Toll-like Receptor Signaling in the Esophageal Epithelium

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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.