Intestinal Lymphocyte Trafficking

肠道淋巴细胞贩运

• 批准号: 9206459
• 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/9206459
• 关键词: 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; Dangerousness; Data; Diarrhea; Enhancers; Eragrostis; FFAR2 gene; Flow Cytometry; Frequencies; GATA3 gene; GPR15 gene; Grant; Gut associated lymphoid tissue; Helper-Inducer T-Lymphocyte; Homeostasis; Homing; Human; Immune; Immune response; Immunity; Immunize; Immunobiology; Immunotherapy; In Vitro; Inflammation; Inflammatory; Injectable; Intestines; Knock-in; Knockout Mice; Lamina Propria; Lectin; Ligands; Lymphocyte; Lymphocyte Homing Receptors; Lymphocyte Subset; Mediating; Modeling; Molecular; Mucosal Immune Responses; Mus; Mutagenesis; Mutation; Pathogenicity; Pathway interactions; Pattern; Play; Proteins; Recruitment Activity; 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; desensitization; drug discovery; experimental study; in vivo; insight; man; microbiome; migration; novel; pathogen; receptor; repaired; 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.

这笔赠款将继续支持我们正在进行的分子发现和功能表征 淋巴细胞归巢至胃肠道的机制。我们提出了以下具体目标： 目的1.GPR15在肠道免疫生物学中的作用及其机制 不同物种表达差异的影响。我们发现效应性T细胞表达GPR15 (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 在人类和小鼠中发现了GPR15表达的主要差异，这可能是 炎症、疫苗接种反应和肠道免疫方面的差异。我们认为异常的GPR15 在小鼠中的表达是由于缺乏调节其他物种中GPR15的GATA3共识结合位点。 我们将利用细胞周期图和流式细胞术来比较GPR15在肠道归巢中的表达和调控 小鼠与人的淋巴细胞亚群。我们将使用标准的报告分析，定点突变和 ChIP-SEQ数据来检验Th和Treg亚集表达的物种差异是 受GATA3与小鼠GPR15增强子结合丧失的控制。最重要的是，我们建议分析 人源化GPR15表达模式对小肠T细胞亚群和T细胞亚群的影响 体内的免疫反应。在自然人类序列控制(BAG)下驱动GPR15的小鼠 转基因；已经产生了5个方正品系)，或者其中引入了GATA3 TFBS或 通过靶向突变或敲击策略修复小鼠增强子序列，将在以下方面进行研究 动态平衡和炎症/免疫状态。GPR15基因表达的改变对人类的影响 将评估肠道淋巴细胞亚群频率和免疫反应的模式。重要意义： 这些研究将直接测试GATA3 TFBS突变在人和小鼠差异中的假设作用 在GPR15的表达和肠道免疫生物学中。如果，如假设的那样，具有人源化Ggr15的小鼠 “增强剂”展示了更接近人类的结肠免疫生物学，研究将有 这对我们理解结肠炎和IBD的药物开发具有重要意义。 目的2：明确CD22作为一种新的肠道淋巴细胞归巢受体的作用和意义 我们的实验表明，CD22在Peyer‘s斑块中作为HEV的B细胞归巢受体发挥作用。短的 将使用术语归巢分析来证实这一点，并对CD22在归巢中的假设作用进行批判性评估 滤泡T辅助细胞亚群也转化为GALT。我们还将评估CD22在淋巴细胞中的作用 向固有层和结肠补片募集。最后，我们观察到显著依赖于SFGA/6的残留 CD22缺陷淋巴细胞归巢到PP：我们假设这可能是由密切相关的 B细胞凝集素，SiglecG，与类似的碳水化合物结合。我们建议使用现成的SiglecG，如果 表明，CD22/SiglecG双KO小鼠来验证这一假说。意义：这一目标将阐明 GALT的特异性B细胞归巢机制，有助于粘膜特化的机制 免疫反应。此外，CD22是自身免疫性疾病的新兴靶点：了解其在 淋巴细胞重新聚集到胃肠道将提供对肠道免疫反应的重要洞察，并且 可能被证明对避免CD22靶向免疫治疗环境中的感染性副作用至关重要。 目的3：GPR43在肠道免疫和免疫动态平衡中的作用：Th17与Treg动态平衡 新型蛋白质配体的鉴定及功能研究。根据我们的初步研究，我们建议使用 GPR43KO/WT混合骨髓嵌合体确定受体在微生物组和短链中的作用 依赖脂肪酸(SCFA)对肠道中Th17和其他效应和调节性T细胞的控制。此外, 我们将阐明GPR43与血管生成素-2(Ang2)的相互作用。在DSS结肠炎模型Ang2中，像GPR43一样， 减轻症状(结肠缩短、腹泻)的严重程度，尽管影响复杂。我们将使用 确定Ang2-GPR43结合和体外黏附的特异性/亲和力的标准生化分析 实验确定Ang2在肠道免疫细胞黏附触发和趋化中的作用。自GPR43以来 调节细胞凋亡，我们还将阐明Ang2-GPR43相互作用对免疫细胞存活和 对促炎信号的反应。意义：GPR43缓解胃肠道炎症， 抑制结肠炎。它的已知配体是肠道微生物群中的短链脂肪酸(SOFA)。 Ang2作为蛋白质配体和GPR43的鉴定表明，有可能有更多的途径 依赖GPR43的肠道免疫反应的控制。由于Ang2调节血管生成和血管 稳定性，其在肠壁修复过程中的诱导可能通过GPR43在缺乏GPR43的情况下抑制炎症 危险的病原体入侵。 其他计划：我们将测试确定的其他候选贩运机制的假设作用 在我们的微阵列中。将静脉注射抗CD63和Bsti单抗以评估对淋巴细胞归巢的影响 高尔特和肠壁。为了检验GPR183介导淋巴细胞渗出的假说 我们将比较体内跨内皮细胞迁移和归巢 正常的B和T细胞对GPR183缺乏或脱敏的淋巴细胞的能力。 意义：这些研究有可能确定其他机制和治疗靶点 肠道淋巴细胞转运的调控。