TLR3-mediated NF-{kappa}B signaling in human esophageal epithelial cells

Abstract

Despite its position at the front line against ingested pathogens, very little is presently known about the role of the esophageal epithelium in host innate immune defense. As a key player in the innate immune response, Toll-like receptor (TLR) signaling has not been well characterized in human esophageal epithelial cells. In the present study, we investigated the inflammatory response and signaling pathways activated by TLR stimulation of human esophageal cells in vitro. Using quantitative RT-PCR, we profiled the expression pattern of human TLRs 1-10 in primary esophageal keratinocytes (EPC2), immortalized nontransformed esophageal keratinocytes (EPC2-hTERT), and normal human esophageal mucosal biopsies and found that TLRs 1, 2, 3, and 5 were expressed both in vivo and in vitro. Using the cytokine IL-8 as a physiological read out of the inflammatory response, we found that TLR3 is the most functional of the expressed TLRs in both primary and immortalized esophageal epithelial cell lines in response to its synthetic ligand polyinosinic polycytidylic acid [poly(I:C)]. Through reporter gene studies, we show that poly(I:C)-induced NF-kappaB activation is critical for the transactivation of the IL-8 promoter in vitro and that nuclear translocation of NF-kappaB occurs at an early time point following poly(I:C) stimulation of esophageal epithelial cells. Importantly, we also show that poly(I:C) stimulation induces the NF-kappaB-dependent esophageal epithelial expression of TLR2, leading to enhanced epithelial responsiveness of EPC2-hTERT cells to TLR2 ligand stimulation, suggesting an important regulatory role for TLR3-mediated NF-kappaB signaling in the innate immune response of esophageal epithelial cells. Our findings demonstrate for the first time that TLR3 is highly functional in the human esophageal epithelium and that TLR3-mediated NF-kappaB signaling may play an important regulatory role in esophageal epithelial homeostasis.

Fig. 1.

mRNA expression profile of Toll-like receptors (TLRs) in human esophageal epithelial cells. Quantitative RT-PCR was performed using RNA isolated from EPC2 cells, EPC2-hTERT cells, and normal human esophageal biopsy samples. A: TLR 1–10 mRNA expression in esophageal epithelial cell lines EPC2 and EPC2-hTERT; changes in mRNA expression are shown relative to TLR1 expression in EPC2-hTERT cells. B: TLR 1–10 mRNA expression in 4 human esophageal biopsy samples; changes in mRNA expression are shown relative to TLR1 expression in patient 1.

Fig. 2.

Preferential induction of IL-8 by the synthetic TLR3 ligand polyinosinic polycytidylic acid [poly(I:C)] through an NF-κB-dependent mechanism in human esophageal epithelial cell lines. A: mRNA expression of IL-8 following stimulation of EPC2 and EPC2-hTERT cells with the following TLR ligands: peptidoglycan (PGN) (TLR2), Pam3CSK4 (TLR1/2), HKLM (TLR2), poly(I:C) (TLR3), and flagellin (TLR5). B: ELISA quantification of secreted IL-8 (pg/ml) by EPC2 and EPC2-hTERT cells stimulated with TLR 1, 2, 3, and 5 agonists. C: NF-κB-dependent transactivation of the human IL-8 promoter. EPC2-hTERT cells were transfected with the promoterless pGL2Basic vector, a wild-type human IL-8 promoter construct [wt(LUC)], and an IL-8 promoter construct containing a site-directed mutation of its NF-κB binding element [mNF-κB(LUC)], followed by stimulation with poly(I:C) at 10 μg/ml for 24 h. D: poly(I:C)-mediated IL-8 mRNA induction in the presence of the NF-κB inhibitor Bay11-7082 vs. vehicle control (DMSO); **P < 0.005; ***P < 0.001. All results shown are representative of three individual experiments. NS, nonsignificant.

Fig. 3.

Kinetics of NF-κB activation following poly(I:C) stimulation of EPC2-hTERT cells. A: Western blot analysis of the cytoplasmic NF-κB inhibitor IκBα, using proteins isolated from EPC2-hTERT cells following poly(I:C) (10 μg/ml) stimulation for various time points. B: immunofluorescent localization of p65/RelA subunit of NF-κB in unstimulated and poly(I:C)-stimulated EPC2-hTERT cells (10 μg/ml) at the 60-min time point; ×40 magnification.

Fig. 4.

TLR3 ligand activation increases the NF-κB-dependent expression of TLR2 and enhances TLR2 ligand responsiveness. A: time course of poly(I:C)-induced TLR2 mRNA expression. B: poly(I:C)-mediated TLR2 induction in the presence or absence of the NF-κB inhibitor, Bay11-7082. C: effect of TLR3 priming on TLR2 ligand responsiveness in EPC2-hTERT cells. During the first 24 h, EPC2-hTERT monolayers were stimulated with either untreated cell culture media (keratinocyte serum-free media), poly(I:C) (10 μg/ml), PGN (10 μg/ml), or poly(I:C)/PGN costimulation. After 24 h, the media was changed, and cells were treated for an additional 24 h with either media or PGN. All results shown are representative of 3 individual experiments; *P < 0.05, **P < 0.005, ***P < 0.001.

Fig. 5.

TLR3 RNA interference leads to reduced responses to poly(I:C) in EPC2-hTERT cells. EPC2-hTERT cells were transfected with either control short inhibitory RNA (siRNA) or TLR3 siRNA. 72 h after transfection, cells were stimulated with poly(I:C) (10 μg/ml) for 24 h. Decrease in mRNA induction of TLR3, IL-8, and TLR2 following poly(I:C) stimulation. Results shown are representative of 2 individual experiments; *P < 0.05, **P < 0.005, ***P < 0.0001.