DataSheet_1_Antibodies to a Citrullinated Porphyromonas gingivalis Epitope Are Increased in Early Rheumatoid Arthritis, and Can Be Produced by Gingival Tissue B Cells: Implications for a Bacterial Origin in RA Etiology.pdf

Based on the epidemiological link between periodontitis and rheumatoid arthritis (RA), and the unique feature of the periodontal bacterium Porphyromonas gingivalis to citrullinate proteins, it has been suggested that production of anti-citrullinated protein antibodies (ACPA), which are present in a majority of RA patients, may be triggered in the gum mucosa. To address this hypothesis, we investigated the antibody response to a citrullinated P. gingivalis peptide in relation to the autoimmune ACPA response in early RA, and examined citrulline-reactivity in monoclonal antibodies derived from human gingival B cells. Antibodies to a citrullinated peptide derived from P. gingivalis (denoted CPP3) and human citrullinated peptides were analyzed by multiplex array in 2,807 RA patients and 372 controls; associations with RA risk factors and clinical features were examined. B cells from inflamed gingival tissue were single-cell sorted, and immunoglobulin (Ig) genes were amplified, sequenced, cloned and expressed (n=63) as recombinant monoclonal antibodies, and assayed for citrulline-reactivities by enzyme-linked immunosorbent assay. Additionally, affinity-purified polyclonal anti-cyclic-citrullinated peptide (CCP2) IgG, and monoclonal antibodies derived from RA blood and synovial fluid B cells (n=175), were screened for CPP3-reactivity. Elevated anti-CPP3 antibody levels were detected in RA (11%), mainly CCP2+ RA, compared to controls (2%), p<0.0001, with a significant association to HLA-DRB1 shared epitope alleles, smoking and baseline pain, but with low correlation to autoimmune ACPA fine-specificities. Monoclonal antibodies derived from gingival B cells showed cross-reactivity between P. gingivalis CPP3 and human citrullinated peptides, and a CPP3+/CCP2+ clone, derived from an RA blood memory B cell, was identified. Our data support the possibility that immunity to P. gingivalis derived citrullinated antigens, triggered in the inflamed gum mucosa, may contribute to the presence of ACPA in RA patients, through mechanisms of molecular mimicry.

基于牙周炎与类风湿关节炎（rheumatoid arthritis, RA）之间的流行病学关联，以及牙周致病菌牙龈卟啉单胞菌（Porphyromonas gingivalis）对蛋白质进行瓜氨酸化的独特特性，有假说提出：多数类风湿关节炎患者体内存在的抗瓜氨酸化蛋白抗体（anti-citrullinated protein antibodies, ACPA），其产生可能在牙龈黏膜中被触发。为验证这一假说，本研究针对早期类风湿关节炎患者体内的自身免疫性ACPA反应，探究了其对牙龈卟啉单胞菌瓜氨酸化肽的抗体应答，并检测了源自人类牙龈B细胞的单克隆抗体的瓜氨酸反应性。本研究通过多重微阵列技术，对2807名类风湿关节炎患者及372名对照人群的血清抗体进行检测，分析其针对牙龈卟啉单胞菌来源的瓜氨酸化肽（命名为CPP3）以及人类瓜氨酸化肽的抗体水平，并探究其与类风湿关节炎危险因素及临床特征的关联。同时，对炎性牙龈组织中的B细胞进行单细胞分选，扩增、测序、克隆并表达了63株重组单克隆抗体（基于免疫球蛋白（immunoglobulin, Ig）基因操作），并通过酶联免疫吸附试验检测其瓜氨酸反应性。此外，本研究还对亲和纯化的多克隆抗环瓜氨酸肽（anti-cyclic-citrullinated peptide, CCP2）IgG，以及源自类风湿关节炎患者血液及滑膜液B细胞的175株单克隆抗体进行CPP3反应性筛选。检测结果显示，类风湿关节炎患者体内抗CPP3抗体水平升高的比例为11%，其中主要为CCP2阳性的类风湿关节炎患者，而对照人群该比例仅为2%（p<0.0001）；抗CPP3抗体水平与HLA-DRB1共享表位等位基因、吸烟史及基线疼痛水平呈显著关联，但与ACPA的自身免疫精细特异性相关性较低。源自牙龈B细胞的单克隆抗体可在牙龈卟啉单胞菌CPP3与人类瓜氨酸化肽之间产生交叉反应性，本研究还鉴定出一株源自类风湿关节炎患者血液记忆B细胞的CPP3+/CCP2+克隆。本研究数据支持以下可能性：在炎性牙龈黏膜中被触发的针对牙龈卟啉单胞菌来源瓜氨酸化抗原的免疫应答，可通过分子模拟机制参与类风湿关节炎患者体内ACPA的产生。