Endocrine Abstracts

Aim: IFNα is a key regulator of the initial dialogue between pancreatic β-cells and the immune system in type 1 diabetes (T1D). IFNα induces endoplasmic reticulum (ER) stress, insulitis and a massive HLA-ABC overexpression in human β-cells, three histological hallmarks of T1D. Against this background we investigated the global role of IFNα on iPSC-derived islet-like cells, used here to mimic islet cells in the early neonatal period when autoimmunity against β-cells starts in many patients.

Methods: Human iPSC were differentiated to islet-like cells following a 7 stage protocol and then treated with 2000 U/mL of IFNα for 24h (dose and timing selected based on time-course and dose-response studies). Bulk and single-cell (sc) RNA-seq were performed. In follow up experiments, dispersed islet-like cells were transfected with a siRNA control or a siRNA targeting NLRC5 and then exposed or not to IFNα for 24h. Gene expression levels were measured by qPCR. NLRC5 protein expression was assessed by western blot. The HLA-ABC expression at the β- cell surface was measured by flow cytometry.

Results: At the end of the differentiation period (stage 7) there were around 50% insulin and 10% glucagon positive cells. Exposure to IFNα induced a predominance of upregulated genes (761 upregulated vs 302 downregulated) as indicated by the bulk RNA-seq data. The upregulated pathways identified were antigen processing and presentation, JAK-STAT signaling and antiviral responses. scRNA-seq of the iPSC-derived islet-like cells identified β- and α-like cells based on their characteristic gene expression. β-like cells exposed to IFNα had higher expression of the ER stress markers CHOP and XBP1, while α-like cells showed higher expression of the protective chaperone BiP, of the anti-apoptotic family member BCL2L1 (Bcl-XL) and of the viral sensor MDA5. However, the expression of HLA-ABC was similar in α-like cells exposed to IFNα as compared to β-like cells, except for the protective HLA- E, which was higher in α-like cells. The transcriptional activator NLRC5 was up-regulated after IFNα treatment in β- and α-like cells and silencing of NLRC5 in iPSC-derived islet-like cells decreased HLA-ABC (gene expression and protein expression at the surface), as well as genes related with antigen presentation such as TAP1 and B2M.

Conclusions: IFNα induces a different response in β- and α-like cells. β-like cells have a more marked expression of ER stress- related genes while α-like cells have higher expression of anti-apoptotic genes, protective chaperones and antiviral mechanisms. These observations suggest that α-like cells can better endure viral infections and ER stress, which could improve their survival in the context to T1D when compared to β-like cells. On the other hand, IFNα induces similar upregulation of HLA-ABC on β- and α-like cells, downstream of the transcriptional regulator NLRC5.