Citrullinated Antigens Bridge Innate and Adaptive Immunity in RA

描述(由申请人提供)： 类风湿关节炎(RA)是自身免疫病因学中最常见的炎症性关节疾病，影响着近1%的人群。可见，获得性免疫系统和先天免疫系统在类风湿关节炎的发病机制中具有重要作用。针对瓜氨酸蛋白的自身抗体是RA特有的，天然免疫受体如Toll样受体(TLRs)在诱导炎症和获得性免疫(如B细胞和T细胞激活)中发挥重要作用。最近发现的抗瓜氨酸蛋白抗体(ACPA)反应的靶点是瓜氨酸纤维蛋白原(CFB)。我们证明了含有CFB-IC的免疫复合体(ICs)存在于RA患者的血液和滑膜组织中，CFB免疫小鼠可诱导炎性关节炎。为什么CFB特异性自身免疫应该介导炎性关节炎仍不清楚。在研究CFB免疫引起炎性关节炎的机制时，我们观察到CFB本身是一种强大的天然佐剂，通过TLR4传递信号，并且CFB-IC可以通过共连接TLR4和Fc3受体(Fc3R)而协同增加巨噬细胞TNF的产生。我们的初步研究还表明，CFB能够以TLR4依赖的方式刺激B细胞的增殖。此外，我们发现在ACPA阳性的RA患者血液中浆母细胞--早期激活的B细胞--的水平显著升高。这些循环中的浆母细胞产生大量的ACPA，包括抗CFB抗体，这表明ACPA阳性的RA与持续的、抗原驱动的外周B细胞激活有关。最后，我们检测到瓜氨酸蛋白的存在，包括瓜氨酸纤维蛋白原，不仅在发炎的关节，而且在RA和非RA受试者的动脉粥样硬化斑块中也存在。我们的首要假设是，RA的炎症是由CFB-IC介导的，CFB-IC通过增加巨噬细胞肿瘤坏死因子的产生来驱动促炎反应，通过激活CFB-IC特异性的B细胞来驱动获得性免疫反应；CFB-IC诱导的固有免疫反应和获得性免疫反应共同传播了RA的滑膜和关节外表现。我们的目的是证明1)抗原驱动的B细胞激活导致浆母细胞的形成，这些浆母细胞是ACPA的主要来源；2)TLR4在慢性炎症部位的B细胞上表达上调，CFB-IC可以通过TLR4和B细胞受体的共同连接来共同刺激这些B细胞。为了实现这些目标，我们将使用新的和建立的免疫复合体关节炎模型，体外细胞培养和流式细胞仪分析，以及一种新的基于微珠的抗原微阵列，用于分析RA患者的自身抗体以及培养的浆母细胞产生的抗体。这些研究的成功将为深入了解类风湿关节炎炎症的中介途径，以及自身抗原驱动的自我反应性B细胞克隆性增殖提供进一步的证据。促炎细胞因子和B细胞都是目前RA治疗的靶点，对RA先天免疫反应和获得性免疫反应机制的深入了解可能会为疾病预测和治疗干预带来新的潜在更好的靶点。

DESCRIPTION (provided by applicant): Rheumatoid arthritis (RA), the most common inflammatory joint disease of autoimmune etiology, affects nearly 1% of the population. It is clear that the adaptive and innate immune systems are important in the pathogenesis of RA. Autoantibodies that target citrullinated proteins are specific to RA, and innate immune receptors such as Toll-like receptors (TLRs) play important roles in the induction of both inflammation and adaptive immunity (e.g. B- and T-cell activation). A recently identified target of the anti-citrullinated protein antibody (ACPA) response in RA is citrullinated fibrinogen (cFb). We demonstrated that immune complexes (ICs) containing cFb (cFb-IC) are present in the blood and synovial tissue of patients with RA, and that immunization of mice with cFb induces inflammatory arthritis. Why cFb-specific autoimmunity should mediate inflammatory arthritis remains unclear. Investigating the mechanisms by which immunization with cFb evokes inflammatory arthritis, we observed that cFb itself is a powerful innate adjuvant that signals through TLR4, and that cFb-IC can synergize to augment macrophage TNF production by co-ligating TLR4 and the Fc3 receptor (Fc3R). Our preliminary studies also indicate that cFb can stimulate proliferation of B cells in a TLR4- dependent manner. In addition, we identified significantly elevated levels of plasmablasts-the early, activated B cells-in the blood of ACPA-positive RA patients. These circulating plasmablasts produce large amounts of ACPA, including anti-cFb antibodies, suggesting that ACPA-positive RA is associated with ongoing, antigen- driven activation of peripheral B cells. Finally, we detected the presence of citrullinated proteins, including citrullinated fibrinogen, not only in the inflamed joint, but also within the atherosclerotic plaque of RA and non- RA subjects. Our overriding hypothesis is that inflammation in RA is mediated by cFb-IC, which drive proinflammatory responses by augmenting macrophage TNF production, and adaptive immune responses by activating B cells specific for cFb; and that the innate and adaptive immune response induced by cFb-IC together propagate the synovial and extra-articular manifestations of RA. We aim to demonstrate 1) that antigen-driven B-cell activation results in plasmablast formation, and that these plasmablasts are a major source of ACPA and 2) that TLR4 is upregulated on B cells at sites of chronic inflammation and that cFb-IC can co-stimulate these B cells via co-ligation of TLR4 and the B-cell receptor. To achieve these aims we will use novel and established models of immune complex arthritis, in vitro cell culture and flow-cytometric analysis,and a novel bead-based antigen microarrayfor profiling of RA patients' autoantibodies as well as antibodies produced by cultured plasmablast. Success of these studies will provide insight into the pathways mediating inflammation in RA, as well as further evidence of autoantigen-driven clonal expansion of self- reactive B cells. Proinflammatory cytokines and B cells are both targeted by current RA therapies, and an improved understanding of the mechanisms of innate and adaptive immune responses in RA could yield new and potentially better targets for disease prognostication as well as therapeutic intervention.