Induction of TLR-2 and TLR-5 Expression by Helicobacter pylori Switches cagPAI-Dependent Signalling Leading to the Secretion of IL-8 and TNF-α

Suneesh Kumar Pachathundikandi,Sabine Brandt,Steffen Backert,Joseph Madassery,Yoshio Yamaoka

doi:10.1371/journal.pone.0019614

Suneesh Kumar Pachathundikandi, Sabine Brandt + Show 3 more

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https://doi.org/10.1371/journal.pone.0019614

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Abstract

Helicobacter pylori is the causative agent for developing gastritis, gastric ulcer, and even gastric cancer. Virulent strains carry the cag pathogenicity island (cagPAI) encoding a type-IV secretion system (T4SS) for injecting the CagA protein. However, mechanisms of sensing this pathogen through Toll-like receptors (TLRs) and downstream signalling pathways in the development of different pathologies are widely unclear. Here, we explored the involvement of TLR-2 and TLR-5 in THP-1 cells and HEK293 cell lines (stably transfected with TLR-2 or TLR-5) during infection with wild-type H. pylori and isogenic cagPAI mutants. H. pylori triggered enhanced TLR-2 and TLR-5 expression in THP-1, HEK293-TLR2 and HEK293-TLR5 cells, but not in the HEK293 control. In addition, IL-8 and TNF-α cytokine secretion in THP-1 cells was induced in a cagPAI-dependent manner. Furthermore, we show that HEK293 cells are not competent for the uptake of T4SS-delivered CagA, and are therefore ideally suited for studying TLR signalling in the absence of T4SS functions. HEK293 control cells, which do not induce TLR-2 and TLR-5 expression during infection, only secreted cytokines in small amounts, in agreement with T4SS functions being absent. In contrast, HEK293-TLR2 and HEK293-TLR5 cells were highly competent for inducing the secretion of IL-8 and TNF-α cytokines in a cagPAI-independent manner, suggesting that the expression of TLR-2 or TLR-5 has profoundly changed the capability to trigger pro-inflammatory signalling upon infection. Using phospho-specific antibodies and luciferase reporter assays, we further demonstrate that H. pylori induces IRAK-1 and IκB phosphorylation in a TLR-dependent manner, and this was required for activation of transcription factor NF-κB. Finally, NF-κB activation in HEK293-TLR2 and HEK293-TLR5 cells was confirmed by expressing p65-GFP which was translocated from the cytoplasm into the nucleus. These data indicate that H. pylori-induced expression of TLR-2 and TLR-5 can qualitatively shift cagPAI-dependent to cagPAI-independent pro-inflammatory signalling pathways with possible impact on the outcome of H. pylori-associated diseases.

Highlights

The innate immune system is evolutionarily conserved in higher eukaryotes and is the first line of defence for protecting hosts from invading microbial pathogens [1,2,3]
We demonstrate that H. pylori infection of either Toll-like receptors (TLRs)-expressing cell line induces the phosphorylation of IRAK-1 and IkB followed by nuclear factor (NF)-kB activation, which is in agreement with the hypothesis that upregulation of both TLR-2 and TLR-5 by H. pylori can switch cag pathogenicity island (cagPAI)-dependent signalling to cagPAI-independent TLR signalling, may changing the outcome of infections substantially
The results indicate the induction of IL-8 and tumour necrosis factor alpha (TNF-a) secretion from THP-1 cells during infection with wild-type H. pylori (Figure 2)

Summary

Introduction

The innate immune system is evolutionarily conserved in higher eukaryotes and is the first line of defence for protecting hosts from invading microbial pathogens [1,2,3]. Toll-like receptors (TLRs) are surface-exposed pattern recognition receptors which can recognize molecular structures on pathogenic microbes associated molecular patterns (PAMPs). Sensing of microorganisms intracellularly can be achieved by nucleotide oligomerization domain (NOD)-like receptors (NLRs) and Retinoic acid inducible gene-1 (RIG-1)-like receptors (RLRs). These two families comprise the intracellular sensors, of which NLRs recognize primarily molecules of bacterial origin while RLRs are involved in antiviral responses [7,8]. Microbial pattern recognition by TLRs in dendritic cells upregulate the expression of co-stimulatory molecules, which is essential for the initiation of adaptive immune responses in the host, linking innate and adaptive immunity [2,10]

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