Abstract
Inflammasomes comprise a group of protein complexes, which activate the protease caspase-1 upon sensing a variety of stress factors. Active caspase-1 in turn cleaves and thereby activates the pro-inflammatory cytokines prointerleukin (IL)-1β and -18, and induces unconventional protein secretion (UPS) of mature IL-1β, IL-18, as well as of many other proteins involved in and required for induction of inflammation. Human primary keratinocytes (HPKs) represent epithelial cells able to activate caspase-1 in an inflammasome-dependent manner upon irradiation with a physiological dose of ultraviolet B (UVB) light. Here, we describe the isolation of keratinocytes from human skin, their cultivation, and induction of caspase-1-dependent UPS upon UVB irradiation as well as its siRNA- and chemical-mediated inhibition. In contrast to inflammasome activation of professional immune cells, UVB-irradiated HPKs represent a robust and physiological cell culture system for the analysis of UPS induced by active caspase-1.
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Acknowledgements
Our work was supported by the Swiss National Science Foundation (SNF 31003A_132450), the Swiss Cancer League, the European Science Foundation (EuroMEMBRANE), the OPO Stiftung, the Center for Clinical Research, University Hospital Zurich and University of Zurich, the Helmut Horten-Stiftung, the Wilhelm Sander-Stiftung and the Novartis Foundation. M.G., J.S., and S.G. are members of the Zurich graduate program in Molecular Life Sciences. We would like to thank Prof. D. Hohl (CHUV, Lausanne) for his help with the establishment of HPK isolation and propagation, and Prof. S. Werner (ETH Zurich) and Prof. L. E. French (University Hospital Zurich) for continuous support.
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Strittmatter, G.E., Garstkiewicz, M., Sand, J., Grossi, S., Beer, HD. (2016). Human Primary Keratinocytes as a Tool for the Analysis of Caspase-1-Dependent Unconventional Protein Secretion. In: Pompa, A., De Marchis, F. (eds) Unconventional Protein Secretion. Methods in Molecular Biology, vol 1459. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3804-9_9
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DOI: https://doi.org/10.1007/978-1-4939-3804-9_9
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