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1.
PLoS One ; 15(12): e0242536, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33301441

RESUMO

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-α-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in 14C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-α-induced 16HBE barrier leak, attenuating 60% of the TNF-α-induced leak to 14C-mannitol and 80% of the leak to 14C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-α resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward "band-shift" in occludin Western immunoblots. The presence of RA partially reversed TNF-α's effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-α-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a "cytokine storm" may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.


Assuntos
Brônquios/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Tretinoína/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Anti-Inflamatórios/farmacologia , Brônquios/citologia , Brônquios/metabolismo , Linhagem Celular , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Permeabilidade/efeitos dos fármacos , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo
2.
mBio ; 11(6)2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33158999

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. Prior studies characterized only short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7 to 10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detected. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of >2.5 × 105 virions per cm2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to >35 million confirmed cases and >1 million fatalities worldwide. SARS-CoV-2 mainly replicates in human airway epithelia in COVID-19 patients. In this study, we used in vitro cultures of polarized human bronchial airway epithelium to model SARS-CoV-2 replication for a period of 21 to 51 days. We discovered that in vitro airway epithelial cultures endure a long-lasting SARS-CoV-2 propagation with recurrent peaks of progeny virus release at an interval of approximately 7 to 10 days. Our study also revealed that SARS-CoV-2 infection causes airway epithelia damage with disruption of tight junction function and loss of cilia. Importantly, SARS-CoV-2 exhibits a polarity of infection in airway epithelium only from the apical membrane; it infects ciliated and goblet cells but not basal and club cells. Furthermore, the productive infection of SARS-CoV-2 requires a high viral load of over 2.5 × 105 virions per cm2 of epithelium. Our study highlights that the proliferation of airway basal cells and regeneration of airway epithelium may contribute to the recurrent infections.


Assuntos
Betacoronavirus/fisiologia , Modelos Biológicos , Mucosa Respiratória/virologia , Brônquios/citologia , Células Cultivadas , Células Epiteliais/patologia , Células Epiteliais/virologia , Humanos , Cinética , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Carga Viral , Tropismo Viral , Liberação de Vírus , Replicação Viral
3.
PLoS One ; 15(10): e0241212, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33095800

RESUMO

Hepatitis B virus (HBV) is a human pathogen of global concern, while a high diversity of viruses related to HBV have been discovered in other animals during the last decade. Recently, the novel mammalian hepadnavirus, tentatively named domestic cat hepadnavirus (DCH), was detected in an immunocompromised cat. Herein, a collection of 209 cat sera and 15 hepato-diseased cats were screened for DCH using PCR, resulting in 12.4% and 20% positivity in the tested sera and necropsied cats, respectively. Among the DCH-positive sera, a significantly high level of co-detection with retroviral infection was found, with the highest proportion being co-detection with feline immunodeficiency virus (FIV). Full-length genome characterization of DCH revealed the genetic diversity between the nine Thai DCH sequences obtained, and that they phylogenetically formed three distinct monophyletic clades. A putative DCH recombinant strain was found, suggesting a possible role of recombination in DCH evolution. Additionally, quantitative PCR was used to determine the viral copy number in various organs of the DCH-moribund cats, while the pathological findings were compared to the viral localization in hepatocytes, adjacent to areas of hepatic fibrosis, by immunohistochemical (IHC) and western blot analysis. In addition to the liver, positive-DCH immunoreactivity was found in various other organs, including kidneys, lung, heart, intestine, brain, and lymph nodes, providing evidence of systemic infection. Ultrastructure of infected cells revealed electron-dense particles in the nucleus and cytoplasm of hepatocytes, bronchial epithelial cells, and fibroblasts. We propose the intracellular development mechanism of this virus. Although the definitive roles of pathogenicity of DCH remains undetermined, a contributory role of the virus associated with systemic diseases is possible.


Assuntos
Coinfecção/veterinária , Síndrome de Imunodeficiência Adquirida Felina/virologia , Infecções por Hepadnaviridae/veterinária , Hepadnaviridae/genética , Animais de Estimação/virologia , Animais , Brônquios/citologia , Brônquios/virologia , Gatos , Coinfecção/virologia , Citoplasma/virologia , Células Epiteliais/citologia , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Síndrome de Imunodeficiência Adquirida Felina/sangue , Feminino , Fibroblastos/citologia , Fibroblastos/virologia , Variação Genética , Genoma Viral/genética , Hepadnaviridae/isolamento & purificação , Infecções por Hepadnaviridae/virologia , Hepatócitos/citologia , Hepatócitos/ultraestrutura , Hepatócitos/virologia , Vírus da Imunodeficiência Felina/isolamento & purificação , Masculino , Microscopia Eletrônica de Transmissão , Filogenia , Recombinação Genética , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Tailândia , Replicação Viral , Eliminação de Partículas Virais
4.
J Pharmacol Exp Ther ; 375(3): 414-429, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33012706

RESUMO

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel that impair airway salt and fluid secretion. Excessive release of proinflammatory cytokines and chemokines by CF bronchial epithelium during airway infection leads to chronic inflammation and a slow decline in lung function; thus, there is much interest in finding safe and effective treatments that reduce inflammation in CF. We showed previously that the cyclic nucleotide phosphodiesterase (PDE) inhibitor ensifentrine (RPL554; Verona Pharma) stimulates the channel function of CFTR mutants with abnormal gating and also those with defective trafficking that are partially rescued using a clinically approved corrector drug. PDE inhibitors also have known anti-inflammatory effects; therefore, we examined whether ensifentrine alters the production of proinflammatory cytokines in CF bronchial epithelial cells. Ensifentrine reduced the production of monocyte chemoattractant protein-1 and granulocyte monocyte colony-stimulating factor (GM-CSF) during challenge with interleukin-1ß Comparing the effect of ensifentrine with milrinone and roflumilast, selective PDE3 and PDE4 inhibitors, respectively, demonstrated that the anti-inflammatory effect of ensifentrine was mainly due to inhibition of PDE4. Beneficial modulation of GM-CSF was further enhanced when ensifentrine was combined with low concentrations of the ß 2-adrenergic agonist isoproterenol or the corticosteroid dexamethasone. The results indicate that ensifentrine may have beneficial anti-inflammatory effects in CF airways particularly when used in combination with ß 2-adrenergic agonists or corticosteroids. SIGNIFICANCE STATEMENT: Airway inflammation that is disproportionate to the burden of chronic airway infection causes much of the pathology in the cystic fibrosis (CF) lung. We show here that ensifentrine beneficially modulates the release of proinflammatory factors in well differentiated CF bronchial epithelial cells that is further enhanced when combined with ß2-adrenergic agonists or low-concentration corticosteroids. The results encourage further clinical testing of ensifentrine, alone and in combination with ß2-adrenergic agonists or low-concentration corticosteroids, as a novel anti-inflammatory therapy for CF.


Assuntos
Brônquios/citologia , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Isoquinolinas/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Pirimidinonas/farmacologia , Linhagem Celular , Quimiocina CCL2/biossíntese , AMP Cíclico/metabolismo , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Células Epiteliais/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Humanos , Interleucina-8/biossíntese , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Regulação para Cima/efeitos dos fármacos
5.
Nat Commun ; 11(1): 5053, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028821

RESUMO

The epithelial-to-mesenchymal transition (EMT) and the unjamming transition (UJT) each comprises a gateway to cellular migration, plasticity and remodeling, but the extent to which these core programs are distinct, overlapping, or identical has remained undefined. Here, we triggered partial EMT (pEMT) or UJT in differentiated primary human bronchial epithelial cells. After triggering UJT, cell-cell junctions, apico-basal polarity, and barrier function remain intact, cells elongate and align into cooperative migratory packs, and mesenchymal markers of EMT remain unapparent. After triggering pEMT these and other metrics of UJT versus pEMT diverge. A computational model attributes effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, those tissues that are subject to development, injury, or disease become endowed with rich mechanisms for cellular migration, plasticity, self-repair, and regeneration.


Assuntos
Movimento Celular/fisiologia , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Regeneração , Mucosa Respiratória/fisiologia , Brônquios/citologia , Brônquios/fisiologia , Plasticidade Celular/fisiologia , Células Cultivadas , Humanos , Cultura Primária de Células , Mucosa Respiratória/citologia
7.
Biomed Pharmacother ; 131: 110653, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32942152

RESUMO

BACKGROUND: Angiotensin receptor blockers (ARBs) reducing inflammation and protecting lung and brain function, could be of therapeutic efficacy in COVID-19 patients. METHODS: Using GSEA, we compared our previous transcriptome analysis of neurons injured by glutamate and treated with the ARB Candesartan (GSE67036) with transcriptional signatures from SARS-CoV-2 infected primary human bronchial epithelial cells (NHBE) and lung postmortem (GSE147507), PBMC and BALF samples (CRA002390) from COVID-19 patients. RESULTS: Hundreds of genes upregulated in SARS-CoV-2/COVID-19 transcriptomes were similarly upregulated by glutamate and normalized by Candesartan. Gene Ontology analysis revealed expression profiles with greatest significance and enrichment, including proinflammatory cytokine and chemokine activity, the NF-kappa B complex, alterations in innate and adaptive immunity, with many genes participating in the COVID-19 cytokine storm. CONCLUSIONS: There are similar injury mechanisms in SARS-CoV-2 infection and neuronal injury, equally reduced by ARB treatment. This supports the hypothesis of a therapeutic role for ARBs, ameliorating the COVID-19 cytokine storm.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Benzimidazóis/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Síndrome da Liberação de Citocina/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Tetrazóis/farmacologia , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Brônquios/citologia , Líquido da Lavagem Broncoalveolar/virologia , Infecções por Coronavirus/complicações , Infecções por Coronavirus/virologia , Síndrome da Liberação de Citocina/virologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/virologia , Transcriptoma
8.
Cells ; 9(9)2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32859121

RESUMO

Natural killer cells are important in the control of viral infections. However, the role of NK cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has previously not been identified. Peripheral blood NK cells from SARS-CoV and SARS-CoV-2 naïve subjects were evaluated for their activation, degranulation, and interferon-gamma expression in the presence of SARS-CoV and SARS-CoV-2 spike proteins. K562 and lung epithelial cells were transfected with spike proteins and co-cultured with NK cells. The analysis was performed by flow cytometry and immune fluorescence. SARS-CoV and SARS-CoV-2 spike proteins did not alter NK cell activation in a K562 in vitro model. On the contrary, SARS-CoV-2 spike 1 protein (SP1) intracellular expression by lung epithelial cells resulted in NK cell-reduced degranulation. Further experiments revealed a concomitant induction of HLA-E expression on the surface of lung epithelial cells and the recognition of an SP1-derived HLA-E-binding peptide. Simultaneously, there was increased modulation of the inhibitory receptor NKG2A/CD94 on NK cells when SP1 was expressed in lung epithelial cells. We ruled out the GATA3 transcription factor as being responsible for HLA-E increased levels and HLA-E/NKG2A interaction as implicated in NK cell exhaustion. We show for the first time that NK cells are affected by SP1 expression in lung epithelial cells via HLA-E/NKG2A interaction. The resulting NK cells' exhaustion might contribute to immunopathogenesis in SARS-CoV-2 infection.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Ativação Linfocitária/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Doadores de Sangue , Brônquios/citologia , Degranulação Celular/genética , Técnicas de Cocultura , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Células Epiteliais/metabolismo , Humanos , Interferon gama/metabolismo , Células K562 , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , RNA Viral/genética , Vírus da SARS/química , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genética , Transfecção
9.
Int J Mol Sci ; 21(15)2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32752138

RESUMO

The COVID-19 pandemic caused by the SARS-CoV-2 virus, overlaps with the ongoing epidemics of cigarette smoking and electronic cigarette (e-cig) vaping. However, there is scarce data relating COVID-19 risks and outcome with cigarette or e-cig use. In this study, we mined three independent RNA expression datasets from smokers and vapers to understand the potential relationship between vaping/smoking and the dysregulation of key genes and pathways related to COVID-19. We found that smoking, but not vaping, upregulates ACE2, the cellular receptor that SARS-CoV-2 requires for infection. Both smoking and use of nicotine and flavor-containing e-cigs led to upregulation of pro-inflammatory cytokines and inflammasome-related genes. Specifically, chemokines including CCL20 and CXCL8 are upregulated in smokers, and CCL5 and CCR1 are upregulated in flavor/nicotine-containing e-cig users. We also found genes implicated in inflammasomes, such as CXCL1, CXCL2, NOD2, and ASC, to be upregulated in smokers and these e-cig users. Vaping flavor and nicotine-less e-cigs, however, did not lead to significant cytokine dysregulation and inflammasome activation. Release of inflammasome products, such as IL-1B, and cytokine storms are hallmarks of COVID-19 infection, especially in severe cases. Therefore, our findings demonstrated that smoking or vaping may critically exacerbate COVID-19-related inflammation or increase susceptibility to COVID-19.


Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Sistema Imunitário/metabolismo , Peptidil Dipeptidase A/metabolismo , Fumar Tabaco , Adulto , Betacoronavirus/isolamento & purificação , Brônquios/citologia , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Pessoa de Meia-Idade , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Regulação para Cima , Adulto Jovem
10.
Methods Mol Biol ; 2203: 119-134, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833209

RESUMO

Well-differentiated primary airway epithelial cell (AEC) cultures have been widely used for the characterization of several human respiratory viruses including coronaviruses. In recent years, there has been an increase in interest toward animal AEC cultures and their application to characterize veterinary viruses with zoonotic potential, as well as studying host-pathogen interactions in animal reservoir host species. In this chapter, we provide a revised and improved protocol for the isolation and establishment of well-differentiated AEC cultures from diverse mammalian species and the use of the cultures for the characterization of veterinary coronavirus. We also describe immunohistochemistry protocols with validated antibodies for the visualization and identification of viral cell tropism in well-differentiated AEC cultures from human, swine, bovine, and feline origin.


Assuntos
Brônquios/citologia , Coronavirus/fisiologia , Células Epiteliais/virologia , Cultura Primária de Células/métodos , Traqueia/citologia , Animais , Gatos , Bovinos , Diferenciação Celular , Células Epiteliais/citologia , Imunofluorescência , Humanos , Imuno-Histoquímica/métodos , Cultura Primária de Células/instrumentação , Suínos , Tropismo Viral
11.
J Virol ; 94(19)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32699094

RESUMO

The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients.IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Células Epiteliais/imunologia , Interferon Tipo I/imunologia , Interferons/imunologia , Pneumonia Viral/imunologia , Animais , Betacoronavirus/fisiologia , Brônquios/citologia , Brônquios/imunologia , Brônquios/virologia , Linhagem Celular , Células Cultivadas , Quimiocinas/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Citocinas/imunologia , Cães , Células Epiteliais/virologia , Humanos , Pulmão/citologia , Pulmão/imunologia , Pulmão/virologia , Células Madin Darby de Rim Canino , Pandemias , Pneumonia Viral/virologia , Células Vero , Replicação Viral
12.
Genes (Basel) ; 11(7)2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32646047

RESUMO

The global spread of COVID-19, caused by pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for an imminent response from medical research communities to better understand this rapidly spreading infection. Employing multiple bioinformatics and computational pipelines on transcriptome data from primary normal human bronchial epithelial cells (NHBE) during SARS-CoV-2 infection revealed activation of several mechanistic networks, including those involved in immunoglobulin G (IgG) and interferon lambda (IFNL) in host cells. Induction of acute inflammatory response and activation of tumor necrosis factor (TNF) was prominent in SARS-CoV-2 infected NHBE cells. Additionally, disease and functional analysis employing ingenuity pathway analysis (IPA) revealed activation of functional categories related to cell death, while those associated with viral infection and replication were suppressed. Several interferon (IFN) responsive gene targets (IRF9, IFIT1, IFIT2, IFIT3, IFITM1, MX1, OAS2, OAS3, IFI44 and IFI44L) were highly upregulated in SARS-CoV-2 infected NBHE cell, implying activation of antiviral IFN innate response. Gene ontology and functional annotation of differently expressed genes in patient lung tissues with COVID-19 revealed activation of antiviral response as the hallmark. Mechanistic network analysis in IPA identified 14 common activated, and 9 common suppressed networks in patient tissue, as well as in the NHBE cell model, suggesting a plausible role for these upstream regulator networks in the pathogenesis of COVID-19. Our data revealed expression of several viral proteins in vitro and in patient-derived tissue, while several host-derived long noncoding RNAs (lncRNAs) were identified. Our data highlights activation of IFN response as the main hallmark associated with SARS-CoV-2 infection in vitro and in human, and identified several differentially expressed lncRNAs during the course of infection, which could serve as disease biomarkers, while their precise role in the host response to SARS-CoV-2 remains to be investigated.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , RNA Longo não Codificante/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Biomarcadores/metabolismo , Brônquios/citologia , Morte Celular , Linhagem Celular , Análise por Conglomerados , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Células Epiteliais/citologia , Células Epiteliais/virologia , Redes Reguladoras de Genes , Humanos , Imunidade Inata , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/virologia , RNA Longo não Codificante/genética , Transcriptoma
13.
Viruses ; 12(6)2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32599823

RESUMO

The respiratory Influenza A Viruses (IAVs) and emerging zoonotic viruses such as Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pose a significant threat to human health. To accelerate our understanding of the host-pathogen response to respiratory viruses, the use of more complex in vitro systems such as normal human bronchial epithelial (NHBE) cell culture models has gained prominence as an alternative to animal models. NHBE cells were differentiated under air-liquid interface (ALI) conditions to form an in vitro pseudostratified epithelium. The responses of well-differentiated (wd) NHBE cells were examined following infection with the 2009 pandemic Influenza A/H1N1pdm09 strain or following challenge with the dsRNA mimic, poly(I:C). At 30 h postinfection with H1N1pdm09, the integrity of the airway epithelium was severely impaired and apical junction complex damage was exhibited by the disassembly of zona occludens-1 (ZO-1) from the cell cytoskeleton. wdNHBE cells produced an innate immune response to IAV-infection with increased transcription of pro- and anti-inflammatory cytokines and chemokines and the antiviral viperin but reduced expression of the mucin-encoding MUC5B, which may impair mucociliary clearance. Poly(I:C) produced similar responses to IAV, with the exception of MUC5B expression which was more than 3-fold higher than for control cells. This study demonstrates that wdNHBE cells are an appropriate ex-vivo model system to investigate the pathogenesis of respiratory viruses.


Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Animais , Brônquios/citologia , Brônquios/virologia , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Cães , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/epidemiologia , Junções Intercelulares , Células Madin Darby de Rim Canino , Modelos Biológicos , Mucina-5AC/metabolismo , Pandemias , Cultura de Vírus
14.
Biochim Biophys Acta Gen Subj ; 1864(10): 129672, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32562736

RESUMO

BACKGROUND: Exposure to PM2.5 has been associated with increased morbidity and mortality of lung diseases although the underlying mechanisms have not been fully uncovered. Airway inflammation is a critical event in the pathogenesis of lung diseases. This study aimed to examine the role of oxidative stress and epidermal growth factor receptor (EGFR) in PM2.5-induced pro-inflammatory response in a human bronchial epithelial cell line, BEAS-2B. METHODS: BEAS-2B cells were exposed to 0, 20, 50, 100 and 150 µg/ml of PM2.5. Secretion of pro-inflammatory mediators including interleukin-6 (IL-6), IL-8 and IL-1ß was determined using enzyme linked immunosorbent assay. Levels of intracellular reactive oxygen species (ROS) were determined using flow cytometry. Phosphorylation of the EGFR was examined with immunoblotting. RESULTS: PM2.5 exposure increased the secretion of IL-6, IL-8, and IL-1ß in a concentration-dependent fashion. Moreover, exposure to PM2.5 elevated intracellular levels of ROS, and phosphorylation of the EGFR (Y1068). Pretreatment of BEAS-2B cells with either an antioxidant or a specific EGFR inhibitor significantly reduced PM2.5-induced IL-6, IL-8 and IL-1ß secretion, implying that both oxidative stress and EGFR activation were involved in PM2.5-induced pro-inflammatory response. Furthermore, pre-treatment of BEAS-2B cells with an antioxidant significantly blunted PM2.5-induced EGFR activation, suggesting that oxidative stress was required for PM2.5-induced EGFR activation. CONCLUSION: PM2.5 exposure induces pro-inflammatory response in human bronchial epithelial cells through oxidative stress-mediated EGFR activation.


Assuntos
Poluentes Atmosféricos/efeitos adversos , Células Epiteliais/metabolismo , Mediadores da Inflamação/metabolismo , Estresse Oxidativo , Material Particulado/efeitos adversos , Brônquios/citologia , Brônquios/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Receptores ErbB/metabolismo , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo
15.
J Med Virol ; 92(11): 2830-2838, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32558946

RESUMO

Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), leads to a series of clinical symptoms of respiratory and pulmonary inflammatory reactions via unknown pathologic mechanisms related to the viral infection process in tracheal or bronchial epithelial cells. Investigation of this viral infection in the human bronchial epithelial cell line (16HBE) suggests that SARS-CoV-2 can enter these cells through interaction between its membrane-localized S protein with the angiotensin-converting enzyme 2 molecule on the host cell membrane. Further observation indicates distinct viral replication with a dynamic and moderate increase, whereby viral replication does not lead to a specific cytopathic effect but maintains a continuous release of progeny virions from infected cells. Although messenger RNA expression of various innate immune signaling molecules is altered in the cells, transcription of interferons-α (IFN-α), IFN-ß, and IFN-γ is unchanged. Furthermore, expression of some interleukins (IL) related to inflammatory reactions, such as IL-6, IL-2, and IL-8, is maintained at low levels, whereas that of ILs involved in immune regulation is upregulated. Interestingly, IL-22, an IL that functions mainly in tissue repair, shows very high expression. Collectively, these data suggest a distinct infection process for this virus in respiratory epithelial cells, which may be linked to its clinicopathological mechanism.


Assuntos
Brônquios/citologia , Células Epiteliais/virologia , Replicação Viral , /metabolismo , Linhagem Celular , Efeito Citopatogênico Viral/imunologia , Células Epiteliais/imunologia , Humanos , Imunidade Inata , Interleucinas/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
16.
Nucleic Acids Res ; 48(13): 7454-7467, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32520327

RESUMO

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5' splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Processamento de RNA , Aminofenóis/farmacologia , Brônquios/citologia , Linhagem Celular Tumoral , Células Cultivadas , Agonistas dos Canais de Cloreto/farmacologia , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Transporte de Íons/efeitos dos fármacos , Mutação , Quinolonas/farmacologia
17.
Toxicol Lett ; 331: 218-226, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32562635

RESUMO

INTRODUCTION: The benchmark dose (BMD) is a dose that produces a predetermined change in the response rate of an adverse effect. This approach is increasingly utilized to analyze quantitative dose-response relationships. To proof this concept, statistical analysis was compared with the BMD approach in order to rank the sensitivity as well as the toxicity and to describe the mode of action. METHODS: Bronchial (BEAS-2B) and alveolar epithelial cells (A549) were exposed to a wide concentration range (0.4-100 µg/mL) of five metal oxide nanoparticles (CeO2, CuO, TiO2, ZnO, ZrO2). Eight toxicity endpoints were determined representing integrity of lysosomal and cell membrane, oxidative stress level, glutathione based detoxification (glutathione S-transferase), oxidative metabolism (cytochrome P450), alteration of the mitochondrial membrane potential, alteration of phase II antioxidative enzyme (NAD(P)H:quinone oxidoreductase), and de novo DNA synthesis. RESULTS: Based on the BMD calculated for the most sensitive test, the toxicity decreased in the following order: ZnO > CuO > TiO2>ZrO2>CeO2 in BEAS-2B. Both statistical evaluation methods revealed a higher sensitivity of BEAS-2B cells. The BMD-derived mode of action for CuO confirmed the existing hypotheses and provided insights into less known mechanisms. CONCLUSION: The findings proofed that BMD analysis is an effective tool to evaluate different aspects of risk assessment.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Brônquios/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Células A549 , Benchmarking , Brônquios/citologia , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Humanos , Pulmão/citologia , Óxidos , Medição de Risco
18.
Sci Rep ; 10(1): 8685, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457348

RESUMO

Extensive use of gallium arsenide (GaAs) has led to increased exposure to humans working in the semiconductor industry. This study employed physicochemical characterization of GaAs obtained from a workplace, cytotoxicity analysis of damage induced by GaAs in 16HBE cells, RNA-seq and related bioinformatic analysis, qRT-PCR verification and survival analysis to comprehensively understand the potential mechanism leading to lung toxicity induced by GaAs. We found that GaAs-induced abnormal gene expression was mainly related to the cellular response to chemical stimuli, the regulation of signalling, cell differentiation and the cell cycle, which are involved in transcriptional misregulation in cancer, the MAPK signalling pathway, the TGF-ß signalling pathway and pulmonary disease-related pathways. Ten upregulated genes (FOS, JUN, HSP90AA1, CDKN1A, ESR1, MYC, RAC1, CTNNB1, MAPK8 and FOXO1) and 7 downregulated genes (TP53, AKT1, NFKB1, SMAD3, CDK1, E2F1 and PLK1) related to GaAs-induced pulmonary toxicity were identified. High expression of HSP90AA1, RAC1 and CDKN1A was significantly associated with a lower rate of overall survival in lung cancers. The results of this study indicate that GaAs-associated toxicities affected the misregulation of oncogenes and tumour suppressing genes, activation of the TGF-ß/MAPK pathway, and regulation of cell differentiation and the cell cycle. These results help to elucidate the molecular mechanism underlying GaAs-induced pulmonary injury.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Gálio/toxicidade , RNA/metabolismo , Regulação para Cima/efeitos dos fármacos , Arsenicais , Brônquios/citologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epitelioides/citologia , Células Epitelioides/efeitos dos fármacos , Células Epitelioides/metabolismo , Humanos , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA/química , Análise de Sequência de RNA , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
19.
Am J Respir Crit Care Med ; 202(7): 962-972, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32459537

RESUMO

Rationale: Puerto Ricans have the highest childhood asthma prevalence in the United States (23.6%); however, the etiology is uncertain.Objectives: In this study, we sought to uncover the genetic architecture of lung function in Puerto Rican youth with and without asthma who were recruited from the island (n = 836).Methods: We used admixture-mapping and whole-genome sequencing data to discover genomic regions associated with lung function. Functional roles of the prioritized candidate SNPs were examined with chromatin immunoprecipitation sequencing, RNA sequencing, and expression quantitative trait loci data.Measurements and Main Results: We discovered a genomic region at 1q32 that was significantly associated with a 0.12-L decrease in the lung volume of exhaled air (95% confidence interval, -0.17 to -0.07; P = 6.62 × 10-8) with each allele of African ancestry. Within this region, two SNPs were expression quantitative trait loci of TMEM9 in nasal airway epithelial cells and MROH3P in esophagus mucosa. The minor alleles of these SNPs were associated with significantly decreased lung function and decreased TMEM9 gene expression. Another admixture-mapping peak was observed on chromosome 5q35.1, indicating that each Native American ancestry allele was associated with a 0.15-L increase in lung function (95% confidence interval, 0.08-0.21; P = 5.03 × 10-6). The region-based association tests identified four suggestive windows that harbored candidate rare variants associated with lung function.Conclusions: We identified common and rare genetic variants that may play a critical role in lung function among Puerto Rican youth. We independently validated an inflammatory pathway that could potentially be used to develop more targeted treatments and interventions for patients with asthma.


Assuntos
Grupo com Ancestrais do Continente Africano/genética , Asma/genética , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 5/genética , Volume Expiratório Forçado/genética , Índios Norte-Americanos/genética , Pulmão/fisiopatologia , Adolescente , Asma/fisiopatologia , Brônquios/citologia , Estudos de Casos e Controles , Linhagem Celular , Criança , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Mucosa Esofágica/metabolismo , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Expressão Gênica , Humanos , Desequilíbrio de Ligação , Pulmão/fisiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Miócitos de Músculo Liso , Mucosa Nasal/metabolismo , Polimorfismo de Nucleotídeo Único , Porto Rico , Locos de Características Quantitativas , Análise de Sequência de RNA , Sequenciamento Completo do Genoma , Adulto Jovem
20.
PLoS One ; 15(5): e0233439, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469934

RESUMO

In epithelial cells, the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl- channel, plays a key role in water and electrolytes secretion. A dysfunctional CFTR leads to the dehydration of the external environment of the cells and to the production of viscous mucus in the airways of cystic fibrosis patients. Here, we applied the quadriwave lateral shearing interferometry (QWLSI), a quantitative phase imaging technique based on the measurement of the light wave shift when passing through a living sample, to study water transport regulation in human airway epithelial CFBE and CHO cells expressing wild-type, G551D- and F508del-CFTR. We were able to detect phase variations during osmotic challenges and confirmed that cellular volume changes reflecting water fluxes can be detected with QWLSI. Forskolin stimulation activated a phase increase in all CFBE and CHO cell types. This phase variation was due to cellular volume decrease and intracellular refractive index increase and was completely blocked by mercury, suggesting an activation of a cAMP-dependent water efflux mediated by an endogenous aquaporin (AQP). AQP3 mRNAs, not AQP1, AQP4 and AQP5 mRNAs, were detected by RT-PCR in CFBE cells. Readdressing the F508del-CFTR protein to the cell surface with VX-809 increased the detected water efflux in CHO but not in CFBE cells. However, VX-770, a potentiator of CFTR function, failed to further increase the water flux in either G551D-CFTR or VX-809-corrected F508del-CFTR expressing cells. Our results show that QWLSI could be a suitable technique to study water transport in living cells. We identified a CFTR and cAMP-dependent, mercury-sensitive water transport in airway epithelial and CHO cells that might be due to AQP3. This water transport appears to be affected when CFTR is mutated and independent of the chloride channel function of CFTR.


Assuntos
Aquaporina 3/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mucosa Respiratória/metabolismo , Água/metabolismo , Aminofenóis/farmacologia , Animais , Aquaporina 3/genética , Transporte Biológico Ativo/efeitos dos fármacos , Fenômenos Biofísicos , Brônquios/citologia , Brônquios/metabolismo , Células CHO , Linhagem Celular , Agonistas dos Canais de Cloreto/farmacologia , Colforsina/farmacologia , Cricetulus , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Humanos , Microscopia de Interferência , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Osmose , Quinolonas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mucosa Respiratória/citologia
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