Toll-like Receptor Signaling in the Esophageal Epithelium

描述（由申请人提供）：胃食管反流病（GERD）在美国影响数百万人。除了严重的社会经济负担外，长期的反流性食管炎也是Barrett食管的危险因素，Barrett食管是一种与食管腺癌发展相关的恶性前病变。因此，了解导致酸致食管炎的机制不仅对这种常见疾病的治疗至关重要，而且与癌症预防领域有关。众所周知，长期暴露于胃酸会导致食管损伤，导致食管上皮棘层细胞坏死。此外，在动物和人类胃食管反流模型中，促炎基因的表达在食管上皮中都有所增强。然而，酸诱导的坏死导致食管上皮中趋化因子和粘附分子诱导的机制尚不完全清楚。在本研究中，我们发现原代和永生化的未转化人食管上皮细胞通过toll样受体（TLR）信号传导诱导与胃食管反流发病相关的促炎基因，自主地作为先天免疫效应细胞发挥功能。我们的初步数据进一步表明，坏死食管上皮细胞释放的物质（损伤相关分子模式，DAMPs）可以在没有致病刺激的情况下，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信号通路使食管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粘附所需的信号转导途径。通过细胞信号传导研究和新的体外GERD器官型细胞培养模型，我们将鉴定坏死食管上皮细胞释放的DAMPs，并确定这些DAMPs激活体外人食管上皮细胞TLR2和TLR3信号传导的机制（Specific Aim 1）。我们还将在体外鉴定纯化内源性DAMPs刺激TLR2和TLR3激活的信号转导途径（Specific Aim 2）。最后，我们将通过体外和体内的方法，利用TLR2、TLR3和下游接头分子TRIF、MyD88和TRAF6靶向缺失的小鼠，确定TLR信号在苛性食管损伤小鼠模型中的作用（Specific Aim 3）。来自这些突变小鼠的原代食管上皮细胞将用于询问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应答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. PUBLIC HEALTH RELEVANCE: Gastroesophageal reflux disease (GERD) is highly prevalent in the United States, and carries a significant socioeconomic burden as well as an increased risk for Barrett's esophagus and associated esophageal adenocarcinoma. The studies in this proposal are based upon the hypothesis that Toll-like receptors (TLRs) enable esophageal epithelial cells to sense danger signals released by dead and dying esophageal epithelial cells, thus providing a mechanistic link between acid-induced injury and esophageal inflammation. The results of our studies will elucidate the mechanisms by which acid- induced damage to the esophageal epithelium leads to esophageal inflammation, and may impact the treatment of common esophageal disorders.