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

Caroline Frørup; Rebekka Gerwig; Cecilie Amalie Søndergaard Svane; Joana Mendes Lopes de Melo; Kristine Henriksen; Tina Fløyel; Flemming Pociot; Simranjeet Kaur; Joachim Størling

doi:10.3389/fendo.2023.1128523

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

Front Endocrinol (Lausanne). 2023 Apr 11:14:1128523. doi: 10.3389/fendo.2023.1128523. eCollection 2023.

Affiliations

¹ Translational Type 1 Diabetes Research, Clinical Research, Steno Diabetes Center Copenhagen, Herlev, Denmark.
² Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
³ Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.

Abstract

Objective: EndoC-βH5 is a newly established human beta-cell model which may be superior to previous model systems. Exposure of beta cells to pro-inflammatory cytokines is widely used when studying immune-mediated beta-cell failure in type 1 diabetes. We therefore performed an in-depth characterization of the effects of cytokines on EndoC-βH5 cells.

Methods: The sensitivity profile of EndoC-βH5 cells to the toxic effects of interleukin-1β (IL-1β), interferon γ (IFNγ) and tumor necrosis factor-α (TNFα) was examined in titration and time-course experiments. Cell death was evaluated by caspase-3/7 activity, cytotoxicity, viability, TUNEL assay and immunoblotting. Activation of signaling pathways and major histocompatibility complex (MHC)-I expression were examined by immunoblotting, immunofluorescence, and real-time quantitative PCR (qPCR). Insulin and chemokine secretion were measured by ELISA and Meso Scale Discovery multiplexing electrochemiluminescence, respectively. Mitochondrial function was evaluated by extracellular flux technology. Global gene expression was characterized by stranded RNA sequencing.

Results: Cytokines increased caspase-3/7 activity and cytotoxicity in EndoC-βH5 cells in a time- and dose-dependent manner. The proapoptotic effect of cytokines was primarily driven by IFNγ signal transduction. Cytokine exposure induced MHC-I expression and chemokine production and secretion. Further, cytokines caused impaired mitochondrial function and diminished glucose-stimulated insulin secretion. Finally, we report significant changes to the EndoC-βH5 transcriptome including upregulation of the human leukocyte antigen (HLA) genes, endoplasmic reticulum stress markers, and non-coding RNAs, in response to cytokines. Among the differentially expressed genes were several type 1 diabetes risk genes.

Conclusion: Our study provides detailed insight into the functional and transcriptomic effects of cytokines on EndoC-βH5 cells. This information should be useful for future studies using this novel beta-cell model.

Keywords: RNA-Seq; apoptosis; inflammation; insulin; model system; pancreatic beta cells; signaling; type 1 diabetes.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Caspase 3 / genetics
Chemokines
Cytokines*
Diabetes Mellitus, Type 1*
Humans
Interferon-gamma / pharmacology
Transcriptome

Substances

Cytokines
Caspase 3
Interferon-gamma
Chemokines

Grants and funding

This research was supported by Skibsreder Per Henriksens, R. og Hustrus Fond. The study funder was not involved in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.