The NOD-like receptor signalling pathway in Helicobacter pylori infection and related gastric cancer: a case-control study and gene expression analyses

Abstract

Background: Currently, it is well established that cancer arises in chronically inflamed tissue. A number of NOD-like receptors (NLRs) form inflammasomes, intracellular multiprotein complexes critical for generating mature pro-inflammatory cytokines (IL-1β and IL-18). As chronic inflammation of the gastric mucosa is a consequence of Helicobacter pylori infection, we investigated the role of genetic polymorphisms and expression of genes involved in the NLR signalling pathway in H. pylori infection and related gastric cancer (GC).

Materials and methods: Fifty-one genetic polymorphisms were genotyped in 310 ethnic Chinese (87 non-cardia GC cases and 223 controls with functional dyspepsia). In addition, gene expression of 84 molecules involved in the NLR signalling pathway was assessed in THP-1 cells challenged with two H. pylori strains, GC026 (GC) and 26695 (gastritis).

Results: CARD8-rs11672725, NLRP3-rs10754558, NLRP3-rs4612666, NLRP12-rs199475867 and NLRX1-rs10790286 showed significant associations with GC. On multivariate analysis, CARD8-rs11672725 remained a risk factor (OR: 4.80, 95% CI: 1.39-16.58). Further, NLRP12-rs2866112 increased the risk of H. pylori infection (OR: 2.13, 95% CI: 1.22-3.71). Statistical analyses assessing the joint effect of H. pylori infection and the selected polymorphisms revealed strong associations with GC (CARD8, NLRP3, CASP1 and NLRP12 polymorphisms). In gene expression analyses, five genes encoding NLRs were significantly regulated in H. pylori-challenged cells (NLRC4, NLRC5, NLRP9, NLRP12 and NLRX1). Interestingly, persistent up-regulation of NFKB1 with simultaneous down-regulation of NLRP12 and NLRX1 was observed in H. pylori GC026-challenged cells. Further, NF-κB target genes encoding pro-inflammatory cytokines, chemokines and molecules involved in carcinogenesis were markedly up-regulated in H. pylori GC026-challenged cells.

Conclusions: Novel associations between polymorphisms in the NLR signalling pathway (CARD8, NLRP3, NLRP12, NLRX1, and CASP1) and GC were identified in Chinese individuals. Our genetic polymorphisms and gene expression results highlight the relevance of the NLR signalling pathway in gastric carcinogenesis and its close interaction with NF-κB.

Figure 1. Helicobacter pylori up-regulates NFKB1 and NF-κB target genes, and down-regulates NF-κB-negative regulators.

A) Gene expression of cytokines and chemokines in H. pylori-challenged THP-1 cells, B) Gene expression of NOD-like receptors in H. pylori-challenged THP-1 cells, C) NFKB1 expression in H. pylori-challenged THP-1 cells and D) PTGS2 and BIRC3 expression in H. pylori-challenged THP-1 cells. Fold-change (2∧(−Delta Delta Ct)) is the normalized gene expression (2∧(−Delta Ct)) in THP-1 cells challenged with H. pylori (GC026 and 26695) divided the normalized gene expression (2∧(−Delta Ct)) in their respective control group. Fold-regulation represents fold-change results in a biologically meaningful way. Fold-change values greater than one indicate up-regulation, and the fold-regulation is equal to the fold-change. Fold-change values less than one indicate down-regulation, and the fold-regulation is the negative inverse of the fold-change. Fold-difference compared to the control group showing a *P-value<0.05 and a **P-value<0.01. P-values were obtained with a Student’s t-test.

Figure 2. Polymorphisms in the NOD-like receptor signalling pathway increase the risk of Helicobacter pylori-related gastric cancer.

Individuals harbouring NLRP12, NLRX1 and CARD8 polymorphisms would have decreased levels or defective NLRP12, NLRX1 and CARD8, and thus, would be more susceptible to acquisition of H. pylori in childhood and present deregulation of NF-κB. Once established, H. pylori infection appears to intensify the decreased expression of these NF-κB-negative regulators. These factors would lead to the production of pro-inflammatory cytokines (IFNB1, IL1, IL-12B, IL6, IL33 and TNF), chemokines (CXCL1, CXCL2, CCL5) and carcinogenesis-related molecules (PTGS2 and BIRC3), among others, which would facilitate the sequence of events that characterises GC development including inflammation, atrophy, intestinal metaplasia, dysplasia, carcinoma in situ, and finally invasive gastric cancer.