Elucidating the role of RXRA in the myeloid lineage in post-infectious basal ganglia encephalitis

项目总结 儿童感染化脓性链球菌(A组链球菌；GAS)与基础 神经节脑炎(BGE)后遗症，表现为运动[Sydenham‘s Chorea(SC)]或精神病 [与链球菌感染相关的儿童自身免疫性神经精神疾病(熊猫)] 症状。人类和动物研究表明，异常细胞(Th17淋巴细胞)和体液 (自身抗体)获得性免疫反应，通常产生以对抗反复发生的化脓性链球菌感染， 靶向大脑并引发神经血管损伤、抗体进入大脑、神经炎症(小胶质细胞 激活和巨噬细胞浸润)和神经回路功能障碍。然而，并不是所有的儿童都有后遗症 传染性BGE，尽管频繁的气体感染表明存在易患遗传风险因素 使他们患上疾病。对82例患者和146例对照的基因组DNA进行全外显子组测序 在生物信息学分析之后，我们已经在大约20个基因中确定了候选遗传变异，这些基因可能 与感染后BGE的疾病风险相关。BGE的顶级风险基因之一，RXRA(编码 视黄醇X受体Alpha)是先天免疫反应和获得性免疫反应的关键调节因子。我们发现了 RXRA mRNA和蛋白在活化的小胶质细胞和浸润性巨噬细胞中表达上调。 小鼠反复鼻腔气体感染后的大脑。在这里，我们将测试RXRA的假设，由 整个外显子组测序，调节髓系中的几种先天免疫反应(小胶质细胞和 巨噬细胞)，包括激活、抗原呈递、细胞因子产生和吞噬，这些都是 反复发作性气体对脑部神经血管损害和神经元回路功能障碍的关键作用 感染。我们已经产生了髓系(小胶质细胞和巨噬细胞)缺乏RXRA功能的小鼠。 为了我们的第一个目标，我们将测试RXRA在小胶质细胞/巨噬细胞中对其激活的需求，抗原 体内呈现，细胞因子的产生和吞噬，使用PET成像，单细胞RNA测序和 在我们的小鼠模型中验证感染后BGE的有效性。同时，我们将对人类进行体外研究 携带SC/PANAS/PANS患者RXRA基因无义突变的小胶质细胞 测试气体暴露后的激活和吞噬能力。对于我们的第二个目标，我们将研究 消除髓系细胞中RXRA功能对神经血管损伤(即血脑屏障)的影响 功能障碍)，利用单细胞RNA消除兴奋性突触和神经元回路功能障碍 反复鼻内气体感染后的测序研究、病理、生理和行为分析 在对照组或髓系特异性RXRA缺陷小鼠中。这些原则证明研究有可能揭示 疾病发病机制的新机制和开发潜在的生物标志物或新的治疗方法来辅助 针对家庭的诊断和治疗策略，目前很少有临床疗效有限的策略。

PROJECT SUMMARY Childhood infections with Streptococcus pyogenes (Group A Streptococcus; GAS) are associated with basal ganglia encephalitis (BGE) sequelae that present with either movement [Sydenham’s chorea (SC)] or psychiatric [Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal infections (PANDAS)] symptoms. Human and animal studies have shown that aberrant cellular (Th17 lymphocytes) and humoral (autoantibodies) adaptive immune responses, generated normally to fight recurrent S. pyogenes infections, target the brain and elicit neurovascular damage, antibody entry into the brain, neuroinflammation (microglial activation and macrophage infiltration) and neural circuit dysfunction. However, not all children develop post- infectious BGE despite frequent GAS infections suggesting the presence of genetic risk factors that predispose them to develop disease. Through whole-exome sequencing of genomic DNA from 82 patients and 146 controls followed by bioinformatics analysis, we have identified candidate genetic variants in ~20 genes that are likely associated with disease risk for post-infectious BGE. One of the top BGE risk genes, RXRA (encoding for Retinoid X Receptor Alpha), is a key regulator of both innate and adaptive immune responses. We have found that Rxra mRNA and proteins levels are upregulated in activated microglia and infiltrating macrophages in the brain after recurrent intranasal GAS infections in mice. Here, we will test the hypothesis that RXRA, identified by whole-exome sequencing, regulates several innate immune responses in the myeloid lineage (microglia and macrophages) ranging from activation, antigen presentation, cytokine production and phagocytosis, that are critical for both neurovascular damage and neuronal circuit dysfunction in the brain after recurrent GAS infections. We have generated mice lacking Rxra function in the myeloid lineage (microglia and macrophages). For our first objective, we will test the requirement of Rxra in microglial/macrophages for their activation, antigen presentation, cytokine production and phagocytosis in vivo using PET imaging, single cell RNA sequencing and validation in our mouse model for post-infectious BGE. In parallel, we will perform in vitro studies with the human microglia cells, carrying the nonsense mutations in the RXRA gene identified in SC/PANDAS/PANS patients, to test activation and phagocytic ability following GAS exposure. For our second objective, we will examine the consequences of eliminating Rxra function in the myeloid cell for neurovascular damage (i.e. blood-brain barrier dysfunction), elimination of excitatory synapses and neuronal circuitry dysfunction using single cell RNA sequencing studies, pathological, physiological and behavioral assays after recurrent intranasal GAS infections in control or myeloid-specific Rxra deficient mice. These proof-of-principle studies have the potential to uncover novel mechanisms of disease pathogenesis and develop potential biomarkers or new therapeutics to assist in diagnostic and treatment strategies for families, of which there are currently few with limited clinical efficacy.