S1648 Modulation of Pellino 1 by Helicobacter pylori Lipopolysaccharide Enhances Toll-Like Receptor 2-Mediated Nuclear Factor-Kappa B Activation and Chemokine Induction

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Background & Aims: Toll-like receptors (TLRs) play a key role in host defense against invading pathogens by functioning as primary sensors for conserved microbial structures known as pathogen-associated molecular patterns (PAMPs). Recognition of PAMPs by a particular TLR results in the activation of transcription factors such as NF-κB, AP-1 and interferon regulatory factors, culminating in the induction of gene expression necessary for the co-ordination of the innate immune response. This study set out to identify novel regulators of TLR signaling in response to the gastric pathogen Helicobacter pylori. We investigated the role of the interacting protein Pellino 1 (PELI1) during the TLR2-mediated response toH. pylori lipopolysaccharide (LPS). PELI1 has recently been suggested to interact with components of the TLR signaling pathway (IRAKs 1 & 4, TRAF6), thereby regulating NF-κB activation.Methods: PELI1 expression in response to H. pylori infection or H. pylori LPS was monitored by quantitative real-time PCR in HEK-TLR2 cells and MKN45 gastric epithelial cells. In order to determine the involvement of PELI1 during TLR2-mediated signaling, HEK-TLR2 cells were co-transfected with increasing quantities of an expression vector for PELI1, and either an NF-κB-reporter construct or promoter-reporter constructs for interleukin 8 (IL-8) and chemokine (C-C) motif ligand 20 (CCL20). Forty-eight hours post-transfection, cells were stimulated with either H. pylori LPS or the synthetic TLR2 ligand Pam2CSK4 for 8 hours. Additionally, co-transfection experiments were carried out using small interfering RNA (siRNA) for PELI1. Results: Stimulation of HEK-TLR2 and MKN45 cells with both intact H. pylori or LPS resulted in a significant increase in PELI1 mRNA expression. PELI1 overexpression in HEK-TLR2 cells resulted in a dose-dependent increase in NF-κB activity in response to both Pam2CSK4 and H. pylori LPS. Additionally, increased PELI1 expression led to transcriptional activation of the IL-8 and CCL20 promoters in H. pylori LPS-treated cells. Furthermore, PELI1 knock-down using siRNA inhibited the TLR2-mediated activating properties of Pam2CSK4 and H. pylori LPS. Conclusions: PELI1 expression positively regulates TLR2-mediated signaling in response toH. pylori LPS, resulting in increased NF-κB activation and pro-inflammatory chemokine induction. Thus, modulation of PELI1 by H. pylori and/ or its products may be an important mechanism during H. pylori-associated pathogenesis.