Association of Specific Haplotype of Tumor Necrosis Factor-ɑ and Interleukin-1β Polymorphisms with Helicobacter pylori Infection and Gastric Carcinogenesis

Abstract

Introduction: The Helicobacter pylori infection and cytokine-mediated inflammatory responses play significant roles in the pathogenesis of gastric cancer (GC). This study was performed to determine the association between the risk of GC and genetic polymorphisms in interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α). Methods: The polymorphisms of IL-1β and TNF-α genes were analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) in 290 patients who underwent endoscopy. Infection with H. pylori was diagnosed by histological analysis, rapid urease test, and PCR of gastric biopsy samples. Quantitative real-time PCR was performed to determine the relative mRNA expression levels. Results: No significant difference was detected in allele frequency and genotype of all studied polymorphisms between chronic gastritis (CG), GC and healthy individuals. IL-1β mRNA was down-regulated in both gastritis (relative quantification (RQ) = 0.447) and the GC groups (RQ = 0.151). In contrast, the expression of TNF-α was up-regulated in the GC group (RQ = 2.817) compared to the gastritis group (RQ = 0.861). Conclusions: The studied single-nucleotide polymorphisms are not risk factors for development of CG and GC. However, H. pylori infection causes a huge increase in the TNF-α expression in GC patients.

Introduction

Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer-related death worldwide [1,2]. The vast majority of GC is attributable to Helicobacter pylori, a persistent and chronic infection that is commonly acquired in childhood. The pathological progression of H. pylori in the gastric mucosa starts with chronic gastritis (CG) and then advances to gastric adenocarcinoma [3]. According to the previous data, GC is somewhat a hereditary disease; the H. pylori infection and the stomach lesions have been shown to occur more frequently in patients with a family history of GC [4].

Several factors can affect the treatment of H. pylori, such as adherence to treatment, lifestyle, bacterial virulence factors, antibiotic resistance, and host genetic polymorphisms [5]. Several gene polymorphisms conceivably influencing the inflammatory process and the immune response to H. pylori are now being investigated to establish risk factors of development of different gastrointestinal diseases (gastritis, intestinal metaplasia or GC), which include; genes encoding toll-like receptor 4 (TLR4), tumor necrosis factor-alpha (TNF-α), interleukins (IL-1β, IL-2, IL-4, IL-6, IL-8 and IL-10), and mannose-binding lectin 2 (MBL2) [6,7].

Interleukin-1β is a pro-inflammatory cytokine induced by H. pylori infection with important proprieties, such as regulation of inflammatory responses to H. pylori infection and avoidance of the secretion of gastric acid [8]. It has been demonstrated that IL-1β gene polymorphisms in the promoter region are related to increased production of IL-1β and are therefore associated with GC development [9,10]. Herein, increased inflammation of the gastric mucosa mediated by IL-1β may inhibit gastric acid secretion and facilitate bacterial clearance, which are both effective mechanisms of response to H. pylori infection [11]. In this way, these polymorphisms can be considered as an expected genetic factor in the progression to gastritis, which shows the risk of malignant transformation.

TNF-α is additionally associated with the development of immune response, and is secreted by macrophages and monocytes. The production of this cytokine is stimulated by bacterial lipopolysaccharide (LPS) that plays a major role in protection against microbial infection. A raised level of TNF-α was identified in the gastric mucosa of patients infected with H. pylori [6,7].

A few examinations have explored the role of IL-1β and TNF-α polymorphisms in the treatment of H. pylori infection, with controversial outcomes in different populations of the world [12,13]. In this way, the present study was performed to assess the frequency of polymorphisms in the IL-1β (–31 C/T, –511 C/T) and TNF-α (–308 G/A, –857 C/T, –863 C/A) genes in patients with H. pylori infection from Tehran. In addition, the association of these polymorphisms with an increased risk of infection, gastritis and GC is being investigated.

Methods

Study population

The patients enrolled in this case-control study were 375 unrelated people who underwent endoscopy at the Milad Medical Centre (Tehran, Iran) from February 2017 to October 2019. The cases (including patients with duodenal ulcer with and without H. pylori infection) were selected from the outpatient department, among those suffering from gastroenterological disorders. The control group included 241 people, without diagnosis of duodenal ulcer and without medical history of stomach diseases including H. pylori infection (H. pylori uninfected patients).

Patients in both groups referred to the clinic only for their medical services under the supervision of a physician. The gastroenterological problems were confirmed by conventional, endoscopic and clinical findings. None of the patients had received medication for H. pylori infection. Informed written consent was obtained before enrolling patients for the examination. An interviewer-based questionnaire was utilized to gather patient information. All procedures were performed in compliance with the ethical standards of the Declaration of Helsinki.

Exclusion criteria

Individuals taking nonsteroidal anti-inflammatory drugs and/or proton pump inhibitors (in the last three months), having received prior antibiotic therapy to eradicate the microorganism, alcohol abusers, smokers or drug users were excluded from this survey. Patients suffering from autoimmune, cardiovascular and osteoarthritis diseases or duodenal-ulcer patients with gastrointestinal bleeding were also excluded from the study.

Biopsy collections and bacterial identification

Biopsies were collected during routine endoscopy from the lesser curvature of the antrum inside two centimeters of the pyloric ring. Two biopsies were used for histopathological assessment. A biopsy from each site was utilized for a rapid urease test (RUT). H. pylori infection was diagnosed when positive results were obtained in two accompanying tests: RUT and histological investigation of gastric biopsy.

Histopathological investigations

The tissues from the gastric mucosa (transferred to the lab in 10% saline) were dehydrated using an alcohol gradient, placed in xylene and embedded in paraffin wax. Consecutive 3-5 μm sections were stained with hematoxylin-eosin (H&E) staining. Histological evaluation was conducted by an experienced pathologist. The amount of gastric mucosa involvement was defined by using microscopic examination and the presence of H. pylori was assessed in carbol fuchsin-stained sections.

DNA extraction and polymorphism genotyping

The DNA extraction from a biopsy of the antrum of each individual was performed using the phenol-chloroform protocol [14]. The genotyping of cytokine gene polymorphisms was conducted by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis in both the case and control groups, as explained in earlier studies [15,16]. For genotyping the polymorphisms of IL-1β (–31 C/T, –511 C/T), each PCR product was digested with Alul and AvaI enzymes, respectively. Likewise, for genotyping the polymorphism of TNF-α (–308 G/A, –857 C/T, –863 C/A), the PCR products were digested with NcoI, HincII and TaiI enzymes, respectively [17,18,19]. The sets of primers (Pishgam LLC, Iran), PCR conditions and the enzymes used in the present study are given in

Table 1. PCR-RFLP conditions: primer sequences, fragment sizes and restriction enzymes.

bp—base pairs; T°—temperature.

.

RNA extraction and cDNA synthesis

Total RNA was extracted from a subset of patients’ biopsies from various groups utilizing an RNA extraction kit (GeneAll Biotechnology, South Korea) as indicated by the manufacturer’s guidelines. The concentration of RNA was assessed by a NanoDrop^® ND1000 spectrophotometer (NanoDrop, USA), and visualized in 1% agarose gel for its integrity. The High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems™, USA) was used for cDNA synthesis adhering to the manufacturer’s guidelines. Two μL of DNase treated RNA was reverse transcribed in a total volume of 20 μL using cDNA synthesis kit, and stored at -20 °C until further use. The integrity of every cDNA preparation was analysed by PCR of the β-actin gene (F: 5′-GGCATCGTGATGGACTCCG-3′ and R: 5′-GCTGGAAGGTGGACAGCG-3′) with visualization in 1% agarose gel.

Quantitative real-time PCR for IL-1β and TNF-α expression

Quantitative cytokine mRNA expression was assessed by real-time PCR method in a Rotor-Gene 6000 real-time PCR cycler (Qiagen Corbett, Germany) using the Taqman technology. The levels of mRNA were normalized according to β-actin housekeeping gene. Relative quantification (RQ) was determined utilizing the comparative cycle threshold method (2⁻ΔΔct). More than 20% of reactions were repeated using the same assay and the results were all completely concordant.

Statistical analysis

The equilibrium of Hardy-Weinberg was examined at all single-nucleotide polymorphisms (SNPs) between cases and controls by the χ² test. The data were analysed using the statistical package for social science (SPSS 21.0 for Windows, USA) and the Fisher’s exact and Chi-square tests were done to analyze the presence of genes and the clinical outcomes. A p value ˂0.05 was considered meaningful.

Results

Clinical and demographic data

Of the 375 patients who underwent endoscopy, 290 patients were included in the study of the IL-1β and TNF-α genes. Other patients were excluded from the study based on the criteria mentioned in the Methods section. The patients’ age was between 15 and 85 years (mean age 47.6±21.3 years, male/female ratio=183/107) and the age of the control group was between 20 and 80 years (mean age 41.1±13.7 years, male/female ratio=165/76).

Prevalence of H. pylori infection

H. pylori infection was identified in 46.9% (136/290) of biopsy specimens. Histopathological examination demonstrated that from the 136 H. pylori-positive patients, 15 (11.0%) had normal mucosa, 91 (66.9%) had CG, and 30 (22.0%) had GC. In the H. pylori-negative patients, 151 (98.1%) cases were determined to have mild CG and three (1.9%) had moderate to severe CG.

Association of polymorphisms with H. pylori infection

The association of TNF-α and IL-1β gene polymorphisms based on the presence of H. pylori infection are detailed in

Table 2. The association of TNF-α and IL-1β polymorphisms based on the presence of H. pylori infection.

^aPercentage calculated from the H. pylori-positive patient population; ^bPercentage calculated from the H. pylori-negative patient population. CI—confidence interval; Hp—H. pylori; OR—odds ratio.

. The genotype distributions of each polymorphism were in Hardy-Weinberg equilibrium inside both the case (p=0.537) and the control (p=0.181) groups. Wild type genotype was detected in the larger part of patients in both H. pylori-positive and negative groups. As shown in Table 2, no statistically significant differences were detected in the allele frequencies and genotypes for all explored polymorphisms between study groups (CG and GC versus healthy individuals).

Gene expression of IL-1β and TNF-α in chronic gastritis and gastric cancer

The relative expression of IL-1β and TNF-α in the CG and GC groups is shown in

Table 3. Comparison of the mRNA expression between the case and control groups.

Hp—Helicobacter pylori; n—number of samples; RQ—relative quantification; SD—standard deviation. *statistical difference when p < 0.05.

. It should be noted that samples from all groups were compared with samples from normal gastric tissue that were negative for H. pylori infection (control group). IL-1β mRNA was down-regulated in both CG (RQ=0.447) and the GC groups (RQ=0.151), with a statistically significant difference (p<0.001). The expression analysis showed that the expression of TNF-α mRNA was up-regulated in the GC group (RQ: 2.817) in comparison with the control group (p<0.001). In the following, the chronic gastritis group was divided based on the presence of H. pylori: both IL-1β and TNF-α expression were down-regulated in patients with gastritis who tested positive for H. pylori (Table 3). In addition, no statistical differences were observed in patients with gastritis without H. pylori.

Discussion

Particular host cytokine reactions to gastric mucosal inflammation caused by H. pylori seem to assume a critical role in clinical results showing the advancement to CG and GC. In view of the need to describe the frequencies of host cytokine gene polymorphisms in mixed populations like Iran, this investigation assessed the impact of host IL-1β, and TNF-α gene polymorphisms in a population with CG and GC.

The IL-1β biological features in the gastric inflammatory response against H. pylori infection and its effect on gastric acid secretion have prompted researchers to find the effect of gene polymorphisms on the treatment of H. pylori infections. Different investigations discovered no relationship of the IL-1β polymorphisms with eradication rates of H. pylori [16]. However, a Japanese report of clarithromycin-susceptible H. pylori-infected patients revealed that the –511 TT (–31 CC) and –511 TC (–31 CT) genotypes were fundamentally related to the H. pylori eradication rates [13]. An investigation utilizing synthetic allele specific oligonucleotides for DNA-protein interactions, addressed the –31 C and –31 T alleles [20]. The formation of the DNA-protein complex did not occur in the presence of the C allele; in any case, DNA-protein interaction with the T allele increased fivefold. A similar investigation was carried out for the –511 T/C polymorphism, a polymorphism specified by T to C transition situated at position –511 of the IL-1β gene, which is in complete reverse linkage disequilibrium with the –31 C/T polymorphism [21]. In this regard, IL-1β production might be up-regulated when the –31 T allele (–31 TT or –31 CT genotypes) is available, causing a strong inflammatory response in the gastric mucosa of infected patients [16].

The results of the studies investigating the link between risk of gastric diseases and TNF-α polymorphism yielded conflicting and inconclusive outcomes. Different studies about the polymorphism TNF-α –308G>A in various populations were collected by Li et al [22]. They identified a weak relationship between the risk of GC and TNF-α –308A variant in Caucasian populations. Additionally, Jang et al. indicated that TNF-α homozygous A/A genotype and –308A allele was highly detected in patients with GC from South Korea [23]. Moreover, the polymorphisms of TNF-α –863 have been explored in a Japanese population and have shown a direct link between TNF-α –863 A allele and the risk of progression to GC [24]. In a study performed by Chakravorty et al. [25]. a fundamentally higher percentage of TNF-α –863A, –857C, –1031T, –308G haplotypes was seen in patients with H. pylori with duodenal ulcer compared with infected patients without ulceration, despite the absence of any connection between disease and the single investigated TNF-α locus. In a Polish study, the TNF-α gene polymorphisms at positions –857 and –863 were assessed in patients with peptic ulcer [26]. There was no significant difference in allele frequency and genotype for both studied polymorphisms between patients with peptic ulcer and healthy individuals. In addition, no relationship was confirmed between the presence of H. pylori infection and the frequency of specific genotypes and alleles for both single nucleotide polymorphisms. A meta-analysis was done to study the relationship between risk of H. pylori infection together with H. pylori-related GC, peptic ulcer disease (PUD), and IL-1β 31C/T, IL-1β –511C/T, IL-8 –251T/A gene polymorphisms [27]. Their results showed that the IL-1β –31C/T gene polymorphisms may increase the risk of infection. The IL-8 –251T/A and IL-1β –511-C/T gene polymorphisms may act as risk factors to H. pylori-associated diseases, including PUD or GC. The current investigation likewise indicated overexpression of TNF-α mRNA in GC patients compared to control group, and the relationship was prominent among H. pylori positive individuals and patients who had cancer. Otherwise, there was no overexpression detected in gastritis patients.

The analyzed sample size is a concerning limitation of the present study, which restricted the statistical power to find differences. Only two pro-inflammatory cytokines have been examined, however, other polymorphisms associated with pro-or anti-inflammatory responses, such as TLR4, IL-6, CXCL8, IL-10 (& more) are likewise implicated in the same disease context.

Conclusions

The results shows that H. pylori infection may induce a large increase in expression of TNF-α in GC patients. These findings propose that the TNF-α pathway may assume a fundamental part in the development of GC. According to the present discoveries on the frequency of TNF-α polymorphisms, more investigations are required to study the association of H. pylori infection with IL-1β and TNF-α gene polymorphisms and disease severity.