Investigation of helicobacter pylori virulence genes and t-cell responses in pediatric gastritis patients

Abstract

Helicobacter pylori is a gram-negative, spiral-shaped and, microaerophilic gastric pathogen that infects more than half of the world. H. pylori is about 2.5-5.0 μm long and about 0.5-1.0 μm wide. H. pylori has 30 μm long and 2.5 nm thick flagella with four to six unipolar sheaths for motility. H. pylori, infects the individual through oral-oral, fecal-oral, and iatrogenic transmission. H. pylori infection is acquired in childhood and remains lifelong if left untreated. Numerous investigations have shown that if this childhood-acquired gastric pathogen is left untreated, it can cause gastritis, peptic ulcers, MALT, and gastritis gastric cancer. Additionally, since 1994, the World Health Organization and the International Agency for Research on Cancer have classified H. pylori, as a class I carcinogen related to the development of gastric cancer. Developed countries have a lower incidence of infection than underdeveloped countries, even though more than 50% of the world's population is infected with H. pylori. This percentage for adults in our country is more than 70%. The incidence of infection in children is lower than in adults. Furthermore, environmental hygiene, dietary conditions, income level, and water resources are some of the main risk factors that affect children's H. pylori infection. The incidence of H. pylori infection is also significantly influenced by these major risk factors. For the diagnosis of H. pylori, invasive, non-invasive, and molecular methods are frequently used nowadays. The diagnosis of H. pylori in children is more difficult than in adults. Non-invasive and molecular methods are preferred because invasive methods need an endoscopy. H. pylori-infected children have been treated with proton pump inhibitors (PPI) and triple antibiotic therapy, the same as adults. For the treatment of H. pylori infection, novel drug combinations or new treatment approaches have been needed due to the increasing antibiotic resistance in the population. Studies have shown that the interactions of virulence genes, host immune response, stomach microenvironment, and other environmental factors have an impact on clinical outcomes such as peptic ulcer, MALT, gastritis, and gastric cancer resulting from H. pylori infection. H. pylori-specific virulence genes are not only involved in inducing inflammatory responses but also maintain chronic inflammation by controlling and regulating immune responses. H. pylori-specific virulence genes allow the bacteria to colonize and survive in the gastric mucosa, causing more immune escape and induction of premalignant changes. Outer membrane proteins, of the virulence genes of H. pylori, ensure permanent colonization of H. pylori through specific interactions with host receptors. Although the genetic strain of H. pylori differs between geographies, there are the most important and common virulence genes. These are; babA2 (blood-group antigen-binding adhesion 2), oipA (outer inflammatory protein A), and sabA (sialic acid-binding adhesin gene A). Virulence genes that cause tissue damage by producing toxins; are cagA (cytotoxin-associated gene A), and vacA (vacuolating cytotoxin gene A). Other virulence genes are ureA (encoding urease enzyme geneA), ureB (encoding urease enzyme gene B), hpaA (putative neuraminyllactose-binding hemagglutinin homolog A), napA (neutrophile- activating gene A), GTT (γ-glutamyl-transpeptidase), iceA (induced by contact with epithelial gene A) and dupA (duodenal ulcer protein gene). The immune response induced by H. pylori infection is also important in determining clinical outcomes. Adult studies have demonstrated that cytokines and the immune response have a role in maintaining the development of chronic inflammation and regulating infection. In particular, T cells as an adaptive immune response are essential for the clinical consequences of H. pylori infection. T helper cells are mediators of the host's immune response. T helper 1 (Th1) cells are mainly involved in protecting the organism from intracellular pathogens. Th1 cells secrete interferon- ɣ (IFN-ɣ). Th2 cells mainly involve defending the organism against extracellular pathogens. Th9 cells play an important role in defense against helminth infections, tumor suppression, allergic responses, and autoimmunity. Th9 cells secrete IL-9. Th17 is very crucial in maintaining mucosal barriers. Th17 cells secrete IL-17. Th22 cells, on the other hand, play a role in autoimmunity, wound healing, and protective mechanisms against pathogens. Th22 cells secrete IL-22. In addition to helper T cells, T regulatory (Treg) cells are part of the adaptive immune system. Treg cells are immunosuppressive and suppress or reduce the proliferation of effector T cells. The main regulatory transcription factor of Treg is FOXP3. PD-1 is a programmed cell death protein, an immune checkpoint inhibitor. It is expressed in gastric tissue and epithelial cell. When PD-1 binds to PDL-1, active T cells convert to the inactive T cell. PDL-1 inhibits T cell activity on PD-1. According to studies, when H. pylori infection occurs, PD-1 and PDL-1 are associated with tumor development and gastric cancer. Expression of PD-1 and PD-L1 in pediatric gastric biopsies with gastritis infected with H. pylori is not well defined. The results obtained in previous scientific studies have demonstrated the relationship between T cells and H. pylori infection. Numerous studies have demonstrated that the T cell response can also be affected by H. pylori-specific virulence genes. However, there is no comprehensive study examining H. pylori-specific virulence genes and immune response in pediatric patients in the Turkish population. In addition, there is no study in the literature examining the cytokine expression levels of IL-9 produced by Th9 and IL-22 produced by Th22 in H. pylori-infected pediatric patients. One of the aims of our study is to characterize H. pylori-specific virulence genes in H. pylori- infected Turkish pediatric patients. Another aim of the study is to investigate T cell responses (Th1, Th9, Th17, Th22, and Treg) in H. pylori-infected pediatric patients with gastritis. Moreover, it is to investigate the expression of PD-1 and PDL-1 immune checkpoint inhibitors, which are known to affect tumor formation, in H. pylori-infected pediatric patients. The study included 80 pediatric patients, aged 5 to 18, who applied to Istanbul Sarıyer Hamidiye Etfal Hospital with a variety of complaints and met the inclusion criteria. There were only 30 pediatric patients with H. pylori infection out of the 80 pediatric patients. As the control group, 23 patients with non-infected with H. pylori were included in the study. Two biopsy samples were taken from the antrum part of gastritis into the tube containing the RNA later from the patients who underwent endoscopy. DNA isolation with one of the biopsy samples and RNA isolation with the other were performed simultaneously. Using the isolated DNAs, H. pylori infection was identified by urease PCR assays. Isolated DNAs were amplified with primers specific to H. pylori-specific virulence genes by PCR, then visualized by agarose gel electrophoresis. Thus, the characterization of fifteen different H. pylori specific-virulence genes were achieved. The correlation of H. pylori-specific virulence genes with each other was determined by the Pearson product-moment correlation coefficient. The isolated RNA was used to determine the expression of transcription factors and cytokines, which are markers of T-cell subsets, at the mRNA level by the Real-Time PCR. Thus, the expression of transcription factors (FOXP3) and cytokines (IFNɣ, IL-9, IL-17, IL-22), which are Th1, Th9, Th17, and Th22 markers, were determined by RT-PCR for H. pylori-infected 30 pediatric patients with gastritis. Additionally, the mRNA expression level of PD1 and PDL-1 in H. pylori-infected and non-infected (control) patients were determined by RT- PCR. The conventional urease PCR assay was applied to all of the patients who were determined to be infected or not infected with H. pylori according to the results of the pathology report. According to the urease PCR assay, all patients infected with H. pylori had at least one of the ureA and ureB are virulence genes. Patients not included in the ureA and ureB virulence genes were used as the control group according to the criteria. After the detection of infection with H.pylori, correlations were detected between 13 different virulence genes; significant positive correlation; vacAs1-napA, vacAs1-sabA,vacAs1-iceA1,vacs1-ureB , dupA-napA, dupA -sabA, dupA-iceA2, dupA-ureB, napA-oipA, napA- sabA, napA-iceA2, napA-ureB, oip iceA1, oipA-iceA2, oipA-ureB, oipA - vacAm2, sabAiceA2 , sabA-ureB, iceA1- ureB, iceA2-ureB, iceA2- GGT, ureB- GGT, and babA2-cagA, a significant negative correlation is detected; vacAs1-vacAs2 and vacAm1-vacAm2. The distribution of virulence genes of H. pylori strains in inactive chronic gastritis and active chronic gastritis, which are different gastritis pathologies, was compared. As a result, the oipA and iceA2 genes were found to be positive at a significantly higher rate in pediatric patients with active chronic gastritis than in pediatric patients with inactive chronic gastritis. mRNA expression levels were determined by RT-PCR using T cell markers. As a result, the expression level of IFN-ɣ, IL-9, IL-17, IL-22, FOXP3, and a PDL-1 was significantly higher in H. pylori-infected pediatric patients than in H. pylori-noninfected pediatric patients. As previously mentioned, some of our H. pylori-infected individuals had inactive chronic gastritis whereas others had active chronic gastritis. The levels of IFN-ɣ, IL-9, IL-17, IL-22, and FOXP3 expression were compared between the two distinct gastritis pathogenesis to determine whether there was a statistically significant difference. Consequently, there was no significant difference between H. pylori-infected patients with active chronic gastritis versus inactive chronic gastritis. Surprisingly, when the expression of T cell markers in patients with inactive chronic gastritis was compared with the control group; had significantly higher expression than patients with active chronic gastritis. Furthermore, the relationship between H. pylori density (less-moderate-high), T cell markers, and PD-1, PDL-1 expression was investigated. No significant correlation was detected between the H. pylori density and expression of T cell markers and the expression of PD-1, PDL-1. In this study, the detection of virulence genes in H. pylori-infected Turkish pediatric patients diagnosed with gastritis was investigated in detail, and the expression of T cell responses, PD-1 and PDL-1 were determined. Our study is the first to investigate the expression levels of IL-9, IL-22, PD-1 and PDL-1 and the relationship between H. pylori density and the expression of these cytokines and immune checkpoint inhibitors in H. pylori-infected diagnoses with gastritis Turkish pediatric patient population.