Neutrophil Extracellular Traps in the Lung and Development of Rheumatoid Arthritis-Related Autoimmunity and Arthritis

项目摘要/摘要 抗瓜氨酸蛋白抗体(ACPA)的形成是类风湿疾病发展的早期阶段 关节炎(RA)。血清ACPA对RA的发展有较高的预测性，并可直接促进关节损害。然而， 在我们对ACPA发展的理解上仍然存在很大的差距，包括它们在哪里以及如何发展 最初是触发的。重要的是，为了实现NIH在RA中预防疾病/残疾的使命，改进了 需要了解导致ACPA的启动因素和途径，因为这些因素/途径 将是预防类风湿关节炎的新靶点。ACPA起源于RA关节炎发病前几年，以及 数据支持它们最初可能在肺粘膜中发育。然而，肺的作用机制 对ACPA发展的贡献尚不清楚。拟议项目的总体假设是中性粒细胞 肺内的细胞外陷阱(Net)可触发ACPA并预测RA。这一假设是基于我们发表的 初步数据包括我们发现在以下情况下受试者肺中净残留物与ACPA相关- 发展RA的风险。重要的是，净网残留物是网形成(网)和网的复合体 通行证。这项研究将确定这两个过程中的一个或两个过程中的异常是否会导致ACPA 肺脏的生成与类风湿关节炎的发展。在这个项目中，诱导痰将从受试者身上收集 谁是RA的高危人群(基于已知的家族性或血清学风险因素)、健康对照和患有 拉。在显微镜下，中性粒细胞在细胞因子作用下发生网织红细胞增多的百分比 刺激将被计算出来。还将测量血清ACPA与痰网上蛋白质的结合。 预计类风湿性关节炎的高危人群会在细胞因子作用下出现痰网织红细胞异常增多 刺激与对照组比较。还预计细胞因子诱导的痰蚊虫增多症将与 痰ACPA水平和痰网将表达与血清ACPA结合的瓜氨酸蛋白- 风险主体。拟议的项目还将衡量痰中脱氧核糖核酸酶对蚊子的降解情况，以及 痰巨噬细胞吞噬清除Net。预计Net的这两种机制 与对照组相比，类风湿性关节炎高危受试者的降解率和清除率将会降低，并将相关 有痰的ACPA。此外，高危受试者将被跟踪3年。痰中ACPA和净残留物 将在基线和每年测量水平，以确定痰净残留物预测 高危人群会发展成类风湿性关节炎。质谱仪也将被用来鉴定存在的瓜氨酸蛋白。 与RA的发展有关的痰中。预计增加的痰净残留物 通过痰中的ACPA和独特的Cit蛋白可以预测RA高危人群中即将发生的RA。 最终，这些发现可以导致针对特定机制的RA预防的新方法 ACPA在初始免疫失调部位的发展(例如净形成和清除)(例如 肺部)是RA的高危人群。

PROJECT SUMMARY/ABSTRACT Formation of anti-citrullinated protein antibodies (ACPA) is an early step in the development of rheumatoid arthritis (RA). Serum ACPA are highly predictive of developing RA and can directly promote joint damage. Yet, there remains a major gap in our understanding of ACPA development, including where and how they are initially triggered. Importantly, to achieve the NIH's mission of disease/disability prevention in RA, an improved understanding of the initiating factors and pathways that lead to ACPA is needed as these factors/pathways would be novel targets for RA prevention. ACPA originate several years prior to the onset of arthritis in RA, and data support that they may initially develop in the lung mucosa. However, the mechanism by which the lung contributes to ACPA development is unclear. The overall hypothesis of the proposed project is that neutrophil extracellular traps (NETs) in the lung trigger ACPA and predict RA. This hypothesis is based on our published and preliminary data including our finding that NET remnants correlate with ACPA in the lung in subjects At- Risk for developing RA. Importantly, NET remnants are a composite of NET formation (NETosis) and NET clearance. This study will establish whether aberrancies in one or both of these processes contribute to ACPA generation in the lung and development of RA. In this project, induced sputum will be collected from subjects who are At-Risk for RA (based on known familial or serologic risk factors), healthy controls and subjects with RA. Using microscopy, the percentage of sputum neutrophils that undergo NETosis following cytokine stimulation will be calculated. The binding of serum ACPA to proteins on sputum NETs will also be measured. It is expected that subjects At-Risk for RA will have abnormally increased sputum NETosis following cytokine stimulation compared to controls. It is also expected that cytokine-induced sputum NETosis will correlate with sputum ACPA levels and that sputum NETs will express citrullinated proteins that bind serum ACPA from At- Risk subjects. The proposed project will also measure the degradation of NETs by DNase in sputum and the clearance of NETs by sputum macrophage phagocytosis. It is expected that both mechanisms of NET degradation and clearance will be decreased in subjects At-Risk for RA compared to controls and will correlate with sputum ACPA. In addition, At-Risk subjects will be followed for 3 years. Sputum ACPA and NET remnant levels will be measured at baseline and yearly to establish the ability of sputum NET remnants to predict which At-Risk subjects will develop RA. Mass spectrometry will also be used to identify citrullinated proteins present in sputum that are associated with development of RA. It is expected that increased sputum NET remnants mediated through sputum ACPA and unique cit-proteins will predict imminent RA in subjects At-Risk for RA. Ultimately, these findings can lead to novel approaches for RA prevention that target specific mechanisms of ACPA development (e.g. NET formation and clearance) at the site of initial immune dysregulation (e.g. the lung) in individuals who are At-Risk for RA.