Background: Despite extensive research on microbiome alterations in ulcerative colitis (UC), the role of the constituent stable microbiota remains unclear. Results: This study, employing 16S rRNA-gene sequencing, uncovers a persistent microbial imbalance in both active and quiescent UC patients compared to healthy controls. Using co-occurrence and differential abundance analysis, the study highlights microbial constituents, featuring Phocaeicola, Collinsella, Roseburia, Holdemanella, and Bacteroides, that are not affected during the course of UC. Co-cultivation experiments, utilizing commensal Escherichia coli and Phocaeicola vulgatus, were conducted with intestinal epithelial organoids derived from active UC patients and controls. These experiments reveal a tendency for a differential response in tight junction formation and maintenance in colonic epithelial cells, without inducing pathogen recognition and stress responses, offering further insights into the roles of these microorganisms in UC pathogenesis. These experiments also uncover high variation in patients’ response to the same bacteria, which indicate the need for more comprehensive, stratified analyses with an expanded sample size. Conclusion: This study reveals that a substantial part of the gut microbiota remains stable throughout progression of UC. Functional experiments suggest that members of core microbiota – Escherichia coli and Phocaeicola vulgatus – potentially differentially regulate the expression of tight junction gene in the colonic epithelium of UC patients and healthy individuals.

Colonic epithelial barrier dysfunction is one of the early events in ulcerative colitis [UC], and microRNAs [miRNAs] participate in its regulation. However, the cell type-specific miRNome during UC remains unknown. Thus, we aimed to explore miRNA expression patterns in colon tissue and epithelial cells during active and quiescent UC. Small RNA-sequencing in colon tissue, crypt-bottom [CD44⁺], and crypt-top [CD66a⁺] colonic epithelial cells from two cohorts of UC patients [n = 74] and healthy individuals [n = 50] was performed. Data analysis encompassed differential expression, weighted gene co-expression network, correlation, and gene-set enrichment analyses. Differentially expressed colonic tissue miRNAs showed potential involvement in the regulation of interleukin-4 [IL-4] and IL-13 signalling during UC. As this pathway plays a role in intestinal barrier regulation, consecutive analysis of spatially distinct colonic epithelial cell populations was performed. Cell-type- [crypt-top and crypt-bottom] specific miRNA expression patterns were identified in both active and quiescent UC. Target genes of differentially expressed epithelial miRNAs under different disease activity were overrepresented in epithelial cell migration and therefore intestinal barrier integrity regulation. The pro-inflammatory miRNA co-expression module M1 correlated with endoscopic disease activity and successfully distinguished active and quiescent UC not only in both epithelial cell populations, but also in the colon tissue. The anti-inflammatory module M2 was specific to crypt-bottom cells and was significantly enriched in quiescent UC patients. miRNA expression was specific to colonic epithelial cell populations and UC state, reflecting endoscopic disease activity. Irrespective of the UC state, deregulated epithelial miRNAs were associated with regulation of intestinal barrier integrity.

Melatonin is a hormone naturally produced by the body that has recently been found to have antiviral properties. However, its antiviral mechanisms are not entirely understood. Using Caco-2 cells, we developed a gastrointestinal organoid model to investigate the impact of melatonin on cellular organoid culture response to Poly I:C-induced viral inflammation in the gastrointestinal tract. Melatonin was found to have different effect when applied as a pretreatment before the induction of viral inflammation or as a treatment after it. Melatonin pretreatment after Poly I:C stimulation did not protect organoids from size reduction but enhanced cell proliferation, especially when lower (1 and 10 µM) melatonin concentrations were used. On the other hand, treatment with melatonin after the induction of viral inflammation helped to maintain the size of the organoids while reducing cell proliferation. In pretreated cells, reduced IFNLR1 expression was found, while melatonin treatment increased IFNLR1 expression and reduced the production of viral cytokines, such as IFNλ1 and STAT1-3, but did not prevent from apoptosis. The findings of this study emphasize the importance of type III IFNs in antiviral defense in epithelial gastrointestinal cells and shed more light on the antiviral properties of melatonin as a potential therapeutic substance.

Background and Aims: Colonic epithelial barrier loss and dysfunction are one of the early events in ulcerative colitis (UC) and microRNAs (miRNAs) participate in its regulation. However, cell type-specific profile of miRNAs during inflammation in UC is still unknown. Thus, we aimed to perform miRNA profiling on colon tissue and epithelial cell levels in active and quiescent UC. Methods: Small RNA-sequencing in colon tissue, crypt-bottom (CD44 ⁺ ), and crypt-top (CD66a ⁺ ) colonic epithelial cell populations from two independent cohorts of UC patients (active and quiescent, n=74), and healthy individuals (n=50) was performed. Data analysis encompassed differential expression, weighted gene co-expression network (WGCNA), correlation, gene-set enrichment analyses (GSEA). Results: In colon tissue of active and quiescent UC, differentially expressed miRNAs were shown to be potentially involved in intestinal barrier integrity regulation. Consecutive analysis of crypt-bottom and crypt-top colonic epithelial cells revealed distinct miRNA expression patterns in response to UC-caused inflammation. GSEA indicated that differentially expressed epithelial miRNAs are commonly involved in inflammation- and intestinal barrier integrity-related processes (such as signalling of interleukin-4 and interleukin-13), while miRNA differences between cell populations might reflect their function, i.e., crypt-bottom cell miRNA target genes involved in regulation of cell differentiation. Finally, pro-inflammatory miRNA co-expression module correlating with endoscopic UC activity was defined not only in both epithelial cell populations, but also in the colon tissue. Conclusions: miRNA expression patterns are colon epithelial cell population- and UC state-specific and correlate with endoscopic UC activity. Irrespective of the UC stage deregulated epithelial miRNAs are potentially involved in regulation of intestinal barrier integrity.

Colorectal cancer (CRC) encompasses biologically diverse cellular phenotypes with distinct microRNA (miRNA) expression patterns. Assessing whether cell-model-derived alterations recur in extracellular vesicles (EVs) and human tumors may help prioritize candidates with broader relevance to CRC. We performed small RNA sequencing of non-malignant HCoEpiC cells, differentiated enterocyte-like Caco-2 cells, less differentiated HCT116 cells, and their corresponding EVs. miRNAs showing concordant dysregulation in both CRC cell lines relative to HCoEpiC, with greater absolute log₂ fold changes in HCT116, were prioritized and evaluated in human colorectal tissues and TCGA-COAD/READ. Seven of 29 prioritized cellular miRNAs met the EV selection criteria. Of the 26 candidates measurable in the patient-tissue dataset, eight showed FDR-significant, directionally concordant differences between CRC and healthy colorectal tissues, including five concordantly dysregulated across cells, EVs, and patient tissues. Analysis of 11 matched TCGA tumor–non-tumor pairs reproduced reduced miR-30a-3p, miR-30c-2-3p, and miR-1271-5p and increased miR-140-5p expression. Target-based enrichment and exploratory miRNA-mRNA correlation analyses identified associations with CRC-relevant signaling and MYC-related, cell-cycle, DNA-repair, and metabolic programs. No significant associations with the evaluated clinicopathological features or overall survival were detected. Collectively, these findings identify a reproducible subset of CRC-associated miRNA alterations and highlight reduced miR-30a-3p and miR-30c-2-3p expression as a recurrent family-level pattern warranting mechanistic investigation.

The gastrointestinal (GI) tract is a major barrier against pathogens, including viruses. The antiviral responses in the GI epithelium have been broadly investigated, but data on the contribution of the stromal cells remain scarce. Melatonin, widely used to treat insomnia, has recently been proposed as an antiviral agent, yet its effect in the GI tract remains poorly understood. We compared the antiviral responses in Caco-2 monocultures and co-cultures with intestinal fibroblasts (HSIFs) and endothelial cells (HUVECs) after stimulation using Poly I:C. We evaluated the apoptosis, proliferation, key antiviral markers (IRF1, IRF3, IFNs, TBK1, STAT3), and mitochondrial and peroxisomal activation with and without melatonin. The Caco-2 cells cultured with the HSIFs and HUVECs demonstrated enhanced proliferation and reduced Poly I:C-induced apoptosis. The co-culture exhibited a more rapid IRF3-IFNλ1 response, higher TBK1 expression, and enhanced peroxisomal activation compared to these properties in the monoculture. Melatonin further reduced apoptosis and modulated organelle-specific antiviral signaling by suppressing peroxisomal activation and promoting mitochondrial activity. Reduced peroxisomal activation was associated with decreased TBK1, IRF3, and IFNλ1 levels and altered STAT3 signaling. These effects were more pronounced when melatonin was applied post-stimulation compared to that under prophylactic use. Fibroblast–endothelial interactions amplify the antiviral responses in the intestinal epithelial cells by activating the TBK1–IRF3–IFNλ1 axis. Melatonin modulates these responses, highlighting its therapeutic potential in viral GI infections.