Keywords

miRNA-155 IL-6 IFN-γ IL-10 Helicobacter pylori chronic gastritis

Abstract

Background/Aims:

Infection with Helicobacter pylori is the most common cause of chronic gastritis and has been linked to persistent inflammation of the stomach. miRNA-155 and inflammatory cytokines play crucial roles in immune regulation but their systemic profiles of expression are not fully understood in H. pylori–associated chronic gastritis. This pilot study was conducted to evaluate peripheral miRNA-155 expression accompanied by circulating levels of IL-6 IFN-γ and IL-10 in chronic gastritis H. pylori –related patients.

Methods:

Twenty-five patients with endoscopically and histologically diagnosed H. pylori–associated chronic gastritis and 25 age-matched healthy subjects were included in this study. The expression of miRNA-155 was determined in peripheral blood by using quantitative real-time PCR • Whereas the levels of serum cytokines were measured by ELISA. Group comparison and an exploratory association were carried out.

Results:

The H. pylori–associated chronic gastritis patients had significantly higher peripheral miRNA-155 levels compared with controls (p < 0.001). Serum IL-6 IFN-γ and IL-10 were all statistically higher in patients (all p < 0.001). Unadjusted logistic regression analyses showed significant correlations between biomarker levels and H.pylori infection status among the study population.

Conclusion:

This exploratory case–control study provides evidence of systemic perturbations in miRNA-155 expression and inflammatory cytokines in subjects with H. pylori–associated chronic gastritis. These data indicate immune-regulatory changes are related in whole blood and justify validation studies on additional cohorts. Due to the exploratory nature and small size of our cohort • The results need to be interpreted with caution and verified by independent cohorts.

Introduction

Helicobacter pylori (H. pylori) is one of the most common bacterial pathogens, infecting over 50% of the world population [1, 2]. The H. pylori infection rate differs widely in different locations and is closely related to the social economy there are more infections in developing countries caused by factors like poor living environments and overcrowding [3]. Chronic H. pylori infection is the principal cause of chronic gastritis and a risk factor for sequential damage to the gastric mucosa leading to atrophic gastritis, intestinal metaplasia and gastric cancer [4]. H. pylori causes a chronic inflammation by its virulence factors secretion, which include cytotoxin associated gene A (CagA) and vacuolating cytotoxin A (VacA), that facilitate disruption of the epithelial barrier and alter local immune signaling [5]. Such chronic inflammatory setting provides the ability of bacteria to persist, together with induction of tissue damage and disease progression [6]. Epidemiological studies in Africa and the Middle East regions have also indicated a significant correlation between H. pylori infection and gastric precancerous lesions, thereby emphasizing its clinical relevance as an obligatory risk factor of global public health importance [7, 8]. Furthermore, H. pylori interactions with the gastric microbiota have been linked to changes in gastric acid secretion and disease progression [9]. Eradication therapy has been shown to cause regression of chronic gastritis whereas the reversibility of advanced gastric lesions is a matter of debate [10], emphasizing the importance for better understanding regarding host-pathogen interaction [11].

MicroRNAs have been identified as critical post-transcription mediators of immunity and inflammation. Among them, miRNA-155 is involved in immune cells activation and cytokine expression and inflammatory regulation. Overexpression of miRNA-155 has been observed in different inflammatory diseases, including H. pylori-associated gastritis [12, 13]. At the mechanistic level, miRNA-155 has been demonstrated to potentially regulate immune-related target genes and potentiate pro-inflammation signaling pathways upon bacterial infection [12, 14]. These data indicate that miRNA-155 could be a marker of systemic immune activation in the context of chronic gastric inflammation.

Cytokines including IL-6, IFN-γ and IL-10 are important factors of immune response against H. pylori. IL-6 as well as IFN-γ are participating in the activation of both innate and adaptive immune cells and creation of chronic inflammation, while IL-10 has a suppressive and anti-inflammatory activity which may counteract exaggerated immune reaction [15–18]. Imbalance of cytokine networks has been associated with the severity and progression of H. pylori-associated gastritis [16–18].

Although miRNA-155 expression (or cytokine responses) has been previously reported individually (all at the levels of gastric tissue or isolated immune cells), little is known regarding their concomitant systematic profiles expressed in peripheral blood from patients with H. pylori-associated chronic gastritis. Accordingly, in this study we investigated the relationship of expression of peripheral miRNA-155 with circulating levels of IL-6, IFN-γ and IL-10 in patients suffering from H. pylori-related chronic gastritis to help elucidate the systemic immunity changes occurring in this disease.

Materials and Methods

Study design
This study was organized as an exploratory case–control study to evaluate the levels of systemic miRNA-155 and circulating cytokines in H. pylori-associated chronic gastritis. This cohort consisted of 25 patients with known H. pylori infection and chronic active gastritis, who were age- and gender-matched to 25 healthy controls without a history of gastrointestinal disease. Biomarker profiles were compared between groups to identify possible relationships among H. pylori infection, miRNA-155 expression, and inflammatory cytokines. Peripheral blood sample instead of sampling at the lesion site was selected as a non-invasive method to evaluate systemic immune response related to chronic gastritis.

Study population
The study population included 25 patients with H. pylori-associated chronic gastricitis and of 25 healthy control persons. H pylori positive patients were enrolled at gastroenterology clinic between April 2023 and October 2023 based on endoscopic and histopathological diagnosis in addition to H. pylori confirmation tests. Inclusion criteria included age between 18 and 65 years, no previous GI surgery, and the absence of other chronic inflammatory diseases. The healthy population had no symptoms of gastrointestinal disease, chronic systemic disease. Participants with acute infections, autoimmune diseases and immunosuppressive treatment were excluded to reduce potential confounds. Demographic and clinical features, such as age, sex and body mass index (BMI), were obtained in all the participants.

Sample Collection
Peripheral venous blood (about 5 mL) was gathered from every patient under aseptic circumstances. The samples were clotted at room temperature and then centrifuged to obtain the plasma and serum layers. The aliquoted plasma and serum samples were frozen at −80 °C until analyzed in order to avoid biomarker degradation.

Ethical considerations
The study protocol was checked and approved by the Local Ethics Committee of Hammurabi College of Medicine, University of Babylon -Iraq (Issue No. 38, dated 26/03/2023). All participants provided written informed consent before they were enrolled. The investigation was performed in compliance with the Declaration Helsinki and all information was treated anonymously.

miRNA-155 Expression Analysis
Plasma total RNA was prepared with miRNeasy Serum/Plasma Kit (Qiagen, Germany) following the manufacturer’s instructions. The RNA concentration and purity were measured by NanoDrop Spectrophotometer. Reverse transcription was conducted with miScript II RT Kit (Qiagen). miRNA-155 and U6 snRNA mRNA expression was measured by quantitative real-time PCR using the miScript SYBR Green PCR Kit with miRNA-155 primers as specific primers, U6 snRNA was used as internal references. The ΔΔCt method was utilized to determine the relative expression of miRNA-155.

IL-6 Cytokine Quantification (IL-6, IFN-γ, IL-10)
Serum levels of IL-6, IFN-γ, and IL-10 were determined by sandwich ELISAs according to the manufacturer's instructions. All samples were done in duplicates, and standard curves were prepared of each recombinant cytokine supplied with kits. Optical density was read at 450 nm on a microtiter plate reader.

Statistical Analysis
All statistical analyses were conducted using SPSS (version 26.0) and GraphPad Prism (version 9.0). Data for continuous variable are mean ± SD. Student’s t-test (for normally distributed variables) and the chi-square test (for categorical variables) were used for group comparisons. Logistic regression analyses were conducted to examine relationships in the sample.

Results

Participant Characteristics
Demographic characteristics of the study population are summarized in Table 1. Patient and control groups were comparable with respect to age and sex distribution. However, body mass index was significantly higher in patients with H. pylori-associated chronic gastritis compared with healthy controls (p < 0.001; Table 1).

miRNA-155 Expression
Peripheral miRNA-155 expression was significantly higher in patients with H. pylori-associated chronic gastritis than in healthy controls (p < 0.001; Table 2). Ct values of the housekeeping gene did not differ significantly between groups, indicating reliable normalization. The relative increase in miRNA-155 expression in the patient group is illustrated in Fig. 1.

Cytokine Levels
The serum levels of IL-6, IFN-γ, and IL-10 in patients were all significantly higher than those in controls  (all p < 0.001; Table 3). These results point to systemic immune activation in the H. pylori-positive population. Cytokine profiles in the two groups are illustrated and compared in Fig. 2.

Exploratory Association Analyses
Exploratory logistic regression analysis was used to examine the relation between miRNA-155 expression, level of circulating cytokines and H. pylori infection among study subjects (Fig. 3 and Table 4). Because of the relatively small number of cases, regression analysis was performed for descriptive and hypothesis-generating reasons only, and not for predictive modeling.

Discussion

In this case–control exploration, higher levels of peripheral expression of miRNA-155 and circulating IL-6, IFN-γ, and IL-10 were observed in H. pylori-infected subjects with chronic gastritis. These results indicate H. pylori chronic infection that leads to global immune modifications. Physiologically, it appears that a combination distinctive of systemic immune modulation may operate during chronic H. pylori infection through the modulation of miRNA-155 expression with cytokines in circulation.

miRNA-155 is a well-documented central player in immune and inflammatory signaling networks. Its higher expression level in peripheral blood might indicate persistent immune activation due to chronic bacterial stimulation. Increased levels of miRNA-155 were found in gastric tissue and immune cells of H. pylori-infected individuals in previous studies [12, 13, 23, 24] and, and the present findings extend these observations to the systemic circulation.

The elevated levels of IL-6 and IFN-γ in the present study are suggestive for a pro-inflammatory immune response whereas an increased level of IL-10 may indicate a compensatory anti-inflammatory mechanism which is beneficial in restricting uncontrolled inflammation [15–18]. Comparable cytokine profiles have been described in other studies of H. pylori-associated gastritis [25–32], lending biological credibility to the reported observations.

Several limitations should be acknowledged. The number of patients is relatively small and the lack of power statistically does lead to overfitting in multivariate analysis. Given the variation in BMI between groups, BMI may be a confounder of the inflammatory marker levels. Moreover, the analysis was limited by being restricted to peripheral blood and no validation in stomach tissues or functional experiments. Lack of independent validation cohort prevents clinical or diagnostic interpretation. The logistic regression analysis is also exploratory and should not be considered as evidence of predictive or diagnostic ability.

These results should not be taken to support their use in the clinical setting but as hypothesis-generating. Additional analyses are needed to dissect the physiological relevance of H. pylori-associated miRNA-155-mediated immune regulation in chronic gastritis, including investigation of more extensive and independent cohorts and experiments examining mechanisms.

Conclusion

In Summary, our findings show that The levels of peripheral miRNA-155 and circulating inflammatory cytokines in H. pylori–related chronic gastritis were up-regulated. The results show system host immune activation in H. pylori infection, and suggest a possibility that miRNA-155 might be associated with cytokine-mediated inflammatory response. Despite the exploratory and small-sample-size nature of the current study, additional work in larger or independent cohorts would be needed, along with mechanistic studies, to establish the biological and clinical significance of these observations.

Acknowledgements

The author acknowledges all participants, particularly those who generously agreed to be part of this study.

Author’s Contributions
S.K.S.A. designed the study, conducted the experiments and analyzed data, and drafted the manuscript.

Funding
The study was not supported by any funding.

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