Lymphotoxin-beta Receptor Pathway in Sjogren's Syndrome: Role of Dendritic Cells

干燥综合征中的淋巴毒素-β 受体途径：树突状细胞的作用

• 批准号: 9275371
• 负责人: ROY A FAVA
• 金额: --
• 依托单位: WHITE RIVER JUNCTION VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-10-01 至 2017-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9275371
• 关键词: Ablation; Advanced Development; Affect; Alpha Cell; Animal Model; Antibodies; Autoimmune Process; Automobile Driving; Awareness; B-Lymphocytes; Backcrossings; Biological Assay; Bone Marrow; CCL19 gene; CCL21 gene; CXCL13 gene; Caring; Cell Separation; Cells; Chimera organism; Contracts; Data; Dendritic Cells; Development; Diagnostic; Disease; Disease Progression; Diverticulitis; Eligibility Determination; Enzyme-Linked Immunosorbent Assay; Event; Exocrine Glands; Eye; Eye diseases; Fluorescence Microscopy; Gene Expression; Gland; Goals; Healthcare; Helicobacter Pylori-Associated Gastritis; Histology; ITGAX gene; Immune response; In Situ Hybridization; Inbred NOD Mice; Interruption; Kinetics; Lacrimal gland structure; Leukocytes; Ligands; Lymphocyte; Lymphoid Follicle; Lymphoid Tissue; Measurement; Messenger RNA; Methods; Modeling; Mouse Strains; Mus; Nature; Pathogenesis; Pathology; Pathway interactions; Phenotype; Play; Population; Production; Recruitment Activity; Reporting; Rheumatoid Arthritis; Risk; Role; Salivary Glands; Services; Signal Transduction; Sjogren' s Syndrome; Source; Speed; Testing; Therapeutic; Therapeutic Effect; Time; Transgenic Mice; Transgenic Organisms; Translations; Tumor Necrosis Factor-Beta; Veterans; Vision; Woman; Women' s Health; cell type; chemokine; chemokine receptor; diphtheria toxin receptor; eye dryness; gene product; high risk; inhibitor/antagonist; insight; leukemia; lymph nodes; lymphotoxin beta receptor; man; men; mouse model; neutralizing monoclonal antibodies; novel therapeutics; pre-clinical trial; public health relevance; simple sequence length polymorphism; therapeutic target

DESCRIPTION (provided by applicant): We have demonstrated that LTBR-pathway blockade has therapeutic effects in NOD mice, a popular model of Sjogren's syndrome. Affymetrix analysis, ELISA and q-PCR analyses showed that CXCL13 increased with disease progression in glands, but was reduced ~5-fold by LTBR-pathway inhibition. Our own preliminary data and a recent report that LTass2 expressed by DC drives most if not all of the LTBR-dependent events in lymph nodes [1] suggest that DC play a critical role in induction of HEV, CXCL13 and gland pathology in Sjogren's syndrome. Until now the evidence connecting DC, HEV, CXCL13 and disease progression is only circumstantial but herein we propose to deplete DC, or to "silence" LT-ass2 expression by DC, and thus definitively verify or refute this concept. We also will determine if dendritic cells are the direc source of CXCL13, a chemokine well known to drive ectopic lymphoid follicles and B-lymphocyte accumulation in diseased glands of mice and man, and evaluate the result of CXCL13 neutralization on disease progression. Our primary goal is to determine definitively whether or not expression of LTass2 by a dendritic cell subset orchestrates much of the pathology in exocrine glands that is associated with reduced gland function and altered gene expression. Aim 1. Determine if direct neutralization of CXCL13 might serve as a therapy in Sjogren's syndrome by interrupting ectopic follicle formation and B-cell accumulation in diseased glands. A neutralizing monoclonal antibody will be used to treat NOD mice in a therapeutic manner. Histology, immunolocalization studies, real- time PCR, Affymetrix chip analyses, direct measurement of the rate of salivary and lacrimal gland secretions and ocular integrity scores will be used to assess the efficacy of direct CXCL13 ablation as a therapeutic target in the NOD mouse model of Sjogren's syndrome. Off target effects on immune responses will be examined. Aim 2. Determine the sources of CXCL13 in lacrimal glands using CXCL13-GFP mice backcrossed to NOD background and/or in situ hybridization and isolation of infiltrating cells by cell-sorting with a FacsAria III. Determine the kinetics, localization, abundance and phenotype of cd11c+ dendritic cell populations of glands of NOD during disease development and progression. Isolation by fluorescent cell sorting, multicolor FACS analyses, real-time PCR, ELISA assays, multicolor fluorescence microscopy and other methods will be employed to define DC populations present in diseased glands at various stages of disease and determine if these dendritic cells are a direct source of CXCL13 and express LTass2, the ligand for LTBR. Aim 3. Determine whether depletion of all CD11c DC or DC-specific ablation of certain gene products (lymphotoxin-alpha, CXCL13) alters the onset or progression of disease or alters the size and nature of the leukocyte infiltrates in in salivary and lacrimal glands in transgenic NOD mouse strains we will create by backcrossing. We will utilize existing mouse strains, namely (1) CD11c-diptheria toxin receptor-NOD mice (NOD.FVB-Tg(Itgax-DTR/EGFP)57Lan/JdkJ), (2) NOD mice, and (3) bone marrow chimeras of various NOD- background transgenic mouse strains. We will create strains in 1.5 years by Simple Sequence Length Polymorphism-assisted "speed-backcrossing" (cd11c-DTR/NOD, LTalpha-/-/NOD, CXCL13-/-/NOD). These will be used to determine whether dendritic cells provide critical signals via the lymphotoxin-beta receptor pathway to drive HEV development, leukocyte accumulation, chemokine production and gland hypofunction.

描述（由申请人提供）： 我们已经证明 LTBR 通路阻断对 NOD 小鼠（一种流行的干燥综合征模型）具有治疗作用。 Affymetrix 分析、ELISA 和 q-PCR 分析表明，CXCL13 随着腺体疾病进展而增加，但由于 LTBR 途径抑制而减少约 5 倍。我们自己的初步数据和最近的一份报告表明，DC 表达的 LTass2 驱动了淋巴结中大多数（如果不是全部）LTBR 依赖性事件 [1]，表明 DC 在诱导 HEV、CXCL13 和干燥综合征的腺体病理学中发挥着关键作用。到目前为止，连接 DC、HEV、CXCL13 和疾病进展的证据只是间接的，但在此我们建议消除 DC，或“沉默”DC 的 LT-ass2 表达，从而明确验证或反驳这一概念。我们还将确定树突状细胞是否是 CXCL13 的直接来源，CXCL13 是一种众所周知的驱动小鼠和人类患病腺体异位淋巴滤泡和 B 淋巴细胞积累的趋化因子，并评估 CXCL13 中和对疾病进展的结果。我们的主要目标是明确确定树突状细胞亚群的 LTass2 表达是否协调了外分泌腺中与腺体功能降低和基因表达改变相关的大部分病理学。目标 1. 确定 CXCL13 的直接中和是否可以通过中断病变腺体中的异位卵泡形成和 B 细胞积累来治疗干燥综合征。中和单克隆抗体将用于以治疗方式治疗 NOD 小鼠。组织学、免疫定位研究、实时 PCR、Affymetrix 芯片分析、唾液和泪腺分泌率的直接测量以及眼部完整性评分将用于评估直接 CXCL13 消融作为干燥综合征 NOD 小鼠模型治疗靶点的功效。将检查对免疫反应的脱靶效应。目标 2. 使用与 NOD 背景回交的 CXCL13-GFP 小鼠和/或通过使用 FacsAria III 进行细胞分选来原位杂交和分离浸润细胞来确定泪腺中 CXCL13 的来源。确定 NOD 腺体 cd11c+ 树突状细胞群在疾病发生和进展过程中的动力学、定位、丰度和表型。通过荧光细胞分选、多色 FACS 分析、实时 PCR、ELISA 测定、多色荧光显微镜和其他方法进行分离，将用于定义疾病不同阶段病变腺体中存在的 DC 群体，并确定这些树突状细胞是否是 CXCL13 的直接来源并表达 LTass2（LTBR 的配体）。目标 3. 确定所有 CD11c DC 的耗竭或某些基因产物（淋巴毒素-α、CXCL13）的 DC 特异性消融是否会改变疾病的发作或进展，或改变我们将通过回交产生的转基因 NOD 小鼠品系中唾液和泪腺中白细胞浸润的大小和性质。我们将利用现有的小鼠品系，即（1）CD11c-白喉毒素受体-NOD小鼠（NOD.FVB-Tg（Itgax-DTR/EGFP）57Lan/JdkJ），（2）NOD小鼠，和（3）各种NOD背景转基因小鼠品系的骨髓嵌合体。我们将通过简单序列长度多态性辅助的“快速回交”（cd11c-DTR/NOD、LTalpha-/-/NOD、CXCL13-/-/NOD）在1.5年内创建菌株。这些将用于确定树突状细胞是否通过淋巴毒素-β受体途径提供关键信号来驱动 HEV 发育、白细胞积累、趋化因子产生和腺体功能减退。