Gene expression in Mouse thrombomoudlin+ and thrombomodulin- dendritic cell

Previously we had shown in a mouse model of bronchial asthma that thrombomodulin (TM; CD141; BDCA3) can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on the cell surface. Thrombomodulin+ dendritic cells are tolerogenic while thrombomodulin- dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin+ and thrombomodulin- dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while cell cycle genes were increased in thrombomodulin-treated and thrombomodulin+ dendritic cells compared to control dendritic cells and thrombomodulin- dendritic cells. We compared the effects on global gene expression of treating dendritic cells with soulble thrombomodulin to no treatment, and dendritic cells separated into thrombomodulin+ and thrombomodulin- dendritic cell subsets. In 4 independent experiments, RNA from mouse bone marrow derived dendritic cells were analyzed on Affymaetrix arrays to determine differences between thrombomodulin+ and thrombomodulin- dendritic cells and if treatment with soluble thrombomodulin altered gene expression.

此前我们在支气管哮喘小鼠模型中证实，血栓调节蛋白（thrombomodulin, TM; CD141; BDCA3）可将免疫原性常规树突状细胞（conventional dendritic cells, cDC）转化为致耐受性树突状细胞（tolerogenic dendritic cells），同时诱导其在细胞表面的自身表达。血栓调节蛋白阳性（thrombomodulin+, TM+）树突状细胞具有致耐受性，而血栓调节蛋白阴性（thrombomodulin-, TM-）树突状细胞则具备促炎活性与免疫原性。本研究推测，对树突状细胞施以血栓调节蛋白处理，可调控其炎症相关基因的表达模式。我们对小鼠骨髓来源的树突状细胞给予可溶性血栓调节蛋白（soluble thrombomodulin, sTM）处理，并检测其表面标志物的表达水平。血栓调节蛋白处理可降低树突状细胞成熟标志物的表达量，同时提升其表面血栓调节蛋白的表达水平。将经血栓调节蛋白处理的树突状细胞与对照组树突状细胞，均分选为血栓调节蛋白阳性与阴性两个亚群，随后通过微阵列（microarray）技术分析其mRNA表达谱。相较于对照组树突状细胞及血栓调节蛋白阴性亚群，经血栓调节蛋白处理的树突状细胞与血栓调节蛋白阳性亚群中，促炎基因及树突状细胞成熟标志物的编码mRNA水平显著下调，而细胞周期相关基因的编码mRNA水平则显著上调。我们对比了可溶性血栓调节蛋白处理树突状细胞与未处理对照组对全局基因表达的影响，同时对比了按血栓调节蛋白表达分选出的阳性与阴性亚群之间的基因表达差异。在4次独立实验中，我们通过Affymetrix基因芯片分析小鼠骨髓来源树突状细胞的RNA，以明确血栓调节蛋白阳性与阴性树突状细胞之间的表达差异，以及可溶性血栓调节蛋白处理是否会改变其基因表达模式。