Centre of Perinatal Medicine and Health, Department of Physiology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
Bioinformatics Core Facility, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
Brain Behav Immun. 2019 Jul;79:216-227. doi: 10.1016/j.bbi.2019.02.004. Epub 2019 Feb 26.
Perinatal infection and inflammation are major risk factors for injury in the developing brain, however, underlying mechanisms are not fully understood. Leukocyte migration to the cerebrospinal fluid (CSF) and brain is a hallmark of many pathologies of the central nervous system including those in neonates. We previously reported that systemic activation of Toll-like receptor (TLR) 2, a major receptor for gram-positive bacteria, by agonist Pam3CSK4 (P3C) resulted in dramatic neutrophil and monocyte infiltration to the CSF and periventricular brain of neonatal mice, an effect that was absent by the TLR4 agonist, LPS. Here we first report that choroid plexus is a route of TLR2-mediated leukocyte infiltration to the CSF by performing flow cytometry and transmission electron microscopy (TEM) of the choroid plexus. Next, we exploited the striking discrepancy between P3C and LPS effects on cell migration to determine the pathways regulating leukocyte trafficking through the choroid plexus. We performed RNA sequencing on the choroid plexus after administration of P3C and LPS to postnatal day 8 mice. A cluster gene analysis revealed a TLR2-specific signature of chemotaxis represented by 80-fold increased expression of the gene Ccl3 and 1000-fold increased expression of the gene Cxcl2. Ingenuity pathway analysis (IPA) revealed TLR2-specific molecular signaling related to cytoskeleton organization (e.g. actin signaling) as well as inositol phospholipids biosynthesis and degradation. This included upregulation of genes such as Rac2 and Micall2. In support of IPA results, ultrastructural analysis by TEM revealed clefting and perforations in the basement membrane of the choroid plexus epithelial cells in P3C-treated mice. In summary, we show that the choroid plexus is a route of TLR2-mediated transmigration of neutrophils and monocytes to the developing brain, and reveal previously unrecognized mechanisms that includes a specific chemotaxis profile as well as pathways regulating cytoskeleton and basement membrane remodeling.
围产期感染和炎症是导致发育中大脑损伤的主要危险因素，但其中的机制尚不完全清楚。白细胞向脑脊液（CSF）和大脑的迁移是包括新生儿在内的许多中枢神经系统疾病的标志。我们之前报道过，革兰氏阳性菌主要受体 Toll 样受体（TLR）2 的系统激活剂 Pam3CSK4（P3C）导致新生小鼠 CSF 和脑室周围脑实质中中性粒细胞和单核细胞的大量浸润，而 TLR4 激动剂 LPS 则没有这种作用。在这里，我们首先通过对脉络丛进行流式细胞术和透射电镜（TEM）检测，报告了 TLR2 介导的白细胞向 CSF 浸润的途径是脉络丛。接下来，我们利用 P3C 和 LPS 对细胞迁移的影响之间的显著差异，来确定调节白细胞通过脉络丛的途径。我们对 P3C 和 LPS 给药后出生后第 8 天的小鼠脉络丛进行了 RNA 测序。聚类基因分析显示，TLR2 特异性趋化基因 Ccl3 的表达增加了 80 倍，基因 Cxcl2 的表达增加了 1000 倍，代表了趋化特征。IPA 分析显示，TLR2 特异性分子信号与细胞骨架组织（如肌动蛋白信号）以及肌醇磷脂生物合成和降解有关。这包括基因如 Rac2 和 Micall2 的上调。支持 IPA 结果，TEM 的超微结构分析显示，在 P3C 处理的小鼠脉络丛上皮细胞的基膜上出现裂孔和穿孔。总之，我们表明脉络丛是 TLR2 介导的中性粒细胞和单核细胞向发育中大脑迁移的途径，并揭示了以前未被识别的机制，包括特定的趋化特征以及调节细胞骨架和基膜重塑的途径。
