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1.
Methods Mol Biol ; 2269: 93-105, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687674

RESUMO

Mesenchymal stem cells (MSCs) have emerged as an attractive candidate for cell-based therapy. In the past decade, many animal and pilot clinical studies have demonstrated that MSCs are therapeutically beneficial for the treatment of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). However, due to the scarcity of adult human MSCs, human-induced pluripotent stem cells mesenchymal stem cells (iPSCs) are now increasingly used as a source of MSCs. iPSCs are derived by reprogramming somatic cells from a wide variety of tissues such as skin biopsies and then differentiating them into iPSC-MSCs. One of the mechanisms through which MSCs exert their protective effects is mitochondrial transfer. Specifically, transfer of mitochondria from iPSC-MSCs to lung cells was shown to protect lung cells against oxidative stress-induced mitochondrial dysfunction and apoptosis and to reduce lung injury and inflammation in in vivo models of lung disease. In this chapter, we detail our methods to visualize and quantify iPSC-MSC-mediated mitochondrial transfer and to study its effects on oxidant-induced airway epithelial and smooth muscle cell models of acute airway cell injury.


Assuntos
Células Epiteliais Alveolares/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo , Células Epiteliais Alveolares/patologia , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Mesenquimais/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/transplante , Miócitos de Músculo Liso/patologia
2.
EMBO J ; 40(5): e107651, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33576058

RESUMO

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.


Assuntos
Células Epiteliais Alveolares/metabolismo , Modelos Biológicos , Organoides/metabolismo , Replicação Viral , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , /patologia , Humanos , Organoides/patologia , Organoides/virologia
3.
Nature ; 591(7850): 451-457, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33561864

RESUMO

All coronaviruses known to have recently emerged as human pathogens probably originated in bats1. Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.


Assuntos
/tratamento farmacológico , Citidina/análogos & derivados , Hidroxilaminas/administração & dosagem , Hidroxilaminas/uso terapêutico , Administração Oral , Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , Quimioprevenção , Quirópteros/virologia , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Citidina/administração & dosagem , Citidina/uso terapêutico , Citocinas/imunologia , Células Epiteliais/virologia , Feminino , Xenoenxertos , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Transplante de Pulmão , Masculino , Camundongos , Profilaxia Pós-Exposição , Profilaxia Pré-Exposição , /patogenicidade , Replicação Viral
4.
Med Sci Monit ; 27: e928837, 2021 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-33580949

RESUMO

BACKGROUND Coronavirus 2 (SARS-CoV-2) was declared a pandemic by the World Health Organization (WHO) in March 2020. To further reveal the pathologic associations between coronavirus and hypoxemia, we report the findings of 4 complete systematic autopsies of severe acute respiratory syndrome coronavirus 2-positive individuals who died of multiple organ failure caused by severe hypoxemia. MATERIAL AND METHODS We examined the donated corpses of 4 deceased patients who had been diagnosed with severe acute respiratory syndrome coronavirus 2. A complete post-mortem examination was carried out on each corpse, and multiple organs were macroscopically examined. RESULTS The 4 corpses were 2 males and 2 females, with an average age of 69 years. Bilateral lungs showed various degrees of atrophy and consolidation, with diffusely tough and solid texture in the sections. A thromboembolism was found in the main pulmonary artery extending into the atrium in 1 corpse, and significant atherosclerotic plaques tagged in the inner wall of the aortic arch were found in 2 corpses. Two corpses were found to have slightly atrophied bilateral renal parenchyma. Atrophic changes in the spleen were found in 2 corpses. Notably, there were significantly expanded alveolar septa and prominent fibroblastic proliferation. CONCLUSIONS The laboratory data of these corpses showed a progressive decrease in blood oxygen saturation, followed by refractory and irreversible hypoxemia. Clinical and laboratory information and autopsy and histologic presentations of multiple organs showed insufficient air exchange due to abnormalities in the respiratory system, and reduced erythropoiesis in bone marrow may play a role.


Assuntos
Autopsia , /virologia , Hipóxia/complicações , Hipóxia/patologia , Pneumonia/patologia , Pneumonia/virologia , /fisiologia , Idoso , Idoso de 80 Anos ou mais , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Agregação Celular , Feminino , Humanos , Pulmão/patologia , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Muco/metabolismo , Miocárdio/patologia , Necrose , Pneumonia/complicações , Cavidade Torácica/patologia
5.
Methods Mol Biol ; 2260: 83-109, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33405032

RESUMO

The respiratory epithelium is the initial point of host contact for inhaled particles, leading to orchestrated, but highly heterogeneous, responses. Human airway epithelial cells (AECs) play a crucial role in host defense by promoting uptake and killing of inhaled microorganisms and concomitant cytokine production in order to recruit professional phagocytes to the site of infection. However, inhaled pathogens can also reside and replicate intracellularly to evade host immune defenses or circulating antimicrobial drugs, ultimately causing apoptosis or cell death of the infected AECs. Imaging flow cytometry (IFC) combines flow cytometry, fluorescent microscopy, and advanced data-processing algorithms to dissect the heterogeneity of the interaction of AECs and inhaled microorganisms and its outcomes at the single-cell level. Here, we describe a novel single-cell approach based on differential fluorescent staining and state-of-the-art IFC to identify, quantify, and analyze individual host-pathogen complexes from cultured AECs infected with spores of the major human fungal pathogen Aspergillus fumigatus.


Assuntos
Células Epiteliais Alveolares/microbiologia , Aspergillus fumigatus/patogenicidade , Citometria de Fluxo , Corantes Fluorescentes/química , Microscopia de Fluorescência , Análise de Célula Única , Células A549 , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Apoptose , Interações Hospedeiro-Patógeno , Humanos , Processamento de Imagem Assistida por Computador , Necrose , Software
6.
Sci Transl Med ; 13(578)2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33431511

RESUMO

Detailed knowledge about the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is important for uncovering the viral and host factors that contribute to coronavirus disease 2019 (COVID-19) pathogenesis. Old-World nonhuman primates recapitulate mild to moderate cases of COVID-19, thereby serving as important pathogenesis models. We compared African green monkeys inoculated with infectious SARS-CoV-2 or irradiated, inactivated virus to study the dynamics of virus replication throughout the respiratory tract. Genomic RNA from the animals inoculated with the irradiated virus was found to be highly stable, whereas subgenomic RNA, an indicator of viral replication, was found to degrade quickly. We combined this information with single-cell RNA sequencing of cells isolated from the lung and lung-draining mediastinal lymph nodes and developed new analysis methods for unbiased targeting of important cells in the host response to SARS-CoV-2 infection. Through detection of reads to the viral genome, we were able to determine that replication of the virus in the lungs appeared to occur mainly in pneumocytes, whereas macrophages drove the inflammatory response. Monocyte-derived macrophages recruited to the lungs, rather than tissue-resident alveolar macrophages, were most likely to be responsible for phagocytosis of infected cells and cellular debris early in infection, with their roles switching during clearance of infection. Together, our dataset provides a detailed view of the dynamics of virus replication and host responses over the course of mild COVID-19 and serves as a valuable resource to identify therapeutic targets.


Assuntos
/epidemiologia , Pulmão/virologia , Análise de Sequência de RNA , Análise de Célula Única , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , Líquido da Lavagem Broncoalveolar/virologia , Chlorocebus aethiops , DNA Viral/genética , Feminino , Genoma Viral/genética , Inflamação/patologia , Pulmão/patologia , Linfonodos/patologia , Macrófagos/patologia , Macrófagos/virologia , Masculino , Mediastino/patologia , Transcrição Genética , Carga Viral , Replicação Viral
7.
Med Hypotheses ; 146: 110412, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33308936

RESUMO

The Corona Virus Disease (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) requires a rapid solution and global collaborative efforts in order to define preventive and treatment strategies. One of the major challenges of this disease is the high number of patients needing advanced respiratory support due to the Acute Respiratory Distress Syndrome (ARDS) as the lung is the major - although not exclusive - target of the virus. The molecular mechanisms, pathogenic drivers and the target cell type(s) in SARS-CoV-2 infection are still poorly understood, but the development of a "hyperactive" immune response is proposed to play a role in the evolution of the disease and it is envisioned as a major cause of morbidity and mortality. Here we propose a theory by which the main targets for SARS-CoV-2 are the Type II Alveolar Epithelial Cells and the clinical manifestations of the syndrome are a direct consequence of their involvement. We propose the existence of a vicious cycle by which once alveolar damage starts in AEC II cells, the inflammatory state is supported by macrophage pro-inflammatory polarization (M1), cytokines release and by the activation of the NF-κB pathway. If this theory is confirmed, future therapeutic efforts can be directed to target Type 2 alveolar cells and the molecular pathogenic drivers associated with their dysfunction with currently available therapeutic strategies.


Assuntos
Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/virologia , /virologia , Modelos Biológicos , NF-kappa B/imunologia , Células Epiteliais Alveolares/patologia , /etiologia , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Heparina de Baixo Peso Molecular/uso terapêutico , Humanos , Inflamação/imunologia , Inflamação/patologia , Ventilação Líquida , Macrófagos/imunologia , Macrófagos/patologia , NF-kappa B/antagonistas & inibidores , Neutrófilos/imunologia , Neutrófilos/patologia , Pandemias , Surfactantes Pulmonares/uso terapêutico , /imunologia , /imunologia , Transdução de Sinais/imunologia
8.
Biomed Pharmacother ; 133: 111026, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378942

RESUMO

The main pathophysiological mechanism of acute respiratory distress syndrome (ARDS) invovles the increase in alveolar barrier permeability that is primarily caused by epithelial glycocalyx and tight junction (TJ) protein destruction. This study was performed to explore the effects of the alveolar epithelial glycocalyx on the epithelial barrier, specifically on TJ proteins, in ARDS. We used C57BL/6 mice and human lung epithelial cell models of lipopolysaccharide (LPS)-induced ARDS. Changes in alveolar permeability were evaluated via pulmonary histopathology analysis and by measuring the wet/dry weight ratio of the lungs. Degradation of heparan sulfate (HS), an important component of the epithelial glycocalyx, and alterations in levels of the epithelial TJ proteins (occludin, zonula occludens 1, and claudin 4) were assessed via ELISA, immunofluorescence analysis, and western blotting analysis. Real-time quantitative polymerase chain reaction was used to detect the mRNA of the TJ protein. Changes in glycocalyx and TJ ultrastructures in alveolar epithelial cells were evaluated through electron microscopy. In vivo and in vitro, LPS increased the alveolar permeability and led to HS degradation and TJ damage. After LPS stimulation, the expression of the HS-degrading enzyme heparanase (HPA) in the alveolar epithelial cells was increased. The HPA inhibitor N-desulfated/re-N-acetylated heparin alleviated LPS-induced HS degradation and reduced TJ damage. In vitro, recombinant HPA reduced the expression of the TJ protein zonula occludens-1 (ZO-1) and inhibited its mRNA expression in the alveolar epithelial cells. Taken together, our results demonstrate that shedding of the alveolar epithelial glycocalyx aggravates the epithelial barrier and damages epithelial TJ proteins in ARDS, with the underlying mechanism involving the effect of HPA on ZO-1.


Assuntos
Células Epiteliais Alveolares/patologia , Barreira Alveolocapilar/patologia , Glicocálix/patologia , Junções Íntimas/patologia , Células A549 , Células Epiteliais Alveolares/metabolismo , Animais , Barreira Alveolocapilar/metabolismo , Líquido da Lavagem Broncoalveolar/química , Modelos Animais de Doenças , Glicocálix/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Permeabilidade , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
9.
Sarcoidosis Vasc Diffuse Lung Dis ; 37(2): 212-217, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33093785

RESUMO

Background: Zonal aggregates of elastic fibres (zonal elastosis) and intraalveolar collagenosis with septal elastosis are histologic components of subpleural fibroelastosis of idiopathic pleuroparenchymal fibroelastosis (IPPFE). Zonal elastosis is considered to result from alveolar collapse, but this mechanism has not been fully justified. Methods: We immunohistochemically attempted to identify epithelial cells in zonal elastosis of 10 patients with IPPFE. The thickness of the zonal elastosis in relation to the total thickness of the fibroelastosis was examined to estimate the influence of zonal elastosis on the occurrence and development of IPPFE. Results: In 9 of the 10 patients, multi-cytokeratin-positive cells were found lining the inner surface of slit-like spaces embedded in the zonal elastosis. Zonal elastosis was predominant when fibroelastosis was < 1 mm thick but less predominant when it was ≥1 mm. Conclusion: The zonal elastosis was proven to result from alveolar collapse, which might be an initial lesion in IPPFE. (Sarcoidosis Vasc Diffuse Lung Dis 2020; 37 (2): 212-217).


Assuntos
Tecido Elástico/patologia , Fibrose Pulmonar Idiopática/patologia , Alvéolos Pulmonares/metabolismo , Adulto , Idoso , Células Epiteliais Alveolares/química , Células Epiteliais Alveolares/patologia , Biomarcadores/análise , Tecido Elástico/química , Feminino , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Imuno-Histoquímica , Queratinas/análise , Masculino , Pessoa de Meia-Idade , Alvéolos Pulmonares/química , Estudos Retrospectivos , Adulto Jovem
10.
Mol Med ; 26(1): 95, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33054759

RESUMO

Pulmonary fibrosis arises from the repeated epithelial mild injuries and insufficient repair lead to over activation of fibroblasts and excessive deposition of extracellular matrix, which result in a mechanical stretched niche. However, increasing mechanical stress likely exists before the establishment of fibrosis since early micro injuries increase local vascular permeability and prompt cytoskeletal remodeling which alter cellular mechanical forces. It is noteworthy that COVID-19 patients with severe hypoxemia will receive mechanical ventilation as supportive treatment and subsequent pathology studies indicate lung fibrosis pattern. At advanced stages, mechanical stress originates mainly from the stiff matrix since boundaries between stiff and compliant parts of the tissue could generate mechanical stress. Therefore, mechanical stress has a significant role in the whole development process of pulmonary fibrosis. The alveoli are covered by abundant capillaries and function as the main gas exchange unit. Constantly subject to variety of damages, the alveolar epithelium injuries were recently recognized to play a vital role in the onset and development of idiopathic pulmonary fibrosis. In this review, we summarize the literature regarding the effects of mechanical stress on the fundamental cells constituting the alveoli in the process of pulmonary fibrosis, particularly on epithelial cells, capillary endothelial cells, fibroblasts, mast cells, macrophages and stem cells. Finally, we briefly review this issue from a more comprehensive perspective: the metabolic and epigenetic regulation.


Assuntos
Infecções por Coronavirus/imunologia , Epigênese Genética/imunologia , Fibrose Pulmonar Idiopática/imunologia , Mecanotransdução Celular/imunologia , Pneumonia Viral/imunologia , Embolia Pulmonar/imunologia , Insuficiência Respiratória/imunologia , Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/patologia , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Fenômenos Biomecânicos , Infecções por Coronavirus/genética , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Citocinas/genética , Citocinas/imunologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/virologia , Pulmão/irrigação sanguínea , Pulmão/imunologia , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/patologia , Mecanotransdução Celular/genética , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Embolia Pulmonar/genética , Embolia Pulmonar/patologia , Embolia Pulmonar/virologia , Insuficiência Respiratória/genética , Insuficiência Respiratória/patologia , Insuficiência Respiratória/virologia , Estresse Mecânico
12.
Ecotoxicol Environ Saf ; 205: 111283, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32977282

RESUMO

Fine particulate matter (PM2.5) airborne pollution increases the risk of chronic respiratory diseases, such as idiopathic pulmonary fibrosis (IPF), which is characterized by non-specific inflammation of the interstitial lung and extensive deposition of collagen fibers. Type 2 alveolar epithelial cells (AEC2s) are alveolar stem cells in the adult lung that contribute to the lung repair process through complex signaling. Our previous studies demonstrated that OGG1, a kind of DNA repair enzyme, have a critical role in protecting cells from oxidative damage and apoptosis induced by PM2.5, but the contribution of OGG1 in proliferation and self-renewal of AEC2s is not known. Here, we constructed OGG1-/-mice to test the effect and mechanism of OGG1 on PM2.5-induced pulmonary fibrosis and injury in vivo. We detected proliferation and self-renewal of OGG1 overexpression or OGG1 knockout AEC2s after PM2.5 injury by flow cytometry and clone formation. We observed that knockout of OGG1 aggravated pulmonary fibrosis, oxidative stress, and AEC2 cell death in PM2.5-injured mice. In addition, OGG1 is required for the proliferation and renewal of AEC2s after PM2.5 injury. Overexpression of OGG1 promotes the proliferation and self-renewal of AEC2s by inhibiting PM2.5-mediated oxidative stress and NF-κB signaling hyperactivation in vitro. Furthermore, NF-κB inhibitors promoted proliferation and self-renewal of OGG1-deficient AEC2s cells after PM2.5 injury, and attenuated PM2.5-induced pulmonary fibrosis and injury in mice. These data establish OGG1 as a regulator of NF-κB signal that serves to regulate AEC2 cell proliferation and self-renewal, and suggest a mechanism that inhibition of the NF-κB signaling pathway may represent a potential therapeutic strategy for IPF patients with low-expression of OGG1.


Assuntos
Poluentes Atmosféricos/toxicidade , Células Epiteliais Alveolares/efeitos dos fármacos , Autorrenovação Celular/genética , DNA Glicosilases/metabolismo , Material Particulado/toxicidade , Fibrose Pulmonar/induzido quimicamente , Células-Tronco/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , DNA Glicosilases/genética , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Transdução de Sinais , Células-Tronco/metabolismo , Células-Tronco/patologia
13.
EBioMedicine ; 60: 102976, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32971472

RESUMO

BACKGROUND: Zoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors. MATERIALS AND METHODS: We systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19. FINDINGS: Our results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19. INTERPRETATION: These results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19.


Assuntos
Células Epiteliais Alveolares/metabolismo , Peptidil Dipeptidase A/genética , Análise de Sequência de RNA/métodos , Adulto , Idoso , Células Epiteliais Alveolares/patologia , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Criança , Pré-Escolar , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Mensageiro/metabolismo , Sistema Respiratório/metabolismo , Sistema Respiratório/patologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Análise de Célula Única , Adulto Jovem
14.
Anesthesiology ; 133(4): 905-918, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32930731

RESUMO

BACKGROUND: Alveolar epithelial cell apoptosis is implicated in the onset of ventilator-induced lung injury. Death-associated protein kinase 1 (DAPK1) is associated with cell apoptosis. The hypothesis was that DAPK1 participates in ventilator-induced lung injury through promoting alveolar epithelial cell apoptosis. METHODS: Apoptosis of mouse alveolar epithelial cell was induced by cyclic stretch. DAPK1 expression was altered (knockdown or overexpressed) in vitro by using a small interfering RNA or a plasmid, respectively. C57/BL6 male mice (n = 6) received high tidal volume ventilation to establish a lung injury model. Adeno-associated virus transfection of short hairpin RNA and DAPK1 inhibitor repressed DAPK1 expression and activation in lungs, respectively. The primary outcomes were alveolar epithelial cell apoptosis and lung injury. RESULTS: Compared with the control group, the 24-h cyclic stretch group showed significantly higher alveolar epithelial cell apoptotic percentage (45 ± 4% fold vs. 6 ± 1% fold; P < 0.0001) and relative DAPK1 expression, and this group also demonstrated a reduced apoptotic percentage after DAPK1 knockdown (27 ± 5% fold vs. 53 ± 8% fold; P < 0.0001). A promoted apoptotic percentage in DAPK1 overexpression was observed without stretching (49 ± 6% fold vs. 14 ± 3% fold; P < 0.0001). Alterations in B-cell lymphoma 2 and B-cell lymphoma 2-associated X are associated with DAPK1 expression. The mice subjected to high tidal volume had higher DAPK1 expression and alveolar epithelial cell apoptotic percentage in lungs compared with the low tidal volume group (43 ± 6% fold vs. 4 ± 2% fold; P < 0.0001). Inhibition of DAPK1 through adeno-associated virus infection or DAPK1 inhibitor treatment appeared to be protective against lung injury with reduced lung injury score, resolved pulmonary inflammation, and repressed alveolar epithelial cell apoptotic percentage (47 ± 4% fold and 48 ± 6% fold; 35 ± 5% fold and 34 ± 4% fold; P < 0.0001, respectively). CONCLUSIONS: DAPK1 promotes the onset of ventilator-induced lung injury by triggering alveolar epithelial cell apoptosis through intrinsic apoptosis pathway in mice.


Assuntos
Células Epiteliais Alveolares/metabolismo , Apoptose/fisiologia , Proteínas Quinases Associadas com Morte Celular/biossíntese , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Células Cultivadas , Proteínas Quinases Associadas com Morte Celular/deficiência , Proteínas Quinases Associadas com Morte Celular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia
15.
Life Sci ; 258: 118166, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32739471

RESUMO

In this paper, a model is proposed of the pathophysiological processes of COVID-19 starting from the infection of human type II alveolar epithelial cells (pneumocytes) by SARS-CoV-2 and culminating in the development of ARDS. The innate immune response to infection of type II alveolar epithelial cells leads both to their death by apoptosis and pyroptosis and to alveolar macrophage activation. Activated macrophages secrete proinflammatory cytokines and chemokines and tend to polarise into the inflammatory M1 phenotype. These changes are associated with activation of vascular endothelial cells and thence the recruitment of highly toxic neutrophils and inflammatory activated platelets into the alveolar space. Activated vascular endothelial cells become a source of proinflammatory cytokines and reactive oxygen species (ROS) and contribute to the development of coagulopathy, systemic sepsis, a cytokine storm and ARDS. Pulmonary activated platelets are also an important source of proinflammatory cytokines and ROS, as well as exacerbating pulmonary neutrophil-mediated inflammatory responses and contributing to systemic sepsis by binding to neutrophils to form platelet-neutrophil complexes (PNCs). PNC formation increases neutrophil recruitment, activation priming and extraversion of these immune cells into inflamed pulmonary tissue, thereby contributing to ARDS. Sequestered PNCs cause the development of a procoagulant and proinflammatory environment. The contribution to ARDS of increased extracellular histone levels, circulating mitochondrial DNA, the chromatin protein HMGB1, decreased neutrophil apoptosis, impaired macrophage efferocytosis, the cytokine storm, the toll-like receptor radical cycle, pyroptosis, necroinflammation, lymphopenia and a high Th17 to regulatory T lymphocyte ratio are detailed.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/fisiopatologia , Pneumonia Viral/fisiopatologia , /fisiopatologia , Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/patologia , Animais , Betacoronavirus/imunologia , Infecções por Coronavirus/complicações , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Humanos , Imunidade Inata , Inflamação/etiologia , Inflamação/imunologia , Inflamação/fisiopatologia , Inflamação/terapia , Ativação de Macrófagos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Ativação de Neutrófilo , Pandemias , Ativação Plaquetária , Pneumonia Viral/complicações , Pneumonia Viral/imunologia , Pneumonia Viral/terapia , /imunologia , Trombofilia/etiologia , Trombofilia/imunologia , Trombofilia/fisiopatologia , Trombofilia/terapia
16.
Int J Legal Med ; 134(6): 2209-2214, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32767018

RESUMO

A 75-year-old man presented to a French hospital with a 4-day fever after returning from a coronavirus disease-19 (COVID-19) cluster region. A reverse-transcription polymerase chain reaction test was positive for severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) using a nasopharyngeal swab sample. After he returned home and a telephone follow-up, he was found deceased 9 days after first showing symptoms. Whole-body, non-enhanced, post-mortem computed tomography (PMCT) and a forensic autopsy were performed approximately 48 h after death, with sanitary precautions. The PMCT showed bilateral and diffuse crazy-paving lung opacities, with bilateral pleural effusions. Post-mortem virology studies detected the presence of SARS-CoV-2 (B.1 lineage) in the nasopharynx, plasma, lung biopsies, pleural effusion and faeces confirming the persistence of viral ribonucleic acid 48 h after death. Microscopic examination showed that severe lung damage was responsible for his death. The main abnormality was diffuse alveolar damage, associated with different stages of inflammation and fibrosis. This case is one of the first to describe complete post-mortem data for a COVID-19 death and highlights the ability of PMCT to detect severe involvement of the lungs before autopsy in an apparently natural death. The present pathology results are concordant with previously reported findings and reinforce the disease pathogenesis hypothesis of combined viral replication with an inappropriate immune response.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/patologia , Pulmão/diagnóstico por imagem , Pulmão/patologia , Pneumonia Viral/patologia , Idoso , Células Epiteliais Alveolares/patologia , Autopsia , Fibrina/metabolismo , Humanos , Hiperplasia , Masculino , Pandemias , Derrame Pleural/diagnóstico por imagem , Derrame Pleural/patologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Tomografia Computadorizada por Raios X
17.
J Stroke Cerebrovasc Dis ; 29(9): 104942, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32807413

RESUMO

BACKGROUND AND OBJECTIVES: Studies implicate the lung in moderating systemic immune activation via effects on circulating leukocytes. In this study, we investigated whether targeted expression of the antioxidant extracellular superoxide dismutase (SOD3) within the lung would influence post-ischemic peripheral neutrophil activation and CNS reperfusion injury. METHODS: Adult, male mice expressing human SOD3 within type II pneumocytes were subjected to 15 min of transient global cerebral ischemia. Three days post-reperfusion, lung and brain tissue was collected and analyzed by immunohistochemistry for inflammation and injury markers. In vitro motility and neurotoxicity assays were conducted to ascertain the direct effects of hSOD3 on PMN activation. Results were compared against C57BL/6 age and sex-matched controls. RESULTS: Relative to wild-type controls, hSOD3 heterozygous mice exhibited a reduction in lung inflammation, blood-brain barrier damage, and post-ischemic neuronal injury within the hippocampus and cortex. PMNs harvested from hSOD3 mice were also resistant to LPS priming, slower-moving, and less toxic to primary neuronal cultures. CONCLUSIONS: Constitutive, focal expression of hSOD3 is neuroprotective in a model of global cerebral ischemia-reperfusion injury. The underlying mechanism of SOD3-dependent protection is attributable in part to effects on the activation state and toxic potential of circulating neutrophils. These results implicate lung-brain coupling as a determinant of cerebral ischemia-reperfusion injury and highlight post-stroke lung inflammation as a potential therapeutic target in acute ischemic cerebrovascular injuries.


Assuntos
Células Epiteliais Alveolares/enzimologia , Isquemia Encefálica/enzimologia , Encéfalo/metabolismo , Neurônios/metabolismo , Ativação de Neutrófilo , Neutrófilos/metabolismo , Pneumonia/prevenção & controle , Traumatismo por Reperfusão/prevenção & controle , Superóxido Dismutase/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Encéfalo/patologia , Isquemia Encefálica/genética , Isquemia Encefálica/imunologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imunidade Inata , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/patologia , Neutrófilos/imunologia , Pneumonia/enzimologia , Pneumonia/genética , Pneumonia/imunologia , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/imunologia , Transdução de Sinais , Superóxido Dismutase/genética
18.
Am J Physiol Cell Physiol ; 319(2): C316-C320, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32639871

RESUMO

Pulmonary surfactant is a heterogeneous active surface complex made up of lipids and proteins. The major glycoprotein in surfactant is surfactant protein A (SP-A), which is released into the alveolar lumen from cytoplasmic lamellar bodies in type II alveolar epithelial cells. SP-A is involved in phospholipid absorption. SP-A together with other surfactant proteins and phospholipids prevent alveolar collapse during respiration by decreasing the surface tension of the air-liquid interface. Additionally, SP-A interacts with pathogens to prevent their propagation and regulate host immune responses. Studies in human and animal models have shown that deficiencies or mutations in surfactant components result in various lung or kidney pathologies, suggesting a role for SP-A in the development of lung and kidney diseases. In this mini-review, we discuss the current understanding of SP-A functions, recent findings of its dysfunction in specific lung and kidney pathologies, and how SP-A has been used as a biomarker to detect the outcome of lung diseases.


Assuntos
Nefropatias/genética , Pneumopatias/genética , Alvéolos Pulmonares/metabolismo , Proteína A Associada a Surfactante Pulmonar/genética , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Citoplasma/genética , Citoplasma/metabolismo , Progressão da Doença , Humanos , Rim/metabolismo , Rim/patologia , Nefropatias/patologia , Pulmão/metabolismo , Pulmão/patologia , Pneumopatias/patologia , Alvéolos Pulmonares/patologia , Surfactantes Pulmonares/metabolismo
19.
Am J Respir Crit Care Med ; 202(8): 1088-1104, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32628504

RESUMO

Rationale: Promoting endogenous pulmonary regeneration is crucial after damage to restore normal lungs and prevent the onset of chronic adult lung diseases.Objectives: To investigate whether the cell-cycle inhibitor p16INK4a limits lung regeneration after newborn bronchopulmonary dysplasia (BPD), a condition characterized by the arrest of alveolar development, leading to adult sequelae.Methods: We exposed p16INK4a-/- and p16INK4a ATTAC (apoptosis through targeted activation of caspase 8) transgenic mice to postnatal hyperoxia, followed by pneumonectomy of the p16INK4a-/- mice. We measured p16INK4a in blood mononuclear cells of preterm newborns, 7- to 15-year-old survivors of BPD, and the lungs of patients with BPD.Measurements and Main Results: p16INK4a concentrations increased in lung fibroblasts after hyperoxia-induced BPD in mice and persisted into adulthood. p16INK4a deficiency did not protect against hyperoxic lesions in newborn pups but promoted restoration of the lung architecture by adulthood. Curative clearance of p16INK4a-positive cells once hyperoxic lung lesions were established restored normal lungs by adulthood. p16INK4a deficiency increased neutral lipid synthesis and promoted lipofibroblast and alveolar type 2 (AT2) cell development within the stem-cell niche. Besides, lipofibroblasts support self-renewal of AT2 cells into alveolospheres. Induction with a PPARγ (peroxisome proliferator-activated receptor γ) agonist after hyperoxia also increased lipofibroblast and AT2 cell numbers and restored alveolar architecture in hyperoxia-exposed mice. After pneumonectomy, p16INK4a deficiency again led to an increase in lipofibroblast and AT2 cell numbers in the contralateral lung. Finally, we observed p16INK4a mRNA overexpression in the blood and lungs of preterm newborns, which persisted in the blood of older survivors of BPD.Conclusions: These data demonstrate the potential of targeting p16INK4a and promoting lipofibroblast development to stimulate alveolar regeneration from childhood to adulthood.


Assuntos
Displasia Broncopulmonar/patologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Fibroblastos/metabolismo , Pulmão/fisiologia , Regeneração/fisiologia , Adolescente , Adulto , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Animais Recém-Nascidos , Apoptose , Displasia Broncopulmonar/metabolismo , Células Cultivadas , Criança , Modelos Animais de Doenças , Fibroblastos/patologia , Humanos , Hiperóxia/complicações , Hiperóxia/metabolismo , Hiperóxia/patologia , Recém-Nascido , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Alvéolos Pulmonares/patologia , Distribuição Aleatória , Amostragem , Adulto Jovem
20.
Mol Immunol ; 125: 15-22, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32619930

RESUMO

PIM1 is serine/threonine protein kinase that is involved in numerous biological processes. Pulmonary fibrosis (PF) is a chronic pathological result of the dysfunctional repair of lung injury without effective therapeutic treatments. In the current study, we investigated whether PIM1 inhibition would improve bleomycin (BLM)-induced pulmonary fibrosis. In a BLM-induced pulmonary fibrosis model, PIM1 was persistently upregulated in fibrotic lung tissues. Furthermore, PIM1 inhibition by the PIM1-specific inhibitor SMI-4a showed protective effects against BLM-induced mortality. Furthermore, SMI-4a suppressed hydroxyproline deposition and reversed epithelial-mesenchymal transition (EMT) formation, which was characterized by E-cadherin and α-SMA expression in vivo. More importantly, the ZEB1/E-cadherin pathway was found to be closely associated with BLM-induced pulmonary fibrosis. After the in vitro treatment of A549 cells, PIM1 regulated E-cadherin expression by dependently modulating the activity of the transcription factor ZEB1. These findings were verified in vivo after SMI-4a administration. Finally, an shPIM1-expressing adeno-associated virus was delivered via intratracheal injection to induce a long-term PIM1 deficiency in the alveolar epithelium. AAV-mediated PIM1 knockdown in the lung tissues alleviated BLM-induced pulmonary fibrosis, as indicated by collagen accumulation reduction, pulmonary histopathological mitigation and EMT reversion. These findings enhance our understanding of the roles of PIM1 in BLM-induced pulmonary fibrosis and suggest PIM1 inhibition as a potential therapeutic strategy in chronic pulmonary injuries.


Assuntos
Células Epiteliais Alveolares/metabolismo , Caderinas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fibrose Pulmonar/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Células A549 , Células Epiteliais Alveolares/patologia , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia
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