Investigation of the relation between Helicobacter pylori and T cell response with examination of PD-L1 expression level in gastric pathogenesis

Gastric cancer is the third most common cancer in the world. The reason of high death rate in this type of cancer is late diagnosis and bad prognosis. Chronic inflammation due to Helicobacter pylori (H. pylori) infection is a key factor in triggering the development of gastric cancer, as well as many factors such as genetic susceptibility, environmental factors, diet and age. Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic, spiral-shaped bacterium that colonizes the stomach of more than half of the world's population during their life span. The bacteria appear around 3μm in length and possess a unipolar bundle of two to six sheathed flagella, about 0,5μm in size, that enable the bacteria to move in the acidic environment of stomach. H. pylori infection is usually acquired in early childhood and persists throughout the life. While most infected individuals remain asymptomatic, H. pylori infection can cause peptic ulcer, chronic gastritis, mucosa-associated lymphoid tissue (MALT) lymphomas and gastric cancer in 20% of the infected ones.H. pylori has several virulence factors that may be associated with diseases. The presence of this virulence factor increases the risk of stomach cancer by causing a higher inflammation in the stomach mucosa. Most studied virulence factor is vacA, which forms vacuoles in cells. H. pylori strains that contains the vacA virulence factor are more likely to develop gastritis. In addition, it increases mucosal inflammation and prognosis of the disease by increasing the colonization of babA and napA virulence factors of bacteria, which are responsible for bacterial adhesion to the gastric epithelium. Another protein found in the outer membrane of bacteria is oipA, which causes IL-8 release together with cagA and it is effective in progressing duodenal ulceration. Also, hpaA, which is found in the outer membrane is effective in colonization of the bacterium. The dupA gene is located in the plasticity zone of the H. pylori genome and has been associated with increased risk of duodenal ulcer. In addition, the ureA and ureB genes are required for the urease activity that protect the bacteria to stomach microenvironment. The stages of development of gastric cancer are classified as chronic gastritis, atrophic gastritis, intestinal metaplasia and dysplasia. In Turkish population, there are patients with gastric precancerous lesions at different levels resulting from a high incidence of H. pylori infection. Some of these patients have potential to develop stomach cancer in the future. However, the identification of whether these patients will develop gastric cancer is not possible because the pathogenesis of the stomach is not fully known from the genetic and immunological aspects. At the same time, the relationship between the characteristics of virulence factors of H. pylori and the response of the infected site is unknown. H. pylori in the gastric mucosa can survive at high degreess for a long time. This suggests that the immune response of the host failed to remove the pathogen. This may be due to a change in the expression of the cytokines that involved in the immune response. H. pylori infection leads to Th1 cells mediated immune response; In contrast, regulatory T cells (Tregs) prevent host inflammation against H. pylori and allow the bacteria to maintain in the stomach for long periods of time. In addition, the link between immune check proteins, programmed death-1 (PD-1) and its ligand-programmed death-ligand-1 (PD-L1), which are found on the surface of active T and B cells leads to negative regulation of T cell response. Therefore, in this study, the correlation between Th1 associated cytokine IFN-γ, Treg transcription factor FOXP3 and PD-L1 expression levels together with H.pylori infection was investigated in patients with gastric precancerous lesions and gastric cancer patients. This study included in total 60 patients; 20 with active chronic gastritis, 3 with inactive chronic gastritis, 9 with intestinal metaplasia and 14 with gastric cancer. In addition, 15 individuals with uneffected and uninfected normal gastric pathology were included as negative control group. These patients were applied to Acıbadem Hospitals with gastrointestinal symptoms and underwent endoscopy gastric cancer surgery.Three tissue samples were taken from the antrum region of patients' stomach who underwent endoscopy and resection. DNA was isolated from one of the received tissue samples and RNA was isolated from the other; the third biopsy specimen was used for hematoxylin and eosin staining to detect stomach pathogenesis and the satatus of Helicobacter pylori (H.pylori). Isolated DNA samples were used for Conventional PCR studies to identify ten different virulence factors of H.pylori. In the characterization of virulence factors, G27 H.pylori strain, which known to carry all virulence factors, was used as a positive control. cDNA synthesized from RNA samples and the expression of Treg-specific transcription factor Forkhead box P3 (FOXP3), Th1 derived cytokine interferon-g (IFN-ɣ) and programmed death ligand-1 (PD-L1) were examined by quantitative Real Time PCR (qRT-PCR) assays . Patients who had high inflammation were selected for optimization of quantitative Real Time PCR (qRT-PCR) assays. Selected patients were examined by conventional PCR assay to generate standard curve for mRNA level expression analysis of FOXP3, IFN-ɣ and PD-L1 by quantitavive Real Time PCR. Characterization of 10 different virulence factors by conventional PCR studies was carried out in patients who known to be H. pylori positive according to the pathology reports. The presence of ureA and ureB virulence factors which required for diagnosis of H. pylori was detected by conventional urease PCR. Conventional urease PCR results was compared with results of histopathological staining of H.pylori which was performed routinely for every patient. Comparison studies have shown a strong correlation between conventional urease PCR and pathological evaluation methods. Virulence factors determined as a result of conventional PCR studies were evaluated in two different groups of patients. The most prevalent virulence factor in patients with active chronic gastritis were; napA, hpaA, and cagA. The most common virulence factor in intestinal metaplasia were; cagA, vacA and hpaA. Expressions of FOXP3, IFN-ɣ, and PD-L1 were evaluated in precancerous and gastric cancer patient groups and expression levels were compared with uneffected and uninfected control individuals. The comparison results were analyzed by student's t test. According to the results of comparison with control group, expression levels of IFN-ɣ and FOXP3 were found to be decreased in patients with gastric precancerous lesions and gastric cancer patients. Otherwise, the level of expression of PD-L1 was higher in gastric cancer and gastric precancerous patients. When compared patients with precancerous lesions and gastric cancer, it was determined that only the expression level of FOXP3 was higher in patients with precancerous lesions. When the patients who included in this study were grouped according to H. pylori infection, only the expression level of IFN-ɣ was found to be higher in H. pylori-infected patients. In this study, the characterization of H. pylori virulence factors, the status of immune responses, and PD-L1 levels were investigated in staircase precancerous lesions to stomach cancer stages progressively for the first time.