In vivo, IL-1ß-producing myeloid cells were found throughout the lung and adjacent to epithelial tips, suggesting that immune cells may direct human lung epithelial development.
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Inmunidad Innata , Pulmón , Humanos , Diferenciación Celular , Células Asesinas Naturales , Células EpitelialesAsunto(s)
Neumotórax/terapia , Guías de Práctica Clínica como Asunto , Neumología/normas , Adolescente , Adulto , Área Bajo la Curva , Tubos Torácicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Neumotórax/diagnóstico por imagen , Curva ROC , Radiografía , Índice de Severidad de la Enfermedad , Sociedades Médicas , Reino Unido , Estados Unidos , Adulto JovenRESUMEN
The European Respiratory Society (ERS) International Congress is the largest respiratory congress and brings together leading experts in all fields of respiratory medicine and research. ERS Assembly 3 shapes the basic and translational science aspects of this congress, aiming to combine cutting-edge novel developments in basic research with novel clinical findings. In this article, we summarise a selection of the scientific highlights from the perspective of the three groups within Assembly 3. In particular, we discuss new insights into the pathophysiology of the human alveolus, novel tools in organoid development and (epi)genome editing, as well as insights from the presented abstracts on novel therapeutic targets being identified for idiopathic pulmonary fibrosis.
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The airways are lined by secretory and multiciliated cells which function together to remove particles and debris from the respiratory tract. The transcriptome of multiciliated cells has been extensively studied, but the function of many of the genes identified is unknown. We have established an assay to test the ability of over-expressed transcripts to promote multiciliated cell differentiation in mouse embryonic tracheal explants. Overexpression data indicated that Fibronectin type 3 and ankyrin repeat domains 1 (Fank1) and JAZF zinc finger 1 (Jazf1) promoted multiciliated cell differentiation alone, and cooperatively with the canonical multiciliated cell transcription factor Foxj1. Moreover, knock-down of Fank1 or Jazf1 in adult mouse airway epithelial cultures demonstrated that these factors are both required for ciliated cell differentiation in vitro This analysis identifies Fank1 and Jazf1 as novel regulators of multiciliated cell differentiation. Moreover, we show that they are likely to function downstream of IL6 signalling and upstream of Foxj1 activity in the process of ciliated cell differentiation. In addition, our in vitro explant assay provides a convenient method for preliminary investigation of over-expression phenotypes in the developing mouse airways.This article has an associated First Person interview with the first author of the paper.
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PURPOSE OF REVIEW: The lung research field has pioneered the use of organoids for the study of cell-cell interactions. RECENT FINDINGS: The use of organoids for airway basal cells is routine. However, the development of organoids for the other regions of the lung is still in its infancy. Such cultures usually rely on cell-cell interactions between the stem cells and a putative niche cell for their growth and differentiation. SUMMARY: The use of co-culture organoid systems has facilitated the in vitro cultivation of previously inaccessible stem cell populations, providing a novel method for dissecting the molecular requirements of these cell-cell interactions. Future technology development will allow the growth of epithelial-only organoids in more defined media and also the introduction of specific non-epithelial cells for the study of cell interactions. These developments will require an improved understanding of the epithelial and non-epithelial cell types present in the lung and their lineage relationships.
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The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated.
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Pulmón/citología , Organoides/crecimiento & desarrollo , Mucosa Respiratoria/citología , Células Madre/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Humanos , RatonesRESUMEN
Heterozygous germ-line mutations in the bone morphogenetic protein type-II receptor (BMPR-II) gene underlie heritable pulmonary arterial hypertension (HPAH). Although inflammation promotes PAH, the mechanisms by which inflammation and BMPR-II dysfunction conspire to cause disease remain unknown. Here we identify that tumour necrosis factor-α (TNFα) selectively reduces BMPR-II transcription and mediates post-translational BMPR-II cleavage via the sheddases, ADAM10 and ADAM17 in pulmonary artery smooth muscle cells (PASMCs). TNFα-mediated suppression of BMPR-II subverts BMP signalling, leading to BMP6-mediated PASMC proliferation via preferential activation of an ALK2/ACTR-IIA signalling axis. Furthermore, TNFα, via SRC family kinases, increases pro-proliferative NOTCH2 signalling in HPAH PASMCs with reduced BMPR-II expression. We confirm this signalling switch in rodent models of PAH and demonstrate that anti-TNFα immunotherapy reverses disease progression, restoring normal BMP/NOTCH signalling. Collectively, these findings identify mechanisms by which BMP and TNFα signalling contribute to disease, and suggest a tractable approach for therapeutic intervention in PAH.