RESUMO
COVID-19 syndrome is characterized by acute lung injury, hypoxemic respiratory failure, and high mortality. Alveolar type 2 (AT2) cells are essential for gas exchange, repair, and regeneration of distal lung epithelium. We have shown that the causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and other members of the ß-coronavirus genus induce an endoplasmic reticulum (ER) stress response in vitro; however, the consequences for host AT2 cell function in vivo are less understood. To study this, two murine models of coronavirus infection were used-mouse hepatitis virus-1 (MHV-1) in A/J mice and a mouse-adapted SARS-CoV-2 strain. MHV-1-infected mice exhibited dose-dependent weight loss with histological evidence of distal lung injury accompanied by elevated bronchoalveolar lavage fluid (BALF) cell counts and total protein. AT2 cells showed evidence of both viral infection and increased BIP/GRP78 expression, consistent with activation of the unfolded protein response (UPR). The AT2 UPR included increased inositol-requiring enzyme 1α (IRE1α) signaling and a biphasic response in PKR-like ER kinase (PERK) signaling accompanied by marked reductions in AT2 and BALF surfactant protein (SP-B and SP-C) content, increases in surfactant surface tension, and emergence of a reprogrammed epithelial cell population (Krt8+ and Cldn4+). The loss of a homeostatic AT2 cell state was attenuated by treatment with the IRE1α inhibitor OPK-711. As a proof-of-concept, C57BL6 mice infected with mouse-adapted SARS-CoV-2 demonstrated similar lung injury and evidence of disrupted surfactant homeostasis. We conclude that lung injury from ß-coronavirus infection results from an aberrant host response, activating multiple AT2 UPR stress pathways, altering surfactant metabolism/function, and changing AT2 cell state, offering a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and acute respiratory failure.NEW & NOTEWORTHY COVID-19 syndrome is characterized by hypoxemic respiratory failure and high mortality. In this report, we use two murine models to show that ß-coronavirus infection produces acute lung injury, which results from an aberrant host response, activating multiple epithelial endoplasmic reticular stress pathways, disrupting pulmonary surfactant metabolism and function, and forcing emergence of an aberrant epithelial transition state. Our results offer a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and respiratory failure.
Assuntos
COVID-19 , Estresse do Retículo Endoplasmático , Endorribonucleases , Homeostase , Vírus da Hepatite Murina , SARS-CoV-2 , Animais , Camundongos , COVID-19/metabolismo , COVID-19/patologia , COVID-19/virologia , COVID-19/complicações , Vírus da Hepatite Murina/patogenicidade , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Células Epiteliais Alveolares/patologia , Chaperona BiP do Retículo Endoplasmático , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Infecções por Coronavirus/complicações , Surfactantes Pulmonares/metabolismo , Resposta a Proteínas não Dobradas , Betacoronavirus , Insuficiência Respiratória/metabolismo , Insuficiência Respiratória/virologia , Insuficiência Respiratória/patologia , Modelos Animais de Doenças , eIF-2 Quinase/metabolismo , HumanosRESUMO
Hermansky-Pudlak syndrome (HPS) is a genetic disorder of endosomal protein trafficking associated with pulmonary fibrosis in specific subtypes, including HPS-1 and HPS-2. Single mutant HPS1 and HPS2 mice display increased fibrotic sensitivity while double mutant HPS1/2 mice exhibit spontaneous fibrosis with aging, which has been attributed to HPS mutations in alveolar epithelial type II (AT2) cells. We utilized HPS mouse models and human lung tissue to investigate mechanisms of AT2 cell dysfunction driving fibrotic remodeling in HPS. Starting at 8 weeks of age, HPS mice exhibited progressive loss of AT2 cell numbers. HPS AT2 cell was impaired ex vivo and in vivo. Incorporating AT2 cell lineage tracing in HPS mice, we observed aberrant differentiation with increased AT2-derived alveolar epithelial type I cells. Transcriptomic analysis of HPS AT2 cells revealed elevated expression of genes associated with aberrant differentiation and p53 activation. Lineage tracing and modeling studies demonstrated that HPS AT2 cells were primed to persist in a Krt8+ reprogrammed transitional state, mediated by p53 activity. Intrinsic AT2 progenitor cell dysfunction and p53 pathway dysregulation are novel mechanisms of disease in HPS-related pulmonary fibrosis, with the potential for early targeted intervention before the onset of fibrotic lung disease.
RESUMO
Epithelial cell organoids have increased opportunities to probe questions on tissue development and disease in vitro and for therapeutic cell transplantation. Despite their potential, current protocols to grow these organoids almost exclusively depend on culture within 3D Matrigel, which limits defined culture conditions, introduces animal components, and results in heterogenous organoids (i.e., shape, size, composition). Here, a method is described that relies on hyaluronic acid hydrogels for the generation and expansion of lung alveolar organoids (alveolospheres). Using synthetic hydrogels with defined chemical and physical properties, human-induced pluripotent stem cell (iPSC)-derived alveolar type 2 cells (iAT2s) self-assemble into alveolospheres and propagate in Matrigel-free conditions. By engineering predefined microcavities within these hydrogels, the heterogeneity of alveolosphere size and structure is reduced when compared to 3D culture, while maintaining the alveolar type 2 cell fate of human iAT2-derived progenitor cells. This hydrogel system is a facile and accessible system for the culture of iPSC-derived lung progenitors and the method can be expanded to the culture of primary mouse tissue derived AT2 and other epithelial progenitor and stem cell aggregates.
Assuntos
Hidrogéis , Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Ácido Hialurônico/metabolismo , Hidrogéis/química , Células-Tronco Pluripotentes Induzidas/metabolismo , Pulmão , Camundongos , Organoides/metabolismoRESUMO
Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3-BDNF-TrkB axis in orchestrating alveolar epithelial regeneration.
Assuntos
Células Epiteliais Alveolares/citologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Lesão Pulmonar/prevenção & controle , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor trkB/metabolismo , Regeneração , Fator de Transcrição STAT3/metabolismo , Células Epiteliais Alveolares/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Feminino , Humanos , Lesão Pulmonar/etiologia , Lesão Pulmonar/patologia , Masculino , Glicoproteínas de Membrana/genética , Proteínas Tirosina Quinases/genética , Receptor trkB/genética , Fator de Transcrição STAT3/genéticaRESUMO
Many lung diseases, such as the acute respiratory distress syndrome (ARDS), display significant regional heterogeneity with patches of severely injured tissue adjacent to apparently healthy tissue. Current mouse models that aim to mimic ARDS generally produce diffuse injuries that cannot reproducibly generate ARDS's regional heterogeneity. This deficiency prevents the evaluation of how well therapeutic agents reach the most injured regions and precludes many regenerative medicine studies since it is not possible to know which apparently healing regions suffered severe injury initially. Finally, these diffuse injury models must be relatively mild to allow for survival, as their diffuse nature does not allow for residual healthy lung to keep an animal alive long enough for many drug and regenerative medicine studies. To solve all of these deficiencies in current animal models, we have created a simple and reproducible technique to selectively induce lung injury in specific areas of the lung. Our technique, catheter-in-catheter selective lung injury (CICSLI), involves guiding an inner catheter to a particular area of the lung and delivering an injurious agent mixed with nanoparticles (fluorescently and/or radioactively labeled) that can be used days later to track the location and extent of where the initial injury occurred. Furthermore, we demonstrate that CICSLI can produce a more severe injury than diffuse models, yet has much higher survival since CICSLI intentionally leaves lung regions undamaged. Collectively, these attributes of CICSLI will allow investigators to better study how drugs act within heterogeneous lung pathologies and how regeneration occurs in severely damaged lung tissue, thereby aiding the development of new therapies for ARDS and other heterogenous lung diseases.
Assuntos
Modelos Animais de Doenças , Lesão Pulmonar , Pulmão , Síndrome do Desconforto Respiratório , Animais , Catéteres/efeitos adversos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Lesão Pulmonar/fisiopatologia , Camundongos , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Síndrome do Desconforto Respiratório/fisiopatologiaRESUMO
BACKGROUND: Systemic Sclerosis (SSc) is a rare connective tissue disorder associated with an increased risk of malignancy including lung cancer. METHODS: A single center review of all cases of lung cancer in patients with SSc was conducted. Clinical, radiographic, and detailed pathologic data was collected. Risk factors were compared with our center's SSc Registry. Cancer characteristics were compared with the National Cancer Institute SEER Cancer Statistics (NCI SEER) data. RESULTS: 17 cases were identified; the majority were females (82%) with the lung cancers diagnosed after the onset of SSc (88%). Tobacco use was identified in 65% of cases. Serologic testing showed 50% of cases were Scl-70 positive. Twelve cases had radiographic evidence of SSc lung involvement, however only 6 had restrictive physiology on pulmonary function testing. Thirteen cases had pulmonary nodules preceding lung cancer. Thirteen of the cancers were adenocarcinoma. Ten underwent molecular mutational profiling: 2/8 had KRAS mutation and 1/10 had EGFR mutation. More of the non-small cell lung cancers were diagnosed at localized disease (56%) than in the NCI SEER database. However, 5 years survival among the stage I cases was 25% versus an expected survival of 54%. CONCLUSIONS: The high proportion of adenocarcinomas seen in our study is different from that reported in the literature. Lung cancers were diagnosed at an early stage, likely due to our center's practice of radiographic screening for SSc associated lung involvement, however this did not confer a survival advantage. A high proportion of patients who developed lung cancer had interstitial lung disease.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Detecção Precoce de Câncer , Doenças Pulmonares Intersticiais/complicações , Neoplasias Pulmonares/diagnóstico , Escleroderma Sistêmico/complicações , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/complicações , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Estudos Transversais , Feminino , Humanos , Doenças Pulmonares Intersticiais/mortalidade , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Escleroderma Sistêmico/mortalidade , Taxa de SobrevidaRESUMO
INTRODUCTION: The demography of healthcare workers (HCWs) and non-HCWs seeking medical care at emergency departments after a non-percutaneous potential exposure to human immunodeficiency virus (HIV) during cardiopulmonary resuscitation (CPR), the types and body locations of their exposures, the time elapsed from exposure to emergency department presentation, and usage of HIV-post-exposure prophylaxis (PEP) for these exposures are described. METHODS: A retrospective study of emergency department patients who were exposed to blood or body fluids during CPR in Rhode Island from January 1995-June 2001 was performed. The demography, characteristics of the exposure, and HIV-PEP usage for these patients were compared, and the elapsed time from exposure to evaluation in the emergency department was calculated. RESULTS: Of the 39 patients exposed to non-percutaneous blood or body fluid during CPR, 22 were healthcare workers (HCWs) and 17 were non-HCWs. Thirty-four patients sustained mucous membrane exposures. Most of the patients (69.2%) were exposed to saliva or sputum (p <0.001), experienced a mouth exposure (71.8%; p <0.0001) and presented to the emergency department within one day of their exposure (84.4%; p <0.0001). Three HCWs and no non-HCWs were offered HIV-PEP for their CPR exposure. Of the three HCWs offered PEP, two actually received it. CONCLUSIONS: Nearly half of the patients who presented with non-percutaneous exposures acquired during CPR were not HCWs. Most of the exposures were to saliva or sputum and occurred on their mucous membranes. Continuing education programs on maintaining universal precautions to prevent blood or body fluid exposures and appreciating the benign nature of most non-percutaneous exposures possible during CPR are needed.