Impaired Ciliogenesis in differentiating human bronchial epithelia exposed to non-Cytotoxic doses of multi-walled carbon Nanotubes

Ryan J Snyder,Stavros Garantziotis,Salik Hussain,Charles J Tucker,Scott H Randell

doi:10.1186/s12989-017-0225-1

Abstract

BackgroundMulti-walled carbon nanotubes (MWCNTs) are engineered nanomaterials used for a variety of industrial and consumer products. Their high tensile strength, hydrophobicity, and semi-conductive properties have enabled many novel applications, increasing the possibility of accidental nanotube inhalation by either consumers or factory workers. While MWCNT inhalation has been previously shown to cause inflammation and pulmonary fibrosis at high doses, the susceptibility of differentiating bronchial epithelia to MWCNT exposure remains unexplored. In this study, we investigate the effect of MWCNT exposure on cilia development in a differentiating air-liquid interface (ALI) model. Primary bronchial epithelial cells (BECs) were isolated from human donors via bronchoscopy and treated with non-cytotoxic doses of MWCNTs in submerged culture for 24 h. Cultures were then allowed to differentiate in ALI for 28 days in the absence of further MWCNT exposure. At 28 days, mucociliary differentiation endpoints were assessed, including whole-mount immunofluorescent staining, histological, immunohistochemical and ultrastructural analysis, gene expression, and cilia beating analysis.ResultsWe found a reduction in the prevalence and beating of ciliated cells in MWCNT-treated cultures, which appeared to be caused by a disruption of cellular microtubules and cytoskeleton during ciliogenesis and basal body docking. Expression of gene markers of mucociliary differentiation, such as FOXJ1 and MUC5AC/B, were not affected by treatment. Colocalization of basal body marker CEP164 with γ-tubulin during days 1–3 of ciliogenesis, as well as abundance of basal bodies up to day 14, were attenuated by treatment with MWCNTs.ConclusionsOur results suggest that a single exposure of bronchial cells to MWCNT during a vulnerable period before differentiation may impair their ability to develop into fully functional ciliated cells.

Highlights

Multi-walled carbon nanotubes (MWCNTs) are engineered nanomaterials used for a variety of industrial and consumer products
Attenuated cilia staining in differentiated air-liquid interface (ALI) culture 28 days after single MWCNT exposure We first analyzed the effect of a single MWCNT exposure on cell differentiation
Cells which were treated with MWCNTs after they had differentiated in ALI for 21 days had ciliary α-tubulin and tight junction actin staining of 32.07 ± 7.32% and 23.83 ± 7.86%, and were not significantly different from control cultures

Summary

Introduction

Multi-walled carbon nanotubes (MWCNTs) are engineered nanomaterials used for a variety of industrial and consumer products. Inhalation of MWCNTs induces pulmonary inflammation and fibrosis [12], and retained fibers have been found in the lungs of exposed rodent models months after MWCNT exposure [13]. These adverse health effects were demonstrated at high doses; even lower, more occupationallyrelevant doses can have significant effects on pulmonary cells [14, 15]. Impaired barrier function in the pulmonary epithelium of MWCNT-exposed individuals may further increase their susceptibility to environmental insult These effects may be important in susceptible individuals with pre-existing lung disease, or in cases of long-term chronic exposure. Denuded epithelium resulting from tobacco smoke [17] or viral infection [18] could expose undifferentiated basal cells to nanoparticles, potentiating further injury or attenuating epithelial regeneration

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