Insitute for Laboratory Animal Science, Hannover Medical School, Hannover, Germany.
Inflamm Bowel Dis. 2009 Dec;15(12):1824-36. doi: 10.1002/ibd.21030. Epub 2009 Jul 27.
By combining QTL and gene expression analyses, we have previously identified Cd14 as a potential candidate gene contributing to the differential IBD susceptibility of C3H/HeJBir (C3/J)-Il10(-/-) mice [carrying IBD-resistance alleles at this QTL (Cdcs6)] and C57BL/6J (B6)-Il10(-/-) mice, corroborating studies that showed an association of a CD14-promoter polymorphism with Crohn's disease and ulcerative colitis. The aim of the present study was to analyze the molecular mechanisms leading to differential intestinal expression of Cd14 and its contribution to IBD development.
Intestinal CD14 expression was assessed by FACS, immunohistochemistry, and ELISA on supernatants of primary epithelial cell and tissue cultures. RAW264.7 cells were stimulated with LPS and PGN in the presence or absence of CD14. Cd14 alleles were sequenced and promoters cloned for luciferase assays in transfected RAW264.7 cells. The severity of typhlocolitis between Cd14(-/-) and wild-type mice was compared in 2 distinct mouse models of IBD (acute DSS and Il10(-/-) ).
In the gut, CD14 was detected mainly in its soluble form (sCD14), with higher expression in C3/J-Il10(-/-) mice. Polymorphisms in C3/J mice caused higher activity of the Cd14 promoter (luciferase assays). Intestinal sCD14 concentrations influenced the LPS and PGN responses of RAW264.7 cells. In vivo, genetic deletion of Cd14 aggravated colitis in both mouse models of IBD.
Our study shows that Cd14-promoter polymorphisms affect CD14 expression and confirms the protective effect of CD14 against experimental IBD, potentially mediated by TLR2- and TLR4-dependent effects on intestinal barrier function. These findings support the concept that human CD14-promoter polymorphisms contribute to disease development.
通过结合 QTL 和基因表达分析，我们之前已经确定 CD14 是一个潜在的候选基因，它导致 C3H/HeJBir（携带该 QTL（Cdcs6）上的 IBD 抗性等位基因）和 C57BL/6J（B6）-Il10（-/-）小鼠对 IBD 的易感性不同[）-Il10（-/-）小鼠，证实了先前的研究表明 CD14 启动子多态性与克罗恩病和溃疡性结肠炎有关。本研究的目的是分析导致肠道 CD14 表达差异的分子机制及其对 IBD 发展的贡献。
通过 FACS、免疫组织化学和 ELISA 评估原发性上皮细胞和组织培养物上清液中的肠道 CD14 表达。用 LPS 和 PGN 刺激 RAW264.7 细胞，在存在或不存在 CD14 的情况下进行刺激。对 Cd14 等位基因进行测序，并克隆启动子进行转染 RAW264.7 细胞的荧光素酶测定。在 2 种不同的 IBD 小鼠模型（急性 DSS 和 Il10（-/-））中比较 Cd14（-/-）和野生型小鼠之间的 typhlocolitis 严重程度。
在肠道中，CD14 主要以其可溶性形式（sCD14）检测到，C3/J-Il10（-/-）小鼠中的表达更高。C3/J 小鼠中的多态性导致 Cd14 启动子的活性更高（荧光素酶测定）。肠道 sCD14 浓度影响 RAW264.7 细胞对 LPS 和 PGN 的反应。在体内，Cd14 的遗传缺失加重了这两种 IBD 小鼠模型的结肠炎。
我们的研究表明，Cd14 启动子多态性影响 CD14 的表达，并证实 CD14 对实验性 IBD 具有保护作用，可能通过 TLR2 和 TLR4 对肠道屏障功能的影响介导。这些发现支持人类 CD14 启动子多态性有助于疾病发展的概念。
