Intestinal Permeability, Nutritional Status and MicroRNA Expression in Children Living in Slum Area of Bangladesh

Abstract

Background: Intestinal barrier dysfunction is a global problem especially in poor settings like Bangladesh where malnutrition and recurrent pathogenic infections are very common. The intestinal epithelial barrier maintenance is the most crucial role of intestinal epithelial cells. Inflammation and immune system instability are linked to intestinal epithelial barrier disruption. In vitro mild irritants, pathogens, toxins, pro-inflammatory cytokines, and other stimuli have been shown to open tight junctions and enhance paracellular permeability. Chronic exposure to toxins and fecal pathogens on a regular basis can cause intestinal inflammation, which opens the barrier to inflammatory molecules including undesirable toxins, colonic bacteria, and bacterial antigens, so enhancing the immune response. Children in Bangladesh who have ongoing infections from Bacteroides fragilis, Campylobacter jejuni, enterotoxigenic Escherichia coli, norovirus, rotavirus, adenovirus, or enteric protozoa (giardiasis, cryptosporidiosis, amebiasis,) are at risk for stunted growth and poor nutritional status. According to these findings, colonization of pathogens may cause persistent inflammation and alterations in the gut microbiota that result in intestinal barrier disruption. MicroRNAs are non-coding, 18–23 nucleotide RNA molecules that are small, highly conserved, and bind to the UTRs of mRNAs to regulate gene expression posttranscriptionally. MicroRNAs have been demonstrated to play critical roles during infection with viruses such the enterovirus, adenovirus and rotavirus, which cause gastroenteritis and alter the bacterial and enterocyte microbiota. Pathogens can directly adhere to and infiltrate the intestinal epithelial barrier when there is intense contact with them during an infection, which results in inflammation. Numerous intestinal disorders have been linked to changes in expression of miRNA, which have been seen in samples that are easily accessible such as faecal and tissue samples. It is thought that nutrition, toxins, and other environmental variables also affect the expression of miRNA. However, further research is needed to fully understand how human miRNAs are modulated. In this study, we looked at the expression levels of two human miRNAs (microRNA-21 and microRNA-122) in faecal samples taken from Bangladeshi children with increased intestinal permeability (IIP). Hypothesis: In this study we hypothesized that dysregulation of microRNA is associated with increased intestinal permeability which could be used as a potential biomarker for Intestinal barrier dysfunction. Aim: The aim of our study was to determine whether persistent contact with several fecal pathogens causes IIP and reduced barrier function, which result in aberrant expression of microRNA-122 and microRNA-21. Methods: This study was a nested observational study which was performed within a longitudinal birth cohort study in the slum areas of Mirpur, Dhaka, Bangladesh. For this study, a total of 442, 2-year-old children were chosen. Mothers who agreed to sign an informed consent form and the absence of any birth defects or congenital abnormalities were the inclusion criteria for the study. Children under or over the age of 2, children without any major congenital defects, and parents who would object to having their child's blood drawn were excluded from this study. After the children (n = 442) ingested the lactulose and mannitol solution, samples of urine were taken from these children over the course of 2 hours. Children were chosen for the analysis of miRNA expression. We examined 120 mg of stool from eighty-five children who were chosen randomly. For gene expression analysis of miRNA-122, we chose 42 children from the NIP (L:M ≤ 0.09) group and 43 children from the IIP (L:M > 0.09) group. Identical fecal samples from thirty-six (36) children— sixteen with IIP and twenty with NIP—were chosen for miRNA-21 gene expression study which was performed by real time quantitative PCR. The remaining forty-nine children, were not included for the miRNA-21 gene expression analysis because their stool volumes were insufficient for qPCR. The fecal biomarkers REG1B and Calprotectin were also assessed in the same stool samples used to determine miRNA-122 and miRNA-21 gene expression and was measured using ELISA kits. Human Bio-Plex Pro Assays were used to measure the levels of IFN-γ, IL-2, IL- 1β, TNF-α, IL-10, IL-5, and IL-13 in serum samples. The TaqMan Array Card (TAC) system real-time polymerase chain reaction was utilized to detect gut enteropathogens from the total nucleic acid isolated from faecal samples. Results: Children with IIP had higher expression levels of miRNA-21 (fold change 10; p < 0.001, 95% CI: 5.05-10.78) and miRNA-122 (fold change 11.6; p < 0.001, 95% CI: 6.14- 11.01) in their fecal samples than those children with normal intestinal permeability (NIP). The inflammatory cytokines IFN-γ, IL-2, IL-1β and TNF-α were found to significantly correlate with miRNA-21 and miRNA-122 levels in faeces (p<0.05). Campylobacter jejuni, rotavirus, Bacteroides fragilis, astrovirus, norovirus, adenovirus and various Escherichia coli strains (ETEC_STp, ETEC_STh, EAEC_aatA EAEC_aaiC,) were frequently found in children with IIP (p < 0.001). Calprotectin and REG1B, two fecal inflammatory markers, strongly correlated with miRNA-122 (p = 0.030, and p = 0.015, respectively), whereas miRNA-21 did not show any correlation with these fecal biomarkers. Conclusion: We conclude by demonstrating that children from Bangladesh who have IIP have significantly changed miRNA expression levels in their feces. Two stool miRNAs were strongly associated with disease activity and readily available surrogate biomarkers such fecal Calprotectin, REG1B, and inflammatory cytokine concentrations in the blood. This work also shows that even after long-term preservation, the miRNA expression levels in feces are quite stable and may be measured in a consistent manner. Overall, our results show microRNAs merit further investigation as possible potential biomarkers of gut barrier diseases, which is consistent with earlier findings that the miRNA expression levels in feces correspond with disease activity.