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

项目总结/摘要 几十年的研究一直无法完全解释为什么女性类风湿性关节炎的发病率较高， 性关节炎（RA）相比，男性或为什么产后期与发病率显着增加 的RA。抗瓜氨酸化（cit）蛋白抗体（ACPA）的形成是瓜氨酸化（cit）蛋白发展的一个公认的早期步骤。 最近的报道支持粘膜部位参与了临床前ACPA的形成， RA发展阶段。迄今为止的研究主要集中在RA的肺、牙龈或肠道粘膜，但这些研究表明， 研究没有帮助解释RA的性别差异。因此，拟议的项目将调查小说 女性生殖道和哺乳期乳腺组织是ACPA产生的粘膜部位的假说 女人独有的。在R61阶段，将采集绝经前女性的宫颈阴道液（CVF） 与RA，一级亲属（FDR）的RA患者和健康对照。母乳将从产后收集- 患有RA、FDR和对照的分娩妇女。将对CVF、母乳和血液进行IgG-ACPA和伊加- ACPA，包括cit和可建立cit特异性的含精氨酸的蛋白质/肽。总IgG 将测量伊加以计算每个部位的ACPA/IG比值。CVF和含高ACPA的母乳样本 水平将经历免疫沉淀，并且结合在免疫复合物中的cit-peptide将通过质量鉴定。 光谱法预计一部分绝经前妇女的CVF和母乳cit升高， 特异性ACPA，与CVF中的血液和免疫复合物相比，CVF和母乳中的ACPA/IG比值更高 和母乳中结合cit-peptides，从而证实在这些粘膜部位产生ACPA。此外，本发明还提供了一种方法， 来自母乳的浆母细胞将通过流式细胞术定量，并且分选的浆母细胞的子集将 进行抗体库测序。母乳浆母细胞中多个克隆家族的存在将 支持与ACPA相关的泌乳乳腺组织中的主动免疫应答。如果ACPA生成在 这些位点在R61阶段得到确认，那么R33阶段将确认ACPA的产生是由以下原因引起的： 炎症起源于这些粘膜部位。具体而言，在实验室中收集的人CVF、母乳和血液 R61期将检测炎性细胞因子并与ACPA相关。预计RA相关 细胞因子将在这些性别特异性粘膜部位与ACPA相关。此外，HLA-DR 4转基因 并且野生型小鼠将用CIT-肽和天然肽对照进行阴道内免疫。一个子集 也会怀孕。将对这些小鼠的CVF、母乳和血液进行ACPA检测。预计在 cit-peptides将直接诱导CV粘膜中的炎症和ACPA产生。在母乳中，它是前- 预期ACPA将由天然存在的cit-peptides诱导。重要的是，了解这一代人 ACPA在这些女性特有的粘膜部位的存在可能会彻底改变我们对性病因学的看法 RA的差异以及我们如何实现RA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.