Epithelial–mesenchymal transition involved in pulmonary fibrosis induced by multi-walled carbon nanotubes via TGF-beta/Smad signaling pathway

Highlights

• MWCNT induced pulmonary fibrosis in a length dependent manner.

• Only long MWCNT produced the observed pulmonary fibrosis in C57Bl/6J mice.

• EMT contributed to the adverse outcome of pulmonary fibrosis induced by MWCNT.

• TGF-β/p-Smad2 signaling pathway played a pivotal role in the occurrence of EMT.

Abstract

Multi-walled carbon nanotubes (MWCNT) are a typical nanomaterial with a wide spectrum of commercial applications. Inhalation exposure to MWCNT has been linked with lung fibrosis and mesothelioma-like lesions commonly seen with asbestos. In this study, we examined the pulmonary fibrosis response to different length of MWCNT including short MWCNT (S-MWCNT, length = 350–700 nm) and long MWCNT (L-MWCNT, length = 5–15 μm) and investigated whether the epithelial–mesenchymal transition (EMT) occurred during MWCNT-induced pulmonary fibrosis. C57Bl/6J male mice were intratracheally instilled with S-MWCNT or L-WCNT by a single dose of 60 μg per mouse, and the progress of pulmonary fibrosis was evaluated at 7, 28 and 56 days post-exposure. The in vivo data showed that only L-MWCNT increased collagen deposition and pulmonary fibrosis significantly, and approximately 20% of pro-surfactant protein-C positive epithelial cells transdifferentiated to fibroblasts at 56 days, suggesting the occurrence of EMT. In order to understand the mechanism, we used human pulmonary epithelial cell line A549 to investigate the role of TGF-β/p-Smad2 signaling pathway in EMT. Our results showed that L-MWCNT downregulated E-cadherin and upregulated α-smooth muscle actin (α-SMA) protein expression in A549 cells. Taken together, both in vivo and in vitro study demonstrated that respiratory exposure to MWCNT induced length dependent pulmonary fibrosis and epithelial-derived fibroblasts via TGF-β/Smad pathway.

Introduction

Multi-walled carbon nanotubes (MWCNT) are fiber-shaped nanomaterials that have a wide range of applications in electrical engineering, computer science and aerospace industry (De Volder et al., 2013). There is raising concern on the potential health effects of carbon nanotubes (CNT) (Zeisberg et al., 2007). Their extremely small size (nano meters in diameter) and light weight make it possible for human inhalation exposure during manufacture, storage, transportation, product application and disposition processes. The fiber-like shape of MWCNT may enable them to exercise toxic effects in a similar manner as asbestos (i.e., lung fibrosis and mesothelioma) (Poland et al., 2008). The National Institute of Occupational Safety and Health (NIOSH, USA) (NIOSH, 2013) has recommended an occupational reference exposure level (REL) of CNT at 1 μg/m³, which is much lower than carbon black (REL = 3500 μg/m³), another carbon-based chemical with different particle size and shape.

The fiber hypothesis places MWCNT in line with asbestos and other pathogenic fibers whose toxicity is closely related to their elongated physical shape and this hypothesis has largely been confirmed by existing studies. Fibrotic responses or multifocal granulomas in the lungs of rats or mice were observed following inhalation, intratracheal instillation and pharyngeal aspiration of MWCNT (Aiso et al., 2010, Carrero-Sanchez et al., 2006, Mercer et al., 2011, Muller et al., 2005, Porter et al., 2010, Reddy et al., 2012, Ryman-Rasmussen et al., 2009, Wang et al., 2011a, Wang et al., 2011b). Poland et al. (2008) demonstrated that intraperitoneal and pleural administration of MWCNT in mice induced asbestos-like pathology in a length-dependent manner. Wang et al. (2013) also reported fibrotic effect of long MWCNT. On the other hand, Muhlfeld et al. (2012) observed alveolar septal fibrosis induced only by exposure to short MWCNT. Thus, the fiber hypothesis with theory of length-dependent fibrotic ability still remains to be confirmed by further investigations.

Fibrotic reaction in the lung interstitium is a common adverse pathologic outcome following inhalation exposure to particles, fibers and metals; however the cellular and molecular mechanisms of MWCNT-induced pulmonary fibrosis remain largely unaddressed. Activated interstitial fibroblasts are considered as the primary responsive cells in the development of pulmonary fibrosis (Strieter and Mehrad, 2009). Recent studies indicated that MWCNT might directly stimulate local fibroblasts activation and collagen production (Mishra et al., 2012) or indirectly induce fibroblast cells collagen production through macrophages activation and transforming growth factor (TGF)-β1 (Wang et al., 2013). While almost all existing studies focused on the local fibroblasts activation, there is growing evidence that fibroblasts that originate from pulmonary epithelial cells via local epithelial–mesenchymal transition (EMT) probably have a great contribution to the local activated fibroblasts (Kim et al., 2006, Willis and Borok, 2007). Epithelial injury can lead to EMT, which represents a gradual cellular transition process in which the epithelial cells acquire mesenchymal features that could culminate in their transition to fibroblasts or myofibroblasts. Chang et al. (2012) estimated that 42.6% of activated fibroblasts originated from epithelial cells during pulmonary fibrosis induced by single walled carbon nanobubes (SWCNT). Recent in vitro studies also showed that asbestos treatment could induce EMT in A549 cells (Tamminen et al., 2012). In human idiopathic pulmonary fibrosis (IPF), markers of EMT have also been identified (Willis and Borok, 2007). These results supported that epithelial cells could serve as a novel surrogate source of fibroblasts during pulmonary fibrosis. This study aims to investigate pulmonary epithelial cells as a source of fibroblasts in MWCNT-induced pulmonary fibrosis and whether pulmonary fibrosis response is dependent on the length of MWCNT.

In fibrotic diseases, cells undergoing EMT will lose epithelial marker proteins including the adherent junction and tight junction proteins such as E-cadherin and zonula occludens (ZO)-1, and instead begin to express mesenchymal proteins including α-smooth muscle actin (α-SMA) and vimentin (Zeisberg and Neilson, 2009). It is well known that TGF-β/Smad signaling pathway can be activated and plays a critical role in EMT process (Kasai et al., 2005, Li et al., 2011, Willis et al., 2005). Here, we hypothesized that MWCNT may induce fibrosis through the TGF-β/Smad signaling pathway. In this study, we performed both in vivo and in vitro study to investigate the occurrence of EMT and the activation of TGF-β/Smad2 signaling pathway during MWCNT-induced pulmonary fibrosis.