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bioRxiv · 10.1101/2022.11.29.518315

Characterisation of the functional and transcriptomic effects of pro-inflammatory cytokines on human EndoC- βH5 beta cells

Frorup, C.·Gerwig, R.·Sondergaard Svane, C. A.·Mendes Lopes de Melo, J.·Floyel, T.·Pociot, F.·Kaur, S.·Storling, J.
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Abstract

ObjectiveEndoC-{beta}H5 is a newly established human beta-cell model which may be superior to previous models of native human beta cells. Exposure of beta cells to proinflammatory cytokines is a widely used in vitro model of immune-mediated beta-cell failure in type 1 diabetes and we therefore performed an in-depth characterisation of the effects of cytokines on EndoC-{beta}H5 cells. MethodsThe sensitivity profile of EndoC-{beta}H5 cells to the toxic effects of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), interferon {gamma} (IFN{gamma}) and tumour necrosis factor- (TNF) was examined in titration and time-course experiments. Cell death was evaluated by caspase 3/7 activity, cytotoxicity, viability, TUNEL assay and immunoblotting. Mitochondrial function was evaluated by extracellular flux technology. Activation of signalling pathways and major histocompatibility complex (MHC) class I expression were examined by immunoblotting, immunofluorescence, and real-time quantitative PCR (qPCR). Glucose-stimulated insulin secretion (GSIS) and cytokine-induced chemokine secretion were measured by ELISA and Meso Scale Discovery multiplexing electrochemiluminescence, respectively. Global gene expression was characterised by stranded RNA sequencing. ResultsCytokines increased caspase activity and cytotoxicity in EndoC-{beta}H5 cells in a time- and dose-dependent manner. The proapoptotic effect of cytokines was primarily driven by IFN{gamma}. Cytokine exposure caused impaired mitochondrial function, diminished GSIS, and induced secretion of chemokines. At the signalling level, cytokines increased the phosphorylation of signal transducer and activator of transcription 1 (STAT1) but not c-jun N-terminal kinase (JNK) and did not cause degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (I{kappa}B). MHC class I was induced by cytokines. Cytokine exposure caused significant changes to the EndoC-{beta}H5 transcriptome including upregulation of HLA genes, endoplasmic reticulum stress markers, and non-coding RNAs. Among the differentially expressed genes were several type 1 diabetes risk genes. ConclusionsOur study provides detailed insight into the functional and transcriptomic effects of cytokines on EndoC-{beta}H5 cells. This knowledge will be helpful for future investigations studying cytokine effects in this cell model.

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Frorup, C., Gerwig, R., Sondergaard Svane, C. A., Mendes Lopes de Melo, J., Floyel, T., Pociot, F., Kaur, S., Storling, J.. 2022-11-29. Characterisation of the functional and transcriptomic effects of pro-inflammatory cytokines on human EndoC- βH5 beta cells. https://doi.org/10.1101/2022.11.29.518315

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