Role of B Cells in Synovial Inflammation & LN Remodeling in RA Arthritis

TNF拮抗剂和B细胞去除疗法（BCDT）在类风湿关节炎（RA）中的疗效已被证实。 从根本上改变了病理生理学模式，并提出了关于疾病机制的新问题。 TNF和B细胞都具有核心重要性的观察结果提供了改善肿瘤细胞增殖的强大动力。 我们对TNF过度产生和炎症途径之间的关系的理解是， B细胞，特别是在疾病爆发和治疗反应不足方面。TNF转基因 （TNF-tg）小鼠品系是唯一的慢性进展性RA模型，具有临床证实的病因，因此对于解决RA中的细胞和免疫事件非常宝贵。我们已经开发了一系列新的小动物成像方法，包括对比增强（CE）MRI，在体内微CT和近红外吲哚菁绿色（NIR-ICG）分析淋巴流，纵向研究与关节炎进展相关的事件。这些研究的结果，结合组织学和流式细胞术，显示在膝关节疾病发作之前和期间，引流腘淋巴结（PLN）以可预测的时间顺序发生了显著变化。在膝关节滑膜炎之前，引流的PLN“扩张”，如通过增加的体积和CE值（即较低的密度）所证明的，并且结节的组织学显示显著扩张、扩大的窦状隙。这种结节扩张的特征还在于PLN B细胞增加 主要是由于具有独特表面表型的B细胞亚群（“B-in”细胞）的积累。这一阶段之后，淋巴结大小和CE迅速下降，B细胞侵入并“堵塞”淋巴窦，淋巴液流经淋巴结减少。PLN塌陷后发生膝关节滑膜炎和局灶性糜烂。BCDT和抗CXCL 13治疗均导致TNF-tg疾病的改善和PLN大小、CE和组织学的恢复，表明B细胞和趋化性的致病作用。因此，我们假设TNF-tg小鼠膝关节炎和PLN功能的演变是从早期阶段开始的，在此期间致病性B细胞被募集并积聚在引流结中，引流结的大小和CE增加。在后期阶段，PLN-内在或外部（例如来自关节）信号诱导扩增的B细胞群迁移到窦状隙，导致淋巴流阻塞和局部细胞因子平衡的改变。这导致促炎介质在膝关节中积聚，触发“耀斑”。在此，我们建议通过以下方式来测试该模型：1）鉴定导致B细胞在PLN中积聚并随后迁移到淋巴空间的机制; 2）证明过继转移的PLN B细胞的致病作用，并测试BCDT功效与窦状隙侵入的B细胞的清除相关的假设; 3）测试TNF-tg B细胞是否可以发挥促炎效应子功能;（4）在BCDT前后评估PLN结构和淋巴功能，并与临床症状相关联的初步临床试验中，研究这些发现对RA患者的适用性。

The efficacy of TNF antagonists and B cell depletion therapy (BCDT) in rheumatoid arthritis (RA) have fundamentally altered pathophysiologic paradigms and raised new quesfions about disease mechanisms. The observation that both TNF and B cells are of central importance provides a strong impetus to improve our understanding ofthe relationship between TNF over-production and infiammation pathways mediated by B cells, particulady in regards to disease flares and inadequate treatment response. The TNF-transgenic (TNF-tg) mouse strain is the only chronically progressive model for RA with a clinically proven etiology, and thus invaluable to address the cellular and immunological events in RA. We have developed a series of novel small animal imaging approaches, including contrast enhanced (CE) MRI, in vivo micro-CT and nearinfrared indocyanin green (NIR-ICG) analysis of lymphatic flow, to study longitudinally the events associated with progression of joint arthritis. The results from these studies, combined with histology and flow cytometry, showed dramatic changes unfolding in a predictable temporal sequence in the draining popliteal lymph-node (PLN) before and duhng the onset of knee disease. Pnor to knee synovitis, the draining PLNs "expand", as evidenced by increased volume and CE values (i.e. lower density), and histology of the node shows markedly dilated, enlarged sinusoids. This nodal expansion is also characterized by increased PLN B cell numbers, primanly due to accumulation of a B cell subset with a unique surface phenotype ("B-in" cells). This phase is followed by a rapid decrease in node size and CE, by invasion and "plugging" ofthe lymphatic sinuses by B cells, and reduced lymphatic flow through the node. The onset of knee synovitis and focal erosions follows PLN collapse. Both BCDT and anti-CXCL13 treatment result in TNF-tg disease ameliorafion and recovery of PLN size, CE and histology, pointing to a pathogenetic role of both B cells and chemotaxis. Thus, we hypothesize that evolution of knee arthritis and PLN function in TNF-tg mice proceed from an early stage during which pathogenetic B cells are recruited to and accumulate in the draining nodes, which increase in size and CE. At a later stage PLN-intrinsic or external (e.g. from the joint) signals induce migration ofthe expanded B cell population to the sinusoidal spaces, resulting in the obstruction of lymph flow and changes in the local cytokine balance. This leads to accumulation of pro-inflammatory mediators in the knee joint, triggering the "flare". Here we propose to test this model by: 1) identifying the mechanisms that lead to B cell accumulation in the PLNs and their subsequent migration to lymphatic spaces; 2) demonstrating the pathogenetic role of adoptively transfered PLN B cells, and testing the hypothesis that BCDT efficacy is associated with clearance of sinusoid-invading B cells; 3) testing whether TNF-tg B cells can exert pro-inflammatory effector functions; and d) investigating the applicability of these finding to RA patients in a pilot clinical tnal in which PLN structure and lymphatic function wil be assessed before and after BCDT, and correlated with clinical svmptoms.