Elucidating the Mechanisms of Arthritic Flare and Developing Treatments

摘要 风湿性关节炎（RA）是一种破坏性的炎症性关节疾病， 发病率和死亡率。虽然生物疗法改善了治疗反应，但未满足的临床 由于RA的难治性和复发性疾病，尽管有积极的治疗， 治疗在之前的资助期间，我们开发了一个多学科的转化研究计划 将吲哚菁绿色（ICG）的纵向近红外（NIR）成像与靶向 阐明肿瘤坏死因子、B细胞和趋化因子如何汇聚在一起触发小鼠关节炎发作的疗法 模型和RA患者。具体来说，我们发现，在RA的体征和症状之前， 淋巴管（LV）收缩频率增加，以增强CD 11b+髓样细胞的流出 从受影响的关节，最终得到不堪重负，在早期发病。类风湿性关节炎疾病的进展， 未活化的多克隆CD 23 +/CD 21 hi/CD 1d hi流入导致关节引流淋巴结扩张 炎症淋巴结（Bin）和淋巴液中的B细胞，直至观察到左心室收缩沿着 同侧轴，这导致淋巴结塌陷和窦的Bin堵塞。有趣的是， TNF-Tg小鼠腘淋巴结（PLN）塌陷后的膝关节滑膜炎可通过抗 CD 20 B细胞耗竭疗法（BCDT），其恢复被动但非主动淋巴流动。最近， 我们证明TNF-Tg小鼠的LV收缩丧失继发于活化的巨噬细胞， 粘附于淋巴管内皮细胞（LEC），随后粘附于LEC和淋巴肌细胞 (LMC)慢性炎症造成的损伤。值得注意的是，这一主要缺陷可以通过反 TNF治疗可改善炎性-糜烂性关节炎，并恢复LEC-LMC的完整性。到 为了进一步了解关节炎发作中关节炎药物的作用，我们提出了三个具体目标。在目标1中， 将证明WT中生长和爆发期间血管周围LMC祖细胞掺入PLV中 生长小鼠（体内平衡），和用抗-TNF-α处理的具有扩张的PLN与塌陷的PLN的TNF-Tg小鼠。 TNF或安慰剂。我们还将在体外过继转移研究中证实它们的LMC祖细胞潜力 和体内。在目标2中，我们将证明PDGF β信号在LMC中的作用， 通过功能基因组研究，关节炎进展的崩溃阶段， 在急性胶原抗体诱导的关节炎和慢性TNF诱导的关节炎中的作用 小鼠为了将这些动物研究与人类疾病联系起来，在目标3中，我们将完成一项临床试验 接受抗TNF治疗手部潮红的RA患者，以正式证明NIR-ICG的效用 成像作为LV恢复的生物标志物，及其与治疗反应的相关性。完成 这些特定的目标将证实我们关于RA耀斑的范式转换假设，并可能提供 对难治性疾病的新见解，可以通过评估传出神经系统来诊断。

Abstract Rheumatoid arthritis (RA) is a destructive inflammatory joint disease associated with increased morbidity and mortality. While biologic therapies have improved treatment response, unmet clinical needs remain due to the refractory nature of RA, and recurrent disease flares despite aggressive treatment. In the prior funding periods, we developed a multidisciplinary translational research program that combined longitudinal near infrared (NIR) imaging of indocyanine green (ICG) with targeted therapies to elucidate how TNF, B cells and lymphatics converge to trigger arthritic flare in murine models and RA patients. Specifically, we found that prior to RA signs and symptoms, there is an increase in lymphatic vessel (LV) contraction frequency to enhance the efflux of CD11b+ myeloid cells from the affected joints, which ultimately gets overwhelmed at early onset. RA disease proceeds with expansion of joint draining lymph nodes from an influx of unactivated-polyclonal CD23+/CD21hi/CD1d hi B cells in inflamed nodes (Bin) and lymph, until a sudden loss of LV contractions is observed along the ipsilateral axis, which results in lymph node collapse and Bin clogging of the sinuses. Interestingly, knee synovitis following popliteal lymph node (PLN) collapse in TNF-Tg mice is ameliorated by anti- CD20 B cell depletion therapy (BCDT), which restores passive but not active lymph flow. Most recently, we demonstrated that loss of LV contractions in TNF-Tg mice is secondary to activated macrophage adherence to the lymphatic endothelial cells (LEC), and subsequent LEC and lymphatic muscle cell (LMC) damage from chronic inflammation. Remarkably, this major defect can be corrected with anti- TNF therapy that ameliorates the inflammatory-erosive arthritis, and restores LEC-LMC integrity. To further understand the role of lymphatics in arthritic flare, we propose three Specific Aims. In Aim 1 we will demonstrate perivascular LMC progenitor incorporation into PLVs during growth and flare in WT growing mice (homeostasis), and TNF-Tg mice with Expanding vs. Collapsed PLN treated with anti- TNF or placebo. We will also confirm their LMC progenitor potential in adoptive transfer studies in vitro and in vivo. In Aim 2 we will demonstrate the role of PDGF signaling in LMC during the Expanding and Collapsed phases of arthritic progression via functional genomic studies, and genetic loss of function in the setting of acute collagen antibody-induced arthritis and chronic TNF-induced arthritis in mice. To correlate these animal studies with human disease, in Aim 3 we will complete a clinical pilot of RA patients receiving anti-TNF therapy for hand flare, to formally demonstrate the utility of NIR-ICG imaging as a biomarker of LV recovery, and its correlation with response to therapy. Completion of these Specific Aims will substantiate our paradigm-shifting hypothesis of RA flare, and may provide novel insights into refractory disease that can be diagnosed by assessing efferent lymphatics.