OC-038 Pellino 1: a novel regulator of Toll-like receptor-mediated signalling in response toHelicobacter pylorilipopolysaccharide

Abstract

Introduction Toll-like receptors (TLRs) play a key role in host defence 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 signalling in response to the gastric pathogen Helicobacter pylori . We investigated the role of the interacting protein Pellino 1 (PELI1) during the TLR2-mediated response to H pylori lipopolysaccharide (LPS). PELI1 has recently been suggested to interact with components of the TLR signalling pathway (IRAKs 1 and 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 signalling, 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 Pam 2 CSK 4 for 8 h. 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 over-expression in HEK-TLR2 cells resulted in a dose-dependent increase in NF-κB activity in response to both Pam 2 CSK 4 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 Pam 2 CSK 4 and H pylori LPS. Conclusion PELI1 expression positively regulates TLR2-mediated signalling in response to H 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.