Human Cellular Models for the Investigation of Lung Inflammation and Mucus Production in Cystic Fibrosis

Abstract

Chronic inflammation, oxidative stress, mucus plugging, airway remodeling, and respiratory infections are the hallmarks of the cystic fibrosis (CF) lung disease. The airway epithelium is central in the innate immune responses to pathogens colonizing the airways, since it is involved in mucociliary clearance, senses the presence of pathogens, elicits an inflammatory response, orchestrates adaptive immunity, and activates mesenchymal cells. In this review, we focus on cellular models of the human CF airway epithelium that have been used for studying mucus production, inflammatory response, and airway remodeling, with particular reference to two- and three-dimensional cultures that better recapitulate the native airway epithelium. Cocultures of airway epithelial cells, macrophages, dendritic cells, and fibroblasts are instrumental in disease modeling, drug discovery, and identification of novel therapeutic targets. Nevertheless, they have to be implemented in the CF field yet. Finally, novel systems hijacking on tissue engineering, including three-dimensional cocultures, decellularized lungs, microfluidic devices, and lung organoids formed in bioreactors, will lead the generation of relevant human preclinical respiratory models a step forward.

Figure 1

Pathophysiology of CF lung disease. (a) In the healthy state, the CFTR protein inhibits the epithelial sodium channel (ENaC), thereby regulating the absorption of sodium and water from the airway lumen, providing the adequate airway surface homeostasis with effective transport of mucus extruding from the airway surface goblet cells and submucosal glands. Physiological bicarbonate and pH regulation facilitates the formation of an airway surface liquid (ASL) that optimizes mucociliary clearance. Moreover, CFTR regulates transepithelial reduced glutathione (GSH) transport, maintaining the redox potential in the airways. (b) In CF, the absence of CFTR on the apical membrane leads to hyperactivity of ENaC, resulting in hyperabsorption of Na⁺ and water and consequently in reduction of the periciliary liquid (PCL) layer. Mucus transport slows down due to the incapacity of cilia beating with disruption of mucociliary clearance, contributing to mucus stasis also given by goblet cell hyperplasia and submucosal gland hypertrophy. Decreased bicarbonate transport contributes to an acidic pH. Moreover, lower levels of GSH contribute to increased concentration of reactive oxygen species (ROS). This oxidative stress leads to a heightened NF-κB translocation in the nuclei of airway epithelial cells. These events contribute to a proinflammatory airway environment characterized by a massive neutrophil infiltration. Dysregulated macrophages contribute to the high inflammatory burden.

Figure 2

Models of AEC culture. 2D (a, b, c) and 3D (d, e, f) models are shown. AECs are traditionally cultured onto filter inserts submerged in media until they polarize (a) or differentiate under ALI condition (b). While secondary cell lines polarize but not differentiate at ALI (e.g., Calu-3), primary AECs differentiate into a pseudostratified epithelium presenting ciliated, basal, and mucus-producing goblet cells. These filter inserts can then be removed and upended to allow the attachment of accompanying cell types before the filters are placed back in the well (c). Fibroblasts are shown; however, they could be other cell types, such as DCs or macrophages. An immunocompetent 3D model of the human upper airway was developed using the epithelium, antigen-presenting cells (APC), such as DCs, and fibroblasts (d). Different cell types can individually grow on support and then layered one on top of the other or directly grow in this way. Another 3D model is based on the deposition of human pulmonary fibroblasts onto a collagen-type IV-coated insert, followed by the seeding of AECs and culturing them first in submerged conditions and then at ALI (e). RWV (rotating wall vessel) technology allows the growth of AECs on the surface of porous collagen-coated microcarrier beads in suspension under low fluid shear and gentle mixed conditions inside cylindrical bioreactors, termed slow-turning lateral vessels (STLV) or high aspect ratio vessels (HARV) (f).

Figure 3

Neutrophil transmigration in AEC culture. AECs are seeded on the opposite side of filter inserts and allowed to attach, then the insert is inverted and can be cultured either submerged or at ALI (not shown). Neutrophils are added on the top chamber representing the basolateral surface of AECs and induced to migrate to the lower chamber against a chemoattractive agent (e.g., fMLP). After 3–4 hours, neutrophils are recovered from the lower chamber and percentage of migrated cells over the total is calculated.