TLR4 and the microbiome in CCM disease

脑海绵状血管畸形（CCM）是一种脑血管疾病， 在美国有20万人，是年轻人中风和癫痫发作的常见原因。 个体目前还没有针对CCM疾病的医学疗法，并且治疗是非常困难的。 仅限于手术切除和抗癫痫药物。我们最近 证明CCM的出现是由于MEKK 3信号的增加， CCM缺陷脑内皮细胞中KLF 2和KLF 4转录因子的表达 细胞（EC）。这些研究揭示了下游信号传导机制的影响， CCM疾病，但他们没有确定强有力的治疗靶点或解决问题 是什么激活了脑内皮细胞中的MEKK 3。我们的初步研究表明，TLR 4 是内皮细胞MEKK 3-KLF信号传导的关键上游激活剂， CCM形成，而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 我们的合作研究者Helen Kim博士独立地对人类CCM患者进行了研究， 将TLR 4及其共受体CD 14鉴定为与CCM病变数量相关的基因， 这表明我们的小鼠发现延伸到人类CCM疾病。这项建议会 进一步测试内皮TLR 4信号传导和肠道微生物组在CCM中的作用 使用小鼠和人类研究的疾病发病机制。目标1将（一）检验以下方面的作用： （ii）确定阻断抗-TLR 4和抗-TLR 4抗体是否在成年小鼠CCM形成过程中起作用， 抗CD 14抗体在小鼠模型中防止CCM形成，（iii）使用小鼠 遗传学方法来确定TLR 4激活MEKK 3的途径， CCM形成，和（iv）应用遗传方法来充分询问TLR 4-MEKK 3- 由于KRIT 1中的常见突变，CCM疾病患者中的KLF通路。 目的2将通过以下方式测试GNB和肠道微生物组在CCM发病机制中的作用：（i） 确定CCM病变是否出现在无菌动物中，（ii）定义微生物组 其赋予小鼠对CCM疾病的抗性或易感性，以及（iii）测试是否 肠上皮屏障的破坏加速了小鼠中CCM的形成。这些 研究将大大扩展我们对分子和生理的理解， CCM发病机制的基础，并为将这些发现转化为 基于阻断TLR 4信号传导的CCM疾病的新疗法（FDA 已经存在批准的药剂）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.