The role of airway epithelial derived extracellular vesicles in allergic sensitisation

Browne, William

The role of airway epithelial derived extracellular vesicles in allergic sensitisation

Publication Date

July 31, 2026

Creator

Browne, William

University of Nottingham

Abstract

The prevalence of allergies has drastically increased over the past 50 years, with an estimated 30% of adults sensitised to an environmental allergen. While this represents a growing public health concern the mechanisms which underpin allergic sensitisation are poorly understood. Recently, it has been suggested that the airway epithelium can influence allergic outcome through the secretion of extracellular vesicle (EVs). These are small lipid bound vesicles, capable of extracellular communication through the simultaneous delivery of different categories of biomolecules to a recipient cell. While EVs provide an exciting new avenue for better understanding the role of the airway epithelium in allergic sensitisation, the existing research is limited and primarily in patient samples and 2D cultures. Consequently, this research aimed to develop and optimise a 3D culture model and mechanism for its stimulation with the indoor allergens Der p 1 and Can f 1, as well as the novel profiling of EV responses with imaging flow cytometry. A systematic review of the literature regarding the role of epithelial-derived EVs in allergic sensitisation was conducted, showing that epithelial-derived EVs can both promote tolerance or allergic sensitisation through the delivery of mRNAs that promote Th2 polarisation and cytokine secretion or promote tolerance through the induction of Tregs (miR34a, miR92b, miR146a-5p and miR210). Additionally, this review highlighted a significant gap in the literature, with only one study using a 3D culture model. Initially, different transformed airway epithelial cell lines were assessed in 2D to identify a suitable candidate for a 3D culture model. Calu-3 cells were selected due to their high expression of key epithelial markers which will be used for the downstream profiling of EVs, as well as their ability to differentiate and form tight junctions when cultured at air liquid interface (ALI). Stimulation of ALI cultures was optimised by assessing submerged exposure, the addition of stimulant to the basal chamber and nebulised exposure. In parallel, a high throughput method for the analysis of EVs using imaging flow cytometry was developed and optimised, allowing for quantification and profiling of markers on EVs. Ultimately, Calu-3 were cultured at ALI and exposed to either nebulised proteolytically active Der p 1, inactive Der p 1 or Can f 1, for 24 and 48 hours. The effects of exposure on epithelial tissues were assessed using confocal microscopy and tight junction staining, as well as scanning electron microscopy. Additionally, alarmin and inflammatory cytokine responses were profiled using ELISA and EV responses were profiled using imaging flow cytometry. Nebulised exposure of active Der p 1 was shown to disrupt epithelial barrier function by reducing tight junction expression. Furthermore, Der p 1 and Can f 1 exposure was shown to induce an increase in basal EV secretion. Additionally, Der p 1 exposure was observed to significantly alter the distribution of tetraspanins CD9, CD63 and CD81, as well as upregulate the expression of the epithelial marker E-Cadherin on EVs. Finally, transcriptomic analysis revealed the upregulation of previously identified miRNA, miR92b, as well as miR101-3p, mir103-3p, miR107, miR148a and miR224a-5p, which could play an important role in early development of Th2 responses. Taken together, the data presented here shows that allergen exposure of epithelial cells can modulate EV responses, increasing secretion of EVs and altering tetraspanin distribution, as well as upregulating miRNA cargos which can promote or prevent sensitisation.

Item Type

ethesis

Thesis Type

PhD

Supervisors

Fairclough, Lucy

University of Nottingham

James, Victoria

University of Nottingham

Onion, David

University of Nottingham

Cochrane, Stella