Pro-inflammatory cytokines induce cell death, inflammatory responses, and endoplasmic reticulum stress in human iPSC-derived beta cells
Pro-inflammatory cytokines induce cell death, inflammatory responses, and endoplasmic reticulum stress in human iPSC-derived beta cells
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DOI：
10.1186/s13287-019-1523-3
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发表时间：
2020-01-03
影响因子：
7.5
通讯作者：
Eizirik, Decio L.
中科院分区：
文献类型：
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作者：
Demine, Stephane;Schiavo, Andrea Alex;Eizirik, Decio L.
背景成人胰腺β细胞是研究糖尿病发病机制的“金标准”，但其应用受到可用性不足和质量不一的限制。最近进行了一项重要的努力，从人类诱导多能干细胞（iPSC）中分化出β细胞，并验证其在糖尿病研究中的用途。我们目前使用7阶段方案从人iPSC产生β细胞，并评估这些细胞是否对促炎细胞因子有反应方法iPSC衍生的胰岛样细胞簇含有40-50%的β细胞和10-15%的α细胞，并表达IFN γ、IL-1 β或IFN α的受体，或IFN α。将细胞暴露于IFN γ（1000 U/mL）+IL-1 β（50 U/mL）或单独的IFN α（2000 U/mL）24/48 h。使用Hoechst/碘化丙啶染色或RealTime Glo Apoptosis Kit（Promega）定量细胞凋亡。处理后，通过ELISA定量CXCL 10分泌。通过RT-qPCR定量多个基因（Ins、Gcg、Nkx2.2、Nkx6.1、Pdx 1、Mafa、BiP、Chop、Atf 3、CXCL 10、CXCL 9、CCL 5和HLA-ABC）的表达。通过蛋白质印迹定量磷酸化状态和STAT 1/STAT 2的总表达，以及PDL 1和ER伴侣BiP的表达。免疫组化检测HLA-ABC、Caspase-3和Ins/Gcg的表达。HLA-ABC在质膜的存在下，通过流式细胞仪测定。ResultsIFN γ + IL-1 β和IFN α诱导的细胞凋亡后48小时的曝光。切割的caspase-3主要但不完全与Ins+细胞共定位。暴露于IFN γ + IL-1 β诱导促炎表型，包括增加的CXCL 10、CXCL 9和CCL 5表达; CXCL 10分泌;和HLA-ABC表达。通过Western印迹和流式细胞术在蛋白水平证实HLA过表达。暴露于IFN γ + IL-1 β（而非IFN α）也诱导β细胞去分化和内质网应激（BiP、Chop和Atf 3 mRNA表达增加）。在IFN γ + IL-1 β和IFN α作用1 h后，STAT 1的磷酸化已被激活，而STAT 2的磷酸化仅在1-4 h被IFN α激活。PDL 1的表达增加IFN γ + IL-1 β和IFN α.ConclusionsOur数据显示，人iPSC衍生的β细胞响应促炎细胞因子IL-1 β + IFN γ和IFN α，通过激活相同的致病过程，作为成年人的初级β细胞。因此，这些细胞是未来研究1型糖尿病发病机制的宝贵工具。
BackgroundAdult 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 gamma, IL-1 beta, or IFN alpha) that play a role in type 1 diabetes.MethodsThe iPSC-derived islet-like cell clusters contained 40-50% beta and 10-15% alpha cells and expressed the receptors for IFN gamma, IL-1 beta, or IFN alpha. Cells were exposed to either IFN gamma (1000U/mL)+IL-1 beta (50U/mL) or IFN alpha alone (2000U/mL) for 24/48h. 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.ResultsIFN gamma + IL-1 beta and IFN alpha induced apoptosis of the cells after 48h of exposure. Cleaved caspase-3 co-localized mostly but not exclusively with Ins+ cells. Exposure to IFN gamma + IL-1 beta 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 gamma + IL-1 beta (but not IFN alpha) also induced beta cell dedifferentiation and endoplasmic reticulum stress (increase in BiP, Chop, and Atf3 mRNA expression). Phosphorylation of STAT1 was stimulated already after 1h by IFN gamma + IL-1 beta and IFN alpha, while phosphorylation of STAT2 was only activated by IFN alpha at 1-4 h. PDL1 expression was increased by both IFN gamma + IL-1 beta and IFN alpha .ConclusionsOur data show that human iPSC-derived beta cells respond to pro-inflammatory cytokines IL-1 beta + IFN gamma and IFN alpha, 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.