In vitro differentiation of ciliated cells in ALI-cultured human airway epithelium – the framework for functional studies on airway differentiation in ciliopathies

The International Thermonuclear Experimental Reactor (ITER) is an international project aimed at the production of carbon-free energy through the use of thermonuclear fusion. During ITER operation, in case of a loss-of-vacuum-accident, tungsten nanoparticles (W-NPs) could potentially be released into the environment and induce occupational exposure via inhalation. W-NPs toxicity was evaluated on MucilAir™, a 3D in vitro cell model of the human airway epithelium. MucilAir™ was exposed for 24 h to metallic ITER-like milled W-NPs, tungstate (WO42−) and tungsten carbide cobalt particles alloy (WC-Co). Cytotoxicity and its reversibility were assessed using a kinetic mode up to 28 days after exposure. Epithelial tightness, metabolic activity and interleukin-8 release were also evaluated. Electron microscopy was performed to determine any morphological modification, while mass spectrometry allowed the quantification of W-NPs internalization and of W transfer through the MucilAir™. Our results underlined a decrease in barrier integrity, no effect on metabolic activity or cell viability and a transient increase in IL-8 secretion after exposure to ITER-like milled W-NPs. These effects were associated with W-transfer through the epithelium, but not with intracellular accumulation. We have shown that, under our experimental conditions, ITER-like milled W-NPs have a minor impact on the MucilAir™ in vitro model.

Download Full-text

In vitro models of the human airway epithelium for inhalation toxicity testing

Toxicology Letters ◽

10.1016/j.toxlet.2016.06.1218 ◽

2016 ◽

Vol 258 ◽

pp. S30-S31

Author(s):

Samuel Constant

Keyword(s):

Airway Epithelium ◽

Toxicity Testing ◽

In Vitro Models ◽

Inhalation Toxicity ◽

Human Airway ◽

Human Airway Epithelium

Download Full-text

Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium

10.1101/2021.04.16.440244 ◽

2021 ◽

Author(s):

Talita B Gagliardi ◽

Monty E Goldstein ◽

Daniel Song ◽

Kelsey M Gray ◽

Jae W Jung ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Airway Epithelium ◽

In Vitro Model ◽

Cytopathic Effects ◽

Human Airway ◽

Human Airway Epithelium ◽

Vitro Model ◽

Rhinovirus C ◽

The Impact

The clinical impact of rhinovirus C (RV-C) is well-documented; yet the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24 hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection and correlating with the formation of large intracellular vesicles. To further define RV-C15 replication sites, we analyzed the expression of giantin, phosphatidylinositol-4-phosphate, and calnexin, as well as the colocalization of these markers with dsRNA. Fluorescence levels of all three cellular markers were elevated during infection and altered giantin distribution further indicated Golgi fragmentation. However, unlike previously characterized RVs, the high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression, facilitating replication, and the induction of incomplete autophagy, a mechanism used by other RVs to promote non-lytic release of progeny virions. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality - aspects of infection that may contribute to pathogenesis in vivo.

Download Full-text