Innate immunity was found to be associated with both persistence of Helicobacter pylori (H. pylori) infection and increased risk of gastric cancer.

To identify the risk factors associated with H. pylori infection and to establish the role of TLR9 rs352140 in suppressing or promoting inflammation related to this infection in children.

We performed a study of 155 children with digestive symptoms, who were divided into two groups according to the histopathological exam: Group 1 – 48 children with H. pylori-induced chronic gastritis, and Group 2 – control group.

Rural area and poor living conditions were significantly associated with H. pylori chronic gastritis (P = 0.0042/P < 0.0001). Both positive immunoglobulin A anti H. pylori and the rapid urease test were significantly associated with H. pylori infection (P < 0.0001). Significantly higher values of leukocytes and neutrophils within the peripheral blood were found in children with H. pylori chronic gastritis (P = 0.111/P = 0.284). We found a significant positive correlation between the variant TT genotype of TLR9 rs352140 polymorphism and both leucocytes and neutrophils (P = 0.0225/P = 0.0292).

Variant TT genotype carriers of the TLR9 rs352140 gene polymorphism might have a more severe degree of inflammation.

It is a well-documented fact that Helicobacter pylori (H. pylori) infection is most commonly acquired during early childhood and that age-related peculiarities are important for delineating the specific features of this infection in children, which are completely different than those encountered in adults. Recent epidemiological studies have proved that the trends are rapidly changing in terms of H. pylori prevalence, indicating a decreasing pattern among children as reported in a study performed in Chinese symptomatic children, which identified a decrease from 25.6% in 2005 to 12.8% in 2017[1]. Although abdominal pain, nausea, vomiting, anorexia, weight loss, or heartburn are commonly encountered in children with H. pylori infection, the clinical picture remains unspecific[2]. Recent studies have also noted several extra-digestive manifestations such as thrombocytopenia, anemia, eosinophilic esophagitis, or otitis media that might be related to the presence of H. pylori in children[3-5]. Nevertheless, taking into account the difficulties related to performing endoscopic studies in children and therefore the scarcity of information in this age group, a precise cause-effect relationship between these manifestations and H. pylori has been difficult to establish. Multiple invasive and non-invasive diagnostic tests are currently available for detecting H. pylori infection in children, but the gold standard remains tissue culture or concordant-positive histopathology test results and the rapid urease test[6]. Although new and improved non-invasive tests such as stool antigen have relatively good sensitivity and specificity in detecting this infection in children, current recommendations do not sustain the use of these tests for the diagnosis of H. pylori infection in pediatric patients[7]. European Society for Pediatric Gastroenterology Hepatology and Nutrition/North American Society For Pediatric Gastroenterology, Hepatology & Nutrition guidelines recommend a 2 wk regimen consisting of an association between a proton pump inhibitor and two antibiotics at high doses for the proper eradication of H. pylori infection in children[7]. Antimicrobial resistance is the main factor that influences the treatment success, which varies depending on the geographic area, but inadequate dose/duration, poor compliance, bacterial virulence factors, improper penetration of antibiotics at the level of gastric mucosa, antibiotic inactivation due to low gastric pH or fast metabolization of certain proton pump inhibitors might also be responsible for therapeutic failure[8].

The persistence of H. pylori infection into adulthood might carry a life-threatening risk since the World Health Organization considered this bacterium as a class I carcinogen[9]. H. pylori was proven to bind gastric epithelial cells activating the host innate immune system via toll-like receptors (TLRs), which subsequently trigger the secretion of pro- and anti-inflammatory cytokines, resulting in gastric atrophy, hypochlorhydria, and a high risk of carcinogenesis[10]. Gastric cancer is the fourth most common type of neoplasia and the second leading cause of death due to cancer among adults[11]. Because H. pylori infection occurs most frequently during early childhood, identifying the risk factors for this infection in pediatric populations might be the cornerstone for the development of further effective prevention strategies that will result in a decrease of H. pylori-related gastric cancer during adulthood. As already mentioned, innate immunity plays an essential role in the eradication of multiple microorganisms by stimulating the adaptive immunity via TLR activation and subsequent induction of inflammatory cytokines, chemokines, costimulatory and antigen-presenting molecules[12]. Multiple TLRs have been identified to date; they express specific receptors for the recognition of different bacterial components such as lipids, lipoproteins, proteins, or microbial nucleic acids[13].

TLR9 recognizes unmethylated CpG oligonucleotides, an important component of bacterial DNA[14]. These oligonucleotides are believed to be transferred to the intracellular domain via non-specific endocytosis since TLR9 itself is located in this compartment[15]. The complex pathway of TLR9 activation is associated with both infectious diseases and a wide spectrum of cancers[16].

The objectives of this study were to identify the risk factors associated with H. pylori infection and to establish the role of TLR9 rs352140 in suppressing or promoting the inflammation related to this infection in children.

H. pylori infection occurs most commonly during early childhood and can result in life-threatening conditions during adulthood if left untreated. Thus, the persistence of acute gastric inflammation associated with this bacterium and its imminent transformation into a chronic process will definitely result in an increased risk for gastric neoplasia[18]. Based on this fact, we assessed children with H. pylori chronic gastritis to delineate the features associated with this disorder in terms of environmental factors, laboratory parameters, and innate immunity since it is of major importance to define and characterize this disorder at young ages. Several environmental factors are associated with an increased risk of H. pylori infection such as low socio-economic status, bed sharing between children and adults, and improper sanitary conditions within the household[19,20]. Moreover, a study performed in children showed that those originating from the rural area were more predisposed to developing gastritis independently of the presence or absence of H. pylori infection[3]. Similarly, our study revealed that both poor living conditions and rural area might be risk factors for H. pylori chronic gastritis in children. Serology and rapid urease tests might be used as additional tests for diagnosing H. pylori infection, but their sole use is not recommended[7]. Similarly, the false-positive results of the rapid urease test and serology were also observed in our study. In terms of the rapid urease test, a possible explanation might be related to the presence of other urease-secreting bacteria within the stomach, such as Proteus spp[21], whereas regarding serology, this fact might be due to a previous infection with this bacteria[7]

Low-grade systemic inflammation associated with H. pylori infection has been the main focus of multiple recent studies. It has been proved that once H. pylori colonizes the gastric mucosa, it exerts chemotactic effects on neutrophils and lymphocytes, which together with other cells present at this level such as mononuclear cells and macrophages as well as several signaling cytokines, result in a low-grade systemic inflammatory status[22]. Other studies have identified a strong association between this inflammatory status associated to H. pylori infection and a wide spectrum of disorders such as cardiovascular diseases, stroke, anemia, idiopathic thrombocytopenic purpura, diabetes, glaucoma, thyroid disease, Alzheimer’s disease, rosacea, eczema, and chronic hives[23]. Moreover, some studies have noted that elevated levels of H. pylori antibodies are significantly correlated with increased systolic blood pressure and arterial stiffness in patients with diabetes[24] and coronary artery disease[25]. Most of the studies that have aimed to assess the markers of this systemic inflammatory status have been performed in adult populations and proved that acute phase reactants are significantly higher in patients with H. pylori infection[26]. Thus, a study that included 50 adult patients with H. pylori-induced gastritis and 50 with H. pylori-negative gastritis showed higher values of leukocytes, lymphocytes, and neutrophils in patients with gastritis induced by this infection compared to those with other types of gastritis[27]. White blood cell subtype along with NLR and platelet/lymphocyte ratio (PLR) also proved their utility in assessing systemic inflammation associated with pediatric obesity[28]. In terms of pediatric gastritis, the scarcity of available information hinders establishment of the precise role of these biomarkers in assessing the systemic inflammatory status related to this infection. Thus, our team has focused on clarifying this issue, and we found that leukocyte and neutrophil counts are important indicators for H. pylori-positive gastritis in children, but we did not identify any association between this infection and NLR, nor PLR[3,29]. Moreover, lymphocyte count might be a better marker for non-H. pylori gastritis[3]. Neutrophils might better reflect the inflammation, while lymphocytes could be a better indicator of the body’s nutritional status and general stress[30]. Similar to the aforementioned findings, our study also showed significantly higher values of both leukocytes and neutrophils in children with H. pylori chronic gastritis compared to the healthy controls, but failed to establish an association with NLR.

TLR9 is an endosome-transmembrane receptor with an essential role in the DNA recognition of both pathogens and damaged host cells via saccharide backbone, a structural component, in a sequence-independent manner[15]. A study that compared H. pylori-infected individuals with healthy controls showed a distinct distribution in these individuals, with predominant expression of this TLR in the apical compartment of the gastric epithelium in the control group compared to H. pylori-positive ones, where this receptor was mainly encountered in the basolateral compartment[31]. Controversial findings have been reported regarding the role of TLR9 in the development of H. pylori infection. Therefore, a study performed in murine models proved that the recognition of H. pylori DNA by TLR9 triggers proinflammatory responses[32], while the study by Otani et al[33] performed in mice indicated that this receptor might act as a suppressor for H. pylori infection during the acute phase of this infection. Based on these findings, Varga et al[34] recently stated that TLR9 might have a dichotomous role, and its role in suppressing or promoting might be influenced by the gastric microenvironment. Thus, the microenvironment that contains inflammatory cells without polarity acts as a trigger for the promotion of proinflammatory cascades via TLR9 resulting in the development of gastric cancer[31,35]. Moreover, cancer tissue expresses upregulates TLR9[15]. Multiple TLR9 single nucleotide polymorphisms (SNPs) were assessed in patients with gastric malignancies. Thus, a study that included 314 Chinese patients with gastric cancer and 314 healthy controls showed that TLR9 -1486C carriers express both an increased risk of gastric cancer and a poorer prognosis[36]. Moreover, a more recent study proved that the 2848A allele of TLR9 is associated with an increased risk for duodenal ulcer, as well as alteration of inflammatory cytokine expression within the gastric mucosa[37]. By contrast, no association was encountered between TLR9 rs5743836 (or -1237T/C) promoter polymorphisms and the risk of gastric cancer[38]. The role of TLR9 rs352140 polymorphism was assessed in patients with different conditions such as renal transplantation[39], systemic lupus erythematous[40], bacterial meningitis[41], primary immune thrombocytopenia[42], cervicitis[43], cervical cancer[44], or placental inflammation[45]. In terms of gastropathy and TLR9 rs352140 SNP, the data are scarce. A recent study performed in adults from India underlined that the heterozygous variant CT genotype of this polymorphism is significantly associated with the persistence of H. pylori infection[46]. Nevertheless, our study found no correlation between TLR9 rs352140 genotypes and H. pylori-induced chronic gastritis, suggesting that this TLR might not influence the persistence of this infection in children. Another study that included patients with chronic gastritis, peptic ulcer disease, and gastric carcinoma showed that the TT genotype was more commonly expressed by patients from the chronic gastritis group[47]. Similarly, the meta-analysis of Zhang et al[48] pointed out a significant association between the TT variant genotype of TLR9 rs352140 SNP and the overall risk of neoplasia, emphasizing that this polymorphism might alter innate immune responses promoting chronic inflammation and subsequent carcinogenesis. This finding was also shown in our study, as it assessed the role of this SNP in children with H. pylori chronic gastritis and proved that the variant TT genotype of TLR9 rs352140 polymorphism was associated with significantly higher values of both leukocytes and neutrophils. The discordance between the lack of difference in terms of TLR9 rs352140 genotype distribution between the two groups, and the aforementioned significant positive association between TT genotype of this polymorphism and increased leukocytes and neutrophils might be explained by a risk for individuals carrying this genotype to express higher values of the parameters as a result of a certain trigger. Another possible explanation might be related to the relatively small sample included in our study. By contrast, a significant association between TT genotype and the increase in both leukocytes and neutrophils might also be influenced by the low number of subjects, but it definitely raises a major concern in this area that requires further studies on larger samples.

Our study had several limitations that must be acknowledged: The relatively small number of patients originating from a single area of our country; the diagnosis of H. pylori infection established only based on the histopathological exam without being confirmed by an additional method; we did not assess the gastric microenvironment, the influence of gastric microbiota or the degrees of gastritis severity; we did not perform esophageal pH monitoring in order to confirm the diagnosis of gastroesophageal reflux; the lack of longitudinal follow-up of the patients endoscopically after the eradication therapy, lack of TLR9 gene expression; and we did not take into account the H. pylori pathogenicity features in the development of chronic gastritis and subsequent increased risk for a more severe degree of systemic inflammation.

The main two strengths of this study were the small age of the assessed population and the fact that the role of TLR9 rs352140 polymorphism was assessed for the first time in children with H. pylori chronic gastritis. Therefore, this study might be considered a pilot study providing valuable results of great multidisciplinary importance that might serve as a solid basis for the development of effective preventive strategies for gastric cancer during adulthood. Other strengths of this study consist of the fact that we established the diagnosis of H. pylori gastritis based on the histological exam of the gastric biopsy, as well as the fact that all upper digestive endoscopies and histological exams were performed by two experienced pediatric gastroenterology and pathology specialists.

Environmental factors, such as poor living conditions and rural area might result in an increased risk for H. pylori chronic gastritis in children. Serological and rapid urease tests could be used as reliable tests for the detection of H. pylori infection in children. Moreover, leukocytes and neutrophils are important non-invasive biomarkers for detecting the low-grade inflammatory status associated with this infection in pediatric patients. In terms of innate immunity, our findings emphasized that variant TT genotype carriers of the TLR9 rs352140 gene polymorphism might express a more severe degree of inflammation. Nevertheless, further studies are required on bigger samples of pediatric patients to identify the precise role of innate immunity and its TLR9 polymorphisms in the development of H. pylori gastritis.

We would like to express our appreciation to Professor Claudia Bănescu, from Center for Advanced Medical and Pharmaceutical Research, “George Emil Palade” University of Medicine, Pharmacy, Sciences and Technology Târgu Mure, Romania for her untiring support and scientific guidance throughout the present research and for critically revising the manuscript.