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Granzyme B disrupts cell-cell adhesion and epithelial barrier function

University of British Columbia

The skin is comprised of multiple layers of keratinocytes which together form a barrier to the external environment, regulating temperature, water loss, and pathogen exposure. As such the skin barrier is vital for health as well as disease prevention. Disruption of the epithelial barrier can result in infection, allergen exposure, and inflammation, culminating into severe conditions. Many autoimmune conditions, such as pemphigus, involve a dysregulation and accumulation of immune cells, this results in a disruption in skin barrier causing a loss of function. Granzyme B (GzmB) is a serine protease that is expressed and secreted by a variety of immune and non-immune cells. It can accumulate in the extracellular milieu and retain its proteolytic functions resulting in chronic inflammation and impaired tissue repair due to extracellular matrix (ECM) remodeling. As such, I hypothesized that GzmB disrupts epithelial barrier function through the proteolytic cleavage of cell junction proteins. The present study investigated the impact of GzmB on epithelial barrier dysfunction using Electric Cell-substrate Impedance Sensing (ECIS) and western blot analyses of intercellular junction cleavage fragments. Human formalin fixed, paraffin embedded blistered skin tissue was assessed for the presence of GzmB. GzmB treatment resulted in a loss of E-cadherin staining on the cell membrane which was supported by western blot analysis of the cell supernatants. Additionally, we observed a dose-dependent increase in E-cadherin fragmentation in GzmB-treated cells compared to controls. HaCaT cells exhibited a significant decrease in barrier function when treated with GzmB while cells treated with GzmB in the presence of a specific GzmB inhibitor remained unaffected. While absent in normal skin, GzmB was observed in abundance within the intra-epidermal blister in addition to the surrounding epithelium. In summary, GzmB contributes to a decline in epithelial barrier function in part through the proteolytic cleavage of cell-cell junctions.

Text

2019-08-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/65537

Pathology and Laboratory Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/