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1.
Cell ; 187(10): 2428-2445.e20, 2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38579712

RESUMEN

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.


Asunto(s)
Células Epiteliales Alveolares , Pulmón , Células Madre , Animales , Ratones , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/citología , Diferenciación Celular , Linaje de la Célula , Pulmón/citología , Pulmón/metabolismo , Pulmón/fisiología , Lesión Pulmonar/patología , Ratones Endogámicos C57BL , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Receptores Notch/metabolismo , Regeneración , Transducción de Señal , Células Madre/metabolismo , Células Madre/citología
2.
Cell ; 186(7): 1478-1492.e15, 2023 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-36870331

RESUMEN

Lungs undergo mechanical strain during breathing, but how these biophysical forces affect cell fate and tissue homeostasis are unclear. We show that biophysical forces through normal respiratory motion actively maintain alveolar type 1 (AT1) cell identity and restrict these cells from reprogramming into AT2 cells in the adult lung. AT1 cell fate is maintained at homeostasis by Cdc42- and Ptk2-mediated actin remodeling and cytoskeletal strain, and inactivation of these pathways causes a rapid reprogramming into the AT2 cell fate. This plasticity induces chromatin reorganization and changes in nuclear lamina-chromatin interactions, which can discriminate AT1 and AT2 cell identity. Unloading the biophysical forces of breathing movements leads to AT1-AT2 cell reprogramming, revealing that normal respiration is essential to maintain alveolar epithelial cell fate. These data demonstrate the integral function of mechanotransduction in maintaining lung cell fate and identifies the AT1 cell as an important mechanosensor in the alveolar niche.


Asunto(s)
Células Epiteliales Alveolares , Mecanotransducción Celular , Células Epiteliales Alveolares/metabolismo , Células Cultivadas , Pulmón , Diferenciación Celular/fisiología , Respiración
3.
Cell ; 184(1): 92-105.e16, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33147445

RESUMEN

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.


Asunto(s)
COVID-19/genética , COVID-19/virología , Interacciones Huésped-Patógeno , SARS-CoV-2/fisiología , Células A549 , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , Enzima Convertidora de Angiotensina 2/metabolismo , Vías Biosintéticas , COVID-19/metabolismo , Colesterol/biosíntesis , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endosomas/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes/métodos , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Interferencia de ARN , SARS-CoV-2/crecimiento & desarrollo , Análisis de la Célula Individual , Carga Viral/efectos de los fármacos , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7
4.
Cell ; 181(5): 1016-1035.e19, 2020 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-32413319

RESUMEN

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.


Asunto(s)
Células Epiteliales Alveolares/metabolismo , Enterocitos/metabolismo , Células Caliciformes/metabolismo , Interferón Tipo I/metabolismo , Mucosa Nasal/citología , Peptidil-Dipeptidasa A/genética , Adolescente , Células Epiteliales Alveolares/inmunología , Enzima Convertidora de Angiotensina 2 , Animales , Betacoronavirus/fisiología , COVID-19 , Línea Celular , Células Cultivadas , Niño , Infecciones por Coronavirus/virología , Enterocitos/inmunología , Células Caliciformes/inmunología , Infecciones por VIH/inmunología , Humanos , Gripe Humana/inmunología , Interferón Tipo I/inmunología , Pulmón/citología , Pulmón/patología , Macaca mulatta , Ratones , Mycobacterium tuberculosis , Mucosa Nasal/inmunología , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/virología , Receptores Virales/genética , SARS-CoV-2 , Serina Endopeptidasas/metabolismo , Análisis de la Célula Individual , Tuberculosis/inmunología , Regulación hacia Arriba
5.
Nature ; 627(8004): 656-663, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38418883

RESUMEN

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8+ alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8+ cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.


Asunto(s)
Adenocarcinoma del Pulmón , Diferenciación Celular , Células Epiteliales , Neoplasias Pulmonares , Animales , Humanos , Ratones , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Aneuploidia , Carcinógenos/toxicidad , Células Epiteliales/clasificación , Células Epiteliales/metabolismo , Células Epiteliales/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación , Organoides/efectos de los fármacos , Organoides/metabolismo , Lesiones Precancerosas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Tasa de Supervivencia , Productos de Tabaco/efectos adversos , Productos de Tabaco/toxicidad
6.
Nature ; 628(8009): 835-843, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38600381

RESUMEN

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.


Asunto(s)
Lesión Pulmonar , Necroptosis , Infecciones por Orthomyxoviridae , Inhibidores de Proteínas Quinasas , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Animales , Femenino , Humanos , Masculino , Ratones , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/virología , Células Epiteliales Alveolares/metabolismo , Virus de la Influenza A/clasificación , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/inmunología , Virus de la Influenza A/patogenicidad , Lesión Pulmonar/complicaciones , Lesión Pulmonar/patología , Lesión Pulmonar/prevención & control , Lesión Pulmonar/virología , Ratones Endogámicos C57BL , Necroptosis/efectos de los fármacos , Infecciones por Orthomyxoviridae/complicaciones , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/virología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Síndrome de Dificultad Respiratoria/complicaciones , Síndrome de Dificultad Respiratoria/patología , Síndrome de Dificultad Respiratoria/prevención & control , Síndrome de Dificultad Respiratoria/virología
7.
Immunity ; 52(2): 275-294.e9, 2020 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-32075728

RESUMEN

Type 3 innate lymphoid cells (ILC3s) are critical for lung defense against bacterial pneumonia in the neonatal period, but the signals that guide pulmonary ILC3 development remain unclear. Here, we demonstrated that pulmonary ILC3s descended from ILC precursors that populated a niche defined by fibroblasts in the developing lung. Alveolar fibroblasts produced insulin-like growth factor 1 (IGF1), which instructed expansion and maturation of pulmonary ILC precursors. Conditional ablation of IGF1 in alveolar fibroblasts or deletion of the IGF-1 receptor from ILC precursors interrupted ILC3 biogenesis and rendered newborn mice susceptible to pneumonia. Premature infants with bronchopulmonary dysplasia, characterized by interrupted postnatal alveolar development and increased morbidity to respiratory infections, had reduced IGF1 concentrations and pulmonary ILC3 numbers. These findings indicate that the newborn period is a critical window in pulmonary immunity development, and disrupted lung development in prematurely born infants may have enduring effects on host resistance to respiratory infections.


Asunto(s)
Inmunidad Innata , Factor I del Crecimiento Similar a la Insulina/metabolismo , Pulmón/inmunología , Linfocitos/citología , Células Epiteliales Alveolares/metabolismo , Animales , Animales Recién Nacidos , Displasia Broncopulmonar/inmunología , Diferenciación Celular , Proliferación Celular , Susceptibilidad a Enfermedades/inmunología , Humanos , Recién Nacido , Recien Nacido Prematuro , Factor I del Crecimiento Similar a la Insulina/deficiencia , Interleucinas/metabolismo , Pulmón/citología , Pulmón/crecimiento & desarrollo , Linfocitos/metabolismo , Ratones , Neumonía/inmunología , Proteína de la Leucemia Promielocítica con Dedos de Zinc/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Interleucina-22
8.
Nature ; 620(7975): 890-897, 2023 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-37558881

RESUMEN

Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1-6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.


Asunto(s)
Células Epiteliales Alveolares , Diferenciación Celular , Linaje de la Célula , Pulmón , Mitocondrias , Estrés Fisiológico , Animales , Ratones , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Pulmón/citología , Pulmón/metabolismo , Pulmón/patología , Mitocondrias/enzimología , Mitocondrias/metabolismo , NAD/metabolismo , NADH Deshidrogenasa/metabolismo , Protones , RNA-Seq , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análisis de Expresión Génica de una Sola Célula
9.
Nature ; 619(7971): 851-859, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37468633

RESUMEN

Lung cancer is the leading cause of cancer deaths worldwide1. Mutations in the tumour suppressor gene TP53 occur in 50% of lung adenocarcinomas (LUADs) and are linked to poor prognosis1-4, but how p53 suppresses LUAD development remains enigmatic. We show here that p53 suppresses LUAD by governing cell state, specifically by promoting alveolar type 1 (AT1) differentiation. Using mice that express oncogenic Kras and null, wild-type or hypermorphic Trp53 alleles in alveolar type 2 (AT2) cells, we observed graded effects of p53 on LUAD initiation and progression. RNA sequencing and ATAC sequencing of LUAD cells uncovered a p53-induced AT1 differentiation programme during tumour suppression in vivo through direct DNA binding, chromatin remodelling and induction of genes characteristic of AT1 cells. Single-cell transcriptomics analyses revealed that during LUAD evolution, p53 promotes AT1 differentiation through action in a transitional cell state analogous to a transient intermediary seen during AT2-to-AT1 cell differentiation in alveolar injury repair. Notably, p53 inactivation results in the inappropriate persistence of these transitional cancer cells accompanied by upregulated growth signalling and divergence from lung lineage identity, characteristics associated with LUAD progression. Analysis of Trp53 wild-type and Trp53-null mice showed that p53 also directs alveolar regeneration after injury by regulating AT2 cell self-renewal and promoting transitional cell differentiation into AT1 cells. Collectively, these findings illuminate mechanisms of p53-mediated LUAD suppression, in which p53 governs alveolar differentiation, and suggest that tumour suppression reflects a fundamental role of p53 in orchestrating tissue repair after injury.


Asunto(s)
Células Epiteliales Alveolares , Diferenciación Celular , Neoplasias Pulmonares , Pulmón , Proteína p53 Supresora de Tumor , Animales , Ratones , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Pulmón/citología , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Ratones Noqueados , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Alelos , Perfilación de la Expresión Génica , Ensamble y Desensamble de Cromatina , ADN/metabolismo , Lesión Pulmonar/genética , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Progresión de la Enfermedad , Linaje de la Célula , Regeneración , Autorrenovación de las Células
10.
Mol Cell ; 80(6): 1104-1122.e9, 2020 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-33259812

RESUMEN

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.


Asunto(s)
Células Epiteliales Alveolares/metabolismo , COVID-19/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , SARS-CoV-2/metabolismo , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/virología , Animales , Antivirales , COVID-19/genética , COVID-19/patología , Chlorocebus aethiops , Efecto Citopatogénico Viral , Citoesqueleto , Evaluación Preclínica de Medicamentos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/virología , Fosfoproteínas/genética , Transporte de Proteínas , Proteoma/genética , SARS-CoV-2/genética , Transducción de Señal , Células Vero , Tratamiento Farmacológico de COVID-19
11.
Proc Natl Acad Sci U S A ; 121(16): e2400077121, 2024 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-38598345

RESUMEN

Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to lower airway repair. Agents that promote the selective expansion of these cells might stimulate regeneration of the compromised alveolar epithelium, an etiology-defining event in several pulmonary diseases. From a high-content imaging screen of the drug repurposing library ReFRAME, we identified that dipeptidyl peptidase 4 (DPP4) inhibitors, widely used type 2 diabetes medications, selectively expand AEC2s and are broadly efficacious in several mouse models of lung damage. Mechanism of action studies revealed that the protease DPP4, in addition to processing incretin hormones, degrades IGF-1 and IL-6, essential regulators of AEC2 expansion whose levels are increased in the luminal compartment of the lung in response to drug treatment. To selectively target DPP4 in the lung with sufficient drug exposure, we developed NZ-97, a locally delivered, lung persistent DPP4 inhibitor that broadly promotes efficacy in mouse lung damage models with minimal peripheral exposure and good tolerability. This work reveals DPP4 as a central regulator of AEC2 expansion and affords a promising therapeutic approach to broadly stimulate regenerative repair in pulmonary disease.


Asunto(s)
Células Epiteliales Alveolares , Diabetes Mellitus Tipo 2 , Animales , Ratones , Células Epiteliales Alveolares/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Pulmón/metabolismo , Modelos Animales de Enfermedad
12.
Am J Hum Genet ; 110(10): 1735-1749, 2023 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-37734371

RESUMEN

Emphysema and chronic obstructive pulmonary disease (COPD) most commonly result from the effects of environmental exposures in genetically susceptible individuals. Genome-wide association studies have implicated ADGRG6 in COPD and reduced lung function, and a limited number of studies have examined the role of ADGRG6 in cells representative of the airway. However, the ADGRG6 locus is also associated with DLCO/VA, an indicator of gas exchange efficiency and alveolar function. Here, we sought to evaluate the mechanistic contributions of ADGRG6 to homeostatic function and disease in type 2 alveolar epithelial cells. We applied an inducible CRISPR interference (CRISPRi) human induced pluripotent stem cell (iPSC) platform to explore ADGRG6 function in iPSC-derived AT2s (iAT2s). We demonstrate that ADGRG6 exerts pleiotropic effects on iAT2s including regulation of focal adhesions, cytoskeleton, tight junctions, and proliferation. Moreover, we find that ADGRG6 knockdown in cigarette smoke-exposed iAT2s alters cellular responses to injury, downregulating apical complexes in favor of proliferation. Our work functionally characterizes the COPD GWAS gene ADGRG6 in human alveolar epithelium.


Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad Pulmonar Obstructiva Crónica , Receptores Acoplados a Proteínas G , Humanos , Células Epiteliales Alveolares/metabolismo , Células Epiteliales/metabolismo , Estudio de Asociación del Genoma Completo , Células Madre Pluripotentes Inducidas/metabolismo , Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Receptores Acoplados a Proteínas G/genética
13.
J Immunol ; 213(5): 600-611, 2024 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-39033086

RESUMEN

The aryl hydrocarbon receptor (AHR) is a receptor/transcription factor widely expressed in the lung. The physiological roles of AHR expressed in the alveolar epithelium remain unclear. In this study, we tested the hypothesis that alveolar epithelial AHR activity plays an important role in modulating inflammatory responses and maintaining alveolar integrity during lung injury and repair. AHR is expressed in alveolar epithelial cells (AECs) and is active. AHR activation with the endogenous AHR ligand, FICZ (5,11-dihydroindolo[3,2-b] carbazole-6-carboxaldehyde), significantly suppressed inflammatory cytokine expression in response to inflammatory stimuli in primary murine AECs and in the MLE-15 epithelial cell line. In an LPS model of acute lung injury in mice, coadministration of FICZ with LPS suppressed protein leak, reduced neutrophil accumulation in BAL fluid, and suppressed inflammatory cytokine expression in lung tissue and BAL fluid. Relevant to healing following inflammatory injury, AHR activation suppressed TGF-ß-induced expression of genes associated with epithelial-mesenchymal transition. Knockdown of AHR in primary AECs with shRNA or in CRISPR-Cas-9-induced MLE-15 cells resulted in upregulation of α-smooth muscle actin (αSma), Col1a1, and Fn1 and reduced expression of epithelial genes Col4a1 and Sdc1. MLE-15 clones lacking AHR demonstrated accelerated wound closure in a scratch model. AHR activation with FICZ enhanced barrier function (transepithelial electrical resistance) in primary murine AECs and limited decline of transepithelial electrical resistance following inflammatory injury. AHR activation in AECs preserves alveolar integrity by modulating inflammatory cytokine expression while enhancing barrier function and limiting stress-induced expression of mesenchymal genes.


Asunto(s)
Células Epiteliales Alveolares , Receptores de Hidrocarburo de Aril , Animales , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Hidrocarburo de Aril/genética , Ratones , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/inmunología , Inflamación/inmunología , Ratones Endogámicos C57BL , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/metabolismo , Línea Celular , Citocinas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico
14.
J Immunol ; 213(3): 268-282, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-38856585

RESUMEN

Recruitment of immune cells to the injury site plays a pivotal role in the pathology of radiation-associated diseases. In this study, we investigated the impact of the chemokine CCL22 released from alveolar type II epithelial (AT2) cells after irradiation on the recruitment and functional changes of dendritic cells (DCs) in the development of radiation-induced lung injury (RILI). By examining changes in CCL22 protein levels in lung tissue of C57BL/6N mice with RILI, we discovered that ionizing radiation increased CCL22 expression in irradiated alveolar AT2 cells, as did MLE-12 cells after irradiation. A transwell migration assay revealed that CCL22 promoted the migration of CCR4-positive DCs to the injury site, which explained the migration of pulmonary CCR4-positive DCs in RILI mice in vivo. Coculture experiments demonstrated that, consistent with the response of regulatory T cells in the lung tissue of RILI mice, exogenous CCL22-induced DCs promoted regulatory T cell proliferation. Mechanistically, we demonstrated that Dectin2 and Nr4a2 are key targets in the CCL22 signaling pathway, which was confirmed in pulmonary DCs of RILI mice. As a result, CCL22 upregulated the expression of PD-L1, IL-6, and IL-10 in DCs. Consequently, we identified a mechanism in which CCL22 induced DC tolerance through the CCR4-Dectin2-PLC-γ2-NFATC2-Nr4a2-PD-L1 pathway. Collectively, these findings demonstrated that ionizing radiation stimulates the expression of CCL22 in AT2 cells to recruit DCs to the injury site and further polarizes them into a tolerant subgroup of CCL22 DCs to regulate lung immunity, ultimately providing potential therapeutic targets for DC-mediated RILI.


Asunto(s)
Antígeno B7-H1 , Quimiocina CCL22 , Células Dendríticas , Lesión Pulmonar , Ratones Endogámicos C57BL , Factores de Transcripción NFATC , Receptores CCR4 , Transducción de Señal , Animales , Ratones , Células Dendríticas/inmunología , Transducción de Señal/inmunología , Lesión Pulmonar/inmunología , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/inmunología , Antígeno B7-H1/inmunología , Tolerancia Inmunológica , Células Epiteliales Alveolares/inmunología , Células Epiteliales Alveolares/metabolismo , Linfocitos T Reguladores/inmunología
15.
Nature ; 588(7839): 670-675, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33238290

RESUMEN

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.


Asunto(s)
COVID-19/virología , Pulmón/citología , Modelos Biológicos , Organoides/citología , Organoides/virología , SARS-CoV-2/fisiología , Técnicas de Cultivo de Tejidos , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , COVID-19/metabolismo , COVID-19/patología , Diferenciación Celular , División Celular , Células Clonales/citología , Células Clonales/metabolismo , Células Clonales/virología , Humanos , Técnicas In Vitro , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Integrina alfa6/análisis , Integrina beta4/análisis , Queratina-5/análisis , Organoides/metabolismo , Neumonía Viral/metabolismo , Neumonía Viral/patología , Neumonía Viral/virología , SARS-CoV-2/crecimiento & desarrollo , Análisis de la Célula Individual , Receptor de TWEAK/análisis
16.
Nature ; 588(7836): 151-156, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33149305

RESUMEN

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.


Asunto(s)
Pulmón/efectos de los fármacos , Pulmón/fisiología , Receptor beta de Linfotoxina/antagonistas & inhibidores , Regeneración/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/agonistas , Inmunidad Adaptativa , Envejecimiento/metabolismo , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Animales , Apoptosis/efectos de los fármacos , Enfisema/metabolismo , Femenino , Humanos , Inmunidad Innata , Pulmón/metabolismo , Receptor beta de Linfotoxina/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Humo/efectos adversos , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
17.
EMBO J ; 40(6): e105543, 2021 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-33586810

RESUMEN

Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces.


Asunto(s)
Proteínas Relacionadas con la Autofagia/genética , Virus de la Influenza A/patogenicidad , Proteínas Asociadas a Microtúbulos/metabolismo , Infecciones por Orthomyxoviridae/genética , Eliminación de Secuencia , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , Animales , Autofagia , Proteínas Relacionadas con la Autofagia/química , Proteínas Relacionadas con la Autofagia/metabolismo , Embrión de Pollo , Citocinas/metabolismo , Perros , Células de Riñón Canino Madin Darby , Ratones , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/mortalidad , Dominios Proteicos , Replicación Viral
18.
EMBO J ; 40(5): e107651, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33576058

RESUMEN

Defining the pulmonary cell types infected by SARS-CoV-2 and finding ways to prevent subsequent tissue damage are key goals for controlling COVID-19. Recent work establishing a human lung organoid-derived air-liquid interface model permissive to SARS-CoV-2 infection identifies alveolar type II cells as the primary cell type infected, reports an infection-induced interferon response and demonstrates the effectiveness of interferon lambda 1 treatment in dampening lung infection.


Asunto(s)
Células Epiteliales Alveolares/metabolismo , COVID-19/metabolismo , Modelos Biológicos , Organoides/metabolismo , SARS-CoV-2/fisiología , Replicación Viral , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/virología , COVID-19/patología , Humanos , Organoides/patología , Organoides/virología , Tratamiento Farmacológico de COVID-19
19.
EMBO J ; 40(5): e105912, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33283287

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interface culture system which was characterized by confocal and electron microscopy and single-cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing fetal lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive alveolar type II-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.


Asunto(s)
Células Epiteliales Alveolares/metabolismo , COVID-19/metabolismo , Modelos Biológicos , Organoides/metabolismo , SARS-CoV-2/fisiología , Replicación Viral , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/virología , Animales , COVID-19/virología , Chlorocebus aethiops , Regulación de la Expresión Génica , Humanos , Interferón Tipo I/biosíntesis , Interferones/biosíntesis , Organoides/patología , Organoides/virología , Células Vero , Interferón lambda
20.
Development ; 149(1)2022 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-34931663

RESUMEN

Alveologenesis requires the coordinated modulation of the epithelial and mesenchymal compartments to generate mature alveolar saccules for efficient gas exchange. However, the molecular mechanisms underlying the epithelial-mesenchymal interaction during alveologenesis are poorly understood. Here, we report that Wnts produced by epithelial cells are crucial for neonatal alveologenesis. Deletion of the Wnt chaperone protein Wntless homolog (Wls) disrupts alveolar formation, resulting in enlarged saccules in Sftpc-Cre/Nkx2.1-Cre; Wlsloxp/loxp mutants. Although commitment of the alveolar epithelium is unaffected, α-SMA+ mesenchymal cells persist in the alveoli, accompanied by increased collagen deposition, and mutants exhibit exacerbated fibrosis following bleomycin challenge. Notably, α-SMA+ cells include a significant number of endothelial cells resembling endothelial to mesenchymal transition (EndMT), which is also present in Ager-CreER; Wlsloxp/loxp mutants following early postnatal Wls deletion. These findings provide initial evidence that epithelial-derived Wnts are crucial for the differentiation of the surrounding mesenchyme during early postnatal alveologenesis.


Asunto(s)
Células Epiteliales Alveolares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Actinas/genética , Actinas/metabolismo , Células Epiteliales Alveolares/citología , Animales , Células Cultivadas , Transición Epitelial-Mesenquimal , Ratones , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/genética
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