Dissecting the mechanisms of A20 and ABIN-1 mediated programmed cell death in intestinal epithelial cells - A crucial step towards understanding intestinal barrier dysfunction in inflammatory bowel diseases

剖析 A20 和 ABIN-1 介导的肠上皮细胞程序性细胞死亡的机制——了解炎症性肠病中肠道屏障功能障碍的关键一步

• 批准号: 433275755
• 负责人: Dr. Dorothea Stibor
• 金额: --
• 依托单位: Helen Diller Family Comprehensive Cancer Center
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/433275755?language=en
• 关键词: Dissecting; mechanisms; A20; ABIN; mediated

Inflammatory bowel diseases (IBDs) are characterized by excessive intestinal epithelial cell (IEC) death, loss of intestinal barrier function and chronic inflammation that promote a circle of chronic disease. The underlying pathophysiology is thereby incompletely understood. The translation of genetic risk loci into causative molecular pathways has illuminated previously unexpected mechanisms in the genesis of IBDs. Mutations in the genes for the ubiquitin-modifying and ubiquitin-binding proteins A20 and ABIN-1 have been associated with IBDs and other autoimmune diseases. Mechanistically, both proteins play a central role in the negative feedback of pro-inflammatory NF-KB activation and in the regulation of programmed cell death. A majority of the preceding studies in this topic has focused on immune cells. The hosting group has recently developed mice harboring IEC specific, tamoxifen-inducible A20 and/or ABIN-1 knock-out and has derived small intestinal organoids (called enteroids) from these mice. Interestingly, individual deletion of A20 or ABIN-1 only had minor effects whereas knock-out of both genes caused massive, spontaneous IEC death constituting a surprisingly powerful, epistatic synergy. These studies demonstrate a protective role of A20 and ABIN-1 in IEC homeostasis and establish a powerful, inducible model of IBD in mice. In this proposal, I will utilize this model to investigate unknown aspects in the geneses of IBDs: (1.) TNF-independent triggers that kill IECs in mice bearing A20 and ABIN-1 deficiency will be studied. The hosting group has shown that TNF is the main IEC-intrinsic death inducing agent, but that other, IEC-extrinsic triggers occur in-vivo. Given that many IBD patients do not respond to anti-TNF agents, the identification of TNF-independent mechanisms is a clinically important goal. I will now screen for potentially harmful ligands from immune cells or microbes and dissect candidate pathways in greater detail. (2.) Molecular mechanisms by which A20 and ABIN-1 synergize to protect IEC survival will also be determined. To this end, known biochemically active genetic motifs of A20 (OTU, ZF4, ZF7) and ABIN-1 (UBAN) will be challenged in terms of their contribution to the regulation of IEC survival. (3.) Lastly, the role of A20 and ABIN-1 in human cells will be investigated. Enteroids from IBD patients will be created and analyzed with regard to expression of A20, ABIN-1 and other biomarkers, in comparison to ex-vivo biopsies and in correlation with the clinical outcome. All aims will be evaluated using enteroid cultures as well as in complementing studies in gene-modified mice or human ex-vivo biopsies. An advantage is, that it will be possible to distinguish between IEC intrinsic or extrinsic mechanisms. Collectively, these investigations can not only expand the current understanding of the pathology behind IBDs but can also create exiting new therapeutic targets with a focus on the preservation of mucosal homeostasis.

炎症性肠病（Inflammatory bowel disease，IBD）是以肠上皮细胞（intestinal epithelial cell，IEC）过度死亡、肠屏障功能丧失和慢性炎症为特征的一种慢性疾病。因此，根本的病理生理学是不完全理解的。将遗传风险位点转化为致病分子途径，阐明了IBD发生中以前意想不到的机制。泛素修饰和泛素结合蛋白A20和ABIN-1的基因突变与IBD和其他自身免疫性疾病有关。从机制上讲，这两种蛋白质在促炎性NF-κ B活化的负反馈和程序性细胞死亡的调节中发挥核心作用。这一主题的大多数先前研究都集中在免疫细胞上。主持小组最近开发了具有IEC特异性、他莫昔芬诱导的A20和/或ABIN-1敲除的小鼠，并从这些小鼠中衍生出小肠类器官（称为肠类器官）。有趣的是，A20或ABIN-1的单独缺失仅具有较小的影响，而两种基因的敲除引起大规模的自发IEC死亡，这构成了令人惊讶的强大的上位协同作用。这些研究证明了A20和ABIN-1在IEC稳态中的保护作用，并在小鼠中建立了强大的可诱导IBD模型。在这个计划中，我将利用这个模型来研究IBD的基因中未知的方面：（1）。将研究杀死A20和ABIN-1缺陷小鼠中IEC的非TNF依赖性触发因素。宿主组已经表明，TNF是主要的IEC-内在死亡诱导剂，但其他IEC-外在触发剂在体内发生。鉴于许多IBD患者对抗TNF药物无反应，因此确定TNF非依赖性机制是临床重要目标。我现在将从免疫细胞或微生物中筛选潜在有害的配体，并更详细地剖析候选途径。（2.）还将确定A20和ABIN-1协同保护IEC存活的分子机制。为此，A20（OTU、ZF 4、ZF 7）和ABIN-1（UBAN）的已知生物化学活性遗传基序将在它们对IEC存活的调节的贡献方面受到挑战。（3.）最后，将研究A20和ABIN-1在人类细胞中的作用。将产生来自IBD患者的肠样组织，并分析A20、ABIN-1和其他生物标志物的表达，与离体活检进行比较，并与临床结果进行相关性分析。将使用肠样培养物以及在基因修饰小鼠或人离体活检的补充研究中评价所有目标。一个优点是，它将有可能区分IEC内在或外在机制。总的来说，这些研究不仅可以扩大目前对IBD背后病理学的理解，而且还可以创建现有的新治疗靶点，重点是保持粘膜稳态。