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: 10483182
• 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/10483182
• 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' 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; 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）是一种谷蛋白诱导的、HLA-DQ 2或-DQ 8依赖性的小细胞炎症性疾病。 没有非饮食疗法可用的肠道。转氨酶2（TG 2）是CeD特异性的靶点。 自身抗体，也参与疾病的发病机制。在CeD患者中，TG 2催化 以高亲和力结合HLA-DQ 2/8并被疾病识别为表位的脱酰胺谷蛋白肽， 特异性CD 4 + T细胞然而，TG 2发挥其致病作用的位置尚不清楚。 我们提出的主要假设是，TG 2来源于肠上皮细胞，脱落到肠腔中， 是CeD致病相关酶的来源。这种管腔TG 2与谷蛋白肽反应形成 Peyer集合淋巴结中TG 2特异性B细胞识别的共价复合物。反过来，这些B细胞将谷蛋白 抗原的疾病特异性T细胞，同时也从这些T细胞获得帮助。这个假设可以解释 TG 2自身抗体在CeD中形成。设计了涉及体外和体内研究的三个目的，以测试 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：将在生物化学水平上检验上述假设的两个关键特征。第一，能力 将探测抗原性谷蛋白肽在TG 2活性位点形成亚稳共价中间体的可能性。 第二，将开发一种新的同位素标记测定来验证小鼠肠中的管腔TG 2可以 识别和脱酰胺的膳食面筋。根据这一目标，我们还将设计一种肠道不可渗透的TG 2抑制剂， 其将用于目的3以验证管腔TG 2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，我们将证明肠上皮细胞具有丰富的催化活性TG 2在 脱落的时间。人肠细胞来源的TG 2与抗原性谷蛋白形成共价加合物的能力 还将验证肽。我们的类器官培养也将用于识别疾病相关的环境 在增加小肠腔中稳态TG 2活性方面最有效的因素。 具体目标3：该目标将在两种小鼠模型中测试肠细胞衍生的管腔TG 2的致病作用 的CeD。在建立的CeD的一个模型中，我们将测试是否TG 2-谷蛋白肽共价复合物中的 肠腔可以刺激TG 2特异性B细胞和麸质特异性T细胞之间的协作。在另一个模型中 其中CeD可以通过喂食麸质、肠细胞中TG 2表达的需要或替代地 将对骨髓室进行检测。 第一代TG 2抑制剂已经在进行人体临床试验。如果我们的总体假设 如果该建议是正确的，它将确定肠腔中的TG 2抑制足以保护免受麸质- 诱导CeD绒毛萎缩。这不仅将激励设计更安全和更有效的疗法， 但我们的肠道不渗透抑制剂也可能成为这种终身疾病的下一代药物原型。