| dc.description.abstract | Lipid depletion due to genetic or environmental factors can impair epidermal integrity. Acylceramides, part of the most abundant lipid fraction in the skin, make up 12% of all epidermal ceramides and link the corneocytes’ outer cornified lipid envelope to the lipid lamellae. They are responsible for maintaining skin moisture and barrier integrity by providing scaffolding for lamellae assembly and preventing hygroscopic leakage. Despite the importance of lipids in skin pathogenesis, there are limited experimental models for studying disease mechanisms highlighting a need for more precise and tuneable in vitro models. Such models of dysregulation could allow us to controllably deplete lipids to provide insights for studying repair processes and testing therapeutics. This thesis describes the development of a novel 3D human skin equivalent (HSE), with tuneable disruption of ceramide synthesis to model lipid deficiency-induced epidermal barrier damage. A genetically engineered N/TERT keratinocyte cell line with inducible knockdown of the ceramide synthase 3 enzyme (CERS3), responsible for the synthesis of the skin-specific acylceramides, was created to model their role in barrier composition, function and repair. The N/TERTs were transduced with either non-targeting control (NTC) or two different CERS3 targeting shRNA sequences. Doxycycline-induced knockdown resulted in significant reductions on the gene and protein levels. These lines were then seeded on decellularised ECM hydrogels to make the HSEs. Histology and immunofluorescence confirmed that all HSE models reproducibly generated well-stratified and terminally differentiated cornified layers. Whilst the knockdown did not affect expression of terminal differentiation proteins, lipid composition was altered across several lipid species. Nile Red staining revealed an overall reduction in the polar lipid content and lipidomics showed significant depletions in several lipid classes including the most abundant acylceramide Cer[EOS] and a shift in abundance from long-chain to shorter chain ceramides. Additionally, in recovery experiments, whilst the protein level was restored there was latency in lipid recovery. In collaboration with the industrial sponsors, the standard non-transduced HSE model successfully showed that upon exposure to an inflammatory cytokine environment akin to atopic dermatitis, epidermal barrier function was perturbed. Barrier function assessments using Transepidermal Water Loss (TEWL) and Transepithelial Electrical Resistance (TEER) showed a significantly dysfunctional epidermal phenotype, particularly from the IL-18 exposed models. | en_US |
