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Macrophage Response to Interleukin-1 Stimulation and the Nature of Cell Surface Interleukin-1 Alpha 10.17863/CAM.58099 Chan, Nuo En 0000000280432190 Clarke, Murray 0000000282158885 Interleukin-1α (IL-1α) is a potent cytokine involved in a range of diseases and can be found expressed on the cell surface (csIL-1α) and in the soluble form, signalling through the type I IL-1 receptor (IL-1R1). IL-1 signalling leads to increased secretion of cytokines, recruitment of immune cells thus perpetuating a highly inflammatory environment. It has been the understanding for years that IL-1R1 is present on many cell types including macrophages and monocytes. We report that there can be false positive staining of target proteins if a permeability stain is not introduced. Thus, we show that when gated on intact cells, there is no IL-1R1 expressed on macrophages or dendritic cells, and when IL-1R1 is overexpressed, IL-1 signalling can occur. We also suggest that the mere presence of IL-1R1 is not sufficient to lead to IL-1 signalling, but that there is a minimum threshold of IL-1R1 expression for that to occur, illustrating a novel checkpoint mechanism to prevent aberrant IL-1 signalling. In the second part of this project, we investigated the nature of csIL-1α, reported to be crucial for the senescence-associated secretory phenotype (SASP). We show that pro-IL-1α is synthesised de novo and trafficked to the membrane following TLR4 stimulation. We report two novel ways by which csIL-1α can be bound, through the type II IL-1 receptor (IL-1R2) and glycosylphosphatidylinositol (GPI) anchors. IL-1R2 is responsible for majority of the csIL-1α binding to the cell membrane with a small portion of csIL-1α being tethered to a GPI- associated protein. We report that csIL-1α is significantly downregulated by IFNγ treatment but intracellular and secreted IL-1α levels are not affected by this. In conclusion, this thesis has contributed to a better understanding of IL-1 signalling through IL-1R1 and its regulation. It has also enhanced the understanding of csIL-1α binding and mechanisms that affect its expression. University of Cambridge 2020-02-01 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/311008 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/e4025663-65a2-411b-aad3-e0648b87dcac/download c1baad5d86dff07f9402e923424048ec https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2c5dda4e-5e90-41f8-bf1c-e1183174051e/download 353adac0d1ebdfd65ab16480263c3c87 https://www.rioxx.net/licenses/all-rights-reserved/ Interleukin-1 cell surface Interleukin-1 alpha