TLR4 and the microbiome in CCM disease

脑海绵状血管瘤（CCM）是一种脑血管疾病，影响超过 美国有 200,000 人，是年轻人中风和癫痫发作的常见原因 个人。目前尚无针对 CCM 疾病的药物治疗方法，治疗方法是 仅限于手术切除和抗癫痫药物。我们最近有 证明 CCM 的出现是由于 MEKK3 信号传导的增强并增加 CCM缺陷脑内皮细胞中KLF2和KLF4转录因子的表达 细胞（EC）。这些研究揭示了影响的下游信号机制 CCM 疾病，但它们没有确定强有力的治疗靶点或解决问题 大脑 EC 中 MEKK3 的激活因素。我们的初步研究表明 TLR4 是内皮 MEKK3-KLF 信号传导的关键上游激活剂，是 CCM 的形成以及肠道微生物组中的革兰氏阴性菌 (GNB) 是 小鼠新生儿 CCM 模型中该途径的关键体内配体。遗传分析 我们的合作研究员 Helen Kim 博士独立对人类 CCM 患者进行了研究 鉴定出 TLR4 及其辅助受体 CD14 是与 CCM 病变数量相关的基因， 表明我们的小鼠研究结果扩展到人类 CCM 疾病。该提案将 进一步测试内皮 TLR4 信号传导和肠道微生物组在 CCM 中的作用 使用小鼠和人体研究来确定疾病发病机制。目标 1 将 (i) 测试 成年小鼠 CCM 形成过程中的 TLR4，(ii) 确定是否阻断 ant-TLR4 和 抗 CD14 抗体可防止小鼠模型中 CCM 的形成，(iii) 使用小鼠 遗传方法确定 TLR4 激活 MEKK3 的途径 CCM 形成，以及 (iv) 应用遗传方法来全面询问 TLR4-MEKK3- 由于 KRIT1 的常见突变，人类 CCM 疾病患者中的 KLF 通路。 目标 2 将通过 (i) 测试 GNB 和肠道微生物组在 CCM 发病机制中的作用 确定无菌动物中是否出现 CCM 病变，(ii) 定义微生物组 赋予小鼠对 CCM 疾病的抵抗力或易感性，以及 (iii) 测试是否 肠道上皮屏障的破坏加速了小鼠 CCM 的形成。这些 研究将显着扩大我们对分子和生理学的理解 CCM发病机制的基础，并为将这些发现转化为基础奠定基础 基于阻断 TLR4 信号传导的 CCM 疾病新疗法（FDA 已批准的药物已经存在）或改变肠道微生物组。

Cerebral cavernous malformation (CCM) is a cerebrovascular disease that affects over 200,000 individuals in the US and is a common cause of stroke and seizure in young individuals. There are presently no medical therapies for CCM disease, and treatment is limited to surgical resection and anti-seizure medications. We have recently demonstrated that CCMs arise due to a gain of MEKK3 signaling and increased expression of the KLF2 and KLF4 transcription factors in CCM-deficient brain endothelial cells (ECs). These studies reveal the downstream signaling mechanism impacted in CCM disease, but they do not identify strong therapeutic targets or address the question of what activates MEKK3 in brain ECs. Our preliminary studies demonstrate that TLR4 is a critical upstream activator of endothelial MEKK3-KLF signaling that is required for CCM formation, and that gram negative bacteria (GNB) in the gut microbiome are the key in vivo ligand for this pathway in the mouse neonatal CCM model. Genetic analysis of human CCM patients by our co-investigator Dr. Helen Kim has independently identified TLR4 and its co-receptor CD14 as genes associated with CCM lesion number, suggesting that our mouse findings extend to human CCM disease. This proposal will further test the role of endothelial TLR4 signaling and the gut microbiome in CCM disease pathogenesis using mouse and human studies. Aim 1 will (i) test the role of TLR4 during CCM formation in adult mice, (ii) determine whether blocking ant-TLR4 and anti-CD14 antibodies prevent CCM formation in the mouse model, (iii) use mouse genetic approaches to determine the pathway by which TLR4 activates MEKK3 during CCM formation, and (iv) apply genetic approaches to fully interrogate the TLR4-MEKK3- KLF pathway in human patients with CCM disease due to a common mutation in KRIT1. Aim 2 will test the role of GNB and the gut microbiome in CCM pathogenesis by (i) determining whether CCM lesions arise in germ-free animals, (ii) defining microbiomes that confer resistance or susceptibility to CCM disease in mice, and (iii) testing whether breakdown of the gut epithelial barrier accelerates CCM formation in mice. These studies will significantly expand our understanding of the molecular and physiologic basis of CCM pathogenesis, and lay the foundation for translating these discoveries to new therapies for CCM disease based on blocking TLR4 signaling (for which FDA approved agents already exist) or altering the gut microbiome.