ACPA Generation in the Female Genital Tract and Lactating Mammary Tissue Mucosae

项目摘要/摘要 几十年的研究一直无法完全解释为什么女性患类风湿性关节炎的几率更高。 类风湿性关节炎(RA)与男性相比，或者为什么产后期与发病率显著增加有关 在RA中。形成抗瓜氨酸(Cit)蛋白抗体(ACPA)是一个公认的早期发展步骤。 RA的进展，最近的报道支持粘膜部位参与了临床前ACPA的形成 风湿性关节炎的发展阶段。到目前为止，研究主要集中在类风湿性关节炎的肺、牙龈或肠道粘膜上，但这些 研究无助于解释类风湿性关节炎的性别差异。因此，拟议的项目将调查这部小说 假设女性生殖道和哺乳期乳腺组织是ACPA世代的粘膜部位 是女性独有的。在R61期，将收集绝经前妇女的宫颈阴道液(CVF) RA患者的一级亲属(FDR)和健康对照。母乳将从邮局收集 患有类风湿性关节炎、FDR和对照组的产后妇女。将对CVF、母乳和血液进行Ig G-ACPA和Ig A检测。 ACPA，包括Cit和可建立Cit特异性的含精氨酸的蛋白质/肽。总免疫球蛋白 并测量IgA以计算每个部位的ACPA/Ig比率。高ACPA的CVF和母乳样本 水平将经历免疫沉淀，结合在免疫复合体中的cit-多肽将通过质量进行鉴定。 光谱分析。预计部分绝经前妇女的CVF和母乳cit将升高。 特异性ACPA，CVF和母乳中的ACPA/Ig比CVF中的血液和免疫复合物高 以及与Cit多肽结合的母乳，从而证实在这些粘膜部位产生了ACPA。此外, 来自母乳的浆母细胞将通过流式细胞仪进行量化，分类后的浆母细胞子集将 进行抗体谱系测序。母乳浆母细胞中存在多克隆家族将 支持与ACPA相关的哺乳期乳腺组织的主动免疫反应。如果ACPA世代在 这些位点在R61阶段得到确认，然后R33阶段将确认ACPA的产生是由 炎症起源于这些粘膜部位的炎症。具体地说，人类CVF、母乳和在 R61期将检测炎性细胞因子，并与ACPA相关。预计与RA相关的 在这些性别特定的粘膜部位，细胞因子将与ACPA相关。此外，人类白细胞抗原-DR4转基因 野生型小鼠将接受Cit多肽和天然多肽对照的阴道免疫。一个子集 也会怀孕。这些小鼠的CVF、母乳和血液将进行ACPA检测。预计 Cit多肽可直接诱导血管黏膜炎症反应和ACPA的产生。在母乳中，它是前- 推测ACPA将由自然产生的Cit多肽诱导。重要的是，理解这一代人 ACPA在这些专属于女性的粘膜部位的研究可能会彻底改变我们对性病因的看法 类风湿性关节炎的差异以及我们如何处理类风湿性关节炎的个性化治疗和预防。

PROJECT SUMMARY/ABSTRACT Decades of research have been unable to fully explain why women have a higher incidence of rheumatoid ar- thritis (RA) compared to men or why the post-partum period is associated with a markedly increased incidence of RA. Formation of anti-citrullinated (cit) protein antibodies (ACPA) is a well-established early step in the devel- opment of RA, and recent reports support that mucosal sites are involved in ACPA formation during a pre-clinical phase of RA development. Studies to date have focused on the lung, gingival or gut mucosae in RA, but these studies have not helped to explain sex differences in RA. As such, the proposed project will investigate the novel hypothesis that the female genital tract and lactating mammary tissue are mucosal sites of ACPA generation unique to women. In the R61 phase, cervicovaginal fluid (CVF) will be collected from pre-menopausal women with RA, first-degree relatives (FDRs) of RA patients and healthy controls. Breast milk will be collected from post- partum women with RA, FDRs and controls. CVF, breast milk and blood will be tested for IgG-ACPA and IgA- ACPA, including both cit and arginine-containing proteins/peptides that can establish cit-specificity. Total IgG and IgA will be measured to calculate ACPA/Ig ratios at each site. CVF and breast milk samples with high ACPA levels will undergo immunoprecipitation, and cit-peptides bound in immune complexes will be identified by mass spectrometry. It is expected that a portion of pre-menopausal women will have elevated CVF and breast milk cit- specific ACPA, higher ACPA/Ig ratios in CVF and breast milk compared to blood and immune complexes in CVF and breast milk that bind cit-peptides, thereby confirming ACPA generation at these mucosal sites. In addition, plasmablasts from breast milk will be quantified by flow cytometry, and a subset of sorted plasmablasts will undergo antibody repertoire sequencing. Presence of multiple clonal families in breast milk plasmablasts will support an active immune response in lactating mammary tissue associated with ACPA. If ACPA generation at these sites is confirmed in the R61 phase, then the R33 phase will confirm that ACPA generation is caused by inflammation originating at these mucosal sites. Specifically, human CVF, breast milk and blood collected in the R61 phase will be tested for inflammatory cytokines and correlated with ACPA. It is expected that RA-related cytokines will be associated with ACPA at these sex-specific mucosal sites. In addition, HLA-DR4 transgenic and wild type mice will undergo intravaginal immunization with cit-peptides and native peptide controls. A subset will also become pregnant. CVF, breast milk and blood in these mice will be tested for ACPA. It is expected that cit-peptides will directly induce inflammation and ACPA generation in the CV mucosa. In breast milk, it is ex- pected that ACPA will be induced by naturally occurring cit-peptides. Importantly, understanding the generation of ACPA at these mucosal sites specific to women could revolutionize the way we think about the etiology of sex differences in RA and how we approach personalized treatment and prevention of RA.