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Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (>60 years) is a significant risk factor for developing severe COVID-19. To better understand the host response of the aged airway epithelium to SARS-CoV-2 infection, we performed an in vitro study using primary human bronchial epithelial cells from donors >67 years of age differentiated on an air–liquid interface culture. We demonstrate that SARS-CoV-2 infection leads to early induction of a proinflammatory response and a delayed interferon response. In addition, we observed changes in the genes and pathways associated with cell death and senescence throughout infection. In summary, our study provides new and important insights into the temporal kinetics of the airway epithelial innate immune response to SARS-CoV-2 in older individuals.
Bharathiraja Subramaniyan; Jason L. Larabee; Manish Bodas; Andrew R. Moore; Anthony W. G. Burgett; Dean A. Myers; Constantin Georgescu; Jonathan D. Wren; James F. Papin; Matthew S. Walters. Characterization of the SARS-CoV-2 Host Response in Primary Human Airway Epithelial Cells from Aged Individuals. Viruses 2021, 13, 1603 .
AMA StyleBharathiraja Subramaniyan, Jason L. Larabee, Manish Bodas, Andrew R. Moore, Anthony W. G. Burgett, Dean A. Myers, Constantin Georgescu, Jonathan D. Wren, James F. Papin, Matthew S. Walters. Characterization of the SARS-CoV-2 Host Response in Primary Human Airway Epithelial Cells from Aged Individuals. Viruses. 2021; 13 (8):1603.
Chicago/Turabian StyleBharathiraja Subramaniyan; Jason L. Larabee; Manish Bodas; Andrew R. Moore; Anthony W. G. Burgett; Dean A. Myers; Constantin Georgescu; Jonathan D. Wren; James F. Papin; Matthew S. Walters. 2021. "Characterization of the SARS-CoV-2 Host Response in Primary Human Airway Epithelial Cells from Aged Individuals." Viruses 13, no. 8: 1603.
Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death in the United States and is primarily caused by cigarette smoking. Increased numbers of mucus-producing secretory (“goblet”) cells defined as goblet cell metaplasia or hyperplasia (GCMH), contributes significantly to COPD pathophysiology. The objective of this study was to determine whether NOTCH signaling regulates goblet cell differentiation in response to cigarette smoke. Primary human bronchial epithelial cells (HBECs) from nonsmokers and COPD smokers were differentiated in vitro on air-liquid interface and exposed to cigarette smoke extract (CSE) for 7 days. NOTCH signaling activity was modulated using (1) the NOTCH/γ-secretase inhibitor Dibenzazepine (DBZ), (2) lentiviral over-expression of the NOTCH3-intracellular domain (NICD3) or (3) NOTCH3-specific siRNA. Cell differentiation and response to CSE were evaluated by qPCR, Western blotting, immunostaining and RNA-Seq. We found that CSE exposure of nonsmoker airway epithelium induced goblet cell differentiation characteristic of GCMH. Treatment with DBZ suppressed CSE-dependent induction of goblet cell differentiation. Furthermore, CSE induced NOTCH3 activation, as revealed by increased NOTCH3 nuclear localization and elevated NICD3 protein levels. Over-expression of NICD3 increased the expression of goblet cell associated genes SPDEF and MUC5AC, whereas NOTCH3 knockdown suppressed CSE-mediated induction of SPDEF and MUC5AC. Finally, CSE exposure of COPD airway epithelium induced goblet cell differentiation in a NOTCH3-dependent manner. These results identify NOTCH3 activation as one of the important mechanisms by which cigarette smoke induces goblet cell differentiation, thus providing a novel potential strategy to control GCMH-related pathologies in smokers and patients with COPD.
Manish Bodas; Andrew R. Moore; Bharathiraja Subramaniyan; Constantin Georgescu; Jonathan D. Wren; Willard M. Freeman; Brent R. Brown; Jordan P. Metcalf; Matthew S. Walters. Cigarette Smoke Activates NOTCH3 to Promote Goblet Cell Differentiation in Human Airway Epithelial Cells. American Journal of Respiratory Cell and Molecular Biology 2021, 64, 426 -440.
AMA StyleManish Bodas, Andrew R. Moore, Bharathiraja Subramaniyan, Constantin Georgescu, Jonathan D. Wren, Willard M. Freeman, Brent R. Brown, Jordan P. Metcalf, Matthew S. Walters. Cigarette Smoke Activates NOTCH3 to Promote Goblet Cell Differentiation in Human Airway Epithelial Cells. American Journal of Respiratory Cell and Molecular Biology. 2021; 64 (4):426-440.
Chicago/Turabian StyleManish Bodas; Andrew R. Moore; Bharathiraja Subramaniyan; Constantin Georgescu; Jonathan D. Wren; Willard M. Freeman; Brent R. Brown; Jordan P. Metcalf; Matthew S. Walters. 2021. "Cigarette Smoke Activates NOTCH3 to Promote Goblet Cell Differentiation in Human Airway Epithelial Cells." American Journal of Respiratory Cell and Molecular Biology 64, no. 4: 426-440.
Basal cells (BC) are the resident stem/progenitor cells of the adult pseudostratified airway epithelium, whose differentiation program is orchestrated by the NOTCH signaling pathway. NOTCH3 receptor mediated signaling regulates BC to club cell differentiation; however, the downstream responses that regulate this process are largely unknown. In the present study we used anin vitroair-liquid interface model of the human pseudostratified airway epithelium to identify the NOTCH3-dependent downstream genes/pathways that regulate human BC to club cell differentiation. Activation of NOTCH3 signaling in BC via lentivirus-mediated over-expression of the active NOTCH3 intracellular domain (NICD3) promoted club cell differentiation. Bulk RNA-seq analysis of controlvsNICD3-transduced cells, identified 692 NICD3 responsive genes enriched for pathways linked to airway epithelial biology and differentiation including Wnt/β-catenin Signaling. Expression of the classical NOTCH target HEYL increased in response to NOTCH3 activation and positively correlated with expression of the club cell marker SCGB1A1. Further, using single-cell RNA-seq, we report that HEYL+cells primarily clustered with SCGB1A1+and NOTCH3+cells. Moreover, HEYL protein co-localized with SCGB1A1 in ALI culturesin vitroand in the human and mouse airway epitheliumin vivo.siRNA-mediated knockdown of HEYL in BC led to changes in epithelial structure including altered morphology and significant reductions in transepithelial electrical resistance and expression of tight junction related genes. Finally, HEYL knockdown significantly reduced the number of SCGB1A1+club cells, along with a corresponding increase in KRT8+BC-intermediate cells. Overall, our data identifies NOTCH3-HEYL signaling as a key regulator of BC to club cell differentiation.
Manish Bodas; Bharathiraja Subramaniyan; Andrew R. Moore; Jordan P. Metcalf; Sarah R. Ocañas; Willard M. Freeman; Constantin Georgescu; Jonathan D. Wren; Matthew S. Walters. The NOTCH3 Downstream Target HEYL Regulates Human Airway Epithelial Club Cell Differentiation. 2021, 1 .
AMA StyleManish Bodas, Bharathiraja Subramaniyan, Andrew R. Moore, Jordan P. Metcalf, Sarah R. Ocañas, Willard M. Freeman, Constantin Georgescu, Jonathan D. Wren, Matthew S. Walters. The NOTCH3 Downstream Target HEYL Regulates Human Airway Epithelial Club Cell Differentiation. . 2021; ():1.
Chicago/Turabian StyleManish Bodas; Bharathiraja Subramaniyan; Andrew R. Moore; Jordan P. Metcalf; Sarah R. Ocañas; Willard M. Freeman; Constantin Georgescu; Jonathan D. Wren; Matthew S. Walters. 2021. "The NOTCH3 Downstream Target HEYL Regulates Human Airway Epithelial Club Cell Differentiation." , no. : 1.
Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death in the United States and is primarily caused by cigarette smoking. Increased numbers of mucus-producing secretory (“goblet”) cells defined as goblet cell metaplasia or hyperplasia (GCMH), contributes significantly to COPD pathophysiology. The objective of this study was to determine whether NOTCH signaling regulates goblet cell differentiation in response to cigarette smoke. Primary human bronchial epithelial cells (HBECs) from nonsmokers and COPD smokers were differentiated in vitro on air-liquid interface and exposed to cigarette smoke extract (CSE) for 7 days. NOTCH signaling activity was modulated using (1) the NOTCH/γ-secretase inhibitor Dibenzazepine (DBZ), (2) lentiviral over-expression of the NOTCH3-intracellular domain (NICD3) or (3) NOTCH3-specific siRNA. Cell differentiation and response to CSE were evaluated by qPCR, Western blotting, immunostaining and RNA-Seq. We found that CSE exposure of nonsmoker airway epithelium induced goblet cell differentiation characteristic of GCMH. Treatment with DBZ suppressed CSE-dependent induction of goblet cell differentiation. Furthermore, CSE induced NOTCH3 activation, as revealed by increased NOTCH3 nuclear localization and elevated NICD3 protein levels. Over-expression of NICD3 increased the expression of goblet cell associated genes SPDEF and MUC5AC, whereas NOTCH3 knockdown suppressed CSE-mediated induction of SPDEF and MUC5AC. Finally, CSE exposure of COPD airway epithelium induced goblet cell differentiation in a NOTCH3-dependent manner. These results identify NOTCH3 activation as one of the important mechanisms by which cigarette smoke induces goblet cell differentiation, thus providing a novel potential strategy to control GCMH-related pathologies in smokers and patients with COPD.
Manish Bodas; Andrew R. Moore; Bharathiraja Subramaniyan; Constantin Georgescu; Jonathan D. Wren; Willard M. Freeman; Brent R. Brown; Jordan P. Metcalf; Matthew S. Walters. Cigarette Smoke Activates NOTCH3 to Promote Goblet Cell Differentiation in Human Airway Epithelial Cells. 2020, 1 .
AMA StyleManish Bodas, Andrew R. Moore, Bharathiraja Subramaniyan, Constantin Georgescu, Jonathan D. Wren, Willard M. Freeman, Brent R. Brown, Jordan P. Metcalf, Matthew S. Walters. Cigarette Smoke Activates NOTCH3 to Promote Goblet Cell Differentiation in Human Airway Epithelial Cells. . 2020; ():1.
Chicago/Turabian StyleManish Bodas; Andrew R. Moore; Bharathiraja Subramaniyan; Constantin Georgescu; Jonathan D. Wren; Willard M. Freeman; Brent R. Brown; Jordan P. Metcalf; Matthew S. Walters. 2020. "Cigarette Smoke Activates NOTCH3 to Promote Goblet Cell Differentiation in Human Airway Epithelial Cells." , no. : 1.
Influenza A virus (IAV) infection is a major cause of morbidity and mortality. Retinoic acid-inducible protein I (RIG-I) plays an important role in the recognition of IAV in most cell types, and leads to the activation of interferon (IFN). We investigated mechanisms of RIG-I and IFN induction by IAV in the BCi-NS1.1 immortalized human airway basal cell line and in the A549 human alveolar epithelial cell line. We found that the basal expression levels of RIG-I and regulatory transcription factor (IRF) 7 were very low in BCi-NS1.1 cells. IAV infection induced robust RIG-I and IRF7, not IRF3, expression. siRNA against IRF7 and mitochondrial antiviral-signaling protein (MAVS), but not IRF3, significantly inhibited RIG-I mRNA expression and IFN induction by IAV infection. Most importantly, even without virus infection, IFN-β alone induced RIG-I, and siRNA against IRF7 did not inhibit RIG-I induction by IFN-β. Similar results were found in the alveolar basal epithelial A549 cell line. RIG-I and IRF7 expression in humans is highly inducible and greatly amplified by IFN produced from virus infected cells. IFN induction can be separated into two phases, that initially induced by the virus with basal RIG-I (the first phase), and that induced by the subsequent virus with amplified RIG-I from the first phase IFN (the second phase). The de novo synthesis of IRF7 is required for the second phase IFN induction during influenza virus infection in human lung bronchial and alveolar epithelial cells.
Wenxin Wu; Wei Zhang; Lili Tian; Brent R. Brown; Matthew S. Walters; Jordan P. Metcalf. IRF7 Is Required for the Second Phase Interferon Induction during Influenza Virus Infection in Human Lung Epithelia. Viruses 2020, 12, 377 .
AMA StyleWenxin Wu, Wei Zhang, Lili Tian, Brent R. Brown, Matthew S. Walters, Jordan P. Metcalf. IRF7 Is Required for the Second Phase Interferon Induction during Influenza Virus Infection in Human Lung Epithelia. Viruses. 2020; 12 (4):377.
Chicago/Turabian StyleWenxin Wu; Wei Zhang; Lili Tian; Brent R. Brown; Matthew S. Walters; Jordan P. Metcalf. 2020. "IRF7 Is Required for the Second Phase Interferon Induction during Influenza Virus Infection in Human Lung Epithelia." Viruses 12, no. 4: 377.