Localizing Pathogenically Relevant Transglutaminase 2 in Celiac Disease

乳糜泻中致病相关转谷氨酰胺酶 2 的定位

基本信息

批准号：
10296119
负责人：
BANA JABRI
金额：
$ 36.4万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-07 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10296119
关键词：
Active Sites Affinity Antigens Autoantibodies B-Cell Antigen Receptor B-Lymphocytes Binding Biochemical Biological Assay CD4 Positive T Lymphocytes Celiac Disease Cells Clinical Trials Collaborations Complex Disease Engineering Enterocytes Environment Environmental Risk Factor Enzymes Epithelial Epithelial Cells Epitopes Generations Gluten Goals HLA-DQ2 Human In Vitro Inflammatory Interferon Type II Intestines Isotope Labeling Laboratories Location Lymphocyte Modeling Mus Myelogenous Organoids Pathogenesis Pathogenicity Patients Peptides Peyer&apos s Patches Pharmaceutical Preparations Play Polymers Proteins Reagent Receptor Cell Recombinant Cytokines Role Series Small Intestines Source T-Cell Receptor T-Lymphocyte TNF gene Testing Time Villous Atrophy absorption adduct cytokine design dietary efficacious treatment feeding in vivo inhibitor/antagonist intestinal epithelium intraepithelial jejunum mouse model neutralizing antibody next generation novel prototype scale up transglutaminase 2 volunteer

项目摘要

Celiac disease (CeD) is a gluten-induced, HLA-DQ2 or -DQ8 dependent inflammatory disorder of the small intestine for which no non-dietary therapy is available. Transglutaminase 2 (TG2) is the target of CeD-specific autoantibodies and is also involved in disease pathogenesis. In a CeD patient TG2 catalyzes the formation of deamidated gluten peptides that bind to HLA-DQ2/8 with high affinity and are recognized as epitopes by disease- specific CD4+ T cells. However, the location where TG2 exerts its pathogenic action is unknown. The overarching hypothesis of our proposal is that TG2 derived from enterocytes shed into the intestinal lumen is the source of pathogenically relevant enzyme in CeD. This luminal TG2 reacts with gluten peptides to form covalent complexes recognized by TG2-specific B cells in Peyer’s patches. In turn, these B cells present gluten antigens to disease-specific T cells, while also deriving help from these T cells. This hypothesis can explain how TG2 autoantibodies are formed in CeD. Three Aims involving in vitro and in vivo studies are designed to test our hypothesis, while taking advantage of the complementary capabilities of the three collaborating laboratories. Specific aim 1: Two key features of the above hypothesis will be tested at a biochemical level. First, the ability of antigenic gluten peptides to form metastable covalent intermediates at the TG2 active site will be probed. Second, a novel isotope labeling assay will be developed to verify that luminal TG2 in the mouse intestine can recognize and deamidate dietary gluten. Under this Aim we will also engineer a gut-impermeable TG2 inhibitor, which will be used in Aim 3 to validate the pathogenic role of luminal TG2. Specific aim 2: Using shed enterocytes collected from the human jejunal lumen as well as human organoid cultures, we will demonstrate that intestinal epithelial cells harbor abundant catalytically competent TG2 at the time of shedding. The ability of human enterocyte-derived TG2 to form covalent adducts with antigenic gluten peptides will also be verified. Our organoid cultures will also be used to identify disease-relevant environmental factors that are most effective at increasing steady-state TG2 activity in the small intestinal lumen. Specific aim 3: This Aim will test the pathogenic role of enterocyte-derived luminal TG2 in two mouse models of CeD. In one model of established CeD, we will test whether TG2-gluten peptide covalent complexes in the gut lumen can stimulate collaboration between TG2-specific B cells and gluten-specific T cells. In another model where CeD can be induced by feeding gluten, the requirement for TG2 expression in enterocytes or alternately in the myeloid compartment will be tested. First-generation TG2 inhibitors are already undergoing human clinical trials. If the overarching hypothesis of our proposal is correct, it will establish that TG2 inhibition in the intestinal lumen is sufficient to protect against gluten- induced villous atrophy in CeD. Not only will this motivate the design of a safer and more efficacious therapies, but our gut-impermeable inhibitors may also serve as next-generation drug prototypes for this lifelong disorder.

乳糜泻 (CeD) 是一种由麸质诱导、HLA-DQ2 或 -DQ8 依赖性的小肠炎症性疾病。没有非饮食疗法可用的肠道，转谷氨酰胺酶 2 (TG2) 是 CeD 特异性的目标。在 CeD 患者中，TG2 催化自身抗体的形成，并且还参与疾病发病机制。脱酰胺面筋肽以高亲和力与 HLA-DQ2/8 结合，并被疾病识别为表位- 然而，TG2 发挥致病作用的位置尚不清楚。我们提议的总体假设是源自肠细胞的 TG2 流入肠腔是 CeD 中致病相关酶的来源。这种管腔 TG2 与麸质肽反应形成。派尔氏斑中 TG2 特异性 B 细胞识别的共价复合物反过来，这些 B 细胞呈递麸质。疾病特异性 T 细胞的抗原，同时也从这些 T 细胞中获得帮助，这一假设可以解释如何做到这一点。 TG2 自身抗体是在 CeD 中形成的，涉及旨在测试的体外和体内研究的三个目标。我们的假设，同时利用三个合作实验室的互补能力。具体目标1：上述假设的两个关键特征将在生化水平上进行检验。首先，能力。将探讨抗原性麸质肽在 TG2 活性位点形成亚稳态共价中间体的过程。其次，将开发一种新的同位素标记测定法来验证小鼠肠道中的管腔 TG2 可以识别膳食麸质并将其脱酰胺化在这一目标下，我们还将设计一种肠道不可渗透的 TG2 抑制剂，其将在目标 3 中用于验证 Luminal TG2 的致病作用。具体目标 2：使用从人空肠腔以及人类器官收集的脱落肠细胞培养物中，我们将证明肠上皮细胞在人肠上皮细胞来源的 TG2 与抗原麸质形成共价加合物的能力。我们的类器官培养物也将用于识别与疾病相关的环境。对增加小肠腔内稳态 TG2 活性最有效的因素。具体目标 3：该目标将在两种小鼠模型中测试肠上皮细胞衍生的腔 TG2 的致病作用在已建立的 CeD 模型中，我们将测试 TG2-麸质肽共价复合物是否存在于 CeD 中。在另一个模型中，肠腔可以刺激 TG2 特异性 B 细胞和麸质特异性 T 细胞之间的协作。其中 CeD 可以通过饲喂麸质、肠上皮细胞中 TG2 表达的要求或其他方式来诱导将在骨髓室中进行测试。如果我们的总体假设是这样的话，第一代 TG2 抑制剂已经正在进行人体临床试验。该提议是正确的，它将确定肠腔中的 TG2 抑制足以防止麸质在 CeD 中诱发绒毛萎缩，这不仅会激发更安全、更有效的疗法的设计，但我们的肠道不可渗透抑制剂也可能作为这种终生疾病的下一代药物原型。