Deciphering Pathways Driving Inflammatory Fibroblast Effector Functions in RA

在类风湿性关节炎(RA)中，关节破坏是由滑膜介导的，滑膜变得炎症， 增生性病变，并被白细胞渗透。成纤维细胞构成滑膜衬里和基质网络。 副线层。目前治疗类风湿性关节炎的方法减少了疾病的活跃度，但很少能缓解。 免疫抑制生物制剂的组合会导致感染并发症增加，限制了它们的使用。 我们和其他人正致力于一种独特的方法，即靶向滑膜成纤维细胞和/或其独特的 消除炎症的途径不依赖于靶向白细胞或炎性细胞因子。在一个 最近发表的报告(Nguyen等人免疫力2017)和初步数据，我们说明了细胞因子如何 肿瘤坏死因子、白介素1b和白介素17(主要信号)均可激活成纤维细胞产生一系列炎性细胞因子。 突出作用的细胞因子(如IL-6)和趋化因子(如CCL2、IL-8)和生长因子(如G-CSF) 成纤维细胞本身在类风湿关节炎中的炎性细胞。重要的是，我们发现要实现强大和 这些炎性细胞因子、趋化因子和生长因子的持续表达是一个关键的放大 回路(二次信号)是必不可少的。这个次级信号是依赖于自分泌正反馈环路的 关于白血病抑制因子受体(LIFR)下游的信号。使该受体沉默会使其丧失。 成纤维细胞产生IL-6和一组共表达的炎症介质，无论是哪种原发的 信号被用来激活成纤维细胞。我们假设LIFR放大环路是一个关键的共享 成纤维细胞激活成分，可能以消除成纤维细胞介导的类风湿关节炎为靶点。 在目标1中，我们通过RNA测序定义了LIFR正反馈扩增环特征 用一系列强有力的激活剂刺激成纤维细胞，包括肿瘤坏死因子、IL-1b、IL-17和内毒素。在目标2中，我们 利用RT-PCR验证构成LIFR正反馈环特征的关键基因和产物 用酶联免疫吸附试验和流式细胞仪进行蛋白质测定。为了验证关键效应器函数，我们检查 LIFR缺失或沉默在体外功能测定中的作用 生存和其他功能。在目标3中，我们评估了活动期人类类风湿关节炎和类风湿关节炎患者的LIFR扩增环活性。 肿瘤坏死因子和甲氨蝶呤不足以使应答者评估这一途径是否为正在进行的 类风湿关节炎的炎症反应。最后，在目标4中，我们确定以LIFR扩增环为目标是否有效 治疗小鼠炎症性关节炎模型。总而言之，这些研究提供了对 成纤维细胞利用的促进类风湿性关节炎炎症的途径及其治疗意义。

In rheumatoid arthritis (RA), joint destruction is mediated by the synovium, which becomes inflamed, hyperplastic, and infiltrated by leukocytes. Fibroblasts constitute the synovial lining and the stromal network of the sublining layer. Current therapies for RA reduce disease activity but rarely achieve remission. Combinations of immunosuppressive biologics result in increased infectious complications limiting their use. We and others are working toward a distinct approach, namely to target synovial fibroblasts and/or their distinct pathways to abrogate inflammation independent of targeting leukocytes or inflammatory cytokines. In a recently published report (Nguyen et al. Immunity 2017) and preliminary data, we illustrate how cytokines like TNF, IL-1b and IL-17 (primary signals) all can activate fibroblasts to produce an array of inflammatory cytokines (e.g. IL-6) and chemokines (e.g. CCL2, IL-8) and growth factors (e.g. G-CSF) that highlight the role of fibroblasts themselves as inflammatory cells in RA. Importantly, we found that to achieve strong and sustained expression of these inflammatory cytokines, chemokines and growth factors, a critical amplification loop (secondary signal) is essential. This secondary signal is an autocrine positive feedback loop dependent on signaling downstream of the leukemia inhibitory factor receptor (LIFR). Silencing this receptor abrogates fibroblast production of IL-6 and a set of co-expressed inflammatory mediators, regardless of which primary signal is used to activate the fibroblasts. We hypothesize that the LIFR amplification loop is a critical shared component of fibroblast activation that may be targeted to abrogate fibroblast mediated inflammation in RA. In Aim 1, we define the LIFR positive feedback amplification loop signature by RNA sequencing after stimulating fibroblasts with a series of potent activators including, TNF, IL-1b, IL-17 and LPS. In Aim 2, we validate the key genes and products that make up the LIFR positive feedback loop signature using RT-PCR and protein determinations by ELISA and flow cytometry. To validate key effector functions, we examine the effects of LIFR deletion or silencing in in vitro functional assays that measure leukocyte recruitment and survival and other functions. In Aim 3, we assess LIFR amplification loop activity in active human RA and in TNF and methotrexate inadequate responders to assess if this pathway is a component of ongoing inflammation in RA. Finally, in Aim 4, we determine if targeting the LIFR amplification loop can be an effective therapy for inflammatory arthritis in mouse models. Together, these studies provide mechanistic insights into the pathways utilized by fibroblasts that drive inflammation in RA with implications for treatment.