Equine bronchial fibroblasts enhance proliferation and differentiation of primary equine bronchial epithelial cells co-cultured under air-liquid interface.

Vanessa Abs,Johannes Kacza,Getu Abraham,Jana Bonicelli,Claudia Zizzadoro,Yohannes Tesfaigzi

doi:10.1371/journal.pone.0225025

Abstract

Interaction between epithelial cells and fibroblasts play a key role in wound repair and remodelling in the asthmatic airway epithelium. We present the establishment of a co-culture model using primary equine bronchial epithelial cells (EBECs) and equine bronchial fibroblasts (EBFs). EBFs at passage between 4 and 8 were seeded on the bottom of 24-well plates and treated with mitomycin C at 80% confluency. Then, freshly isolated (P0) or passaged (P1) EBECs were seeded on the upper surface of membrane inserts that had been placed inside the EBF-containing well plates and grown first under liquid-liquid interface (LLI) then under air-liquid interface (ALI) conditions to induce epithelial differentiation. Morphological, structural and functional markers were monitored in co-cultured P0 and P1 EBEC monolayers by phase-contrast microscopy, scanning and transmission electron microscopy, hematoxylin-eosin, immunocytochemistry as well as by measuring the transepithelial electrical resistance (TEER) and transepithelial transport of selected drugs. After about 15–20 days of co-culture at ALI, P0 and P1 EBEC monolayers showed pseudo-stratified architecture, presence of ciliated cells, typically honeycomb-like pattern of tight junction protein 1 (TJP1) expression, and intact selective barrier functions. Interestingly, some notable differences were observed in the behaviour of co-cultured EBECs (adhesion to culture support, growth rate, differentiation rate) as compared to our previously described EBEC mono-culture system, suggesting that cross-talk between epithelial cells and fibroblasts actually takes place in our current co-culture setup through paracrine signalling. The EBEC-EBF co-culture model described herein will offer the opportunity to investigate epithelial-mesenchymal cell interactions and underlying disease mechanisms in the equine airways, thereby leading to a better understanding of their relevance to pathophysiology and treatment of equine and human asthma.

Highlights

Within the pulmonary tissues of all mammalian species including horses, the epithelial and the striking distance to mesenchymal layers of the airway bronchioles provide a physiologically balanced cellular environment and conduct important interactions with the external environment [1]
Electron microscopic examination of P0 and passage 1 (P1) equine bronchial epithelial cells (EBECs) monolayers revealed the presence of typically structured cilia and microvilli on the cell apical surface starting from day 7 of co-culture at air-liquid interface (ALI) (Fig 2A and 2B), with the proportion of ciliated cells increasing with increasing days of ALI (Fig 2B)
In “mature” P0 and P1 EBEC monolayers, after about 15–20 days of co-culture at ALI, histological and histochemical examination revealed the formation of pseudo-stratified columnar epithelium, consisting of a mixed population of basal and differentiated cells, the latter including both pseudo-stratified and ciliated (Fig 2E)

Summary

Introduction

Within the pulmonary tissues of all mammalian species including horses, the epithelial and the striking distance to mesenchymal layers of the airway bronchioles provide a physiologically balanced cellular environment and conduct important interactions with the external environment [1]. The underlying connective tissue with mesenchymal cells, i.e., fibroblasts, modulates the airway function through extracellular matrix (ECM) deposition and secretion of soluble factors participating in airway inflammation and remodelling [5,6]. With this background in mind, epithelial cells can regulate fibroblast proliferation and attraction [7], and fibroblasts can regulate epithelial cell proliferation, differentiation and wound healing [8,9,10,11]. Recent investigations suggest that during inflammatory airway disorders extensive injury of the airway epithelium might result in shedding of damaged epithelial cells in the airway lumen but with parallel activation of the remaining surviving epithelial cells and of the underlying fibroblasts in the airways [13,14,15]

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