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

项目总结/摘要 抗瓜氨酸蛋白抗体（ACPA）的形成是类风湿性关节炎发展的早期步骤。 关节炎（RA）。血清ACPA是发展RA的高度预测因素，并可直接促进关节损伤。然而， 我们对ACPA发展的理解仍然存在重大差距，包括它们在哪里以及如何发展 最初触发。重要的是，为了实现NIH在RA中预防疾病/残疾的使命， 需要了解导致ACPA的启动因素和途径，因为这些因素/途径 将成为预防类风湿性关节炎的新靶点。ACPA起源于RA关节炎发作前数年， 数据支持它们最初可能在肺粘膜中发育。然而，肺的机制 对ACPA发展的贡献尚不清楚。拟议项目的总体假设是， 肺中的细胞外陷阱（NET）触发ACPA并预测RA。这一假设是基于我们发表的 以及初步数据，包括我们的发现，即NET残留与受试者肺中的ACPA相关， 发生RA的风险。重要的是，NET残余物是NET形成（NETosis）和NET的复合物。 间隙这项研究将确定是否异常在一个或两个这些过程有助于ACPA 在肺中的生成和RA的发展。本项目将采集受试者诱导痰 有RA风险的受试者（基于已知的家族或血清学风险因素）、健康对照和 RA.使用显微镜检查，在细胞因子刺激后， 将计算刺激。还将测量血清ACPA与痰NET上的蛋白质的结合。 预计RA风险受试者在接受细胞因子治疗后，痰液NETosis将异常增加。 与对照组相比，还预期尼古丁诱导的痰液NETosis将与 痰ACPA水平，并且痰NET将表达瓜氨酸化蛋白，其结合来自At- 风险主体。拟议的项目还将测量唾液中DNase对NET的降解， 通过痰巨噬细胞吞噬作用清除NET。预计NET的两种机制 与对照组相比，RA风险受试者的降解和清除率将降低， 痰ACPA。此外，将对处于风险中的受试者进行3年随访。痰液ACPA和NET残留 将在基线和每年测量水平，以确定痰液NET残留物预测 有风险的受试者将发生RA。质谱法也将用于鉴定存在的瓜氨酸化蛋白质 与类风湿关节炎的发生有关。预计增加的痰净残留物 通过痰液ACPA和独特的cit-protein介导，可以预测RA高危受试者即将发生的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NET形成和清除）（例如 肺）在个体中谁是风险的RA。

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.