Project 4 - TLR2-mediatecl control of inflammatory arthritis

类风湿性关节炎（RA）是一种使人衰弱的慢性炎症性疾病，影响全世界约1%的人口。虽然固有免疫反应被认为对风湿性关节炎患者慢性关节炎症和骨质流失的发展至关重要，但目前对涉及RA的分子和细胞事件的了解有限。胶原诱导关节炎（CIA）是RA最常用的动物模型之一；更好地了解CIA的免疫发病机制有可能为人类RA的机制提供重要信息。先天免疫反应是toll样受体（TLRs）识别不同的病原体相关分子模式（PAMPs）并对各种病原体施加后续免疫反应的第一道防线。事实上，先天免疫细胞上通过tlr的信号传导诱导关键细胞因子的产生，这些细胞因子负责辅助性T细胞极化和随后的适应性免疫反应。已知用于CIA诱导的分枝杆菌佐剂富含这些tlr的配体，特别是TLR2。该项目的总体目标是更好地了解TLR2促进关节炎发展的机制。在早期RA中，抗原呈递细胞如树突状细胞、巨噬细胞和B细胞响应pamp介导的信号并产生炎症细胞因子。在临床上，抑制这些细胞因子可显著改善症状，并可有效预防RA疾病进展。最近，产生标志性细胞因子IL-17的Th17细胞被认为与RA的发病有关。我们的初步研究结果表明，IL-6和IL-17介导了CIA的许多慢性炎症变化，包括骨质流失，TLR2-缺陷小鼠（TLR2- k0）对这一过程相对有抵抗力。我们假设TLR2信号在触发CIA发病机制导致关节炎症和骨质流失的免疫反应激活中起关键作用。我们将通过以下具体目标来验证这一假设：1)利用流式细胞术、ImageStream技术、qRT-PCR和ELISA技术评估TLR2依赖性诱导il -17产生细胞在CIA发病中的作用；2)表征TLR2表达在CIA发生过程中滑膜细胞反应中的作用；3)鉴定骨髓来源的树突状细胞和滑膜细胞中tlr2依赖的磷酸化蛋白（使用磷酸化蛋白组学），作为鉴定宿主来源的CIA“激活因子”的手段。短期内，我们的研究结果将扩大对CIA的理解。从长远来看，这种性质的研究将有助于确定支持开发治疗类风湿性关节炎的新疗法的途径。

Rheumatoid arthritis (RA) is a debilitating chronic inflammatory disease affecting approximately 1% of the population worldwide. Present understanding of molecular and cellular events involved in RA is limited, though innate immune responses are considered critical to development of chronic joint inflammation and bone loss seen in RA patients. Collagen-induced arthritis (CIA) is one of the most commonly used animal models of RA; better understanding of the immunopathogenesis of CIA has the potential to provide important information on the mechanisms of human RA. The innate immune response is the first-line of defense in which Toll-like receptors (TLRs) recognize distinct pathogen-associated molecular patterns (PAMPs) and exert subsequent immune responses against a variety of pathogens. Indeed, signaling through TLRs on innate immune cells induces the production of critical cytokines responsible for T helper (Th) cell polarization and subsequent adaptive immune responses. Mycobacterial adjuvants used for CIA induction are known to be rich in ligands for these TLRs, particularly TLR2. The overall goal of this project is to better understand the mechanism by which TLR2 contributes to the development of arthritis. During early RA, antigenpresenting cells such as dendritic cells, macrophages, and B cells respond to PAMP-mediated signaling and produce inflammatory cytokines. Clinically, inhibition of these cytokines dramatically improves symptoms and can be effective in preventing RA disease progression. Recently, Th17 cells, which produce the signature cytokine IL-17, have been implicated in the etiopathogenesis of RA. Our preliminary findings show that IL-6 and IL-17 mediate many of the chronic inflammatory changes in CIA including bone loss, and that TLR2- deficient mice (TLR2-K0) are relatively resistant to this process. We hypothesize that TLR2 signaling plays a pivotal role in the immune response activation that triggers CIA pathogenesis leading to joint inflammation and bone loss. We will test this hypothesis via the following Specific Aims: 1) Assess the role of TLR2- dependent induction of IL-17-producing cells in the pathogenesis of CIA using flow cytometry, ImageStream technology, qRT-PCR, and ELISA; 2) Characterize the role of TLR2 expression in synoviocyte responses during the development of CIA; 3) Identify TLR2-dependent phosphoproteins in both bone marrow-derived dendritic cells and synoviocytes (using phosphoproteomics) as a means of identifying host-derived "activators" of CIA. Near term, our study results will broaden understanding of CIA. Long term, studies of this nature will help identify pathways to support development of novel therapies to treat RA.