Introduction

Ulcerative colitis (UC) is an inflammatory bowel disease marked by chronic inflammation of the colon's inner lining. Its pathogenesis involves a multifactorial interplay between genetic and environmental factors, dysregulated immune responses, and alterations in the gut microbiome. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression implicated in UC pathogenesis. Understanding the differential expression of miRNAs in colonic epithelial cells and their extracellular vesicles (EVs) during bacterial exposure could offer potential insights into the interplay between gut microbiota and epithelial cell-mediated inflammatory responses.

Aims & Methods

The objective of this study was to investigate the expression of UC-associated miRNAs in colonic epithelial cells and their EVs, using UC patients and control subjects-derived colonic epithelial cells co-cultures with Escherichia coli and Phocaeicola vulgatus bacteria.
Colonic stem cells from healthy controls (HC) (n=10) and patients with active UC (n=10) were used to generate 3D colonic epithelial organoids, which subsequently served as a cell source for constructing polarized monolayers of colonic epithelial cells. These monolayers were co-cultured with commensal E. coli or P. vulgatus bacteria for 2 hours, washed, replenished with fresh culture medium, and further incubated for an additional 24 hours. EVs from cell-conditioned media were isolated using precipitation method and characterized by ELISA (CD63, HSP70, Apo-A1), Dynamic Light Scattering and cryo-TEM. RNAs from colonic epithelial cells and precipitated EVs were isolated using silica-column based method. MiRNAs expression was evaluated by TaqMan qPCR. Statistical analysis and visualization of the results were performed using R studio.

Results

Although statistically insignificant, but the expression of miR-183-5p and miR-135b-5p in epithelial cells of HC group was tended to decrease after contact with E. coli compared to untreated cells (resp. FC=0.84 (p=0.574) and FC=0.87 (p=0.798)), while miR-146a-5p increased (FC=1.37 (p=0.798)). In the EVs samples of HC, the same trends were observed. Interestingly, in the UC patients’ cells, after incubation with E. coli, the expression of all studied miRNAs - miR-183-5p, miR-135b-5p, miR-146a-5p - tended to increase (resp. FC=1.53 (p=0.730), FC=1.71 (p=0.666), and FC=2.39 (p=0.258)). Similar expression changes were observed in the EVs of colonic epithelial cells from UC patients following stimulation with E. coli (resp. FC=1.32 (p=0.730) (miR-183-5p), FC=1.32 (p=0.666) (miR-135b-5p), and FC=2.07 (p=0.258) (miR-146a-5p)).
After culturing colonic epithelial cells with P. vulgatus the expression of all investigated miRNAs, compared to unstimulated cells, remained almost unchanged, both in HC and in UC patients' monolayers. Meanwhile, in both the HC and UC groups, a decrease in miR-183-5p and miR-135b-5p expression was observed in EVs following contact with P. vulgatus, compared to control conditions (in the HC group resp. FC=0.59 (p=0.382) and FC=0.51 (p=0.234); in the UC group resp. FC=0.70 (p=0.605) and FC=0.73 (p=0.489)). However, the expression of miR-146a showed a decreasing trend only in the HC group compared to unstimulated cells (FC=0.53 (p=0.161)).

Conclusion

Due to the considerable variation in the response between study subjects, the observed expression changes did not reach statistical significance. The experimental study revealed a tendency that the expression of miRNAs associated with UC can be modulated by commensal gut bacteria, particularly E. coli, both in colonic epithelial cells and their EVs.

Introduction

A compromised intestinal epithelial barrier plays a central role in inflammatory bowel diseases (IBD) by increasing intestinal permeability and promoting inflammation. Therefore, the restoration of intestinal barrier has become critical in IBD management ¹. Methylxanthine caffeine and its metabolites are widely consumed bioactive compounds that are known to influence various physiological processes ². However, their impact on gut health, particularly in modulating inflammation and epithelial permeability, remains underexplored.

Aims & Methods

The aim of this study was to investigate potential effects of caffeine (CAF) and its primary metabolites paraxanthine (PRX), theobromine (TBR) and theophylline (TPHL) on inflammatory responses and barrier integrity in colonic epithelial organoids, providing insights into their potential role in IBD management. Primary screening of different concentrations of CAF (1/2.5/5 mM) and its metabolites PRX (10/50/100 µM), TBR (10/20/50 µM) and TPHL (25/100/500 µM) was performed in Caco-2 cells. Selected metabolites and concentrations were further tested on inflamed 3D intestinal epithelial organoids derived from ulcerative colitis patient (n = 1) and non-IBD individual (n = 1). Both Caco-2 cells and organoids were pre-treated with metabolites for 1 hour before inflammation induction and were further co-treated with TNF-α/IFN-γ cytokine mix (10 ng/ml each) and metabolites for 24 hours. The effects on inflammation status and epithelial barrier integrity were tested through targeted gene expression analysis using RT-PCR and FITC-Dextran (4kDa) assay, while the effects on generation of reactive oxygen species were investigated by Flow cytometry and DHE assay. The study was approved by the Kaunas Regional Biomedical Research Ethics Committee (protocol no. BE-2-31).

Results

Primary screening revealed that both CAF and its metabolite PRX affected the expression of genes encoding inflammatory cytokines (TNF, CXCL9; p ≤ 0.05) and tight junction proteins (OCLN, TJP1, CLDN1; p ≤ 0.05) and were selected for further analysis in 3D intestinal organoids. Neither CAF, nor PRX reduced the expression of inflammatory cytokine-encoding genes in inflamed intestinal organoids. However, both CAF and PRX increased expression of tight junction proteins in inflamed intestinal organoids derived from control non-IBD (TJP1 – 3.16-fold by CAF and 1.33-fold by PRX (p ≤ 0.05); OCLN – 1.57-fold by CAF and 1.38-fold by PRX (p ≤ 0.05)) and ulcerative colitis (TJP1 – 1.32-fold by PRX; OCLN - 1.14-fold by CAF and 1.27-fold by PRX (p ≤ 0.05)) patient-derived organoids. Mean percentages of FITC-Dextran positive organoids were 43 %, 51 %, and 34 % in untreated, inflamed, and inflamed + PRX groups, respectively, revealing a trend in barrier restoration, though it did not reach statistical significance (p = 0.16 in inflamed vs. inflamed + PRX). A tendency of reduction in reactive oxygen species production was also observed after exposure to caffeine or paraxanthine (by 17 % and 11 %, respectively (p > 0.05)).

Conclusion

Methylxanthine paraxanthine might have a positive effect on gut epithelial barrier function through restoration of the expression of tight junction protein-coding genes.
The study was funded by the European Union and the State Secretariat for Education, Research and Innovation (SERI) (project: miGut-Health, grant no. 101095470).

References

1. Neurath MF et al. (2025) The intestinal barrier: a pivotal role in health, inflammation, and cancer. The Lancet Gastroeneterology & Hepatology, S2468-1253(24)00390-X, https://doi.org/10.1016/S2468-1253(24)00390-X
2. Reddy VS et al. (2024) Pharmacology of caffeine and its effects on the human body. European Journal of Medicinal Chemistry Reports, 10 (100138), https://doi.org/10.1016/j.ejmcr.2024.100138.

Introduction

The usage of intestinal stem cell-derived organoids for intestinal diseases studies is emerging. The main reason for this is that they can be generated from the intestinal tissue and cultured in vitro for prolonged period. It has been shown that long-term cultured intestinal organoids maintain stable genetic and transcriptome profiles, spatial and compositional cellular arrangement and reflect the in vivostate of the tissue of origin [1-3]. However, the data on DNA methylation levels and its stability in colon organoids derived and sub-cultured from adult intestinal stem cells remain very scarce, especially in the context of ulcerative colitis (UC) [4-5].

Aims & Methods

The aim of the study was to evaluate the epigenetic stability of human intestinal organoids obtained and cultured from colonic biopsies of UC patients and healthy controls during long-term culture by assessing quantitative methylation level of LINE-1.
Study included control subjects (CON, n=6), patients with active UC (aUC, n=6) and quiescent UC (qUC, n=7) recruited at the Department of Gastroenterology, Hospital of Lithuanian University of Health Sciences. Bisulfite conversion of DNA samples of colon biopsies, crypts primary and sub-cultured organoids was performed (in total, n=95 DNA samples), followed by PCR amplification using biotin-labeled LINE-1 primers. Quantitative determination of methylation of LINE-1 CpG islands was performed by pyrosequencing on PyroMark Q24 machine. Pyrograms were analyzed using the PyroMark Q24 software (v. 2.0.8, Qiagen), statistical analysis of quantitative methylation data (Wilcox-test) and data visualization were performed using R studio (R version 4.0.3).

Results

LINE-1 region was highly methylated in all studied biological sample groups of all conditions. The average LINE-1 methylation level when assessing all collected biological samples of corresponding group was identical in CON (66.4% ± 5.1) and qUC (66.6% ± 4.0) groups while it was slightly lower in the group of aUC (64.5% ± 4.7). Further, the tendency indicating that regardless the condition, methylation level of the initial colon biopsy samples differ significantly when comparing them to the respective epithelial colon organoid cultures was observed. In CON group, LINE-1 methylation level of late-passage organoids decreased significantly when compared to colon biopsy (by 8.1%), crypts (by 8.0%), and passage 0 (by 6.3%) and passage 1 (by 5.9%) organoids. Similarly, methylation levels of passage 1 and passage 5 organoids also differed from primary biological material in the groups of qUC (by 4.0 and 5.0%, respectively) and aUC (by 4.9 and 4.6%, respectively). Finally, LINE-1 methylation level comparisons of organoids-derived data revealed significant differences between health conditions. Methylation level of LINE-1 region was lower in the passage 1 organoids of aUC patients when compared to either CON or qUC group (by 4.7 and 3.7%, respectively). Interestingly, sizable decrease in LINE-1 methylation level was observed in the late-passage organoids of CON group, which, in turn, resulted in significant difference of 3.8% when compared to passage 5 organoids of qUC.

Conclusion

LINE-1 region of both healthy control and UC patients colon tissues and corresponding intestinal organoids is highly methylated, and long-term culturing of these organoids results in decrease of LINE-1 methylation level which proceeds at different pace depending on the inflammation status of primary tissue.

References

1. Dotti I, Salas A. Potential use of human stem cell-derived intestinal organoids to study inflammatory bowel diseases. Inflamm Bowel Dis. 2018;24(12):2501–9.
2. Middendorp S, Schneeberger K, Wiegerinck CL, Mokry M, Akkerman RDL, van Wijngaarden S, et al. Adult stem cells in the small intestine are intrinsically programmed with their location-specific function: Intestinal stem cells are intrinsically programmed. Stem Cells. 2014;32(5):1083–913.
3. van der Hee B, Madsen O, Vervoort J, Smidt H, Wells JM. Congruence of transcription programs in adult stem cell-derived jejunum organoids and original tissue during long-term culture. Front Cell Dev Biol. 2020;8:375.
4. Howell KJ, Kraiczy J, Nayak KM, Gasparetto M, Ross A, Lee C, et al. DNA methylation and transcription patterns in intestinal epithelial cells from pediatric patients with inflammatory bowel diseases differentiate disease subtypes and associate with outcome. Gastroenterology. 2018;154(3):585–98.
5. Edgar RD, Perrone F, Foster AR, Payne F, Lewis S, Nayak KM, et al. Culture-associated DNA methylation changes impact on cellular function of human intestinal organoids. Cell Mol Gastroenterol Hepatol. 2022;14(6):1295–310.

Introduction

Microscopic colitis (MC) is an inflammatory bowel disease (IBD) that causes chronic non-bloody watery diarrhea and abdominal pain that significantly reduces the quality of life¹. MC can be subdivided into collagenous colitis (CC) and lymphocytic colitis (LC), which are clinically indistinguishable but differ histologically². The activation of immune responses and the alteration of the intestinal epithelial barrier via water channel aquaporin downregulation and crypt hyperproliferation are characteristic features of MC³, often shared by other gastrointestinal disorders, such as “classical” IBD (Crohn’s disease and ulcerative colitis, UC), or irritable bowel syndrome (IBS). Enteric glial cells (EGC) are key regulators of gut homeostasis and epithelial permeability, that respond to inflammation by entering a reactive state, potentially driving epithelial dysfunction. Whether EGC promote resolution or perpetuate inflammation remains unclear.⁴

Aims & Methods

MC is poorly understood and EGC role in MC is still unknown. Therefore, our aim is to investigate the involvement of EGC – due to their neuroimmune functions and involvement in intestinal dysmotility – in patients with MC. In addition, mast cells will be investigated since altered interactions with EGC have been demonstrated during IBS⁵.
Paraffin-embedded colonic biopsies from patients with active CC, LC, UC, IBS-diarrhea (D) and healthy controls (HC) were collected (n=13-26 per cohort). In addition, a subset of CC patients was also sampled during successful treatment with glucocorticosteroid 9mg budesonide (n=13). The amount of EGC was quantified using glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S100β). Mast cells were quantified using tryptase.

Results

Immunofluorescence analysis of EGC revealed an increased amount of EGC^GFAP+ in CC compared to HC (p<0.001). Of note, CC EGC^GFAP+ levels were comparable to those shown by biopsies from patients suffering from IBS-D and UC. Among the 13 CC patients with paired samples before and under budesonide treatment, we observed a trend toward reduced EGC^GFAP+ levels during treatment (p = 0.08). In contrast, LC patients displayed similar EGC^GFAP+ levels as to HC, but lower than those observed in biopsies from patients with CC, IBS-D, and UC (p<0.001). On the other hand, EGC^S100β+ were increased only in active CC (p<0.0001). Yet, UC patients displayed the highest level of EGC^S100β+ (p<0.0001). Mast cells analysis showed an increased counts for tryptase in CC before and under treatment compared to HC (p<0.01).

Conclusion

Our findings indicate that EGC and mast cells are involved in CC pathogenesis due to an increased reactivity of EGC and mast cells in the colonic mucosa. CC patients exhibit signs of EGC^GFAP+ levels comparable to those observed in UC and IBS-D. Oppositely, LC seems to be independent of neuroinflammation as EGC and mast cells presence was comparable to HC. These preliminary findings support the theory that CC and LC are distinct disease entities. Future work will include RNA sequencing of EGC-related genes, counts of eosinophils as well as in situ analysis of mast cells and eosinophils degranulation, and assessment of budesonide effects on EGC in vitro.

References

1. Nyhlin N, Wickbom A, Montgomery SM, et al. Long-term prognosis of clinical symptoms and health-related quality of life in microscopic colitis: a case-control study. Aliment Pharmacol Ther 2014; 39: 963-972. 20140311. DOI: 10.1111/apt.12685.
2. Carrasco A, Esteve M, Salas A, et al. Immunological Differences between Lymphocytic and Collagenous Colitis. J Crohns Colitis 2016; 10: 1055-1066. 20160229. DOI: 10.1093/ecco-jcc/jjw058.
3. Escudero-Hernandez C, Munch A, Ostvik AE, et al. The Water Channel Aquaporin 8 is a Critical Regulator of Intestinal Fluid Homeostasis in Collagenous Colitis. J Crohns Colitis 2020; 14: 962-973. DOI: 10.1093/ecco-jcc/jjaa020.
4. Gonzales J and Gulbransen BD. The Physiology of Enteric Glia. Annu Rev Physiol 2024 20241115. DOI: 10.1146/annurev-physiol-022724-105016.
5. Meira de-Faria F, Casado-Bedmar M, Marten Lindqvist C, et al. Altered interaction between enteric glial cells and mast cells in the colon of women with irritable bowel syndrome. Neurogastroenterol Motil 2021; 33: e14130. 20210402. DOI: 10.1111/nmo.14130.