The role of the pathogen recognition molecule, NOD1, in the induction of gastric epithelial cell signalling during Helicobacter pylori infection.
2017-01-13T01:26:51Z (GMT) by
Helicobacter pylori colonises a large proportion of the world’s population, with infection invariably leading to chronic, life-long gastritis. While the infection often persists undiagnosed and without causing severe pathology, there are a number of host, bacterial and environmental factors that can influence disease outcome. In particular, the H. pylori cag pathogenicity island (cagPAI) encodes a Type IV secretion system (T4SS), which facilitates the translocation of bacterial effectors, such as peptidoglycan, into the host cell. The cytosolic pathogen recognition molecule, Nucleotide Oligomerisation Domain 1 (NOD1), senses the peptidoglycan of H. pylori and other Gram-negative organisms, resulting in the rapid initiation pro-inflammatory responses that are critical for the induction of innate and eventually adaptive immune responses. H. pylori is generally considered to be an extracellular pathogen and gastric epithelial cells are the pathogen's primary point of contact with the host. Therefore, complete characterisation of H. pylori-induced signalling in these cells, particularly with respect to the role of NOD1 and the cagPAI, will offer further insights into the mechanisms involved in the activation and recruitment of host immune effectors. Furthermore, these studies were designed to investigate the existence of a positive feedback mechanism between inflammatory responses and gastric epithelial cell signalling once infection is established. In order to ascertain the involvement of NOD1 in host signalling responses to H. pylori, this study employed an in vitro co-infection model using various isogenic H. pylori mutants and a gastric epithelial cell line stably expressing siRNA to knock-down NOD1 expression. In addition, gastric biopsies from patients with differing degrees of gastritis or gastric cancer were analysed to assess the expression of various immune-related genes involved in inflammation and disease. Initially, we addressed the respective contributions of NOD1 and H. pylori virulence factors to transcription factor activation and the expression of signalling molecules during infection. Accordingly, this study confirmed the role of NOD1 in the activation and nuclear translocation of "Nuclear Factor-kappa B" (NF-кB). Furthermore, we found that H. pylori induced the rapid phosphorylation of p38 and Extracellular-signal Regulated Kinase (ERK) Mitogen Activated Protein Kinases (MAPKs) in a NOD1-dependent manner and that this was essential for the downstream activation of "Activating Protein-1" (AP-1), an important pro-inflammatory transcription factor involved in host responses to infection. Intriguingly, while NOD1 was essential for "c-Jun N-terminal Kinase" (JNK) MAPK phosphorylation in Shigella flexneri-infected cells, it was not required for the cagPAI-dependent phosphorylation of JNK, nor was it involved in the induction of a characteristic cell scattering and elongation phenotype observed in H. pylori-stimulated epithelial cells. These findings were extended to investigate potential cross-talk between NOD1 and the chronic inflammatory responses induced during H. pylori infection. We show that H. pylori is capable of activating and enhancing certain components of the IFN-y signalling pathway in gastric epithelial cells, specifically through NOD1 and the cagPAI. Many of these responses are known to be involved in the recruitment of immune cells to the site of infection, which would further exacerbate inflammation. In agreement with this hypothesis, the expression of several key factors involved in NOD1 or IFN-y signalling pathways were found to be significantly upregulated in gastric biopsies from patients infected with H. pylori, particularly in those exhibiting either severe gastritis or tumour formation. Taken together, the findings demonstrate a mechanism through which virulent strains of H. pylori are able to exacerbate inflammatory responses via activation of NOD1-dependent signalling pathways other than those previously reported. Furthermore, the recruitment of immune cells to the gastric mucosa may enhance epithelial responses in a cagPAI-dependent manner, offering insight into how virulent H. pylori isolates perpetuate the cycle of chronic inflammation. This work has identified a number of novel signalling and inflammatory responses not previously reported for H. pylori or associated gastric pathologies, which will form the basis for exciting future research.