Islet-Autonomous Inflammatory Signaling Propagates Autoimmunity and Promotes Diabetes in Nonobese Diabetic Mice. Islet-Autonomous Inflammatory Signaling Propagates Autoimmunity and Promotes Diabetes in Nonobese Diabetic Mice

Type 1 diabetes (T1D) is an immune-mediated disease that leads to β cell dysfunction and death. However, the contribution of β cells in this process remains unclear. To understand how islet-derived pro-inflammatory signaling pathways contribute to diabetes pathogenesis, we generated inducible islet-specific Alox15 knockout mice on the NOD background. We report that islet-specific deletion of Alox15 induced a substantial increase of β-cell mass and decreased islet insulitis, and ultimately lead to a protection against spontaneous autoimmune diabetes in NOD mice. Single cell RNA-sequencing and mass spectrometry analysis revealed that islet-specific knockout of Alox15 leads to an increase of β cells expressing Pd-l1 and promotes an anti-inflammatory phenotype in myeloid cells and T cells inside the islets. Furthermore, islet- specific Alox15 deletion enhances the expansion of M2-like macrophages and regulatory T cells in the pancreatic islets and pancreatic lymph nodes. Together, these results lead to the conclusion that proinflammatory signals produced by β cells allows these cells to express immunoregulators that protects these cells of immune cell attack and promote β cell survival. Overall design: Mouse islets were isolated from collagenase-perfused pancreata and cultured in RPMI medium as previously describe (Stull et al. 2012). After isolation, islets were handpicked and digested with Accutase (EMD Millipore Corporation) containing 2U/ml of DNAse during 5min at 37C sob agitation (1000rpm). Then cells were washed several times with PBS+2%FBS to eliminate DNAse and then filtered using a cell strainer (40m). Samples with more than 90% viability were used for scRNAseq.

1型糖尿病（Type 1 diabetes, T1D）是一种免疫介导性疾病，可导致β细胞功能障碍与死亡。然而，β细胞在此过程中的作用仍不明确。为阐明胰岛源性促炎信号通路如何参与糖尿病发病机制，我们在非肥胖糖尿病（Non-obese diabetic, NOD）小鼠遗传背景中构建了可诱导性胰岛特异性Alox15基因敲除小鼠。本研究发现，胰岛特异性敲除Alox15可显著提升β细胞质量，减轻胰岛炎程度，并最终保护NOD小鼠免受自发性自身免疫性糖尿病的侵袭。单细胞RNA测序（single cell RNA-sequencing, scRNA-seq）与质谱分析显示，胰岛特异性敲除Alox15可增加表达程序性死亡配体1（Programmed death-ligand 1, PD-L1）的β细胞比例，并促进胰岛内髓系细胞与T细胞向抗炎表型转化。进一步研究表明，胰岛特异性Alox15基因敲除可增强胰腺胰岛及胰腺淋巴结中M2样巨噬细胞与调节性T细胞（regulatory T cells, Tregs）的扩增。综上，本研究结果证实：β细胞产生的促炎信号可促使该细胞表达免疫调节分子，从而抵御免疫细胞的攻击并促进β细胞存活。
实验设计：我们通过胶原酶灌注法分离小鼠胰腺胰岛，并参照此前报道的方法（Stull等，2012）在RPMI培养基中培养。胰岛分离完成后，经手工挑选，使用含2U/ml脱氧核糖核酸酶（Deoxyribonuclease, DNAse）的Accutase（EMD密理博公司，EMD Millipore Corporation）在37℃、1000rpm轨道振荡条件下孵育5分钟。随后用含2%胎牛血清（Fetal Bovine Serum, FBS）的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）多次洗涤细胞以去除DNA酶，再通过40μm细胞筛过滤。活细胞率高于90%的样本将用于单细胞RNA测序。