AN IN VITRO IMMUNOCOMPETENT HUMAN TISSUE-ENGINEERED MODEL OF ATOPIC DERMATITIS FOR DRUG TESTING

Jun 29, 2022, 2:00 PM
10m
Room: S1

Room: S1

Speaker

Barragan Vazquez, Inmaculada (School of Clinical Dentistry, University of Sheffield )

Description

Introduction: Atopic dermatitis (AD) is a chronic inflammatory and common skin disorder that is frequently associated with other atopic diseases such as allergic rhinitis and asthma. The immunopathogenesis of AD is a complex process, as both innate and adaptive immune systems are involved in the development of eczema in those patients. Keratinocytes, mast cells, dendritic cells, and T cells, among others, are involved in skin inflammation, however, in AD, there is a tendency towards T-helper 2 (Th2) responses. There is not a complete cure for AD, and most of the current treatments focus on symptom relief. Therefore, new treatments to improve the long-term control of AD are necessary. Murine models have been developed to mimic aspects of AD pathophysiology; however, they usually show markedly different responses to drug treatments that may lead to high failure rate of drug development.
Methodology: Purified peripheral blood human monocytes were differentiated into monocyte-derived dendritic cells (Mo-DC) using GM-CSF and IL-4. Purified naïve CD4+ T cells were CD3/CD28 activated, stimulated with IL-2 and polarised into Th2 cells using IL-4 and anti-IFNγ. Cells were characterised by qPCR and flow cytometry for key cell markers. To develop in vitro human skin models, type I collagen matrix populated with human dermal fibroblasts were seed on the apical surface with N/TERT immortalised human skin keratinocytes and Mo-DC, and cultured at an air-to-liquid interface before analysis. Th2 cells were incorporated into the dermal component.
Results: Mo-DC displayed successful differentiation by expression of cell-specific markers including CD1a, CD11c and CD207, and also high levels of proinflammatory markers such as IL-18 in response to allergens. Th2 cells showed increased CD119, D154, CD4 and CCR4, and secreted increased levels of IL-4, IL-5, IL-6, IL-13 and thymic stromal lymphopoietin (TSLP). Tissue-engineered skin models based on dermal fibroblasts and keratinocytes showed a keratinised, stratified squamous epidermis on top of a well-populated fibroblast containing dermis that histologically mimicked human skin. T cells were successfully incorporated into the dermis and Mo-DC into the epidermis, as determined by histological analysis.
Conclusion: Multiple immune cells can be differentiated from peripheral blood human monocytes and cultured in a 3D environment together with other human skin cells. The next aim is to show immune cell functionality within a 3D model in response to human allergens.

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