Human Gut Commensal Cross-reactivity in Antiphospholipid Syndrome

 描述(申请人提供)：抗磷脂综合征(APS)是一种潜在的致命性自身免疫性凝血障碍，会导致血栓栓子事件和产科并发症，经常与狼疮和其他系统性自身免疫性疾病有关。APS的治疗目前仅限于终生抗凝，以防止未来的血栓栓子事件。APS的原因尚不清楚，但感染性触发因素已被认为与抗磷脂抗体的瞬时诱导有关。人类肠道微生物区系的数量超过宿主真核细胞数量的10倍，为抗原变异和持久免疫激活提供了主要来源。这种共生变异性从未被探索过在慢性自身免疫性疾病中进行分子模仿的可能性。我们假设，良性肠道微生物区系内的共生体通过这种机制在遗传易感个体中持续诱导致病性自身抗体。我们有初步数据支持肠道微生物区系在APS小鼠模型中的基本作用。重要的是，我们基于与APS中主要自身抗原的关键T和B细胞自身抗原表位的高度序列同源性，确定了潜在的交叉反应人类共生体(？2-糖蛋白I；？2GPI)，并培养了一个候选共生体。我们计划使用合成肽和培养的共生蛋白提取物来测试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，从抗-2GPI阳性的APS中收集外周血液和粪便，并纵向在三个时间点对患者进行对照。我们将定义APS患者CD4+T和B细胞靶向的自身抗原表位，并设计基于PCR的策略，针对携带与这些患者目标自身表位同源氨基酸序列的候选共生体的粪便微生物群进行靶向筛选。我们还将采取公正的方法，对整个粪便微生物组进行高通量16S rRNA测序，以发现以前未知的候选基因，这些候选基因追踪ç2GPI的免疫活性。这种方法已经揭示了另一个具有交叉反应潜力的候选者。我们认为，APS患者自身抗原模拟共生体的波动将与粪便或血液中的抗？2GPI抗体滴度以及自身抗原特异性T细胞相关。最后，我们克隆了2GPI特异性的CD4 Memory T细胞，并将测试与培养的关键候选细胞和合成肽的交叉反应。对于同时具有T和B细胞表位同源性的候选人，我们还将使用ELISA法和Western印迹法测试自身抗体的交叉反应性。综上所述，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区系的全身性自身免疫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.