Toll-like Receptor Signaling in the Esophageal Epithelium

描述(申请人提供)：胃食道反流病(GERD)在美国影响数以百万计的人。除了严重的社会经济负担外，长期存在的GERD食管炎也是Barrett‘s食道的危险因素，Barrett’s食道是一种与食管腺癌发展相关的癌前疾病。因此，了解酸源性食管炎的发病机制，不仅对这一常见病的治疗至关重要，而且对癌症预防领域也具有重要意义。众所周知，长期暴露在胃酸中会导致食道损伤，导致食道上皮棘层细胞坏死。此外，在GERD动物和人类模型中，促炎症基因在食道上皮中的表达均增强。然而，酸诱导的坏死导致食道上皮细胞产生趋化因子和黏附分子的机制还完全不清楚。在这项研究中，我们现在展示了原代和永生化的未转化的人食道上皮细胞通过Toll样受体(TLR)信号通路诱导与GERD发病相关的促炎基因，作为先天免疫效应细胞自主发挥功能。我们的初步数据进一步表明，在没有致病刺激的情况下，坏死的食道上皮细胞释放的物质(损伤相关分子模式或DAMPS)可以通过TLR2和TLR3依赖的信号转导途径诱导IL-8、ICAM-1和RANTES的表达。因此，我们的总体假设是TLR2和TLR3信号使食道上皮细胞能够感知与细胞坏死相关的分子模式，激活中性粒细胞趋化和黏附所需的信号转导通路。通过GERD的细胞信号转导研究和新的器质性细胞体外培养模型，我们将鉴定坏死的食道上皮细胞释放的DAMs，并确定这些DAMP在体外激活人食道上皮细胞TLR2和TLR3信号的机制(特异性目标1)。我们还将在体外通过纯化的内源性DAMs识别TLR2和TLR3刺激激活的信号转导通路(特定目标2)。最后，我们将使用体外和体内两种方法(特定目标3)，通过靶向缺失TLR2、TLR3和下游适配分子TRIF、MyD88和TRAF6的小鼠，确定TLR信号在腐蚀性食道损伤小鼠模型中的作用。来自这些突变小鼠的原代食道上皮细胞将被用于询问AIMS 1和2中研究的信号通路。总之，这些研究结果将为食道上皮细胞作为先天性免疫效应细胞响应食道损伤的机制提供新的见解，并可能影响高度流行的人类食道疾病的诊断和治疗。

DESCRIPTION (provided by applicant): Gastroesophageal reflux disease (GERD) affects millions of individuals in the United States. In addition to its significant socioeconomic burden, longstanding GERD esophagitis is a risk factor for Barrett's esophagus, a premalignant condition associated with the development of esophageal adenocarcinoma. Thus, an understanding of the mechanisms which lead to acid-induced esophagitis is not only critical for the treatment of this common disease, and is also relevant to the field of cancer prevention. It is known that prolonged exposure to gastric acid leads to esophageal injury, resulting in cell necrosis in the stratum spinosum of the esophageal epithelium. In addition, the expression of proinflammatory genes is enhanced in the esophageal epithelium in both animal and human models of GERD. However, the mechanisms by which acid-induced necrosis leads to the induction of chemokines and adhesion molecules in the esophageal epithelium are completely unknown. In this proposal, we now show that primary and immortalized non-transformed human esophageal epithelial cells function autonomously as innate immune effector cells through the induction of proinflammatory genes relevant to the pathogenesis of GERD, via Toll-like receptor (TLR) signaling. Our Preliminary Data further demonstrates that substances released by necrotic esophageal epithelial cells (damage-associated molecular patterns, or DAMPs) can induce the expression of IL-8, ICAM-1, and RANTES through TLR2 and TLR3-dependent signal transduction pathways in the absence of pathogenic stimulation. Therefore, our overall hypothesis is that TLR2 and TLR3 signaling enables esophageal epithelial cells to sense molecular patterns associated with cell necrosis, activating signal transduction pathways required for neutrophil chemotaxis and adhesion. Through cell-signaling studies and novel organotypic cell culture models of GERD in vitro, we will identify the DAMPs released by necrotic esophageal epithelial cells and determine the mechanisms by which these DAMPs activate TLR2 and TLR3 signaling in human esophageal epithelial cells in vitro (Specific Aim 1). We will also identify the signal transduction pathways activated by TLR2 and TLR3 stimulation by purified endogenous DAMPs in vitro (Specific Aim 2). Finally, we will determine the role of TLR signaling in a mouse model of caustic esophageal injury using mice with targeted deletions of TLR2, TLR3, and downstream adaptor molecules TRIF, MyD88, and TRAF6, using both ex vivo and in vivo methods (Specific Aim 3). Primary esophageal epithelial cells from these mutant mice will be used to interrogate the signaling pathways studied in Aims 1 and 2. Together, the results of these studies will provide new insights into the mechanisms by which esophageal epithelial cells function as innate immune effector cells in response to esophageal damage, and may impact the diagnosis and treatment of highly prevalent human esophageal diseases.