Determining the role of oxysterols in lymphocyte homing to lymph nodes in homeostasis and inflammation

确定氧甾醇在淋巴细胞归巢至淋巴结的稳态和炎症中的作用

• 批准号: 10677408
• 负责人: KEVIN Y CHEN
• 金额: $ 3.98万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677408
• 关键词: 25-hydroxycholesterol; Adhesions; Adoptive Transfer; Antibody Response; Antigens; Area; Autoimmunity; Automobile Driving; B-Cell Activation; B-Lymphocytes; BLR1 gene; Basal lamina; Binding; Biological Assay; Blood; Blood Circulation; Blood capillaries; CCL19 gene; CCL21 gene; CD4 Positive T Lymphocytes; CXCR4 gene; Capillary Endothelial Cell; Carbohydrates; Cells; Cellular Immunity; Chemotactic Factors; Cholesterol; Chronic; Circulation; Complement; Complement 3d Receptors; Coupled; Data; Defect; Dendritic Cells; Dependence; Development; Disease; Drops; Endothelium; Enzymes; Exhibits; Extravasation; Fibroblasts; G-Protein-Coupled Receptors; Generations; Genes; Genetic Transcription; Goals; High Endothelial Venule; Homeostasis; Homing; Hour; Human Herpesvirus 4; Hydroxylation; Immune; Immunization; Immunologic Surveillance; Immunotherapy; In Vitro; Infection; Inflammation; Interferons; Invaded; Knock-out; Knockout Mice; Knowledge; Ligands; Lymphatic; Lymphocyte; Lymphocytic Infiltrate; Lymphoid Tissue; Malignant Neoplasms; Mediating; Memory; Migration Assay; Mixed Function Oxygenases; Mus; Organ; Peripheral; Peyer' s Patches; Process; Property; Publishing; Reporting; Role; Scanning; Signal Transduction; Site; Skin; Stains; Stimulus; Stromal Cells; Surface; T-Lymphocyte; Testing; Toll-like receptors; Virus Diseases; Visualization; Work; acute infection; adaptive immune response; antigen test; cell motility; cell type; chemokine; design; draining lymph node; experimental study; fMet-Leu-Phe receptor; improved; in vivo; intravital imaging; lymph nodes; lymphatic circulation; migration; novel; novel therapeutics; pathogen; postcapillary venule; protein expression; receptor; reconstitution; recruit; selective expression; sialomucins; trafficking; tumor; tumorigenesis; two photon microscopy; vascular addressins

PROJECT SUMMARY/ABSTRACT Lymphocyte entry into lymph nodes (LNs) from blood is a key homeostatic process for efficient initiation of an adaptive immune response to pathogens. Naïve B and T cells traffic through LNs to scan for foreign antigens delivered to and concentrated in these organs from diverse sites of potential infection. If no antigens are encountered, lymphocytes exit LNs into lymphatic circulation before returning to the blood and beginning the cycle again. This constant lymphocyte recirculation requires large scale extravasation into lymphoid tissue under non-inflammatory conditions, and the specialized vessels supporting this process in LNs are called high endothelial venules (HEVs). HEVs express and luminally present several vascular ‘addressins’ and chemokines that together support a multi-step adhesion cascade for lymphocyte entry into LNs. The signals promoting lymphocyte arrest on HEV walls are well-elucidated, but the chemoattractants driving transmigration across the endothelium are not fully understood. Elucidating the ligands and receptors that mediate this fundamental step of entry is essential for understanding and manipulating immune cell trafficking in diseases such as cancer and autoimmunity. The enzyme Ch25h, which produces 25-hydroxycholesterol (25HC) from cholesterol, is highly and selectively expressed in HEVs compared to capillary endothelium in LNs. Furthermore, Cyp7b1, which hydroxylates 25HC to generate 7𝛼𝛼,25-dihydroxycholesterol (7,25HC), is expressed in stromal cells surrounding LN HEVs. 7,25HC is a potent ligand for EBI2, a GPCR that helps guide activated B cell movement within LNs and is also highly expressed in naïve B and T cells. The capability for oxysterol synthesis by HEVs suggests EBI2 and 7,25HC may play a role in mediating lymphocyte entry into LNs. We hypothesize that a gradient of 7,25HC across HEVs supports post-adhesion transendothelial migration of naïve B and T cells. In preliminary studies, EBI2 KO B cells and CD4 T cells displayed a recruitment defect to LNs in adoptive transfers, and a similar homing defect was evident in mice lacking Ch25h or Cyp7b1. Furthermore, dependency on EBI2 for lymphocyte entry into LNs increased in a viral infection setting. We hypothesize this is due to elevated 7,25HC levels and reduced chemokine levels in inflamed LNs. From these results, we aim to determine the step of LN entry that EBI2 directs via a combination of in vivo homing assays, intravital imaging, and in vitro migration assays (Aim 1). In addition, we will examine EBI2’s role in maintaining lymphocyte homing in inflamed LNs by characterizing the changes, and signals mediating these changes, in LN oxysterol and chemokine levels during infection (Aim 2). Overall, this work will define a novel chemoattractant driving lymphocyte migration into LNs and advance our understanding of the transmigration process. As well as their role in LNs, HEVs form in tumor associated tertiary lymphoid tissues and at sites of chronic inflammation. Our findings could inform development of novel therapeutics that modulate lymphocyte infiltration in these disease settings.

项目摘要/摘要 血液中淋巴细胞进入淋巴结（LNS）是有效启动的关键稳态过程 对病原体的自适应免疫反应。幼稚的B和T细胞通过LNS流动以扫描外国抗原 从潜在感染的不同部位传递到这些器官中并集中在这些器官中。如果没有抗原 遇到的，淋巴细胞退出淋巴循环，然后返回血液并开始 再次循环。这种恒定的淋巴细胞再循环需要将大规模渗入到淋巴组织中 非炎症条件和支持该过程的专业船只称为高 内皮静脉（HEVS）。 HEVS Express并发表了几个血管“地址”和趋化因子 这共同支持多步胶级联反应，以进入淋巴细胞进入LNS。信号促进 在HEV墙上停滞的淋巴细胞被良好检测，但趋于跨越移民的化学吸引力 内皮尚未完全理解。阐明调解这一基本步骤的配体和接收器 进入对于理解和操纵癌症和疾病中的免疫细胞运输至关重要 自身免疫性。从胆固醇产生25-羟基胆固醇（25hc）的酶CH25H高度高 与LNS中的毛细血管内皮相比，在HEV中有选择地表达。此外，CYP7B1 羟基盐25HC生成7𝛼𝛼，25-二羟基胆固醇（7,25HC），在周围的基质细胞中表达 LN HEVS。 7,25HC是EBI2的潜在配体，EBI2是一种GPCR，可帮助指导LNS中激活的B细胞运动 并且在幼稚的B和T细胞中也高度表达。 HEV的氧蛋白酶合成的能力表明 EBI2和7,25HC可能在介导淋巴细胞进入LNS中发挥作用。我们假设是 跨HEV的7,25HC支持幼稚的B和T细胞的粘附后跨内皮迁移。在初步 研究，EBI2 KO B细胞和CD4 T细胞在适应性转移中显示出招募缺陷，A 类似的归巢缺陷是缺乏CH25H或CYP7B1的小鼠的证据。此外，对EBI2的依赖 在病毒感染环境中LNS增加。我们假设这是由于7,25hc升高 发炎的LN的水平和降低的趋化因子水平。从这些结果中，我们旨在确定LN的步骤 EBI2通过体内归巢测定，插入式成像和体外迁移的组合指示的条目 测定（目标1）。此外，我们将研究EBI2在维持LNS中淋巴细胞归巢中的作用 在LN氧化酚和趋化因子水平中表征这些变化，并信号介导这些变化 感染（目标2）。总体而言，这项工作将定义一种新型的趋化剂驱动淋巴细胞迁移到LNS 并提高我们对传输过程的理解。以及它们在LN中的作用，HEV在肿瘤中形成 相关的三级淋巴组织和慢性感染部位。我们的发现可以告知发展 在这些疾病环境中调节淋巴细胞浸润的新型疗法。