Title: Characterisation of epithelial cells from small airways in asthma
Funder: Asthma UK

Abstract: The airway epithelium is the critical interface between airborne particles and airway tissue. The airway epithelial barrier is especially important in asthma as multiple airborne triggers including viruses, allergens and fungi can activate an abnormal inflammatory response. Due to limited sampling techniques little is currently known about the airway epithelium in the small airways in asthma, and whether this differs from the large airways. This novel proposal uses a new small bronchial brush technique to directly address the hypothesis that the epithelial barrier of the small airways is altered in asthma versus control subjects. It will also assess whether the airway epithelial barrier differs between the large and small airways; this is more pertinent following the introduction of small particle steroids. We intend to both validate the small brush approach to isolate small airway epithelial cells from well phenotyped patients with asthma and then determine the capacity of asthma and control small and large airway epithelial cells to develop a differentiated barrier representative of in vivo. Acutely dissociated cells will be obtained from patients with asthma and controls using both small and large brush techniques. Cells will then be prepared for morphological characterisation including differential cell counts and epithelial cell markers; CK14 (basal cell marker), CC26 (Clara cell specific marker), CCSP (Clara cell secretary proteins) using immunofluorescence. We will then assess the differentiation potential by monitoring the development of transepithelial resistance during the air liquid interface differentiation period. Once differentiated we will extensively characterise the differentiated layers with respect to cellular composition by assessing the proportion of goblet cells and ciliated cells using MUC5AC and alpha tubulin immunofluorescence respectively. We will then quantify tight junction proteins claudins 1 and 4, occludin and ZO-1 using a combination of qPCR, Western blotting and confocal imaging. This novel pilot work may allow development of new treatments that prevent or suppress the airway inflammatory response by modifying epithelial barrier function and could help development of small airway epithelial models for pre-clinical drug discovery. This research will directly address two of the questions raised in the Basic Research Asthma Strategy 2, namely: a) How can we use our existing knowledge of mechanisms to prevent or treat asthma and how can we increase the knowledge base to develop novel therapies? b) How are the inflammatory mechanisms in severe asthma different from those in mild asthma and how do these mechanisms relate to asthma symptoms and why are these mechanisms less sensitive to corticosteroids in people with severe asthma? This work builds upon other epithelial projects being performed by Drs Shaw and Sayers, and if we succeed in culturing small airway cells using the small brush technique we will write an MRC grant to further explore the role of the distal epithelial barrier with reference to asthma precipitants such as viruses and bacteria with the focus on identifying targetable cell markers to prevent and restore epithelial damage.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RESPIRATORY