Emerging evidence suggests that several of the lysosomal cathepsin proteases are genetically associated with type 1 diabetes (T1D) and participate in immune-mediated destruction of the pancreatic β cells. We previously reported that the T1D candidate gene cathepsin H is downregulated by pro-inflammatory cytokines in human pancreatic islets and regulates β-cell function, apoptosis, and disease progression in children with new-onset T1D. In the present study, the objective was to investigate the expression patterns of all 15 known cathepsins in β-cell model systems and examine their role in the regulation of cytokine-induced apoptosis. Real-time qPCR screening of the cathepsins in human islets, 1.1B4 and INS-1E β-cell models identified several cathepsins that were expressed and regulated by pro-inflammatory cytokines. Using small interfering RNAs to knock down (KD) the cytokine-regulated cathepsins, we identified an anti-apoptotic function of cathepsin C as KD increased cytokine-induced apoptosis. KD of cathepsin C correlated with increased phosphorylation of JNK and p38 mitogen-activated protein kinases, and elevated chemokine CXCL10/IP-10 expression. This study suggests that cathepsin C is a modulator of β-cell survival, and that immune modulation of cathepsin expression in islets may contribute to immune-mediated β-cell destruction in T1D.

The type 1 diabetes (T1D) risk locus on chromosome 15q25.1 harbors the candidate gene CTSH (cathepsin H). We previously demonstrated that CTSH regulates β-cell function in vitro and in vivo. CTSH overexpression protected insulin-secreting INS-1 cells against cytokine-induced apoptosis. The purpose of the present study was to identify the genes through which CTSH mediates its protective effects. Microarray analysis identified 63 annotated genes differentially expressed between CTSH-overexpressing INS-1 cells and control cells treated with interleukin-1β and interferon-γ for up to 16h. Permutation test identified 10 significant genes across all time-points: Elmod1, Fam49a, Gas7, Gna15, Msrb3, Nox1, Ptgs1, Rac2, Scn7a and Ttn. Pathway analysis identified the "Inflammation mediated by chemokine and cytokine signaling pathway" with Gna15, Ptgs1 and Rac2 as significant. Knockdown of Rac2 abolished the protective effect of CTSH overexpression on cytokine-induced apoptosis, suggesting that the small GTPase and T1D candidate gene Rac2 contributes to the anti-apoptotic effect of CTSH.

Circular RNAs (circRNAs) have recently been implicated in impaired β-cell function in diabetes. Using microarray-based profiling of circRNAs in human EndoC-βH1 cells treated with pro-inflammatory cytokines, this study aimed to investigate the expression and possible regulatory roles of circRNAs in human β cells. We identified ~5000 β-cell-expressed circRNAs, of which 84 were differentially expressed (DE) after cytokine exposure. Pathway analysis of the host genes of the DE circRNAs revealed the enrichment of cytokine signaling pathways, indicative of circRNA transcription from inflammatory genes in response to cytokines. Multiple binding sites for β-cell-enriched microRNAs and RNA-binding proteins were observed for the highly upregulated circRNAs, supporting their function as ‘sponges’ or ‘decoys’. We also present evidence for circRNA sequence conservation in multiple species, the presence of cytokine-induced regulatory elements, and putative protein-coding potential for the DE circRNAs. This study highlights the complex regulatory potential of circRNAs, which may play a crucial role during immune-mediated β-cell destruction in type 1 diabetes.

Objective: EndoC-βH5 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-βH5 cells. Methods: The sensitivity profile of EndoC-βH5 cells to the toxic effects of the pro-inflammatory cytokines interleukin-1β (IL-1β), interferon γ (IFNγ) and tumour 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. 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. Results: Cytokines increased caspase activity and cytotoxicity in EndoC-βH5 cells in a time- and dose-dependent manner. The proapoptotic effect of cytokines was primarily driven by IFNγ. 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 α (IκBα). MHC class I was induced by cytokines. Cytokine exposure caused significant changes to the EndoC-βH5 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. Conclusions: Our study provides detailed insight into the functional and transcriptomic effects of cytokines on EndoC-βH5 cells. This knowledge will be helpful for future investigations studying cytokine effects in this cell model.