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，滑膜发炎， 增生，并被白细胞浸润。成纤维细胞构成滑膜衬里和基质网络， 下层衬层。目前RA的治疗方法减少了疾病活动，但很少达到缓解。 免疫抑制生物制剂的组合导致感染性并发症增加，限制了它们的使用。 我们和其他人正在朝着一种独特的方法努力，即靶向滑膜成纤维细胞和/或其独特的细胞因子。 消除炎症的途径，不依赖于靶向白细胞或炎性细胞因子。中 最近发表的报告（Nguyen et al. Immunity 2017）和初步数据，我们说明了细胞因子，如 TNF、IL-1b和IL-17（主要信号）都可以激活成纤维细胞产生一系列炎性细胞因子。 细胞因子（例如IL-6）和趋化因子（例如CCL 2、IL-8）和生长因子（例如G-CSF），其突出了 成纤维细胞本身作为炎性细胞在类风湿关节炎中的作用。重要的是，我们发现，要实现强大的， 这些炎性细胞因子、趋化因子和生长因子的持续表达， 循环（次要信号）是必不可少的。这个次级信号是一个自分泌正反馈环依赖 白血病抑制因子受体（LIFR）下游信号传导。沉默这种受体， 成纤维细胞产生IL-6和一组共表达的炎症介质，无论哪种原发性 信号用于激活成纤维细胞。我们假设LIFR放大环是一个关键的共享 成纤维细胞活化的组分，其可以靶向消除RA中成纤维细胞介导的炎症。 在目标1中，我们通过RNA测序定义了LIFR正反馈扩增环签名， 用包括TNF、IL-1b、IL-17和LPS的一系列有效活化剂刺激成纤维细胞。在目标2中， 使用RT-PCR验证构成LIFR正反馈环签名的关键基因和产物 以及通过ELISA和流式细胞术测定蛋白质。为了验证关键效应器功能，我们检查 LIFR缺失或沉默在测量白细胞募集的体外功能测定中的作用， 生存和其他功能。在目标3中，我们评估了活动性人RA和正常人RA中LIFR扩增环的活性。 TNF和甲氨蝶呤应答者不足，无法评估该途径是否是正在进行的 RA炎症。最后，在目标4中，我们确定靶向LIFR扩增环是否可以是有效的 治疗小鼠模型中的炎性关节炎。总之，这些研究提供了机械的见解， 成纤维细胞利用的驱动RA炎症的途径与治疗的意义。

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.