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

• 批准号: 10704104
• 负责人: BANA JABRI
• 金额: $ 35.13万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 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/10704104
• 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 Cells; Epithelium; 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' s Patches; Pharmaceutical Preparations; Play; Polymers; Proteins; Reaction; Reagent; Recombinant Cytokines; Role; Series; Small Intestines; Source; T-Cell Receptor; T-Lymphocyte; TNF gene; Testing; Time; Villous Atrophy; absorption; adduct; cytokine; deamidation; design; dietary; efficacious treatment; feeding; in vivo; inhibitor; interleukin-21; intestinal epithelium; intraepithelial; jejunum; mouse model; neutralizing antibody; next generation; novel; prototype; scale up; transglutaminase 2; volunteer

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)是一种面筋蛋白诱导的、依赖于HLADQ2或DQ8的小规模炎症性疾病 没有非饮食疗法可用的肠道。转谷氨酰胺酶2(TG2)是CED特异性的靶标 自身抗体也参与了疾病的发病机制。在CED患者中，TG2催化形成 与人类白细胞抗原-DQ2/8高亲和力结合并被疾病识别为表位的去胺化面筋多肽- 特异性的CD4+T细胞。然而，TG2在哪里发挥致病作用尚不清楚。 我们提出的最重要的假设是TG2来源于肠道细胞进入肠腔。 是CED致病相关酶的来源。这种流明的TG2与面筋多肽反应形成 Peyer‘s斑块中TG2特异性B细胞识别的共价复合体。反过来，这些B细胞提供面筋 疾病特异性T细胞的抗原，同时也从这些T细胞获得帮助。这一假说可以解释 CED形成TG2自身抗体。涉及体外和体内研究的三个目标旨在测试 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：上述假说的两个关键特征将在生化水平上进行检验。第一，能力 将探索在TG2活性部位形成亚稳态共价中间体的抗原性谷蛋白多肽。 其次，将开发一种新的同位素标记分析方法来验证小鼠肠道中的流明TG2可以 识别和去除膳食面筋。在这个目标下，我们还将设计一种肠道不渗透的TG2抑制剂， 它将在AIM 3中用于验证鲁米那TG2的致病作用。 具体目标2：使用从人空肠腔和人类器官收集的脱落肠细胞 培养后，我们将证明肠上皮细胞在 脱毛时间。人肠上皮源性TG2与抗原性面筋蛋白形成共价加合物的能力 多肽也将得到验证。我们的有机培养物也将用于鉴定与疾病相关的环境 在增加肠腔内稳态TG2活性方面最有效的因素。 具体目标3：该目标将在两个小鼠模型中测试肠源性鲁米那TG2的致病作用 CED的。在一个已建立的CED模型中，我们将测试TG2-谷蛋白多肽共价复合体在 肠腔可以刺激TG2特异性B细胞和面筋特异性T细胞之间的协作。在另一个模型中 在可以通过饲喂面筋蛋白诱导CED的情况下，对TG2在肠细胞中表达的要求或替代 将在髓系隔室中进行测试。 第一代TG2抑制剂已经在进行人体临床试验。如果我们最重要的假设是 建议是正确的，它将确立TG2在肠腔中的抑制足以防止面筋- 诱导CED绒毛萎缩。这不仅将激励设计一种更安全、更有效的疗法， 但我们的肠道不渗透抑制剂也可能成为这种终生疾病的下一代药物原型。