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

描述(由申请人提供)： 我们已经证明了LTbR通路的阻断对NOD小鼠有治疗作用，NOD小鼠是一种流行的干燥综合征模型。Affymetrix分析、ELISA和Q-PCR分析表明，CXCL13在腺体中随着疾病的进展而增加，但由于LTbR途径的抑制，CXCL13的表达减少了约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LTbR依赖的大部分事件[1]，表明DC在干燥综合征的HEV、CXCL13和腺体病理的诱导中发挥了关键作用。到目前为止，将DC、HEV、CXCL13与疾病进展联系起来的证据仅是间接的，但在此，我们建议耗尽DC，或通过DC“沉默”LT-ass2的表达，从而明确地验证或驳斥这一概念。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，可推动异位淋巴滤泡和B淋巴细胞在小鼠和人类病变腺体中聚集，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表达LTass2是否协调了与腺体功能降低和基因表达改变相关的外分泌腺的大部分病理。目的1.确定直接中和CXCL13是否可以通过阻断异位卵泡的形成和病变腺体中B细胞的积聚来治疗干燥综合征。一种中和性单抗将被用来治疗NOD小鼠。组织学、免疫定位研究、实时聚合酶链式反应、Affymetrix芯片分析、唾液和泪腺分泌率的直接测量以及眼睛完整性评分将用于评估直接CXCL13消融作为干燥综合征NOD小鼠模型的治疗靶点的有效性。我们将研究目标外对免疫反应的影响。目的：采用CXCL13-GFP小鼠与NOD背景回交和/或原位杂交及FacsAria III细胞分选分离浸润性细胞的方法，确定泪腺中CXCL13的来源，并测定NOD腺体中CD11c树突状细胞群在疾病发生发展过程中的动态、定位、丰度和表型。通过荧光细胞分选、多色流式细胞仪分析、实时聚合酶链式反应、酶联免疫吸附试验、多色荧光显微镜和其他方法进行分离，以确定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。目的3.通过回交建立转基因NOD小鼠品系，确定去除所有CD11c DC或DC特异性消融某些基因产物(淋巴毒素-α，CXCL13)是否会改变疾病的发生或发展，或改变唾液和泪腺中白细胞的大小和性质。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，(3)各种NOD背景转基因小鼠的骨髓嵌合体。我们将在1.5年内通过简单序列长度多态辅助的“快速回交”(CD11c-DTR/NOD，LTalpha-/-/NOD，CXCL13-/-/NOD)创造菌株。这些将被用来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的发展、白细胞聚集、趋化因子的产生和腺体功能低下。

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.