No abstract

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.

Type 1 diabetes is an immune-driven disease, where the insulin-producing beta cells from the pancreatic islets of Langerhans becomes target of immune-mediated destruction. Several studies have highlighted the implication of circulating and exosomal microRNAs (miRNAs) in type 1 diabetes, underlining its biomarker value and novel therapeutic potential. Recently, we discovered that exosome-enriched extracellular vesicles carry altered levels of both known and novel miRNAs in breast milk from lactating mothers with type 1 diabetes. In this study, we aimed to characterize exosomal miRNAs in the circulation of lactating mothers with and without type 1 diabetes, hypothesizing that differences in type 1 diabetes risk in offspring from these groups are reflected in the circulating miRNA profile. We performed small RNA sequencing on exosome-enriched extracellular vesicles extracted from plasma of 52 lactating mothers around 5 weeks postpartum (26 with type 1 diabetes and 26 age-matched controls), and found a total of 2,289 miRNAs in vesicles from type 1 diabetes and control libraries. Of these, 176 were differentially expressed in plasma from mothers with type 1 diabetes (167 upregulated; 9 downregulated, using a cut-off of abs(log2FC) >1 and FDR adjusted p-value <0.05). Extracellular vesicles were verified by nanoparticle tracking analysis, transmission electron microscopy and immunoblotting. Five candidate miRNAs were selected based on their involvement in diabetes and immune modulation/beta-cell functions: hsa-miR-127-3p, hsa-miR-146a-5p, hsa-miR-26a-5p, hsa-miR-24-3p and hsa-miR-30d-5p. Real-time qPCR validation confirmed that hsa-miR-146a-5p, hsa-miR-26a-5p, hsa-miR-24-3p, and hsa-miR-30d-5p were significantly upregulated in lactating mothers with type 1 diabetes as compared to lactating healthy mothers. To determine possible target genes and affected pathways of the 5 miRNA candidates, computational network-based analyses were carried out with TargetScan, mirTarBase, QIAGEN Ingenuity Pathway Analysis and PantherDB database. The candidates showed significant association with inflammatory response and cytokine and chemokine mediated signaling pathways. With this study, we detect aberrant levels of miRNAs within plasma extracellular vesicles from lactating mothers with type 1 diabetes during the postpartum period, including miRNAs with associations to disease pathogenesis and inflammatory responses.

No abstract

The single nucleotide polymorphism rs7804356 located in the Src kinase-associated phosphoprotein 2 ( SKAP2 ) gene is associated with type 1 diabetes (T1D), suggesting SKAP2 as a causal candidate gene. The objective of the study was to investigate if SKAP2 has a functional role in the β-cells in relation to T1D. In a cohort of children with newly diagnosed T1D, rs7804356 predicted glycemic control and residual β-cell function during the 1st year after diagnosis. In INS-1E cells and rat and human islets, proinflammatory cytokines reduced the content of SKAP2. Functional studies revealed that knockdown of SKAP2 aggravated cytokine-induced apoptosis in INS-1E cells and primary rat β-cells, suggesting an antiapoptotic function of SKAP2. In support of this, overexpression of SKAP2 afforded protection against cytokine-induced apoptosis, which correlated with reduced nuclear content of S536-phosphorylated nuclear factor-κB (NF-κB) subunit p65, lower nitric oxide production, and diminished CHOP expression indicative of decreased endoplasmic reticulum stress. Knockdown of CHOP partially counteracted the increase in cytokine-induced apoptosis caused by SKAP2 knockdown. In conclusion, our results suggest that SKAP2 controls β-cell sensitivity to cytokines possibly by affecting the NF-κB–inducible nitric oxide synthase–endoplasmic reticulum stress pathway.

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.

ObjectiveEndoC-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.MethodsThe 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.ResultsCytokines 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.ConclusionOur 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.

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.