Establishment and validation of red fox (vulpes vulpes) airway epithelial cell cultures at the air-liquid-interface

Abstract

The airway epithelium represents a central barrier against pathogens and toxins while playing a crucial role in modulating the immune response within the upper respiratory tract. Understanding these mechanisms is particularly relevant for red foxes (Vulpes vulpes), which serve as reservoirs for various zoonotic pathogens like rabies or the fox tapeworm (Echinococcus multilocularis). The study aimed to develop, establish, and validate an air-liquid interface (ALI) organoid model of the fox respiratory tract using primary airway epithelial cells isolated from the tracheas and main bronchi of hunted red foxes. The resulting ALI cultures exhibited a structurally differentiated, pseudostratified epithelium, characterised by ciliated cells, mucus secretion, and tight junctions, as confirmed through histological and immunohistochemical analysis. Functional assessments using a paracellular permeability assay and measurement of transepithelial electrical resistance, demonstrated a tight epithelial barrier. The potential of model's utility for studying innate immune responses to respiratory infections was validated by exposing the cultures to lipopolysaccharide, phorbol-12-myristate-13-acetate and ionomycin, and nematode somatic antigens. Quantitative PCR revealed notable changes in the expression of pro-inflammatory cytokines TNF and IL-33. This in vitro model represents a significant advancement in respiratory research for non-classical species that may act as important wildlife reservoirs for a range of zoonotic pathogens.

Keywords: In vitro model; Fox; Host-pathogen interaction; Respiratory tract; Wildlife reservoir.

Fig. 1

Scatter plot of number of isolated cells against cell viability in respiratory epithelial cells of twelve foxes.

Fig. 2

Red fox primary airway cells maintained at the air-liquid interface present a ciliated pseudostratified columnar epithelium. Representative HE staining of (a) three weeks old red fox WD-AECs and (b) primary tracheal red fox tissue isolated from afreshly obtained, hunted red fox (Vulpes vulpes) Scale bar: 10 μm; 60x magnification.

Fig. 3

Differentiated primary red fox well-differentiated airway epithelial cells (left) in comparison to fox primary tracheal tissue (right). (a) Immunohistochemical staining of pancytokeratin; (b) Immunohistochemical staining of tight junction (ZO-1); (c) Immunohistochemical staining of the ciliary brush border (β-tubulin); (d) Periodic acid Schiff staining of mucus secreting goblet cells. White arrowhead: mucus-secreting goblet cell. Scale bar: 10 μm; 60x magnification.

Fig. 4

Expression levels of TNF and IL-33 mRNAs upon stimulation of fox ALI cultures with selected agonists. LPS lipopolysaccharide from E. coli; PMA phorbol-12-myristat-13-acetat; Iono ionomycin, nematode somatic antigen:Angiostrongylus vasorum first-stage larval full somatic antigen. Data from three individual donor foxes.