Gene expression profiling reveals mast cell-dependent inflammation in the meninges in early EAE

The meninges are generally considered relatively inert tissues that house the CSF and provide protection for the brain and spinal cord. However, our previous studies using Kit mutant (Kit W/Wv) mast cell-deficient mice demonstrated that mast cells residing in the dura mater and pia mater exacerbate the severity of experimental autoimmune encephalomyelitis (EAE), the rodent model of the CNS demyelinating disease, multiple sclerosis. These data suggest that the meninges are sites of active immune responses in disease. Gene expression profiles of meningeal tissue from wild type and mast cell deficient mice prior to and at day 6 post-EAE induction were found highly distinct. Increases in both mast cell- and neutrophil-associated transcripts were among the notable disease-related changes observed in wild type mice. Kinetic analyses show that meningeal mast cells are activated within 24 hours of disease induction to express multiple mediators including IL-1b and TNF as well as the neutrophil chemoattractant, CXCL2, an observation corresponding with an influx of neutrophils to the meninges. Neutrophil recruitment as well as the disease-related loss of BBB integrity is dependent on mast cell-derived TNF. These data provide unequivocal evidence that the meninges are sites of early inflammatory events in EAE. Mast cells residing within these tissues promote disease by orchestrating an early and efficient immune cell co-localization resulting in a robust local inflammatory response and a breach of the proximal BBB. We hypothesize that these events reflect an aberrant manifestation of the normal immune surveillance role of the meninges in infection settings. Immunized WT and Kit W/Wv mice were sacrificed on Day 6 post-immunization and perfused with PBS as were naive littermate control mice. The dura mater was immediately removed from the calvarium of the skull and pooled (10 mice/group, 4 groups). RNA was isolated using SV Total RNA Isolation System (Promega). Each pool was analyzed in technical triplicates. Briefly, cRNA was synthesized and amplified/labeled using the Affymetrix Express Kit, then fragmented and hybridized to the The GeneChip® Mouse Genome 430 2.0 Array in accordance to the Affymetrix GeneChip expression analysis technical manual (Affymetrix, Santa Clara, CA). After hybridization, arrays were washed and stained with Affymetrix fluidics protocol FS450_0001 and scanned with a 7G Affymetrix GeneChip Scanner. Image data were analyzed with Affymetrix Expression Console software and normalized with Robust Multichip Analysis (RMA; www.bioconductor.org/) to determine signal log ratios (CITE: Gentleman, R.C., Carey, V.J., Bates, D.M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., et al. (2004). Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5, R80.). The mean fold change was calculated from 3 independent technical replicates for each of the four experimental conditions and assessed by a non-parametric rank product test (CITE: Hong, F., Breitling, R., McEntee, C.W., Wittner, B.S., Nemhauser, J.L., and Chory, J. (2006). RankProd: a bioconductor package for detecting differentially expressed genes in meta-analysis. Bioinformatics 22, 2825-2827). Heat maps were generated with Genesis (Cite: Sturn, A., Quackenbush, J. and Trajanoski, Z. (2002) Genesis: cluster analysis of microarray data. Bioinformatics, 18, 207-208).脑膜通常被认为是相对惰性的组织，其容纳脑脊液（cerebrospinal fluid, CSF）并为脑与脊髓提供保护。然而，本团队此前利用Kit突变（Kit W/Wv）肥大细胞缺陷小鼠开展的研究证实，定位于硬脑膜与软脑膜的肥大细胞会加重实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）的病情严重程度——该疾病是中枢神经系统（CNS）脱髓鞘疾病多发性硬化的啮齿类动物模型。上述数据表明，脑膜是疾病发生时活跃免疫应答的发生位点。野生型（wild type, WT）与肥大细胞缺陷小鼠的脑膜组织，在EAE诱导前及诱导后第6天的基因表达谱存在显著差异。野生型小鼠中观察到的显著疾病相关变化包括：肥大细胞相关转录本与中性粒细胞相关转录本的表达均出现上调。动力学分析显示，脑膜肥大细胞在疾病诱导后24小时内即被激活，表达包括白细胞介素1β（IL-1β）、肿瘤坏死因子（TNF）以及中性粒细胞趋化因子CXCL2在内的多种介质；这一观察结果与中性粒细胞向脑膜的浸润相吻合。中性粒细胞的招募以及疾病相关的血脑屏障（BBB）完整性丧失均依赖于肥大细胞来源的TNF。上述数据提供了明确的证据，表明脑膜是EAE早期炎症事件的发生位点。定位于这些组织中的肥大细胞通过介导早期且高效的免疫细胞共定位，进而触发强烈的局部炎症反应并破坏邻近的血脑屏障，从而促进疾病进展。本团队提出假说：上述事件反映了脑膜在感染情境下正常免疫监视功能的异常表现。于免疫后第6天处死免疫后的WT与Kit W/Wv小鼠，并以磷酸盐缓冲液（PBS）进行灌流；同窝未免疫对照小鼠亦采用相同处理流程。即刻从颅骨颅盖处分离硬脑膜，并将样本进行混合（每组10只小鼠，共4组）。采用SV总RNA提取系统（Promega）分离RNA。每个混合样本设置3次技术重复进行分析。简言之，采用Affymetrix Express试剂盒合成并扩增/标记互补RNA（cRNA），随后按照Affymetrix GeneChip表达分析技术手册（Affymetrix，美国加利福尼亚州圣克拉拉市）将cRNA片段化并与GeneChip®小鼠基因组430 2.0阵列进行杂交。杂交完成后，按照Affymetrix流体学方案FS450_0001对芯片进行洗涤与染色，并采用7G Affymetrix GeneChip扫描仪进行扫描。采用Affymetrix Expression Console软件分析图像数据，并通过稳健多芯片分析（Robust Multichip Analysis, RMA；www.bioconductor.org/）进行归一化处理，以计算信号对数比（引用文献：Gentleman, R.C.等，2004，Bioconductor：面向计算生物学与生物信息学的开源软件开发，Genome Biology 5, R80）。针对4种实验条件分别从3次独立技术重复中计算平均倍数变化，并通过非参数秩积检验进行显著性评估（引用文献：Hong, F.等，2006，RankProd：用于荟萃分析中差异表达基因检测的Bioconductor包，Bioinformatics 22, 2825-2827）。采用Genesis软件绘制热图（引用文献：Sturn, A.等，2002，Genesis：微阵列数据的聚类分析，Bioinformatics, 18, 207-208）。