Immunomodulatory Effect of H. Pylori CagA Genotype and Gastric Hormones On Gastric Versus Inflammatory Cells Fas Gene Expression in Iraqi Patients with Gastroduodenal Disorders

AIM: To evaluate the Immunomodulatory effects of CagA expression; pepsinogen I, II & gastrin-17 on PMNs and lymphocytes Fas expression in inflammatory and gastric cells; demographic distribution of Fas molecule in gastric tissue and inflammatory cells. METHODS: Gastroduodenal biopsies were taken from 80 patients for histopathology and H. pylori diagnosis. Serum samples were used for evaluation of pepsinogen I (PGI); (PGII); gastrin-17 (G-17). RESULTS: Significant difference (p < 0.001) in lymphocytes & PMNs Fas expression; epithelial & lamina propria Fas localization among H. pylori associated gastric disorders. No correlation between grade of lymphocytes & PMNs Fas expression in gastric epithelia; lamina propria and types of gastric disorder. Significant difference (p < 0.001) in total gastric Fas expression, epithelial Fas; lamina propria and gastric gland Fas expression according to CagA, PGI; PGII; PGI/PGII; Gastrin-17. Total gastric Fas expression has significant correlation with CagA, PGII levels. Gastric epithelial and gastric lamina propria Fas expression have significant correlation with CagA, PGI; PGII levels. Significant difference (p < 0.001) was found in lymphocytes & PMNs Fas expression; epithelial & lamina propria localization of lymphocytes & PMNs Fas expression according to CagA, PGI; PGII; PGI/PGII; Gastrin-17. Lymphocytes Fas expression have correlation with PGI, PGII, PGI/PGII. PMNs Fas expression have correlation with PGI, PGII. CONCLUSION: Fas gene expression and localization on gastric and inflammatory cells affected directly by H. pylori CagA and indirectly by gastric hormones. This contributes to progression of various gastric disorders according to severity of CagA induced gastric pathology and gastric hormones disturbance throughout the course of infection and disease.


Introduction
Helicobacter pylori infection is associated with several benign and malignant human diseases. Most infected individuals remain asymptomatic. If untreated, infection lasts for decades .The prevalence of infection ranging between 50% in developed countries and 90% in developing countries [1].
H. pylori occupies a unique niche, extremely acidic environment [2]. The urease of H. pylori is essential for its colonization and survival at extremely low pH, to ensure cytoplasmic homeostasis during large pH changes that occur during feeding. H. pylori can use molecular hydrogen as energy source; thus, its growth depends to some extent on the hydrogen excreted [3]. H. pylori infected gastric mucosa evolves through stages of chronic gastritis, intestinal metaplasia, glandular atrophy, and dysplasia before carcinoma develops.
H. pylori Infection is associated mostly with chronic antral gastritis, characterized by a mucosal infiltration of polymorphonuclear (PMNs) and mononuclear leukocytes [4]. Some studies have reported that H. pylori infection is suggested by the presence of active inflammation [3]. Neutrophil infiltration is a hallmark of active inflammation [4]. It is unknown whether neutrophil infiltration may be a marker of H. pylori infection.
It is now established that Helicobacter pylori causes more than 80% of duodenal ulcers and up to 60% of gastric ulcers [3]. The link between Helicobacter pylori infection and subsequent gastritis and peptic ulcer disease has been established through studies of human volunteers, antibiotic treatment studies and epidemiological studies. In some individuals, Helicobacter pylori also infect the corpus region of the stomach. This results in a more widespread inflammation that predisposes not only to ulcer in the corpus region, but also to stomach cancer [3]. Because of extremely low pH, the stomach is a hostile environment to most other microorganisms. The ability of H. pylori to flourish in the stomach has been attributed to protective mechanisms such as its production of urease, protecting the bacterium from gastric acidity by creating a basic microenvironment [5]. However, we reasoned that H. pylori might have evolved away to gain growth advantage in this niche, possibly by exploiting a gastric factor. A logical candidate would be one up regulated by H. pylori infection.
One such factor is the gastric hormone gastrin. Gastrin is produced as a prohormone by G cells located within the gastric antrum. The prohormone processed to shorter peptides, the most abundant of which is 17 amino acids long, termed gastrin-17 (G17). The major role attributed to gastrin within gastric tissue is the regulation of acid secretion [6]. After infection, gastrin levels are found to be consistently elevated and normal physiological negative feedback control of secretion is lost. Furthermore, after H. pylori eradication, gastrin levels are reduced and normal feedback control of gastrin secretion is restored [6,7].
A number of studies have investigated the pathogenicity of H. pylori in relation to cytotoxic products, including urease, Cag, and vacuolating toxin (VacA) [8]. Potential apoptosis-inducing activity was reported in VacA [8] and urease [9]. Apoptosis in H. pylori-associated gastritis accompanies the activation of Fas and the Fas ligand system [10] in epithelial cells. Fas is a member of the tumor necrosis factor receptor family, which, when bound by its ligand, activates caspase-8, an initiator of the downstream apoptotic process that includes the cleavage of other death substrates, cellular and nuclear morphological changes and, ultimately, cell death [11]. Variations in host responses might cause the H. pylori mediated pathogenesis to result in a variety of clinical outcomes.
The aim of the present study is to evaluate the Immunomodulatory effects of CagA gene expression and gastric secretions (pepsinogen I, -II, Gastrin17) on inflammatory response mainly PMNs and lymphocytes infiltrations as well as expression of Fas gene in inflammatory and gastric cells under influence of CagA and gastric hormones and demographic distribution of Fas molecule in gastric tissue and inflammatory cells.

Patients
In this cross sectional study, (80) patients, age range 16-80 years, mean (47.24 ± 18.82) years, with clinical indications for upper gastrointestinal tract endoscopy during June 2013 to January 2015 were studied. Males represent 44 (55%) versus 36 (45%) females. This study was conducted according to the principles of Helsinki declaration. Before endoscopy, a full explanation about the purpose of this study to all patients was done. Dully-filled consent form obtained from all patients that agree to participate in the study. Approval of ethical review Committee of College of medicine -Diyala University -Iraq, was taken prior to initiation of the work at gastroenterology department of Baqubah teaching hospital in Diyala province -Iraq. Any patient under antibiotics or colloidal bismuth compounds for past one month treatment; having a history of previous gastric surgery and recent or active gastrointestinal bleeding was excluded from this study.

Methods
After topical pharyngeal anesthesia for overnight fasted Patients, A sterile flexible endoscope was introduced for full investigation of Stomach and duodenum. Six biopsy samples from congested, inflamed or erosive lesions were picked via sterile biopsy forceps. Samples were placed in Serim® PyloriTek® Test Kit for detection of urease activity. Each PyloriTek strip has a built-in positive analyte control and a negative control, which run concurrently with the test specimen. The PyloriTek positive control automatically appears with every test within the normal 1-hour time. With competitive tests the positive control is run after waiting 24 hours then inserting a urease positive control material [12].
Biopsy sample was placed in sterile glass slide with a drop of normal saline and teased with sterile scalpel to make smaller fragments of tissue then another sterile glass slide was placed over the teased first tissue and the tissue was crushed between the two glasses then stain by Gram's staining. Existence of Gram negative spiral bacteria embedded in the tissue cells was diagnostic for H. pylori [13].
True positive results were considered if a combination of urease test and Gram stain give positive results for a single biopsy specimen [14].
Human Fas gene and H. pylori CagA gene expression detected by insitu hybridization procedure in 5µm thickness serial gastric mucosal sections fixed on positively charged slides using biotinylated long DNA probe for Human Fas gene; Cat. No. IH-60047 (fas-6001-B); H. pylori/ CagA gene, Cat. No. IH-60061 (HPY-6001-B) (Maxim biotech-USA) and the DNA Probe hybridization/Detection System -In Situ Kit (Maxim biotech-USA), according to Maxim biotech instruction manual [15].The examination and scoring were done under light microscope by pathologists at power X 400 according to the scoring system [16]. For serological assay blood was drawn from each patient during the visit to the endoscopy unit. Separated serum samples were stored at 27˚C until analyses. Serum pepsinogen I(PGI) and II(PGII) and gastrin-17 (G-17) were assayed with ELISA using monoclonal antibodies to pepsinogen I and II and gastrin-17 (Biohit Diagnostics, Biohit, Devon, UK). All procedures were carried out according to the manufacturer's instructions and results of pepsinogen I and II reported in µg/l and pmol/l for gastrin-17. The pepsinogen I: II ratio was calculated and reported in fraction [17].

Statistical analysis
Frequency of variables express as percentage. PG I, II and G-17 values express as mean ± standard deviation (Mean ± SD). Pearson test for correlation was used for non-categorical data. Chitest used to compare the PG I, PGII, and G17 according to CagA gene expression.
The level of significance was 0.05 (two-tail) in all statistical testing; significant of correlations (Pearson, spearman) include also 0.01 (two-tail). Statistical analysis was performed using SPSS for windows TM version 17.0, and Microsoft Excel for windows 2010.

* Spearman Correlation
As shown in Table 4, Fas overexpression appear to be more frequent (53.75%) than moderate (41.25%) and mild (5%) grade of expression in gastric glands. Significant difference without correlation between CagA expression and grade of Fas in gastric glands (p value = 0.001, p value = 0.743).
Significant difference in grade of Fas expression (p value < 0.001) without significant correlation between serum PGI and grade of gastric glands Fas expression (p value = 0.296).Significant difference among disorders in grade of Fas (p value < 0.001) without correlation between serum PGII and grade of gastric glands Fas expression (p value = 0.501). Significant difference in grade of gastric gland Fas expression (p value < 0.001) without correlation between PGI/PGII serum level and grade of gastric cells Fas (p value = 0.947). Significant difference among disorders in grade of Fas gene expression (p value < 0.001) without correlation between gastrin17 and grade of gastric glands Fas expression (p value = 0.286).
As shown in Table 6 As shown in Table 6, significant difference with marginal correlation between CagA gene expression and grade of PMNs & lymphocytes Fas (p value = 0.001, p value = 0.056). Significant difference in grade of PMNs & lymphocytes Fas (p value < 0.001) without significant correlation between serum level of PGI and grade of PMNs &lymphocytes Fas (p value = 0.737). Significant difference among disorders in grade of PMNs & lymphocytes Fas (p value < 0.001) without correlation with PGII levels (p value = 0.191).
As shown in Table 6
In current study high grade of Fas gene expression was not detected at all. Mild lymphocytes expression (5-25%) reported in (61.25%), followed by moderate grade (26-50%) of expression (31.25%) among different disorders. In PMNs, mild Fas expression was reported in (46.25%) versus (3.75%) moderate the frequency of Fas gene expression more frequently reported in gastritis and gastropathy. Even there was significant difference in lymphocytes Fas expression among disorders, there was no correlation between this expression and type of gastric disorder. This is a logical finding because the induction of Fas expression via infiltrating lymphocytes and PMNs is associated and attributed to the induction via H. pylori infection [10]. Low Fas expression in infiltrating PMNs compared with infiltrating lymphocytes may attributed to the chronic active inflammatory reaction among different disorders [11].
One of interesting points that the lymphocytes and PMNs Fas expression in epithelial lining range from mild (gastric ulcer; gastritis; gastropathy) to moderate ( gastritis ; DU; gastropathy) with significant difference. While in lamina propria, infiltrating lymphocytes and PMNs among all disorders have high grade of Fas gene expression. This may attributed to the ability of H. pylori to invade deeply in gastric tissue gastric beyond mucosal epithelia, which indicates initiation of interaction between H. pylori and inflammatory cells in lamina propria leading to activation of NFkB gene in gastric as well as Lymphocytes and PMNs, finally IL8 production and increase oxidative stress in gastric tissue leading final to increase Fas gene production which is more obvious in lamina propria than in gastric epithelia, reflecting the underling pathology in near future [1,10,11].
In current study CagA genotype have a positive effect and correlation on the gastric cells Fas over expression in (43.75%). Mild expression detected in (10%). This results come in line with [1,10]. This result come in concordance with opinion of [10,18] stated that whenever H. pylori have potent Cag PAI, bacterial adhesion, good signals will be received by the gastric epithelial cells which reflect its response by increasing of expression of MHCII to play as antigen presenting cell (APC) and strong Th1 response will be occur with obvious IFNγ secretion which acts as good stimulator for up-regulation of FAS and even FASL in gastroduodenal tissue and tissue infiltrating lymphocytes (TILs).
One of exciting things in this study, the fluctuation of gastric Fas expression independently from a status of gastric hormones. All cases with hyposecretion of PGI; PGI/PGII characterized by gastric Fas overexpression, (7.5%) vs. (27.5%). On the other hand all cases with normal gastric hormone level also associated with gastric Fas overexpress, PGI (31.25%), PGII (15%), PGI/PGII (33.75%), G17 (56.25%) without any significant correlation between a status of PGI, PGI/PGII, Gastrin 17 and gastric Fas overexpression. PGII have positive correlation with gastric Fas overexpression among different disorders.
These results indicating a H. pylori induced pangastric type of mucosal inflammation even to duodenal region that reflects the disruption in gastric hormones secretion mainly over production of PGI (31.25%), PGII (76.25%), G17 (5%) [19]. Gastric Fas expression and gastric hormones have indirect correlation. Mucosal colonization by the H. pylori ignites a cascade of events that result in large increases in inflammatory cytokines in the infected tissue which were originated from the gastric mucosa as well as from infiltrating inflammatory cells. Among these inflammatory cytokines, IL-1, IL-2, TNF-α, and IFNγ that have been shown to up-regulate the expression of Fas antigen in gastric as well as inflammatory cells. T-helper type 1 (Th1) cells are selectively increased during H. pylori infection [20]. Th1 cytokines, such as IFNγ and TNF-α, can increase the release of proinflammatory cytokines, such as IL-8 from the epithelium as well as Fas and Fas ligand (FasL) 34. Furthermore, these cytokines can also increase the expression of MHC class II molecules by gastric epithelial cells, thereby increasing the binding of H. pylori to the gastric epithelium [21].
One of frustrating things that there was no clinical study to evaluates the correlation between gastric Fas expression and the level of gastric hormones. One of remarkable finding in current study when making topographical analysis for the correlation between gastric hormones and gastric Fas expression in gastric epithelia, lamina propria and gastric glands. This study reported a positive correlation between gastric Fas expression and PGI;PGII in gastric epithelia, lamina propria but not in gastric gland even with presence of significant difference in gastric hormones according to Fas gene expression.
Attachment of H. pylori on specific molecules expressed and acts as a receptor in the gastric epithelia, MHCII and CD74 using multiple adhesins that have been identified on the outer membrane of H. pylori such as BabA, SabA. BabA and SabA bind to fucosylated and sialylated blood group antigens, respectively. While the attachment of H. pylori using BabA as an adhesin does not appear to induce signaling or immune responses from host cells, SabA appears to be required for activation of neutrophils and the resulting oxidative burst by binding to sialylated neutrophil receptors [22]. This inflammatory signal extended throughout gastric epithelia to lamina propria also via the effect of neutrophil activating protein and urease as well as CagA production by H. pylori which in turn leads to activation of lymphocytes and gastric NFkB causing production of proinflammatory cytokines, mainly IL8 and hence the gastric epithelia and lamina propria sinking with inflammatory reaction and cells leading to increase of Fas expression on gastric epithelia and lamina propria as well as inflammatory cells [1,11,23]. Because CagA interacts with important signaling mediators in the gastric cells, it is considered responsible for changes in cell morphology, adhesion and turnover [23]. CagA cause increase in Fas expression independently or in conjunction with other virulence factors such as urease [10,11]. At the same time as a results of pan gastric inflammatory reaction this leads to direct effects on PGII producing cells causing increase in PGII production and as a results of neuroendocrine activity through vagus nerve and gastrin, PGI also affected. Fas expression on gastric glands has no correlation with gastric hormones even in the presence of significant differences in hormones according to grade of Fas expression may be associated with fact that gastric glands appear to be Immunological privileged site.
In current study, H. pylori influence on apoptosis of lymphocytes in gastric mucosa .lymphocytes Fas expression has positive correlation with PGI; PGII, PGI/PGII. Obvious fluctuation of lymphocytes Fas according to gastric hormones from mild to moderate expression as in Table 5. One of remarkable finding was the mild to moderate expression of Fas on PMNS which has positive correlation with PGI serum level only. This may attributed to chronic active pangastric inflammation affecting the PGI, PGII producing chief cells within gastric mucosa, reflecting by (3.75%) mild expression of PMNs, (6.25%) in lymphocytes and clinically patients suffered from atrophic corpus gastritis. The mild to moderate expression of Fas on gastric tissue infiltrating lymphocytes give an indication about the immunopathological role of these cells in underling clinical diseases .by relatively low expression of apoptotic marker (Fas) in lymphocytes as well as PMNs this will leads to delay spontaneous apoptosis and prolong their survival. It results in the prolonged activity of cytokines secreted by these cells and therefore augments their damaging action upon gastric mucosa [24,25]. On the other hand the mild to moderate expression of Fas receptors on lymphocytes and PMNs act in delay of their proliferation and affects its ability to get rid of H. pylori from gastric tissue, and at sum this will cause persistence infection and a protective strategy for H. pylori [24,26].
When making topographical analysis for the correlation between gastric hormones and inflammatory cells (lymphocytes & PMNs) Fas expression in gastric epithelia, lamina propria; No correlation was reported between inflammatory cells (lymphocytes & PMNs) Fas expression and gastric hormones secretion in gastric epithelia, lamina propria even with presence of significant difference in gastric hormones according to Fas gene expression as in Table 6. This finding reflect the role of; and give an indication that H. pylori acts as inducer for Fas gene expression on gastric cells as well as lymphocytes & PMNs, directly through its virulence factors, mainly products of Cag pathogenicity island, CagA and others, and indirectly through cytokines produce by TH1 cells such as IFNγ as well as the oxidative stress produced by reactive oxygen species from inflammatory cells infiltrated into gastric tissue due to H. pylori infection [10,11,24].
In conclusion, Fas gene expression and localization on gastric and inflammatory cells affected directly by H. pylori CagA and indirectly by gastric hormones. This contributes to progression of various gastric disorders according to severity of CagA induced gastric pathology and gastric hormones disturbance throughout the course of infection and disease.