The human B cell toll-like receptor 4 complex

描述(由申请人提供)：粘膜上的宿主-微生物相互作用需要一种微妙的、尽管知之甚少的平衡，其中共生和致病细菌的存在需要歧视性反应。虽然肠道的正常微生物如果留在肠道微生态位内不会致病，但如果它们越过上皮屏障，就会引发炎症反应。易位细菌的持续存在在炎症性肠病(IBD)的病因和发病机制中起着重要作用。关于细菌感应基因在IBD中的作用的文献中的不一致表明，这种宿主-微生物相互作用的复杂性比人们所认识的更高。我们在知识上的一些差距可能是由于对粘膜中最丰富的白细胞之一--B细胞--的作用定义不清。B细胞表达脂多糖受体Toll样受体(TLR)4，在IBD中可能对微生物起重要作用。矛盾的是，尽管TLR4表达，但患者B细胞对大肠杆菌脂多糖的反应通常很差。相反，TLR4+B细胞对内毒素分子的反应具有低酰化脂质A的特征，这是一组被证明对髓系TLR4具有拮抗作用的配体。本应用的目的是确定人类B细胞表达的潜在的新的和动态的TLR4复合体，以及B细胞TLR4反应在IBD中的功能意义。我们的中心假设是B细胞TLR4是一个动态的复合体，允许通过一种新的TLR4共受体TRIL和结构修饰TLR4识别低酰化的内毒素。我们的假设是基于我们关于B细胞TLR4复合体疾病特异性变化的令人兴奋的数据，以及我们最近的报告表明，IBD患者的B细胞通过分泌IgA来响应低酰化的内毒素。因此，我们进一步假设TLR4刺激一个限制性的IgA谱系，它表现出广泛的反应性，并有助于保护粘膜。我们将通过两个具体目标来检验我们的假设。在第一个目标中，我们将通过使用患者的原始B细胞进行消融和添加研究来确定所需的辅助受体，从而定义人类B细胞TLR4复合体。我们还将通过对已识别的B细胞TLR4变体进行定向测序和克隆，来确定TLR4潜在的结构修饰(S)的影响，这可能会影响对内毒素的识别。这些数据将与IBD的临床参数和TLR4基因的多态性相关。在第二个目标中，我们将通过评估基因和激酶的激活来确定TLR4介导的免疫球蛋白A产生的机制(S)，并通过对免疫球蛋白A的V区进行定向测序来确定B细胞反应的克隆性。该提案中产生的数据将通过确定改变疾病进程的改进治疗方法的开发的关键路径，具有直接的临床意义。此外，通过定义B细胞如何识别内毒素，我们还将推进全基因组关联研究、人类B细胞免疫学以及与许多慢性病有关的人-微生物界面的机制(S)。 公共卫生相关性：炎症性肠病(IBD)是一种慢性胃肠道疾病，我们无法治愈。在这项提案中，我们希望确定我们的免疫系统与肠道友好细菌相互作用的新方式，希望为IBD患者开发治疗方法。

DESCRIPTION (provided by applicant): The host-microbial interactions at the mucosa require a delicate, although poorly understood, balance where the presence of commensal and pathogenic bacteria requires discriminatory responses. Although the normal microbes of the gut are not pathogenic if they remain within their gut micro-niche, they will induce an inflammatory response if they cross the epithelial barrier. The persistence of translocated bacteria is thought to play an important role in the etiology and pathogenesis of inflammatory bowel disease (IBD). Inconsistencies in the literature regarding the role of bacteria-sensing genes in IBD suggest a higher level of complexity to this host-microbe interaction than has been appreciated. Some of our gaps in knowledge may be explained by the poorly defined role of one of the most abundant leukocytes in the mucosa, the B cell. B cells express Toll-like receptor (TLR) 4, the receptor for lipopolysaccharide (LPS), in IBD and likely have an important role in sensing microbes. Paradoxically, despite TLR4 expression, patient B cells generally respond poorly to E. coli LPS. In contrast, TLR4+ B cells respond to LPS molecules with characteristics of hypo-acylated lipid A, a group of ligands shown to be antagonistic for myeloid TLR4. The objective of this application is to define the potentially novel and dynamic TLR4 complex expressed by human B cells and the functional significance of B cell TLR4 responses in IBD. Our central hypothesis is that B cell TLR4 is a dynamic complex that allows recognition of hypo-acylated LPS through TRIL, a novel TLR4 co-receptor, and structural modification TLR4. Our hypothesis is based on our exciting data on disease-specific changes in the B cell TLR4 complex and our recent report demonstrating that B cells from IBD patients respond to hypo-acylated LPS by secreting IgA. We thus further hypothesize that TLR4 stimulates a restricted IgA repertoire, which exhibits broad reactivity and helps to protect the mucosa. We will test our hypothesis through two Specific Aims. In the first aim, we will define the human B cell TLR4 complex by identifying the required co-receptors through ablation and addition studies using primary B cells from patients. We will also define the effect of potential structural modification(s) of TLR4 that may affect recognition of LPS through targeted sequencing and cloning of identified B cell TLR4 variants. Data will be correlated with clinical parameters of IBD and polymorphisms in TLR4. In the second aim, we will define the mechanism(s) of TLR4-mediated IgA production by evaluating gene and kinase activation and the clonality of the B cell response through targeted sequencing of the V region of IgA. The data generated in this proposal will have direct clinical relevance by identifying critical pathways for the development of improved treatments that change the course of disease. Further, by defining how B cells recognize LPS, we will also advance the field of genome-wide association studies, human B cell immunology, and mechanism(s) of the human-microbial interface implicated in many chronic diseases. PUBLIC HEALTH RELEVANCE: Inflammatory bowel disease (IBD) is a chronic gastrointestinal disease for which we have no cure. In this proposal, we hope to identify new ways in which our immune system interacts with the friendly bacteria of the intestines in hopes of developing treatments for patients with IBD.