The Mechanisms of Heterogenic Necrotic and Apoptotic Cell Death Fates in Human Epidermal Keratinocytes Following UVB Exposure

UVB radiation is a carcinogen that triggers inflammation and contributes to skin carcinogenesis. Although UVB exposure can induce apoptosis, this form of cell death is non-inflammatory and thus cannot explain the inflammatory response observed following UVB. Necrotic regulated cell death (RCD) pathways, namely necroptosis, pyroptosis, and PANoptosis, are inherently pro-inflammatory, and thus this study focused on characterizing these in irradiated human epidermal keratinocytes. Keratinocytes were irradiated, treated with cell death inhibitors, and analyzed for apoptotic and necrotic RCD markers using immunofluorescence (IF). Despite uniform UVB exposure, keratinocytes displayed heterogeneous cell death responses, and these findings were validated in ex vivo human skin explants. Clonal analysis of keratinocytes from the HaCaT cell line revealed that clonal populations varied in their cell death fates, indicating that inflammatory cell death is not a random process, but is guided by intrinsic cellular factors unique to each clone. CRISPR-Cas9 gene editing generated MLKL knockouts verified with IF, western blot, and cell viability assays and found that cells are capable to switch inflammatory cell death pathways upon UVB exposure when necroptosis was inhibited. Co-localization and fluorescence intensity quantification demonstrated overlapping activation of apoptotic and necrotic pathways, including mixed PANoptotic phenotypes. Together, these findings support a model in which UVB-induced keratinocyte death is governed by a balance between immunologically silent and pro-inflammatory mechanisms. This research identifies insights into UVB-induced RCD heterogeneity and potential therapeutic targets for skin cancer chemoprevention.