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

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.

What Have In Vitro Co-Culture Models Taught Us about the Contribution of Epithelial-Mesenchymal Interactions to Airway Inflammation and Remodeling in Asthma?

“…The co-culture models used to assess cellular communication in the lung EMTU have included conditioned medium-exposure experiments, epithelial-mesenchymal co-cultures, and 3D-ECM (co-culture) models. These models have been established both with lung epithelial and mesenchymal cell lines [ 19 ] and primary cells isolated either from animal models [ 20 ] or human asthmatic and control lungs [ 21 ].…”

Immunologic and Non-Immunologic Mechanisms Leading to Airway Remodeling in Asthma

“…Fibroblasts promoted epithelial cell proliferation by stimulating the transforming growth factor (TGF)-β2 signaling pathway in severe asthma [80]. A similar stimulating effect of fibroblasts on bronchial epithelial cells was reported in horses [71]. In contrast, the secretion of Wnt-5a/b by mesenchymal cells repressed the function of alveolar epithelial progenitor cells, and may therefore indicate a novel strategy to control lung remodeling in chronic inflammatory diseases [81].…”

“…This interaction between the two cell layers of the airway wall was regarded as a major regulator of the homeostasis of the airway wall structure, and was described over two decades ago [69,70]. The importance of the EMTU for the maintenance and function of the airway came back into focus recently [71,72]. However, most studies that investigated the interaction between the epithelial cells and sub-epithelial mesenchymal cells focused on a one-way direction from epithelial cells to fibroblasts.…”

Bone Marrow Derived Mesenchymal Stromal Cells Promote Vascularization and Ciliation in Airway Mucosa Tri-Culture Models in Vitro

“…Most studies dealing with airway epithelial co-cultures use fibroblasts as supporting cell type (Hiemstra et al, 2019). Their ability to promote epithelial cell differentiation has been confirmed in several studies (Albers et al, 2015;Abs et al, 2019). In our first study that established the tri-culture system based on HNEC, we therefore used HNF as supporting cell type.…”