Project 4 - TLR2-mediatecl control of inflammatory arthritis

类风湿关节炎(RA)是一种使人衰弱的慢性炎症性疾病，影响全球约1%的人口。目前对RA所涉及的分子和细胞事件的了解有限，尽管先天免疫反应被认为是RA患者慢性关节炎症和骨丢失发生的关键。胶原诱导性关节炎(CIA)是目前最常用的RA动物模型之一，对CIA免疫发病机制的深入了解有助于为人类RA的发病机制提供重要信息。Toll样受体(TLRs)识别不同的病原体相关分子模式(PAMPs)，并对多种病原体实施后续免疫反应，先天免疫反应是Toll样受体的第一道防线。事实上，通过先天免疫细胞上的TLRs信号诱导产生关键的细胞因子，这些细胞因子负责T辅助(Th)细胞极化和随后的适应性免疫反应。用于诱导CIA的分枝杆菌佐剂已知富含这些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，树突状细胞、巨噬细胞和B细胞等抗原提呈细胞对PAMP介导的信号作出反应并产生炎性细胞因子。在临床上，抑制这些细胞因子可以显著改善症状，并可以有效地防止RA疾病的进展。最近，产生标志性细胞因子IL-17的Th17细胞与RA的发病机制有关。我们的初步发现表明，IL-6和IL-17介导了CIA的许多慢性炎症变化，包括骨丢失，而TLR2缺陷小鼠(TLR2-K0)相对对这一过程具有抵抗力。我们假设TLR2信号在免疫反应激活中发挥关键作用，免疫反应激活触发CIA发病机制，导致关节炎症和骨丢失。我们将通过以下具体目标来验证这一假说：1)使用流式细胞仪、ImageStream技术、qRT-PCR和ELISA法评估TLR2依赖的IL-17产生细胞的诱导在CIA发病机制中的作用；2)表征TLR2表达在CIA发生发展过程中滑膜细胞反应中的作用；3)在骨髓来源的树突状细胞和滑膜细胞中鉴定依赖TLR2的磷酸蛋白(使用磷酸蛋白质组学)，作为识别宿主来源的CIA“激活物”的手段。近期，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.