Citrullinated Antigens Bridge Innate and Adaptive Immunity in RA

描述（由申请人提供）： 类风湿性关节炎（RA）是最常见的自身免疫性关节炎，影响近1%的人口。很明显，适应性和先天性免疫系统在RA的发病机制中是重要的。靶向瓜氨酸化蛋白的自身抗体对RA具有特异性，先天免疫受体如Toll样受体（TLR）在诱导炎症和适应性免疫（例如B细胞和T细胞活化）中起重要作用。最近确定的RA中抗瓜氨酸化蛋白抗体（ACPA）反应的靶点是瓜氨酸化纤维蛋白原（cFb）。我们证明了含有cFb的免疫复合物（IC）（cFb-IC）存在于RA患者的血液和滑膜组织中，并且用cFb免疫小鼠诱导炎性关节炎。为什么cFb特异性自身免疫应该介导炎性关节炎仍然不清楚。研究用cFb免疫引起炎性关节炎的机制，我们观察到cFb本身是通过TLR 4发出信号的强大先天佐剂，并且cFb-IC可以通过共连接TLR 4和Fc 3受体（Fc 3R）协同增加巨噬细胞TNF产生。我们的初步研究还表明，cFB可以刺激增殖的B细胞在TLR 4依赖的方式。此外，我们发现ACPA阳性RA患者血液中的成浆细胞（早期活化的B细胞）水平显著升高。这些循环浆母细胞产生大量ACPA，包括抗cFB抗体，表明ACPA阳性RA与外周B细胞的持续抗原驱动活化相关。最后，我们检测到瓜氨酸化蛋白，包括瓜氨酸化纤维蛋白原的存在，不仅在发炎的关节中，而且在RA和非RA受试者的动脉粥样硬化斑块中。我们最重要的假设是，RA中的炎症是由cFb-IC介导的，cFb-IC通过增加巨噬细胞TNF的产生来驱动促炎反应，并通过激活对cFB特异性的B细胞来驱动适应性免疫反应;并且由cFb-IC诱导的先天性和适应性免疫反应一起传播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细胞活化导致浆母细胞形成，并且这些浆母细胞是ACPA的主要来源，以及2）TLR 4在慢性炎症部位的B细胞上上调，并且cFb-IC可以通过TLR 4和B细胞受体的共连接来共刺激这些B细胞。为了实现这些目标，我们将使用新的和已建立的免疫复合物关节炎模型，体外细胞培养和流式细胞术分析，以及一种新的基于珠的抗原微阵列来分析RA患者的自身抗体以及培养的浆母细胞产生的抗体。这些研究的成功将提供对RA中介导炎症的途径的深入了解，以及自身抗原驱动的自身反应性B细胞克隆扩增的进一步证据。促炎细胞因子和B细胞都是目前RA治疗的靶点，对RA中先天性和适应性免疫应答机制的进一步理解可以产生新的和潜在的更好的疾病诊断和治疗干预的靶点。 公共卫生相关性： 风湿性关节炎（RA）是一种慢性自身免疫性关节炎，影响近1%的美国人口，是退伍军人残疾和医疗保健支出的主要原因。拟议实验的成功将证明，瓜氨酸化-身体蛋白质的特定修饰-以及靶向瓜氨酸化蛋白质的自身抗体的发展不仅对关节炎症引起的发病率至关重要，而且还可能增加与RA相关的心血管死亡率。拟议的实验将提供深入了解疾病的发病机制，可能导致新的治疗方法的RA及其相关的残疾的发展。

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. PUBLIC HEALTH RELEVANCE: Rheumatoid arthritis (RA) is a chronic autoimmune arthritis that affects nearly 1% of the United States population and is a leading cause of disability and health-care expenditures among veterans. Success of the proposed experiments would demonstrate that citrullination-a specific modification of the body's proteins- and the development of autoantibodies that target citrullinated proteins are crucial not only to morbidity caused by inflammation of the joints, but potentially also to the increased cardiovascular mortality associated with RA. The proposed experiments will provide insights into diseaes pathogenesis that could lead to development of new therapeutic approaches for RA and its associated disability.