Berberine and obatoclax inhibit SARS-CoV-2 replication in primary human nasal epithelial cells in vitro

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and has infected an estimated 10% of the global population 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, 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 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, in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of a specific set 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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Berberine and Obatoclax Inhibit SARS-Cov-2 Replication in Primary Human Nasal Epithelial Cells In Vitro

Viruses ◽

10.3390/v13020282 ◽

2021 ◽

Vol 13 (2) ◽

pp. 282

Author(s):

Finny S. Varghese ◽

Esther van Woudenbergh ◽

Gijs J. Overheul ◽

Marc J. Eleveld ◽

Lisa Kurver ◽

...

Keyword(s):

Epithelial Cells ◽

Early Stage ◽

Antiviral Drugs ◽

Target Cells ◽

Nasal Epithelial Cells ◽

Cytokines And Chemokines ◽

Human Nasal Epithelial Cells ◽

Apoptotic Protein ◽

Air Liquid Interface

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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Major Complex Trait for Early De Novo Programming ‘CoV-MAC-TED’ Detected in Human Nasal Epithelial Cells Infected by Two SARS-CoV-2 Variants Is Promising to Help in Designing Therapeutic Strategies

Vaccines ◽

10.3390/vaccines9121399 ◽

2021 ◽

Vol 9 (12) ◽

pp. 1399

Author(s):

José Hélio Costa ◽

Shahid Aziz ◽

Carlos Noceda ◽

Birgit Arnholdt-Schmitt

Keyword(s):

Epithelial Cells ◽

Data Collection ◽

De Novo ◽

Complex Trait ◽

Cell System ◽

Target Cells ◽

Transcriptome Data ◽

Nasal Epithelial Cells ◽

Human Nasal Epithelial Cells

Background: Early metabolic reorganization was only recently recognized as an essentially integrated part of immunology. In this context, unbalanced ROS/RNS levels connected to increased aerobic fermentation, which is linked to alpha-tubulin-based cell restructuring and control of cell cycle progression, were identified as a major complex trait for early de novo programming (‘CoV-MAC-TED’) during SARS-CoV-2 infection. This trait was highlighted as a critical target for developing early anti-viral/anti-SARS-CoV-2 strategies. To obtain this result, analyses had been performed on transcriptome data from diverse experimental cell systems. A call was released for wide data collection of the defined set of genes for transcriptome analyses, named ‘ReprogVirus’, which should be based on strictly standardized protocols and data entry from diverse virus types and variants into the ‘ReprogVirus Platform’. This platform is currently under development. However, so far, an in vitro cell system from primary target cells for virus attacks that could ideally serve for standardizing the data collection of early SARS-CoV-2 infection responses has not been defined. Results: Here, we demonstrate transcriptome-level profiles of the most critical ‘ReprogVirus’ gene sets for identifying ‘CoV-MAC-TED’ in cultured human nasal epithelial cells infected by two SARS-CoV-2 variants differing in disease severity. Our results (a) validate ‘Cov-MAC-TED’ as a crucial trait for early SARS-CoV-2 reprogramming for the tested virus variants and (b) demonstrate its relevance in cultured human nasal epithelial cells. Conclusion: In vitro-cultured human nasal epithelial cells proved to be appropriate for standardized transcriptome data collection in the ‘ReprogVirus Platform’. Thus, this cell system is highly promising to advance integrative data analyses with the help of artificial intelligence methodologies for designing anti-SARS-CoV-2 strategies.

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FcRn-Dependent Transcytosis of Monoclonal Antibody in Human Nasal Epithelial Cells In Vitro: A Prerequisite for a New Delivery Route for Therapy?

International Journal of Molecular Sciences ◽

10.3390/ijms20061379 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1379 ◽

Cited By ~ 2

Author(s):

Emilie Bequignon ◽

Christine Dhommée ◽

Christelle Angely ◽

Lucie Thomas ◽

Mathieu Bottier ◽

...

Keyword(s):

Epithelial Cells ◽

Chronic Inflammatory Disease ◽

Nasal Administration ◽

Basal Cells ◽

Nasal Epithelial Cells ◽

Human Nasal Epithelial Cells ◽

Culture Model ◽

And Function ◽

Air Liquid Interface

Monoclonal antibodies (mAbs) are promising therapies to treat airway chronic inflammatory disease (asthma or nasal polyps). To date, no study has specifically assessed, in vitro, the potential function of neonatal Fc receptor (FcRn) in IgG transcytosis through the human nasal airway epithelium. The objective of this study was to report the in vitro expression and function of FcRn in nasal human epithelium. FcRn expression was studied in an air–liquid interface (ALI) primary culture model of human nasal epithelial cells (HNEC) from polyps. FcRn expression was characterized by quantitative RT-PCR, western blot, and immunolabeling. The ability of HNECs to support mAb transcytosis via FcRn was assessed by transcytosis assay. This study demonstrates the expression of FcRn mRNA and protein in HNEC. We report a high expression of FcRn in the cytosol of ciliated, mucus, and basal cells by immunohistochemistry with a higher level of FcRn proteins in differentiated HNEC. We also proved in vitro transepithelial delivery of an IgG1 therapeutic mAb with a dose–response curve. This is the first time that FcRn expression and mAb transcytosis has been shown in a model of human nasal respiratory epithelium in vitro. This study is a prerequisite for FcRn-dependent nasal administration of mAbs.

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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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Real-time measurement of cellular bioenergetics in fully differentiated human nasal epithelial cells grown at air-liquid-interface

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00414.2019 ◽

2020 ◽

Vol 318 (6) ◽

pp. L1158-L1164

Author(s):

Emily Mavin ◽

Bernard Verdon ◽

Sean Carrie ◽

Vinciane Saint-Criq ◽

Jason Powell ◽

...

Keyword(s):

Epithelial Cells ◽

Real Time ◽

Aerobic Glycolysis ◽

Airway Epithelial Cells ◽

Respiratory Epithelium ◽

Liquid Interface ◽

Human Nasal Epithelial Cells ◽

Respiratory Epithelial ◽

Air Liquid Interface

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes.

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