416 Pyroptosis of keratinocyte in Stevens-Johnson syndrome / toxic epidermal necrolysis

Abstract

Stevens Johnson syndrome (SJS)/ toxic epidermal necrolysis (TEN) are characterized by the remarkable cell death of keratinocytes. Our previous studies have revealed that keratinocyte death pathway involve apoptosis and necroptosis. However, in recent years, it has been found that there is a crosstalk between apoptosis, necroptosis and pyroptosis. Pyroptosis is an inflammatory programmed cell death characterized by caspase 1-mediated cleavage of GSDMD and the release of IL-1b and IL-18. The aim of our study is to reveal whether pyroptosis is associated with the pathogenesis of SJS/TEN and develop specific therapeutic agents that can suppress these cell death programs in SJS/TEN. Expression of cleaved GSDMD and cleaved caspase1, which were key players of pyroptosis, were observed in the keratinocytes of SJS/TEN patients by immunofluorescent staining. To prove that pyroptosis can occur in keratinocytes, we stimulated HaCaT cells with pyroptosis inducers, LPS and nigertin, and evaluated pyroptosis reaction by live/dead staining assay, observation of morphological changes and cleaved GSDMD expression. HaCaT cells after LPS and nigertin stimulation formed large bubbles and a punctate positive reaction of cleaved GSDMD on the cell membrane, occurring as a result of membrane pore-forming activity of the gasdermin-N domain. Pyroptosis occurred even after stimulating HaCaT cells with SJS serum. Our study showed that pyroptosis occurred in keratinocytes of SJS/TEN patients and substances that induce pyroptosis to keratinocyte were present in the serum of the patients with SJS/TEN. We have investigated SJS/TEN-specifically elevated proteins by selected reaction monitoring. We are currently exploring the substances that induce pyroptosis. It is expected that understanding the mechanisms of keratinocyte death in SJS/TEN lead us to develop a specific therapeutic drug that can suppress cell death in SJS/TEN.