Human Gut Commensal Cross-reactivity in Antiphospholipid Syndrome

 描述（由申请人提供）：抗磷脂综合征（APS）是一种潜在致死性自身免疫性凝血障碍，可导致血栓栓塞事件和产科并发症，通常与狼疮和其他全身性自身免疫性疾病相关。APS的治疗目前仅限于终身抗凝，以防止未来的血栓栓塞事件。APS的原因尚不清楚，但感染性触发因素与抗磷脂抗体的短暂诱导有关。人类肠道微生物群超过宿主真核细胞数量的10倍，为抗原变异和持续免疫激活提供了主要来源。在慢性自身免疫性疾病中，从未探讨过这种细胞变异性是否可能是分子模拟。我们假设良性肠道微生物群中的细菌通过这种机制持续诱导遗传易感个体的致病性自身抗体。我们有初步的数据来支持APS小鼠模型中肠道微生物群的基本作用。重要的是，基于与APS中主要自身抗原（β 2-糖蛋白I; β 2 GPI）的关键T和B细胞自身抗原表位的高序列同源性，我们已经鉴定了潜在的交叉反应性人自体抗原，并培养了候选自体抗原。我们计划使用合成肽和培养的唾液蛋白提取物测试APS患者的自身反应性T和B细胞的交叉反应潜力。为此，我们提出了一项研究，收集外周血和粪便从抗β 2 GPI阳性APS和对照组患者纵向在三个时间点。我们将确定APS患者的CD 4 + T和B细胞靶向的自身抗原表位，并设计基于PCR的策略，用于靶向筛选粪便微生物组，以寻找携带与这些患者中靶向的自身表位同源的氨基酸序列的候选微生物。我们还将采用无偏的方法对整个粪便微生物组进行高通量16 S rRNA测序，以发现以前未知的与β 2GPI免疫反应性相关的候选物。该方法已经揭示了具有交叉反应潜力的另外的候选物。我们认为自身抗原模拟抗体的波动与APS患者粪便或血液中抗β 2GPI抗体的滴度以及自身抗原特异性T细胞相关。最后，我们已经克隆了β 2GPI特异性CD 4记忆T细胞，并将测试与培养的关键候选物和合成肽的交叉反应性。对于具有T和B细胞表位同源性的候选物，我们还将使用ELISA和蛋白质印迹测试自身抗体的交叉反应性。总之，我们的目标是发现APS中致病性自身抗体产生的持久触发因素。这些研究将代表人类自身免疫如何产生的新范式，并将作为开发针对肠道微生物群的全身性自身免疫的全新治疗途径的基础。

 DESCRIPTION (provided by applicant): The antiphospholipid syndrome (APS) is a potentially lethal autoimmune clotting disorder that leads to thromboembolic events and obstetric complications and is frequently associated with lupus and other systemic autoimmune diseases. Treatment for APS is currently limited to life-long anticoagulation in order to prevent future thromboembolic events. The cause of APS is unknown but infectious triggers have been implicated in transient induction of antiphospholipid antibodies. The human gut microbiota exceeds 10-fold the number of the host eukaryotic cells, providing a major source for antigenic variation and persistent immune activation. This commensal variability has never been explored for the possibility of molecular mimicry in chronic autoimmune diseases. We hypothesize that commensals within the benign gut microbiota persistently induce pathogenic autoantibodies in genetically predisposed individuals via this mechanism. We have preliminary data to support a fundamental role of the gut microbiota in a murine model of APS. Importantly, we have identified potentially cross-reactive human commensals based on high sequence homologies with both the key T and B cell autoantigenic epitopes of the major autoantigen in APS (ß2-glycoprotein I; ß2GPI) and cultured a candidate commensal. We plan to test the cross-reactive potential of autoreactive T and B cells from APS patients using synthetic peptides and cultured commensal protein extracts. To this end, we propose a study to collect peripheral blood and stool from anti-ß2GPI-positive APS and control patients longitudinally at three time points. We will define the autoantigenic epitopes targeted by CD4+ T and B cells from APS patients and design PCR- based strategies for targeted screening of the fecal microbiomes for the candidate commensals that carry homologous amino acid sequences to the autoepitopes targeted in these patients. We will also take the unbiased approach of high-throughput 16S rRNA sequencing of the entire fecal microbiome in order to discover previously unknown candidates that track with ß2GPI immunoreactivities. This approach already revealed an additional candidate with cross-reactive potential. We propose that fluctuations of autoantigen- mimicking commensals will correlate with titers of anti- ß2GPI antibodies in stool or blood and with autoantigen- specific T cells in APS patients. Finally, we have cloned ß2GPI-specific CD4 memory T cells and will test + cross-reactivity with cultured key candidates and synthetic peptides. For candidates with both T and B cell epitope homologies, we will also test cross-reactivity of autoantibodies using ELISA and western blot. In summary, we aim to discover the persistent triggers of pathogenic autoantibody production in APS. These studies will represent a novel paradigm for how human autoimmunity can arise and will serve as the basis for development of entirely novel therapeutic avenues in systemic autoimmunity that are aimed at the gut microbiota.