Combination of Helicobacter pylori infection and the interleukin 8 –251 T > A polymorphism, but not the mannose-binding lectin 2 codon 54 G > A polymorphism, might be a risk factor of gastric cancer

The Helicobacter pylori infection rate is about 50% among the worldwide adult population [ 1]. In Korea, the adult H. pylori infection rate was 66.9% in 1998, 59.6% in 2005, and dropped to 54.4% in 2011 [ 2]. The main cause of this decrease in infection rate is the improvement in unsanitary environmental conditions. Besides environmental factors, bacterial and host factors are involved in the pathogenesis of H. pylori infection. With regard to bacterial factors, H. pylori strains possessing the virulence factors cagA, vacA s1a/m1, and iceA1 are known to be particularly virulent, and are frequently associated with severe gastric epithelial damage [ 3]. In contrast to Western populations, the cagA protein is commonly found in Korean patients with gastric cancer (GC) and duodenal ulcer (DU) [ 4]. However, there have been no associations reported between different H. pylori genotypes and clinical outcome in Korean patients [ 5, 6].

With regard to host factors, host genetic variants may influence susceptibility to H. pylori and the pathogenesis of gastroduodenal diseases. Host factors are mainly related to two processes: recognition of H. pylori by the innate immune system, and the level of the cytokine response [ 7, 8]. Polymorphisms in pro- and anti-inflammatory cytokines are associated with the risk of atrophic gastritis (AG) and GC. Interleukin 1 beta (IL-1β), tumor necrosis factor-alpha, IL-6, and IL-8 are up-regulated in response to H. pylori infection [ 9]. Several anti-inflammatory cytokines such as IL-Rα and IL-10 are also related to H. pylori infection [ 10, 11].

IL-8 is a major neutrophil-activating cytokine and plays a central role in the immuno-pathogenesis of H. pylori-induced gastric mucosal injury. IL-8 levels are 10-fold higher in GC specimens than in normal gastric tissues [ 12]. The IL-8 -251 T > A polymorphism has been reported to be associated with increased production of IL-8 protein, and higher risks of AG, gastric ulcer, and GC [ 13– 17]. However, many other reports are inconsistent with these findings [ 18– 23], and a meta-analysis of epidemiological studies revealed no overall association [ 24].

The innate immune response to H. pylori infection is a further candidate host factor. Toll-like receptors (TLRs) recognize conserved pathogen-associated molecular patterns expressed by many pathogens, including H. pylori [ 25]. Mannose-binding lectin (MBL), a pattern recognition receptor encoded by the MBL2 gene, recognizes lipopolysaccharide in the cell wall of gram-negative bacteria such as H. pylori [ 26, 27]. H. pylori activates MBL in vitro, resulting in complement deposition [ 28, 29]. Some studies have found a possible association of MBL2 haplotype with susceptibility to H. pylori infection, as well as with risk of GC [ 30, 31]. However, other studies did not find any significant association between MBL genotype and H. pylori infection prevalence or GC risk [ 32, 33].

Serum MBL levels vary widely between healthy individuals, mainly due to genetic variation [ 34– 36]. The variation in serum MBL levels is correlated with point mutations in the coding and promoter regions of MBL2. Three mutations within exon 1 (in codons 52, 54, and 57) interfere with MBL function and are associated with low serum levels of MBL. In African populations, point mutations at codons 52 and 57 occur frequently [ 36, 37]. In Caucasians, mutations at codons 52 and 54 are common [ 38]. In Chinese, Japanese, and Korean populations, mutations are predominantly common in codon 54, but not in codons 52 or 57 [ 39– 41]. Polymorphisms within the promoter and 5′-untranslated regions of MBL2 also affect serum levels of MBL, but the effects were found to be lower than those of the exon 1 polymorphisms [ 41].

The aims of this study were: 1) to examine the influence of the polymorphisms in codons 52, 54, and 57 of MBL2 (related to innate immunity) on susceptibility to H. pylori infection; 2) to evaluate the association of the IL-8 -251 T > A polymorphism with the risk of gastroduodenal diseases in a Korean population; and 3) to analyze our and other investigators’ large-scale data regarding the IL-8 -251 T > A polymorphism and GC risk in Korean, Japanese, Chinese, and Caucasian populations.

From January 2012 to May 2015, H. pylori-negative healthy control subjects (control, n = 176), H. pylori-positive non-atrophic gastritis patients (NAG, n = 108), H. pylori-positive mild AG patients ( n = 52), H. pylori-positive severe AG patients ( n = 61), DU patients ( n = 175), and GC patients ( n = 283) were consecutively enrolled.

All participants ( n = 855) underwent upper gastrointestinal endoscopy and routine laboratory tests. The controls were asymptomatic subjects who visited the Health Screening Center for a health status check-up, and their endoscopic findings were normal. Exclusion criteria were H. pylori eradication history; use of antibiotics, proton pump inhibitors, nonsteroidal anti-inflammatory drugs, or anticoagulant drugs; and severe systemic illnesses. Age, sex, alcohol consumption (current or never), smoking habits (current or never), salt intake (high, low-moderate), and family history of GC (first-degree relatives) were recorded. Informed consent was obtained from all included subjects. The Institutional Review Board of the Kyung Hee University Hospital approved the study protocol (KMC IRB 1523–04).

Table 1 shows clinical features of the control and disease groups. Age, sex ratio, and alcohol consumption were similar among all groups. Risk factors for GC, such as smoking, high salt intake, and family history of GC, were more frequently observed in the H. pylori-positive GC group than in the control group. The differences were statistically significant.

The frequencies of the MBL2 codon 54 and IL-8 -251 polymorphisms in the control group did not deviate significantly from those expected under Hardy–Weinberg equilibrium ( p = 1.000 and p = 0.184, respectively). In this study population, MBL exon 1 polymorphic variants were found only in codon 54. There were no variants at codons 52 or 57; only the wild type was observed.

The innate immune response is the first line of defense against H. pylori infection in the human stomach. TLR and MBL are recognized as important proteins in innate immunity. Several studies have demonstrated that TLR4 and TLR2 polymorphisms are associated with the risk of GC [ 45– 47]. However, some of the associations are controversial, and there are discrepancies between the results for Asian and Western populations [ 48]. A recent study in the Netherlands found that only the TLR1 polymorphism is associated with the prevalence of H. pylori seropositivity [ 49]. Further studies are needed in other populations worldwide to confirm these associations.

MBL binds to bacteria, yeasts, and viruses via specific repeated oligosaccharide moieties on the cell surface. MBL activates the complement-lectin pathway, facilitates opsonization and phagocytosis, and induces direct cellular lysis. MBL deficiency or a low serum MBL level has been associated with several infectious and autoimmune diseases, including meningococcal meningitis, pneumonia, arterial thrombosis, systemic lupus erythematosus, and celiac disease [ 50, 51].

At the time of its discovery, H. pylori was considered an extracellular bacterium that mainly colonized the gastric mucus layer or attached to gastric epithelial cells. However, it has since been demonstrated that H. pylori invades the lamina propria and gastric epithelial cells [ 52]. Therefore, H. pylori might be a target of phagocytosis by MBL activation. There have been few clinical studies regarding the role of MBL in H. pylori infection. Various microorganisms such as H. pylori, Neisseria meningitidis groups B and C, Nocardia farcinica, and Legionella pneumophila induce MBL activity in vitro [ 28]. Activated complements are found in the epithelium of patients with H. pylori-associated gastritis [ 29]. One pediatric study reported that MBL2 mRNA expression in gastric biopsy specimens was higher in H. pylori-positive chronic gastritis than in H. pylori-negative chronic gastritis patients [ 53]. However, the study had two weaknesses in terms of its ability to reach conclusions regarding the role of MBL2 expression in the development of H. pylori-infected chronic gastritis. The first weakness is the small number of biopsy specimens that were obtained, with only five H. pylori-positive children and four control children included. The second weakness is that they could not find any association between MBL2 genotype and the risk of H. pylori-infected chronic gastritis.

The association between the MBL2 haplotype and the risk of GC has been studied previously [ 30, 31]. A study conducted in Southern Italy found that the HYP + D haplotype (H/Y promoter region mutation + P untranslated region mutation + codon 52 mutation) may be a genetic marker for H. pylori-positive GC risk [ 30]. Another study performed in Warsaw, Poland found that the HY + D haplotype (H/Y promoter region mutation + codon 52 mutation) was related to an increased risk of GC compared with the HY+ A haplotype (H/Y mutation + codon 52 wild type) [ 31]. Therefore, the codon 52 D variant (cysteine > arginine) was specifically related to the risk of GC in two populations. In contrast to the above studies, which reported positive associations, Australian researchers evaluated healthy individuals for H. pylori infection, MBL2 genotype, mannan binding level, and complement 4 level in plasma, and found that MBL deficiency, defined by either genotype or plasma activity, was not associated with higher susceptibility to H. pylori infection [ 33]. In a Japanese study, they could no significant differences were found in MBL2 genotypes between GC patients and healthy controls [ 32]. Instead, the investigators found that the MBL2 codon 54 polymorphism was weakly associated with severe AG and advanced GC [ 32, 54]. In the present study, we first demonstrated that the codon 54 polymorphism did not increase susceptibility to H. pylori infection in a Korean population. Secondly, we did not find any evidence of a role for MBL2 in the development of gastroduodenal diseases. Thirdly, we did not find a higher risk of advanced GC or severe AG compared to early GC or mild AG, respectively, associated with MBL2 genotype.

With regard to interracial differences, the Korean population differs from the European (Italian and Polish) and African populations reported previously. However, the results for the Korean population are very similar to those reported for the Chinese and Japanese populations [ 35, 38– 40]. The frequencies of point mutations in European populations are in between those of the East Asian and African populations.

In the present study, serum levels of MBL, an indicator of the functional activity of MBL, differed significantly according to the genotype. However, serum MBL levels were not significantly different between the control and disease groups, because the frequency of each genotype was similar in these groups.

H. pylori infection stimulates IL-8 gene expression and increases the IL-8 cytokine level in gastric epithelial cells. A significant correlation between a high level of IL-8 in the gastric mucosa and the risk of GC has been reported [ 13]. Our previous study found that the IL-8 level in gastric mucosal tissues was significantly higher in H. pylori-infected subjects compared with that in H. pylori non-infected subjects, irrespective of their gastroduodenal disease phenotype. After H. pylori eradication, the IL-8 level decreased dramatically, to the same level observed in non-infected subjects [ 55]. In this study, we confirmed once again that the IL-8 level in gastric mucosal tissues is mainly dependent on H. pylori-positive status.

It has been reported that the IL-8 -251 T > A polymorphism is related to higher levels of IL-8 and to an increased risk of AG, gastric ulcer, and GC [ 13, 14]. In this study, we also demonstrated that the IL-8 -251 T > A polymorphism increased IL-8 production, and was significantly associated with the risk of GC and severe AG. However, many other epidemiological studies have reported negative associations between the IL-8 -251 polymorphism and GC risk (18–23), and a meta-analysis revealed no overall association (24). In this study, we analyzed large-scale raw data from controls and GC patients from Korean, Japanese, Chinese, and Caucasian (Poland, Finland, and Portugal) populations (13–23). Korean results, including ours, were consistent with Japanese results, but not with Chinese or Caucasian results. The concordance between the Korean and Japanese results might be explained by genetic similarities. In a large study of single nucleotide polymorphism (SNP) maps covering the human genome performed in African Americans, Asians (Japanese-Chinese-Korean), and European Americans (Caucasians) [ 56], SNP differences in autosomes were only 5.86% between Korean and Japanese populations. Therefore, the Korean population is very similar to the Japanese population with respect to the pattern of SNPs [ 56].

The MBL2 codon 54 G > A polymorphism does not influence susceptibility to H. pylori infection and does not increase the risk of gastroduodenal diseases. We suggest that a combination of the IL-8 -251 T > A polymorphism and increased IL-8 production in response to H. pylori infection may be a risk factor for severe AG and GC development in a Korean population.