目标:血管炎,自身免疫性1型糖尿病之前的炎症标志,导致功能β细胞的最终丧失。然而,即使面对持续的胰岛炎,功能β细胞也可以持续存在。尽管免疫抑制治疗取得了进展,维持功能β细胞以防止胰岛炎进展和高血糖仍然是一个挑战。大麻素1型受体(CB1R),存在于免疫细胞和β细胞中,调节炎症和β细胞功能。这里,我们开创了一个反映人类胰岛炎的离体模型,以研究CB1R在这一过程中的作用。
方法:从1型糖尿病发病的男性和女性个体和非糖尿病个体的外周血单核细胞(PBMC)中分离CD4+T淋巴细胞,提取RNA并通过实时PCR分析mRNA表达。从数据挖掘获得来自患有1型糖尿病的供体的单个β细胞表达。将来自男性和女性尸体供体的患者来源的人胰岛在溶解的细胞外基质凝胶中3D培养,与相同的供体PBMC共培养,并与细胞因子(IL-1β,TNF-α,IFN-γ)在存在媒介物或增加浓度的CB1R阻断剂JD-5037的情况下持续24-48小时。使用CRISPR/Cas9技术消除CNR1(编码CB1R)的表达。活力,通过活细胞探测和实时PCR测定细胞内应激和信号传导。在灌注系统中确定了以葡萄糖刺激的胰岛素分泌测量的胰岛功能。通过显微镜监测免疫细胞向胰岛中的浸润。将7周龄的非肥胖糖尿病小鼠用JD-5037治疗1周,然后安乐死。通过流式细胞术对胰岛中浸润的免疫细胞进行分析。
结果:CNR1表达在1型糖尿病发病个体的循环CD4+T细胞中上调(比健康个体高6.9倍),在1型糖尿病供体的分选胰岛β细胞中上调(比健康个体高3.6倍)。外周限制性CB1R反向激动剂JD-5037可阻止人和小鼠胰岛炎的发生。机械上,CB1R阻断阻止了胰岛NO的产生,并改善了未折叠蛋白反应的ATF6臂。因此,人胰岛细胞/趋化因子表达降低,导致持续的胰岛细胞活力和功能。
结论:这些结果表明,CB1R可能是1型糖尿病的一个有趣的靶标,同时突出了胰岛炎的调节机制。此外,这些发现可能适用于2型糖尿病,胰岛炎症也是一种病理生理因素。
方法:分选的人β细胞的转录组学分析来自基因表达综合数据库,加入编号GSE121863,可在https://www上获得。ncbi.nlm.nih.gov/geo/query/acc。cgi?acc=GSM3448161。
OBJECTIVE: Insulitis, a hallmark of inflammation preceding autoimmune type 1 diabetes, leads to the eventual loss of functional beta cells. However, functional beta cells can persist even in the face of continuous insulitis. Despite advances in immunosuppressive treatments, maintaining functional beta cells to prevent insulitis progression and hyperglycaemia remains a challenge. The cannabinoid type 1 receptor (CB1R), present in immune cells and beta cells, regulates inflammation and beta cell function. Here, we pioneer an ex vivo model mirroring human insulitis to investigate the role of CB1R in this process.
METHODS: CD4+ T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) from male and female individuals at the onset of type 1 diabetes and from non-diabetic individuals, RNA was extracted and mRNA expression was analysed by real-time PCR. Single beta cell expression from donors with type 1 diabetes was obtained from data mining. Patient-derived human islets from male and female cadaveric donors were 3D-cultured in solubilised extracellular matrix gel in co-culture with the same donor PBMCs, and incubated with cytokines (IL-1β, TNF-α, IFN-γ) for 24-48 h in the presence of vehicle or increasing concentrations of the CB1R blocker JD-5037. Expression of CNR1 (encoding for CB1R) was ablated using CRISPR/Cas9 technology. Viability, intracellular stress and signalling were assayed by live-cell probing and real-time PCR. The islet function measured as glucose-stimulated insulin secretion was determined in a perifusion system. Infiltration of immune cells into the islets was monitored by microscopy. Non-obese diabetic mice aged 7 weeks were treated for 1 week with JD-5037, then euthanised. Profiling of immune cells infiltrated in the islets was performed by flow cytometry.
RESULTS: CNR1 expression was upregulated in circulating CD4+ T cells from individuals at type 1 diabetes onset (6.9-fold higher vs healthy individuals) and in sorted islet beta cells from donors with type 1 diabetes (3.6-fold higher vs healthy counterparts). The peripherally restricted CB1R inverse agonist JD-5037 arrested the initiation of insulitis in humans and mice. Mechanistically, CB1R blockade prevented islet NO production and ameliorated the ATF6 arm of the unfolded protein response. Consequently, cyto/chemokine expression decreased in human islets, leading to sustained islet cell viability and function.
CONCLUSIONS: These results suggest that CB1R could be an interesting target for type 1 diabetes while highlighting the regulatory mechanisms of insulitis. Moreover, these findings may apply to type 2 diabetes where islet inflammation is also a pathophysiological factor.
METHODS: Transcriptomic analysis of sorted human beta cells are from Gene Expression Omnibus database, accession no. GSE121863, available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3448161 .