Characterization of Innate immune receptors

描述（由申请人提供）：乳糜泻（Cd）是一种T细胞介导的小肠疾病。它是由谷物中的膳食面筋蛋白引发的，每130名美国公民中就有1人受到影响。虽然镉可导致严重的吸收不良，但大多数患者表现为轻微或非典型（非消化性）症状。镉与继发性自身免疫有关，如1型糖尿病或自身免疫性甲状腺炎，长期镉没有得到严格的无麸质饮食治疗可能导致（胃肠道）恶性肿瘤。已脱酰胺的镉自身抗原组织转氨酶谷蛋白肽强烈结合HLA-DQ 2或-DQ 8，镉的基本遗传易感性。这导致肠道Th 1 T细胞活化和粘膜破坏（适应性免疫）。除了引发适应性免疫外，最近的数据表明谷蛋白（麦胶蛋白）还可以刺激先天免疫，即，对常见外来抗原（如细菌多糖或病毒RNA）的直接和相对非特异性免疫反应。先天免疫对麦醇溶蛋白的可变贡献可以解释为什么携带HLA-DQ 2或-DQ 8的个体中只有2- 5%最终发展为Cd。负责的麦胶蛋白肽和介导对麸质的先天免疫的受体仍有待鉴定。 我们的初步研究结果表明，麦胶蛋白，其中包含大约1000种不同的麦胶蛋白肽的肽-胰蛋白酶消化触发了显着的先天性免疫反应，在人类树突状细胞，但也肠上皮细胞，评估释放的炎症介质白细胞介素8和MCP-1。基于这些数据，我们计划：1。通过使用树突状细胞（肠上皮细胞）作为指示剂和MALDI-TOF质谱法进行色谱分离后，鉴定驱动先天免疫的麦胶蛋白肽。2.通过使用标记的细胞膜在鉴定的固定刺激性麦胶蛋白肽上的亲和层析和MALDI-TOF质谱法，分离并表征这些细胞上触发对麦胶蛋白的先天免疫应答的先天免疫受体。受体的身份和功能将通过使用功能阻断抗体、siRNA和信号传导途径抑制剂进一步确认。 我们预计，识别的受体（S）负责的先天免疫反应麦醇溶蛋白将导致更好地了解镉的发病机制，并可能以新的非饮食疗法来治疗这种常见的肠道疾病。 乳糜泻（Cd）是一种常见的小肠免疫性疾病，每130名美国公民中就有1名受到影响。它是由小麦和相关谷物的储藏蛋白（麦谷蛋白，特别是麦醇溶蛋白）的摄入引发的。镉如果不通过严格的饮食麸质排除来治疗，会导致严重的吸收不良和恶性肿瘤，并与继发性自身免疫有关，如1型糖尿病。虽然对谷蛋白肽的适应性（T细胞介导的）免疫应答导致再吸收肠粘膜的破坏是很好理解的，但对麦胶蛋白的先天性免疫应答（对外来抗原如细菌多糖或病毒RNA的直接且相对非特异性的反应）还不清楚。负责的麦胶蛋白肽和介导对麸质的先天免疫的受体仍有待鉴定。我们计划通过使用蛋白质组学技术、亲和层析和功能研究来鉴定在人树突细胞和肠上皮细胞中驱动先天免疫的麦胶蛋白肽，并表征其应答性先天免疫受体。我们预计，识别的受体（S）负责的先天免疫反应麦醇溶蛋白将导致更好地了解镉的发病机制，并可能以新的非饮食疗法来治疗这种常见的肠道疾病。

DESCRIPTION (provided by applicant): Celiac disease (Cd) is a T cell mediated disease of the small intestine. It is triggered by dietary gluten proteins from cereals and affects 1 in 130 US citizens. While Cd can lead to severe malabsorption, most patients present with minor or atypical (nondiarrheal) symptoms. Cd is associated with secondary autoimmunity, such as type 1 diabetes or autoimmune thyroiditis, and longterm Cd that has not been treated with a strict gluten free diet can lead to (gastrointestinal) malignancy. Gluten peptides that have been deamidated by the Cd autoantigen tissue transglutaminase bind strongly to HLA-DQ2 or -DQ8, the essential genetic predisposition for Cd. This results in intestinal Th1 T cell activation and mucosal destruction (adaptive immunity). Apart from triggering adaptive immunity, recent data suggest that gluten (gliadin) can also stimulate innate immunity, i.e., the immediate and relatively nonspecific immune response to common foreign antigens, such as bacterial polysaccharide or viral RNA. The variable contribution of innate immunity to gliadin could explain why only 2- 5% of those individuals that carry HLA-DQ2 or -DQ8 finally develop Cd. The responsible gliadin peptide(s) and the receptors mediating innate immunity to gluten remain to be identified. Our preliminary results show that a peptic-tryptic digest of gliadin which contains roughly 1000 different gliadin peptides triggers a marked innate immune response in human dendritic but also intestinal epithelial cells, as assessed by release of the inflammatory mediators interleukin 8 and MCP-1. Based on these data we plan to: 1. Identify the gliadin peptide(s) that drive innate immunity after their chromatographic separation by using dendritic (intestinal epithelial) as indicators and MALDI-TOF mass spectrometry. 2. Isolate and characterize the innate immune receptor(s) on these cells that trigger(s) the innate immune response to gliadin by use of affinity chromatography of labeled cell membranes on the identified immobilized stimulatory gliadin peptide(s) and MALDI-TOF mass spectrometry. Identity and function of the receptor(s) will further be confirmed by use of function blocking antibodies, siRNA and signaling pathway inhibitors. We anticipate that identification of the receptor(s) responsible for the innate immune response to gliadin will lead to a better understanding of the pathogenesis of Cd, and possibly to novel nondietary therapies to treat this common intestinal disorder. PUBLIC HEALTH RELEVANCE Celiac disease (Cd) is a common immune disease of the small intestine that affects 1 in 130 US citizens. It is triggered by ingestion of the storage proteins of wheat (glutenins and especially gliadins) and related cereals. Cd that is not treated by strict dietary gluten exclusion can lead to severe malabsorption and malignancy, and is associated with secondary autoimmunity, such as type 1 diabetes. While the adaptive (T cell mediated) immune response to gluten peptides that leads to destruction of the resorptive intestinal mucosa is well understood, there is a yet ill defined innate immune response (the immediate and relatively nonspecific reaction to foreign antigens, such as bacterial polysaccharide or viral RNA) to gliadins. The responsible gliadin peptide(s) and the receptors mediating innate immunity to gluten remain to be identified. We plan to identify the gliadin peptide(s) that drive innate immunity in human dendritic and intestinal epithelial cells and characterize their responsive innate immune receptor(s) by use of proteomic techniques, affinity chromatography and functional studies. We anticipate that identification of the receptor(s) responsible for the innate immune response to gliadin will lead to a better understanding of the pathogenesis of Cd, and possibly to novel nondietary therapies to treat this common intestinal disorder.