Identification of the molecular pathways that mediate cytokine induced immunopathology of the gut epithelium

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Saini Rokade, Nisha
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University College Cork
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Although the phenomenon of synergistic cell death induced by the combination of IFN-γ and TNF-α has been known for more than 30 years, the underlying mechanisms are still not clear. The main aim of my PhD was to identify the mechanisms which are required for the killing of human intestinal epithelial cells (Chapter-I) by this cytokine combination. A human kinome RNAi screen and a drug repurposing screen identified the Janus kinases (JAKs) JAK1 and JAK2 as the major kinases required for IFN-γ + TNF-α dependent synergistic cell death. While TNF receptor-1 (TNFR1) was also required, there was no direct dependency of cell death on TNFR1-downstream canonical apoptotic and necroptotic signalling pathways. Furthermore, we identified a non-canonical role for caspase-8 and receptor-interacting serine/threonine-protein kinase 3 (RIPK3) in the cell death signalling pathway (Chapter-II). For the first time we report that IFN-γ and IFN-γ + TNF-α induced JAK1/2-STAT1 signalling followed a dose-to-duration pattern of activation whereby increasing concentration of cytokines resulted in sustained JAK-STAT signalling leading to epithelial cell death. This dose-to-duration signalling pattern allowed cells to respond appropriately to increasing levels of inflammatory cytokines. RNA-sequencing revealed that sustained activation of the JAK1/2-STAT1 pathway resulted in the expression of a unique set of ‘late’ genes associated with different cell death pathways (Chapter-III). Phosphoproteomics identified potential target substrates for sustained JAK1 and JAK2 kinase activity. These JAK1/2 substrate proteins are associated with epithelial junctions, cell adhesion and cell-cell interactions, highlighting the importance of JAK1/2-STAT1 signalling in regulating the integrity of the epithelial barrier. Overall, our results identify the central underlying mechanisms underpinning IFN-γ + TNF-α induced cell death of intestinal epithelial cells and disruption of the intestinal epithelial barrier during intestinal inflammatory diseases.
Interferon gamma , Tumour necrosis factor alpha , Cytokines , Synergism , Cell death , Signalling , Dose-to-duration , Janus kinase , Organoids , RNA sequencing
Saini Rokade, N. 2020. Identification of the molecular pathways that mediate cytokine induced immunopathology of the gut epithelium. PhD Thesis, University College Cork.