Choosing the Right Differentiation Medium to Develop Mucociliary Phenotype of Primary Nasal Epithelial Cells In Vitro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and it has infected over 100 million people in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures, including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, which was originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but it strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, which is in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells that were cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of specific sets of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.

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In vitro exposure of nasal epithelial cells to atmospheric dust

Biomechanics and Modeling in Mechanobiology ◽

10.1007/s10237-017-0999-y ◽

2018 ◽

Vol 17 (3) ◽

pp. 891-901 ◽

Cited By ~ 1

Author(s):

David Elad ◽

Uri Zaretsky ◽

Sharon Avraham ◽

Ruthie Gotlieb ◽

Michael Wolf ◽

...

Keyword(s):

Epithelial Cells ◽

Atmospheric Dust ◽

Nasal Epithelial Cells ◽

In Vitro Exposure

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Targeting of the Nasal Mucosa by Japanese Encephalitis Virus for Non-Vector-Borne Transmission

Journal of Virology ◽

10.1128/jvi.01091-18 ◽

2018 ◽

Vol 92 (24) ◽

Cited By ~ 6

Author(s):

Obdulio García-Nicolás ◽

Roman O. Braun ◽

Panagiota Milona ◽

Marta Lewandowska ◽

Ronald Dijkman ◽

...

Keyword(s):

Epithelial Cells ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Ex Vivo ◽

Encephalitis Virus ◽

Nasal Epithelium ◽

Virus Shedding ◽

Nasal Epithelial Cells ◽

Vector Borne

ABSTRACTThe mosquito-borne Japanese encephalitis virus (JEV) causes severe central nervous system diseases and cycles betweenCulexmosquitoes and different vertebrates. For JEV and some other flaviviruses, oronasal transmission is described, but the mode of infection is unknown. Using nasal mucosal tissue explants and primary porcine nasal epithelial cells (NEC) at the air-liquid interface (ALI) and macrophages asex vivoandin vitromodels, we determined that the nasal epithelium could represent the route of entry and exit for JEV in pigs. Porcine NEC at the ALI exposed to with JEV resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines, indicating infection and replication in macrophages. Moreover, macrophages stimulated by alarmins, including interleukin-25, interleukin-33, and thymic stromal lymphopoietin, were more permissive to the JEV infection. Altogether, our data are important to understand the mechanism of non-vector-borne direct transmission of Japanese encephalitis virus in pigs.IMPORTANCEJEV, a main cause of severe viral encephalitis in humans, has a complex ecology composed of a mosquito-waterbird cycle and a cycle involving pigs, which amplifies virus transmission to mosquitoes, leading to increased human cases. JEV can be transmitted between pigs by contact in the absence of arthropod vectors. Moreover, virus or viral RNA is found in oronasal secretions and the nasal epithelium. Using nasal mucosa tissue explants and three-dimensional porcine nasal epithelial cells cultures and macrophages asex vivoandin vitromodels, we determined that the nasal epithelium could be a route of entry as well as exit for the virus. Infection of nasal epithelial cells resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines and therefore infection and replication in macrophages, which is favored by epithelial-cell-derived cytokines. The results are relevant to understand the mechanism of non-vector-borne direct transmission of JEV.

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Identification of urban particulate matter-induced disruption of human respiratory mucosa integrity using whole transcriptome analysis and organ-on-a chip

Journal of Biological Engineering ◽

10.1186/s13036-019-0219-7 ◽

2019 ◽

Vol 13 (1) ◽

Cited By ~ 2

Author(s):

Junhyoung Byun ◽

Boa Song ◽

Kyungwoo Lee ◽

Byoungjae Kim ◽

Hae Won Hwang ◽

...

Keyword(s):

Endothelial Cells ◽

Particulate Matter ◽

Epithelial Cells ◽

Respiratory Mucosa ◽

Nasal Epithelial Cells ◽

Tissue Microenvironment ◽

Human Nasal Epithelial Cells ◽

Whole Transcriptome Analysis ◽

Whole Transcriptome

Abstract Background Exposure to air particulate matter (PM) is associated with various diseases in the human respiratory system. To date, most in vitro studies showing cellular responses to PM have been performed in cell culture using a single cell type. There are few studies considering how multicellular networks communicate in a tissue microenvironment when responding to the presence of PM. Here, an in vitro three-dimensional (3D) respiratory mucosa-on-a-chip, composed of human nasal epithelial cells, fibroblasts, and endothelial cells, is used to recapitulate and better understand the effects of urban particulate matter (UPM) on human respiratory mucosa. Results We hypothesized that the first cells to contact with UPM, the nasal epithelial cells, would respond similar to the tissue microenvironment, and the 3D respiratory mucosa model would be a suitable platform to capture these events. First, whole transcriptome analysis revealed that UPM induced gene expression alterations in inflammatory and adhesion-related genes in human nasal epithelial cells. Next, we developed an in vitro 3D respiratory mucosa model composed of human nasal epithelial cells, fibroblasts, and endothelial cells and demonstrated that the model is structurally and functionally compatible with the respiratory mucosa. Finally, we used our model to expose human nasal epithelial cells to UPM, which led to a disruption in the integrity of the respiratory mucosa by decreasing the expression of zonula occludens-1 in both the epithelium and endothelium, while also reducing vascular endothelial cadherin expression in the endothelium. Conclusions We demonstrate the potential of the 3D respiratory mucosa model as a valuable tool for the simultaneous evaluation of multicellular responses caused by external stimuli in the human respiratory mucosa. We believe that the evaluation strategy proposed in the study will move us toward a better understanding of the detailed molecular mechanisms associated with pathological changes in the human respiratory system.

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Effects of IL-1β, TNF-α, and TGF-β on Proliferation of Human Nasal Epithelial Cells in Vitro

American Journal of Rhinology ◽

10.2500/105065898781390064 ◽

1998 ◽

Vol 12 (4) ◽

pp. 279-282 ◽

Cited By ~ 6

Author(s):

Yang-Gi Min ◽

Chae-Seo Rhee ◽

Sam-Hyun Kwon ◽

Kang Soo Lee ◽

Ja Bock Yun

Keyword(s):

Epithelial Cells ◽

Collagen Gel ◽

Time Dependent ◽

Dependent Manner ◽

Primary Cells ◽

Nasal Epithelial Cells ◽

Human Nasal Epithelial Cells ◽

Tnf Α ◽

Collagen Gel Matrix

Previous reports suggest that cytokines may be involved in proliferation of the epithelium. The aim of this study was to determine the effects of cytokines, IL-1β, TNF-α, and TGF-β on proliferation of human nasal epithelial cells (HNECs) in vitro. Primary cells were cultured from HNECs on collagen gel matrix. Subcultured HNECs were incubated in a medium with recombinant human (rh) cytokines, rhIL-1β, rhTNF-a, and rhTGF-β at different concentrations of 0.01 ng/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, and 100 ng/mL. After 2-day incubation with these cytokines, daily cell proliferation was measured by MTT assay for 6 days. While rhIL-1β inhibited proliferation of HNECs in concentration-dependent and time-dependent manners, rhTNF-a stimulated HNEC growth at concentrations ranging from 0.01 ng/mL to 10 ng/mL in concentration-dependent and time-dependent manner. In contrast, rhTGF-b inhibited HNEC growth irrespective of concentration and incubation time. This study suggests that IL-1β, TNF-α, and TGF-β may have an important role in the repair of the nasal mucosa by regulating proliferation of the nasal epithelium.

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Basic and clinical immunology – 3023. Influence of fexofenadine hydrochloride on uteroglobin production from nasal epithelial cells in vitro and in vivo

World Allergy Organization Journal ◽

10.1186/1939-4551-6-s1-p199 ◽

2013 ◽

Vol 6 ◽

pp. P199

Author(s):

Kenichi Kanai ◽

Kazuhito Asano ◽

Atsuko Furuta ◽

Takeyuki Sanbe ◽

Harumi Suzaki

Keyword(s):

Epithelial Cells ◽

Clinical Immunology ◽

Nasal Epithelial Cells

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Alpha 1-adrenergic signaling in human airway epithelial cells involves inositol lipid and phosphate metabolism

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1992.262.2.l183 ◽

1992 ◽

Vol 262 (2) ◽

pp. L183-L191 ◽

Cited By ~ 1

Author(s):

C. M. Liedtke

Keyword(s):

Epithelial Cells ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Nasal Epithelial Cells ◽

Human Airway ◽

Adrenergic Antagonist ◽

Human Airway Epithelial Cells

A role for phospholipase C (PLC) hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) as a mechanism of alpha 1-adrenergic signal transduction in human airway epithelial cells (AEC) was investigated in isolated normal tracheal and cystic fibrosis (CF) nasal epithelial cells grown in in vitro culture and prelabeled with 3 muCi myo-[3H]inositol/ml for 72 h. Breakdown of polyphosphoinositides was measured using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and PIP2. Inositol phosphates were separated by ion-exchange column chromatography. In normal AEC, the addition of the endogenous catecholamine l-epinephrine produced a rapid, transient accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2) and breakdown of PIP and PIP2. IP3 increased 1.7-fold and IP2 1.6-fold after 20 and 40 s, respectively. A maximal decrease of 35% PIP2 and 30% PIP is observed after 20 and 40 s, respectively. The effects of l-epinephrine were not blocked by the beta-adrenergic antagonist dl-propranolol but were mimicked by the alpha 1-adrenergic agonist methoxamine. Prazosin, an alpha 1-adrenergic antagonist, and pertussis toxin (PTX) blocked the effects of l-epinephrine and methoxamine. Addition of l-epinephrine and methoxamine to CF nasal epithelial cells also induced prazosin-sensitive polyphosphoinositide breakdown and inositol phosphate accumulation. A 2.2-fold accumulation of IP3 was observed after 10 s and 2.0-fold increase in IP2 after 20 s. Maximal decreases of 32% PIP2 and 23% PIP levels were observed after 20-s incubation with l-epinephrine. PTX reduced the effects of l-epinephrine and significantly blocked the effects of methoxamine.(ABSTRACT TRUNCATED AT 250 WORDS)

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