Introduction

Intestinal stem cells (ISCs), located at the intestinal crypt base, are exposed to diverse signals that contribute to their proliferation and differentiation. These signals originate from the stem cell niche, which is comprised of epithelial cell types, enteric neurons, myofibroblasts or endothelial cells. Acetylcholine (ACh) is an important neurotransmitter of the enteric nervous system (ENS), which activates muscarinic (mAChRs) and nicotinic receptors (nAChRs). Mammalian mAChRs consist of five main subtypes (M1–M5) which are coupled to G-proteins. In addition to cells of the ENS, mAChRs are prominently expressed by intestinal epithelial cell (IEC) types including Lgr5-positive ISC. We could previously show that the modulation of mAChRs altered small intestinal differentiation and the cellular activity of Lgr5+ ISCs.

Aims & Methods

We investigated the role of mAChRs in colonic epithelial homeostasis and regeneration by utilizing male and female Vil-Cre × M3R fl/fl mice with epithelial ablation of M3R. The colonic Lgr5-expressing ISC number was assessed in Vil-Cre × M3R fl/fl and C57bl/6 mice by in situ hybridization (ISH). Intestinal inflammation was induced by treating C57bl/6 and Vil-Cre × M3R fl/fl mice with 1% (v/v) DSS in drinking water for 7 days, followed by 5 days of normal drinking water. Epithelial cell proliferation rate and numbers of Lgr5+ ISC were assessed in colonic organoids treated with 250 nM bethanechol (Beth), a muscarinic agonist, for 4 days.

Results

Epithelial deletion of M3R in vivo revealed sex-based differences in colonic Lgr5- expressing ISC cell numbers. While male Vil-Cre × M3R fl/fl mice exhibited a severe reduction in colonic Lgr5-expressing ISCs, female mice showed the opposite phenotype with a moderate expansion of Lgr5-expressing cells. This translated into exacerbated DSS-induced colitis in male Vil-Cre x M3R fl mice as compared to very mild inflammation in female Vil-Cre x M3R fl mice normalized to respective age- and gender matched C56BL WT controls. In vitro, pharmacologic muscarinic agonism using Bethanechol similarly induced sex-specific changes to epithelial proliferation as well as Lgr5-positive ISC counts employing murine colonic organoids. Interestingly, Bethanechol treatment induced increased proliferation and an expansion of Lgr5-positive ISC in vitro in colonoids from female mice, which did not occur in treated organoids from male mice.

Conclusion

In summary, our study highlights the significant role of mAChRs, particularly M3, in ISCs and colonic health. The sex-based disparities observed in Lgr5+ ISC populations and responses to inflammatory stimuli emphasize the potential intricate interplay between mAChRs and sex hormones in intestinal physiology. These findings suggest potential therapeutic targets for intestinal disorders, emphasizing the importance of understanding sex-specific mAChR signaling for personalized treatments.

Introduction

Acetylcholine (Ach) is the main transmitter in the enteric nervous system, which is considered an essential component of the intestinal stem cell (ISC) niche. Ach modulates epithelial homeostasis and secretion predominantly via cholinergic, muscarinic G-protein coupled receptors such as M3R and M1R.

Aims & Methods

In our previous work, genetic ablation of M3R in the murine small intestine induced a significant reduction in Lgr5+ ISC and was sensed by Prox1+ progenitor cells, which orchestrated the selective, compensatory expansion of DCLK1+ tuft cells. Surprisingly, this phenotype did not occur in the colon of the same mice, hence we here aimed to elucidate potential regional differences in the modulation of ISC homeostasis by cholinergic signaling in the murine intestine.
Immunohistochemical analysis and RT-PCR analysis of colonic tissues from transgenic Vil-Cre x M3R fl/fl mice and Vil-Cre x M1R fl/fl mice compared to WT mice were performed. Further experiments included in-situ hybridization for Lgr5, flow cytometry analysis of transgenic Lgr5-EGFP-IRES-CreERT2 and Prox1-CreERT2 mice, bulk RNA sequencing analysis and immunoblot analysis of key signaling pathways.

Results

Immunohistochemical analysis of small intestinal and colonic segments from Vil-Cre x M3R fl/fl mice showed significant differences in differentiation. In contrast to an expansion of DCLK1+ tuft cells in small intestine, these showed significantly reduced in the colon of Vil-Cre x M3R fl/fl mice, while p-EGFR+ tuft cells expanded. This pointed to potential differences in sensing cholinergic signaling interruption by ISC subtypes, which we currently investigate employing Lgr5-EGFP-IRES-CreERT2 x M3R fl/fl and Prox1-CreERT2 x M3R fl/fl mice. Similar to small intestine, however, colonic Vil-Cre x M3R fl/fl tissues revealed a pronounced decrease in Lgr5+ ISC concomitant to increased mucosal Ach levels. Also, Vil-Cre x M3R fl/fl tissues showed a prominent upregulation of PI3K, which was abrogated in Vil-Cre x M3R fl/fl x M1R fl/fl mice. In addition, RT-PCR and bulk RNA sequencing results from WT tissues or organoids showed more prominent expression of Chrm1 (gene encoding M1R) in colonic epithelia than Chrm3 (gene encoding M3R), and analysis of Vil-Cre x M1R fl/fl colonic tissues revealed more prominent changes to differentiation and Lgr5+ ISC compared to Vil-Cre x M3R fl/fl mice.

Conclusion

Our data point to prominent regional differences in the modulation of ISC by cholinergic muscarinic niche signaling and local ISC subtypes sensing cholinergic signaling interruption. Moreover, the receptor subtypes M3R and M1R appear to modulate distinct intracellular pathways, and M1R more prominently modulates colonic ISC. Hence, our data point to potentially distinct roles of cholinergic receptor subtype signaling in intestinal homeostasis and regeneration in small and large intestine.

References

Barker, N., van Es, J., Kuipers, J. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).
Barker, N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration. Nat Rev Mol Cell Biol 15, 19–33 (2014).
Haber, A. L. et al. A single-cell survey of the small intestinal epithelium. Nature 551, 333–339 (2017).
McKinley, E. T. et al. Optimized multiplex immunofluorescence single-cell analysis reveals tuft cell heterogeneity. JCI Insight 2, 11 (2017).
Middelhoff, M., Nienhüser, H., Valenti, G. et al. Prox1-positive cells monitor and sustain the murine intestinal epithelial cholinergic niche. Nat Commun 11, 111 (2020).

Introduction

The intestinal epithelium is the most regenerative tissue in the human body and an essential interface between the microbial ecosystem and the underlying mucosa, orchestrating both immune-mediated and metabolic functions. Mitochondrial signaling and metabolism controls the phenotype and function of intestinal epithelial cells (IECs) and mitochondrial perturbation is associated with chronic inflammation. We hypothesize that disruption of microbial-metabolic circuits and impaired metabolic flexibility of IECs contribute to the initiation and progression of tissue injury underlying chronic inflammation.

Aims & Methods

To address the role of mitochondrial impairment in epithelial homeostasis, conditional deletion of Heat shock protein 60 (Hsp60) was induced in the epithelium of Hsp60^Δ/ΔIEC and Hsp60^Δ/ΔIEC;Il10^-/- mice under specific pathogen free and germfree conditions. Blinded histological analysis was used to assess metabolic inury and tissue inflammation. Additional 16S rRNA and shallow shotgun sequencing as well as metaproteomic analysis were performed to identify microbiome changes at the compositional and functional levels. Nanostring analysis and single-cell RNA sequencing (scRNA-seq) were used to identify epithelial cell population-specific transcriptional responses and to validate these gene signatures in resected tissues from Crohn’s disease patients.

Results

Hsp60 deletion in IECs leads to the disruption of mitochondrial metabolism and the development of irregular crypt architecture, referred to as metabolic injury. Tissue lesions and IBD-related colitis are accelerated in Hsp60^Δ/ΔIEC;Il10^-/- mice. Germfree mice are protected from injury and inflammation, demonstrating a causal contribution of bacteria to metabolic injury. Mitochondrial perturbation in Hsp60^Δ/ΔIEC mice is associated with microbial dysbiosis, including an increased abundance of Bacteroides spp., which are maintained during sustained inflammation in Hsp60^Δ/ΔIEC;Il10^-/- mice. Consistently, B. caecimuris triggers tissue aberration in monocolonized-Hsp60^Δ/ΔIEC mice, supporting its deleterious role in tissue homeostasis. Furthermore, metaproteomic analysis revealed a metabolic adaptation of B. caecimuris in response to tissue injury. Importantly, the clinical relevance of Bacteroides spp. is confirmed in a combined analysis of three IBD cohorts (N=560 patients). Transcriptional profiling of the epithelium identified a gene signature in injury-associated sub-populations, including Ido1, Nos2 and Duox2. Remarkably, the identified metabolic injury gene signature discriminates inflamed (N=196) versus non-inflamed (N=147) mucosa in CD patients, thus validating the human relevance of epithelial mitochondrial disruption in IBD.

Conclusion

Bacterial presence is a key driver of intestinal injury and metabolic injury associates with dysbiotic changes, notably expansion of Bacteroides spp. and their metabolic adaptation. Furthermore, mitochondrial perturbation induces a metabolic injury signature, which differentiates inflamed versus non-inflamed mucosa in IBD patients, strengthening the hypothesis of unresolved metabolic injury as a novel concept in the pathogenesis of intestinal inflammation.

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

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

Metaplasia refers to the emergence of cells not normally present in a given organ, typically as a result of chronic inflammation. While often linked to cancer development, its role in inflamed epithelium remains unclear. Paneth cell metaplasia (PCM) is a phenomenon in which Paneth cells, normally restricted to the small intestine, appear ectopically in the inflamed colonic epithelium of patients with ulcerative colitis (UC). However, the roles and characteristics of PCM in the inflamed colonic mucosa remain poorly understood. This study aimed to characterize the clinical and molecular features of colonic PCM and investigate its functional role in the inflamed epithelium.

Aims & Methods

We first analyzed the clinical characteristics of PCM using biopsy samples and clinical data from UC patients who underwent endoscopic evaluation at our institution. To elucidate the molecular features of colonic PCM, we employed spatial transcriptomics to compare PCM in the colon with Paneth cells in the small intestine from the same individuals. These findings were validated in a knockout mouse model, murine colonic organoids, and patient-derived human colonic organoids. This multi-model approach allowed us to investigate the mechanisms underlying PCM formation and assess its functional relevance in intestinal epithelial biology.

Results

PCM was predominantly observed in the left-sided colon and was associated with longer disease duration and the presence of pathological inflammation. Spatial transcriptomic HD analysis suggested that colonic PCM emerges within an inflammatory microenvironment distinct from that of the small intestine. Notably, the expression of the Wnt target genes HD5 and HD6, which are typically required for Paneth cell differentiation in the mouse small intestine, was reduced in colonic PCM. In contrast, expression of REG3A, a gene known to be induced by the inflammation-associated cytokine IL-22, was maintained. Consistent with previous reports highlighting the role of IL-22 in the differentiation and maturation of human Paneth cells, these findings suggest that IL-22 may contribute to the induction of PCM under inflammatory conditions. Mechanistically, IL-22 was shown to suppress Notch signaling in human colonic organoids, thereby promoting PCM formation and inducing REG3A expression in metaplastic cells. In vitro experiments using mouse colonic organoids demonstrated that Reg3a enhances epithelial cell proliferation and promotes wound healing. In vivo, Reg3a^ΔIEC mice exhibited impaired wound healing in both acute and chronic DSS-induced colitis models. Furthermore, REG3A treatment of human colonic organoids derived from UC patients stimulated epithelial proliferation and accelerated wound closure.

Conclusion

PCM contributes to epithelial protection in UC by promoting REG3A-mediated regeneration. These findings suggest that PCM may play a compensatory role in mucosal healing during colonic inflammation.

Introduction

Patient-derived organoid culture is a powerful and versatile system for investigating the molecular mechanisms underlying a wide range of gastrointestinal diseases, including inflammatory bowel disease (IBD) [1]. In the context of IBD, intestinal organoids derived from patients retain key genetic, epigenetic, and disease-specific features, offering a unique platform to model individual responses to inflammatory triggers [2, 3]. This approach also shows promise in predicting personalized responses to biologic and small-molecule therapies, advancing precision medicine strategies for IBD [4]. Although intestinal organoids represent a major advancement in personalized ex vivo cellular modeling, the detailed effects of differentiation and inflammation on the cellular landscape remain poorly understood.

Aims & Methods

In this study, we analyzed differentiated and undifferentiated intestinal epithelial organoids derived from both an IBD patient with active ulcerative colitis and a healthy donor (non-IBD) (n = 4). Inflammation was induced by treating organoids with the pro-inflammatory cytokines TNF-α (10 ng/mL) and IFN-γ (10 ng/mL). Organoids were dissociated using TrypLE Express Enzyme supplemented with Y27632, and viable cells were enriched by fluorescence-activated cell sorting (FACS) using 7-AAD staining. Cell viability and concentration were assessed using the Countess 3 automated cell counter and Trypan Blue staining. Single-cell suspensions were then processed using the 10x Genomics GEM-X Universal 3’ Gene Expression application, and libraries were sequenced on the NovaSeq X Plus platform. Primary data processing was performed using Cell Ranger (10X Genomics), followed by downstream visualization in Loupe Browser (10X Genomics) and an in-house pipeline.

Results

The mean number of recovered cells per sample was 1,957, with an average of 142,015 reads per cell and 27,127 genes detected across all samples. Single-cell transcriptome analysis revealed five distinct clusters in undifferentiated organoids (both treated and untreated), and six to seven clusters in differentiated organoids (treated and untreated). The majority of cells expressed the epithelial marker EPCAM, confirming their epithelial origin. Inflammatory stimulation led to a significant upregulation of TNF-α and IFN-γ gene expression (p < 0.05) in both differentiated and undifferentiated samples. Furthermore, differentiated organoids displayed a significant increase in specific epithelial subtypes, including Paneth cells, enteroendocrine cells, and proliferative cells (p < 0.05), compared to their undifferentiated counterparts.

Conclusion

To conclude, single-cell transcriptomic profiling of patient-derived intestinal organoids revealed a dynamic and heterogeneous cellular landscape shaped by both differentiation and inflammatory signals. The observed upregulation of inflammatory markers underscores the organoid model's utility in mimicking inflammatory responses. Additionally, the potential expansion of distinct epithelial subpopulations in differentiated conditions highlights the model's ability to faithfully recapitulate the complexity of the intestinal epithelium.

References

1. Günther, C., Winner, B., Neurath, M. F., & Stappenbeck, T. S. (2022). Organoids in gastrointestinal diseases: from experimental models to clinical translation. Gut, 71(9), 1892-1908.\
2. Dotti, I., Mora-Buch, R., Ferrer-Picón, E., Planell, N., Jung, P., Masamunt, M. C., ... & Salas, A. (2017). Alterations in the epithelial stem cell compartment could contribute to permanent changes in the mucosa of patients with ulcerative colitis. Gut, 66(12), 2069-2079.
3. Sato, T., Stange, D. E., Ferrante, M., Vries, R. G., Van Es, J. H., Van Den Brink, S., ... & Clevers, H. (2011). Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology, 141(5), 1762-1772.
4. Arnauts, K., Verstockt, B., Santo Ramalho, A., Vermeire, S., Verfaillie, C., & Ferrante, M. (2020). Ex vivo mimicking of inflammation in organoids derived from patients with ulcerative colitis. Gastroenterology, 159(4), 1564-1567.