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: 8967088
• 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/8967088
• 关键词: Ablation; Advanced Development; Affect; Animal Model; Antibodies; Autoimmune Process; Automobile Driving; 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; 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; 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; Reporting; Rheumatoid Arthritis; Risk; Role; Salivary Glands; Services; Signal Transduction; Sjogren' s Syndrome; Source; Speed; Staging; Testing; Therapeutic; Therapeutic Effect; Thinking; 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）： 我们已经证明了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。亲和素分析、ELISA和q-PCR分析显示，CXCL13随着腺体中疾病的进展而增加，但通过LTBR途径抑制而减少~5倍。我们自己的初步数据和最近的报告表明，DC表达的LTass2驱动淋巴结中的大多数（如果不是全部的话）LTBR依赖性事件[1]，这表明DC在诱导HEV、CXCL13和干燥综合征中的腺体病理中起关键作用。到目前为止，将DC、HEV、CXCL13和疾病进展联系起来的证据只是间接的，但在本文中，我们提出耗尽DC，或通过DC "沉默" LT-ass2表达，从而明确地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是一种众所周知的趋化因子，可驱动小鼠和人类患病腺体中的异位淋巴滤泡和B淋巴细胞积聚，并评估CXCL13中和疾病进展的结果。我们的主要目标是明确确定是否由树突状细胞亚群的LTass2的表达编排与腺体功能降低和基因表达改变相关的外分泌腺中的大部分病理。目标1.确定CXCL13的直接中和是否可以通过中断异位卵泡形成和患病腺体中的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。将使用组织学、免疫定位研究、真实的-时间PCR、Affyssin芯片分析、唾液腺和泪腺分泌速率的直接测量以及眼完整性评分来评估直接CXCL 13消融作为舍格伦综合征NOD小鼠模型中的治疗靶标的功效。将检查对免疫应答的脱靶效应。目标2.使用与NOD背景回交的CXCL13-GFP小鼠和/或原位杂交并通过FacsAria III细胞分选分离浸润细胞来确定泪腺中CXCL13的来源。确定疾病发展和进展过程中NOD腺体cd11c+树突状细胞群的动力学、定位、丰度和表型。将采用荧光细胞分选、流式细胞术分析、实时PCR、ELISA测定、荧光显微镜和其他方法进行分离，以确定在疾病的各个阶段存在于患病腺体中的DC群体，并确定这些树突状细胞是否是CXCL 13的直接来源并表达LTass2（LTBR的配体）。目标3.在我们将通过回交建立的转基因NOD小鼠品系中，确定所有CD11c DC的耗竭或某些基因产物（CXCL13）的DC特异性消融是否会改变疾病的发作或进展，或改变唾液腺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。我们将在1.5年内通过简单序列长度多态性辅助的"快速回交"（cd11c-DTR/NOD，LT alpha-/-/NOD，CXCL13-/-/NOD）创建菌株。这些将用于确定树突状细胞是否通过光敏素β受体途径提供关键信号，以驱动HEV发育，白细胞积累，趋化因子产生和腺体功能减退。