Pro-inflammatory cytokines induce cell death, inflammatory responses, and endoplasmic reticulum stress in human iPSC-derived beta cells.

ABSTRACT:

Background

Adult human pancreatic beta cells are the "gold standard" for studies on diabetes pathogenesis, but their use is limited by insufficient availability and variable quality. An important effort has recently taken place to differentiate beta cells from human induced pluripotent stem cells (iPSCs) and validate their use for diabetes research. We presently used a 7-stage protocol to generate beta cells from human iPSC and evaluated whether these cells are responsive to the pro-inflammatory cytokines (IFN?, IL-1?, or IFN?) that play a role in type 1 diabetes.

Methods

The iPSC-derived islet-like cell clusters contained 40-50% beta and 10-15% alpha cells and expressed the receptors for IFN?, IL-1?, or IFN?. Cells were exposed to either IFN? (1000?U/mL)?+?IL-1? (50?U/mL) or IFN? alone (2000?U/mL) for 24/48?h. Apoptosis was quantified using Hoechst/propidium iodide staining or the RealTime Glo Apoptosis Kit (Promega). After treatment, CXCL10 secretion was quantified by ELISA. The expression of multiples genes (Ins, Gcg, Nkx2.2, Nkx6.1, Pdx1, Mafa, BiP, Chop, Atf3, CXCL10, CXCL9, CCL5, and HLA-ABC) was quantified by RT-qPCR. Phosphorylation state and total expression of STAT1/STAT2, as well as expression of PDL1 and of the ER chaperone BiP, were quantified by Western blotting. The co-localization of HLA-ABC or cleaved caspase-3 and Ins/Gcg expression was assessed by immunohistochemistry. The presence of HLA-ABC at the plasma membrane was measured by flow cytometry.

Results

IFN? + IL-1? and IFN? induced apoptosis of the cells after 48?h of exposure. Cleaved caspase-3 co-localized mostly but not exclusively with Ins+ cells. Exposure to IFN? + IL-1? induced a pro-inflammatory phenotype, including increased CXCL10, CXCL9, and CCL5 expression; CXCL10 secretion; and HLA-ABC expression. HLA overexpression was confirmed at the protein level by Western blotting and flow cytometry. Exposure to IFN? + IL-1? (but not IFN?) also induced beta cell dedifferentiation and endoplasmic reticulum stress (increase in BiP, Chop, and Atf3 mRNA expression). Phosphorylation of STAT1 was stimulated already after 1?h by IFN? + IL-1? and IFN?, while phosphorylation of STAT2 was only activated by IFN? at 1-4 h. PDL1 expression was increased by both IFN? + IL-1? and IFN?.

Conclusions

Our data show that human iPSC-derived beta cells respond to pro-inflammatory cytokines IL-1? + IFN? and IFN?, by activating the same pathogenic processes as adult human primary beta cells. These cells thus represent a valuable tool for future research on the pathogenesis of type 1 diabetes.

SUBMITTER: Demine S 

PROVIDER: S-EPMC6942385 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature