Intestinal Lymphocyte Trafficking

肠道淋巴细胞贩运

基本信息

批准号：
9894708
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
项目类别：
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/9894708
关键词：
Adhesions Angiopoietin-2 Apoptosis Autoimmune Diseases Avidity B-Lymphocytes Binding Binding Sites Biochemical Biological Assay Blood Vessels Bone Marrow CD22 gene 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 Genetic Enhancer Element Grant Helper-Inducer T-Lymphocyte Homeostasis Homing Human Immune Immune response Immunity Immunize 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 Pathogenicity Pathway interactions Pattern Peyer&apos s Patches Play Proteins Regulation Regulatory T-Lymphocyte Reporter Residual state Role Severities Signal Transduction Site-Directed Mutagenesis Small Intestines Specificity Symptoms T-Lymphocyte Testing Transgenic Organisms Vaccination Volatile Fatty Acids angiogenesis base desensitization drug discovery effector T cell experimental study gut microbiota in vivo insight man microbiome migration novel pathogen receptor recruit repaired response side effect species difference therapeutic target trafficking

项目摘要

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.

该赠款将继续支持我们正在进行的发现ANTD功能特征淋巴细胞归巢的机制。我们提出以下具体目标：目标1。GPR15在肠道免疫生物学中：肠道免疫反应和机制的作用物种在表达上的特定差异的影响。我们通过效应T细胞显示了GPR15的表达（Teff）在结肠炎中，并证明这种新型结肠归巢受体可以发挥致病作用。我们也是发现了人类与小鼠的GPR15表达的主要差异，这些差异有潜力炎症，疫苗接种反应和肠道免疫的差异。我们建议异常的GPR15 小鼠中的表达是由于缺乏调节其他物种中GPR15的GATA3共有结合位点。我们将使用细胞和流式细胞仪比较肠道归巢上GPR15的表达和调节小鼠与男子中的淋巴细胞子集。我们将使用标准的记者分析，网站定向诱变和 CHIP-seq数据以检验以下假设：Th与Treg子集表达中的物种差异是由GATA3与小鼠GPR15增强子的结合丧失控制。最重要的是，我们建议分析 “人性化” GPR15表达模式对小肠Teff与Treg子集的影响以及肠体内免疫反应。 GPR15在本地人类序列控制下驱动的小鼠（袋转基因；已经生成了5个创始人线），或者引入GATA3 TFB或通过靶向诱变或敲除策略在小鼠增强子序列中修复，将是在稳态和发炎/免疫状况。改变的“类人” GPR15表达的影响将评估肠道淋巴细胞子集频率和免疫反应的模式。意义：研究将直接测试GATA3 TFBS突变在人与小鼠差异中的假设作用在GPR15表达和肠道免疫生物学中。如假设的那样，具有“人源性GGR15的小鼠” 增强剂“显示结肠免疫生物学，更紧密地反映了人类的结肠免疫生物学，研究将具有对我们对结肠炎和IBD的药物发现的理解的基本意义。目标2：将CD22的作用和意义定义为一种新型的肠道淋巴细胞寄养受体我们的实验表明，CD22在Peyer斑块中充当HEV的B细胞归巢受体。短的术语归巢测定法将用于确认这一点，并评估CD22在归宿中的批判性作用卵泡T辅助细胞集也到Galt。我们还将评估CD22在淋巴细胞中的作用招募了固有层和结肠斑块。最后，我们观察到明显的残留SFGA/6依赖性 CD22缺乏淋巴细胞的归纳为PP：我们假设这可能是由密切相关的 B细胞凝集素Siglecg，结合了相似的碳水化合物。我们建议使用随时可用的siglecg和指出CD22/Siglecg双KO小鼠以检验该假设。意义：这个目标将阐明 galt的特定B细胞归巢机制，有助于粘膜专业化的机制免疫反应。此外，CD22是自身免疫性疾病的新兴目标：了解其在淋巴细胞募集到GL区域将为肠道免疫反应提供重要的见解，并对于避免在CD22靶向免疫疗法的环境中避免传染性副作用而言至关重要。 AIM 3：GPR43在肠道免疫和免疫稳态中：在TH17与Treg稳态中的作用，以及新型蛋白质配体的鉴定和功能。根据我们的初步研究，我们建议使用 GPR43 KO /WT混合骨髓嵌合体以定义受体在微生物组和短链中的作用脂肪酸（SCFA）依赖性控制Th17以及肠道中的其他效应子和调节性T细胞。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，例如GPR43，尽管影响很复杂，但降低了症状的严重程度（结肠缩短，腹泻）。我们将使用标准的生化测定，以定义ANG2-GPR43结合的特异性/亲和力和体外粘附定义Ang2在肠道免疫细胞粘附触发和趋化性中的作用的测定。自GPR43以来调节凋亡，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响应。显着性：GPR43适度GL道中的炎症，抑制结肠炎。它已知的配体是肠道菌群的短链脂肪酸（沙发）。 ANG2鉴定为蛋白质配体，对于GPR43，这表明有可能采取其他途径 GPR43依赖于肠道免疫反应的控制。由于Ang2调节血管生成和血管稳定性，其在肠道壁修复过程中的诱导可能会通过GPR43作用，以抑制炎症危险的病原体入侵。其他计划：我们将测试已确定的其他候选贩运机制的假设作用在我们的微阵列中。将向抗CD63和BSTI mABS注入IV，以评估对淋巴细胞归巢的影响戈尔特和肠墙。为了测试GPR183介导淋巴细胞dipedesis的假设其氧化酚配体的反式HEV梯度，我们将比较体内跨内皮迁移和归巢正常B和T细胞对GPR183缺乏或脱敏的淋巴细胞的能力。意义：这些研究有可能定义其他机制和治疗靶标的肠道淋巴细胞运输的调节。