Intestinal Lymphocyte Trafficking

肠道淋巴细胞贩运

• 批准号: 8849684
• 负责人: EUGENE C BUTCHER
• 金额: $ 34.73万
• 依托单位: PALO ALTO VETERANS INSTIT FOR RESEARCH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8849684
• 关键词: ANGPTL2 gene; Adhesions; Adverse effects; Angiopoietin-2; Apoptosis; Autoimmune Diseases; Avidity; B-Lymphocytes; Binding; Binding Sites; Biochemical; Biological Assay; Blood Vessels; Bone Marrow; CD22 gene; CD4 Positive T Lymphocytes; Carbohydrates; Cell Adhesion; Cell Survival; ChIP-seq; Chemotaxis; Chimera organism; Colitis; Colon; Complex; Consensus; Data; Diarrhea; Enhancers; Eragrostis; Flow Cytometry; Frequencies; GATA3 gene; GPR15 gene; Grant; Gut associated lymphoid tissue; Helper-Inducer T-Lymphocyte; Homeostasis; Homing; Human; Immune; Immune response; Immunity; Immunobiology; Immunotherapy; In Vitro; Inflammation; Inflammatory; Intestines; Knock-in; Knockout Mice; Lamina Propria; Lectin; Ligands; Lymphocyte; Lymphocyte Homing Receptors; Lymphocyte Subset; Mediating; Modeling; Molecular; Mucosal Immune Responses; Mus; Mutagenesis; Mutation; Pathway interactions; Pattern; Play; Proteins; Regulation; Regulatory T-Lymphocyte; Reporter; Residual state; Role; Severities; Signal Transduction; Site-Directed Mutagenesis; Specificity; Structure of aggregated lymphoid follicle of small intestine; Symptoms; T-Lymphocyte; Testing; Transgenic Organisms; Vaccination; Volatile Fatty Acids; angiogenesis; base; drug discovery; in vivo; insight; man; microbiome; migration; novel; pathogen; receptor; repaired; research study; response; species difference; therapeutic target; trafficking

This grant will continue to support our ongoing discovery antd functional characterization of molecular mechanisms of lymphocyte homing to the Gl tract. We propose the following specific Aims: Aim 1. GPR15 in intestinal immunobiology: role in intestinal immune responses and mechanisms and effects of species specific differences in expression. We showed expression of GPR15 by effector T cells (Teff) in colitis, and demonstrated that this novel colon homing receptor can play a pathogenic role. We also discovered major differences in GPR15 expression in humans vs mice that have the potential to underlie differences in inflammation, vaccination responses, and intestinal immunity. We propose that aberrant GPR15 expression in mice is due to absence of GATA3 consensus binding sites that regulate GPR15 in other species. We will use CyTOF and flow cytometry to compare the expression and regulation of GPR15 on gut homing lymphocyte subsets in mouse vs man. We will use standard reporter assays, site directed mutagenesis and Chip-seq data to test the hypothesis that the species differences in Th vs Treg subset expression are controlled by loss of GATA3 binding to the mouse Gpr15 enhancer. Most importantly, we propose to analyze the effects of "humanized" Gpr15 expression patterns on small intestinal Teff vs Treg subsets, and on intestinal immune responses in vivo. Mice in which GPR15 is driven under native human sequence control (BAG transgenics; 5 founder lines have been generated already), or in which GATA3 TFBS are introduced or repaired in the mouse enhancer sequence by targeted mutagenesis or knockin strategies, will be studies under homeostatic and inflamed/immunized conditions. The effects of altered, 'human-like' GPR15 expression patterns on intestinal lymphocyte subset frequencies and immune responses will be assessed. Significance: The studies will directly test the hypothesized role of GATA3 TFBS mutations in human vs mouse differences in GPR15 expression and in intestinal immunobiology. If, as hypothesized, mice with a "humanized Ggr15 enhancer" display colon immunobiology that much more closely reflects that of humans, the studies will have fundamental implications for our understanding of colitis and for drug discovery for IBD. Aim 2: To define the role and significance of CD22 as a novel intestinal lymphocyte homing receptor Our experiments show that CD22 functions as a B cell homing receptor for HEV in Peyer's patches. Short term homing assays will be used to confirm this, and to assess critically a hypothesized role of CD22 in homing of follicular T helper cell subsets to GALT as well. We will also assess the role of CD22 in lymphocyte recruitment to the lamina propria and colon patches. Finally, we observe significant residual Sfga/6-dependent homing of CD22-deficient lymphocytes to PP: we hypothesize that this may be mediated by the closely related B cell lectin, SiglecG, which binds similar carbohydrates. We propose to use readily available SiglecG and if indicated, CD22/SiglecG double KO mice to test this hypothesis. Significance: This Aim will elucidate specific B cell homing mechanisms for GALT, mechanisms that contribute to the specialization of mucosal immune responses. Moreover, CD22 is an emerging target for autoimmune diseases: Understanding its role in lymphocyte recruitment to the Gl tract will provide important insight into intestinal immune responses, and could prove critical to avoid infectious side effects in settings of CD22 targeted immunotherapy. Aim 3: GPR43 in intestinal immunity and immune homeostasis: role in Th17 vs Treg homeostasis, and identification and function of novel protein ligands. Based on our preliminary studies, we propose to use GPR43 KO /WT mixed bone marrow chimeras to define the role of the receptor in microbiome- and short chain fatty acid (SCFA)-dependent control of Th17 and other effector and regulatory T cells in the gut. In addition, we will elucidate GPR43 interactions with angiopoietin-2 (Ang2). In the DSS colitis model Ang2, like GPR43, reduces the severity of symptoms (colon shortening, diarrhea), although the effects are complex. We shall use standard biochemical assays to define the specificity/avidity of Ang2-GPR43 binding, and in vitro adhesion assay to define the role of Ang2 in intestinal immune cell adhesion triggering and chemotaxis. Since GPR43 regulates apoptosis, we will also elucidate the effects of Ang2-GPR43 interaction on immune cell survival and responses to pro-inflammatory signals. Significance: GPR43 moderates inflammation in the Gl tract, dampening colitis. Its known ligands are short chain fatty acids (SOFA) from intestinal microflora. Identification of Ang2 as a protein ligand and for GPR43 suggests the potential for additional pathways to GPR43-dependent control of intestinal immune responses. Since Ang2 regulates angiogenesis and vascular stability, its induction during gut wall repair may act through GPR43 to suppress inflammation in the absence of dangerous pathogen invasion. Additional Plans: We will test the hypothesized roles of additional candidate trafficking mechanisms identified in our microarrays. Anti-CD63 and Bsti MAbs will be injected iv to assess effects on lymphocyte homing to GALT and gut wall. To test the hypothesis that GPR183 mediates lymphocyte diapedesis in response to a trans-HEV gradient of its oxysterol ligands, we will compare the in vivo transendothelial migration and homing ability of normal B and T cells to those of lymphocytes in which GPR183 is deficient or desensitized. Significance: These studies have the potential to define additional mechanisms and therapeutic targets for regulation of intestinal lymphocyte trafficking.

这项资助将继续支持我们正在进行的发现和分子的功能表征， 淋巴细胞归巢到胃肠道的机制。我们提出以下具体目标： 目标1.肠道免疫生物学中的GPR 15：在肠道免疫应答和机制中的作用， 物种特异性表达差异的影响。我们发现效应T细胞表达GPR 15， （Teff）在结肠炎中的作用，并证明这种新的结肠归巢受体可以发挥致病作用。我们也 发现人类与小鼠GPR 15表达的主要差异， 炎症、疫苗接种反应和肠道免疫的差异。我们认为异常的GPR 15 在小鼠中的表达是由于缺乏在其它物种中调节GPR 15的GATA 3共有结合位点。 我们将使用CyTOF和流式细胞术来比较GPR 15在肠道归巢中的表达和调控 我们将使用标准的报告基因测定、定点诱变和 Chip-seq数据用于检验Th与Treg亚群表达的种属差异是 通过GATA 3与小鼠Gpr 15增强子结合的丧失来控制。最重要的是，我们建议分析 “人源化”Gpr 15表达模式对小肠Teff相对于Treg亚群的影响，以及对小肠Teff相对于Treg亚群的影响。 体内免疫反应。其中GPR 15在天然人序列控制（BAG）下驱动的小鼠 转基因;已经产生了5个创始系），或其中引入了GATA 3 TFBS，或 通过靶向诱变或敲入策略在小鼠增强子序列中修复，将在 稳态和发炎/免疫状况。改变的“类人”GPR 15表达的影响 将评估肠淋巴细胞亚群频率和免疫应答的模式。重要性： 这些研究将直接测试GATA 3 TFBS突变在人类与小鼠差异中的假设作用。 GPR 15表达和肠道免疫生物学。如果像假设的那样，具有“人源化Ggr 15”的小鼠 增强子”显示结肠免疫生物学，更密切地反映了人类，研究将有 对我们理解结肠炎和IBD药物发现的基本意义。 目的2：明确CD 22作为一种新型肠道淋巴细胞归巢受体的作用和意义 我们的实验表明，CD 22的功能作为B细胞归巢受体的戊型肝炎病毒在派尔集合淋巴结。短 将使用术语归巢测定来证实这一点，并严格评估CD 22在归巢中的假设作用 滤泡性T辅助细胞亚群对GALT的影响。我们还将评估CD 22在淋巴细胞中的作用。 募集到固有层和结肠补丁。最后，我们观察到显著的残余Sfga/6依赖性。 CD 22缺陷型淋巴细胞归巢PP：我们假设这可能是由与PP密切相关的 B细胞凝集素，SiglecG，其结合类似的碳水化合物。我们建议使用现成的SiglecG，如果 如图所示，使用CD 22/SiglecG双KO小鼠来测试该假设。意义：本目标将阐明 GALT的特异性B细胞归巢机制，这种机制有助于粘膜上皮细胞的特化， 免疫反应。此外，CD 22是自身免疫性疾病的一个新兴靶点：了解其在 淋巴细胞向胃肠道的募集将提供对肠道免疫应答的重要了解， 可能证明在CD 22靶向免疫治疗的环境中避免感染性副作用至关重要。 目的3：肠道免疫和免疫稳态中的GPR 43：在Th 17 vs Treg稳态中的作用， 新蛋白质配体鉴定和功能。根据我们的初步研究，我们建议使用 GPR 43 KO /WT混合骨髓嵌合体，以确定受体在微生物组和短链中的作用 脂肪酸（SCFA）依赖性控制肠道中的Th 17和其他效应和调节T细胞。此外，本发明还提供了一种方法， 我们将阐明GPR 43与血管生成素-2（Ang 2）的相互作用。在DSS结肠炎模型Ang 2中，与GPR 43一样， 减轻症状的严重程度（结肠缩短，腹泻），尽管效果很复杂。我们将使用 标准生物化学测定，以确定Ang 2-GPR 43结合的特异性/亲合力和体外粘附 测定以确定Ang 2在肠免疫细胞粘附触发和趋化性中的作用。自GPR 43以来 调节细胞凋亡，我们还将阐明Ang 2-GPR 43相互作用对免疫细胞存活的影响， 对促炎信号的反应。意义：GPR 43缓和胃肠道中的炎症， 抑制结肠炎其已知的配体是来自肠道微生物菌群的短链脂肪酸（SOFA）。 Ang 2作为蛋白质配体和GPR 43的鉴定表明可能存在其他途径， GPR 43依赖性控制肠道免疫应答。由于Ang 2调节血管生成和血管生成， 稳定性，其在肠壁修复过程中的诱导可能通过GPR 43起作用，以在缺乏 危险的病原体入侵 其他计划：我们将测试所确定的其他候选贩运机制的假设作用 在我们的微阵列中。将静脉内注射抗CD 63和Bsti MAb以评估对淋巴细胞归巢的影响， GALT和肠壁。为了验证GPR 183介导淋巴细胞渗出的假设， 我们将比较其氧固醇配体的体内跨内皮迁移和归巢， 正常B和T细胞的能力与其中GPR 183缺陷或脱敏的淋巴细胞的能力相比。 意义：这些研究有可能确定其他机制和治疗靶点， 肠淋巴细胞运输的调节。