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

炎症性肠病（IBDs）的特征是肠上皮细胞（IEC）过度死亡，肠屏障功能丧失和慢性炎症，促进慢性疾病循环。因此，潜在的病理生理学尚不完全清楚。遗传风险位点转化为致病分子途径已经阐明了ibd发生中以前意想不到的机制。泛素修饰蛋白和泛素结合蛋白A20和ABIN-1基因突变与ibd和其他自身免疫性疾病有关。从机制上讲，这两种蛋白在促炎NF-KB激活的负反馈和程序性细胞死亡的调节中发挥核心作用。在此之前的大多数研究都集中在免疫细胞上。宿主组最近培育了具有IEC特异性、他莫昔芬诱导的A20和/或ABIN-1敲除的小鼠，并从这些小鼠中获得了小肠类器官（称为类肠）。有趣的是，A20或ABIN-1的个体缺失只会产生轻微的影响，而这两个基因的敲除会导致大量自发的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，并建立了一个强大的小鼠IBD诱导模型。在本提案中，我将利用该模型来研究ibd基因的未知方面：(1)。将研究在A20和ABIN-1缺乏小鼠中杀死IECs的不依赖于tnf的触发因素。宿主组的研究表明，TNF是主要的iec内源性死亡诱导剂，但其他iec外源性触发因素也发生在体内。鉴于许多IBD患者对抗tnf药物没有反应，确定与tnf无关的机制是一个重要的临床目标。现在，我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)。A20和ABIN-1协同保护IEC存活的分子机制也将被确定。为此，已知的A20 （OTU, ZF4, ZF7）和ABIN-1 （urban）的生物化学活性遗传基序在IEC存活调控中的作用将受到挑战。(3)。最后，我们将探讨A20和ABIN-1在人类细胞中的作用。将创建IBD患者的肠样，并分析A20、ABIN-1和其他生物标志物的表达，与离体活检相比较，并与临床结果相关联。所有目的都将通过肠道培养以及基因修饰小鼠或人类离体活检的补充研究进行评估。一个优点是，它将有可能区分IEC的内在或外在机制。总的来说，这些研究不仅可以扩大目前对ibd背后病理的理解，而且还可以创建新的治疗靶点，重点是保持粘膜稳态。

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.