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

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

Vasoactive intestinal peptide (VIP) is a neuropeptide that plays a crucial role in the regulation of intestinal physiology. However, while previous studies hint at its role in the regulation of intestinal proliferation and differentiation, its detailed effect on intestinal stem cells (ISC) in homeostasis and regeneration remains poorly understood. Hence, we here aimed to investigate the effects of VIP on ISC proliferation, differentiation and intracellular signaling in intestinal homeostasis and in a radiation-induced injury model.

Aims & Methods

To study the role of VIP on ISC homeostasis and regeneration, we employed an in vitro 3D organoid model. Intestinal crypts were isolated from the murine proximal small intestine and cultured in vitro with or without VIP treatment. Its effect on ISC differentiation was analyzed by RT-PCR and immunocytochemical analysis. Flow cytometry analysis of organoids derived from Lgr5‐EGFP‐CreERT2 and induced Lgr5-IRES-CreERT2/R26R-TdTomato mice, respectively, allowed for the analysis of the effect of VIP on Lgr5+ ISC and their progeny. Further experiments included EdU and CTG assays as well as bulk RNA sequencing analysis.

Results

Administration of VIP promoted secretory cell differentiation in treated organoids as evidenced by expansion of Lysozyme 1- and Mucin 2-positive cells and increased expression of secretory cell transcripts. The promotion of secretory cell differentiation, interestingly, appeared to be mediated mainly by p38 MAPK and MEK1 signaling. Further, VIP treatment induced an expansion of Lgr5+ ISC, while it prominently reduced overall Edu+ cell proliferation in homeostatic conditions. This coincided with upregulated hallmark gene sets associated with cell cycle such as HALLMARK_MITOTIC_SPINDLE or HALLMARK_G2M_CHECKPOINT in sorted, VIP-treated Lgr5+ ISC. Importantly, in the context of radiation-induced acute injury, we could observe the promotion of organoid viability as well as a marked increase in overall Edu+ cell proliferation with concurrent VIP treatment. This was accompanied by the prominent expansion of Lgr5+ ISC as well as upregulated Lgr5+ ISC activity.

Conclusion

Our findings provide evidence that VIP prominently modulates ISC activity and differentiation in homeostasis. Moreover, VIP may have potential in mitigating radiation-induced damage to the intestinal epithelium and promoting tissue regeneration after injury.

Introduction

Sepsis is defined as an organic dysfunction that threatens the life of patients due to an abnormally regulated response to infection [1]. The initial phase of sepsis is dominated by an increased production of proinflammatory cytokines, which leads to augmented capillary permeability, extravasation, hypercoagulability and myelopoiesis. One of the main sources of infection in sepsis is believed to be the intestinal microbiota via traslocation through the mucosa to the bloodstream. Systemic inflammation weakens intestinal barrier function (IBF) in animal models, resulting in increased bacterial traslocation [2]. Even if the management of sepsis has advanced in the last decades, mortality is still high and there is room for improvement. Thus, the search for effective treatments is clearly justified. Glucocorticoids (GC) constitute a therapeutic modality in sepsis, but they have only shown moderate benefit. This may be accounted for by the harmful effects of GCs on IBF, which may facilitate translocation of luminal microorganisms and molecules. Besides, GC treatment impairs epithelial healing in experimental colitis in mice [3]. Previous results of our research group have shown that mice with induced deletion of the GC receptor (GR) in intestinal epithelial cells (i.e. NR3C1^ΔIEC mice) are protected against dextran sulphate sodium (DSS)-induced colitis [4].

Aims & Methods

Our aim is to understand the role of the intestinal epithelial GR and its involvement in IBF regulation in experimental sepsis, with the ultimate goal of improving the management of sepsis with GCs.
The cecal ligation and puncture (CLP) model of sepsis was applied to WT C57BL/6J and NR3C1^ΔIEC mice. Caecum-exposed mice were used as control (sham). Mice were sacrificed 24 hours after surgery. Four hours before sacrifice, mice were administered 4 kD FITC-dextran, a fluorescent marker of permeability. Colon, jejunum, adrenes, kidney and liver RT-qPCRs were performed as well as determination of corticosterone plasma levels.

Results

After 24 h, CLP mice exhibited hypoglycemia and splenomegaly with elevated corticosterone plasma levels, which were associated to increased Cyp11a1 in the adrenes. Of note, both parameters were less pronounced in KO mice. Intestinal barrier function was weakened, as indicated by downregulation of Tjp1 and increased FITC-dextran plasma levels, which were similar in WT and KO mice. A modest increase in inflammatory markers was noted in the colon and jejunum. However, KO mice exhibited dampened inflammatory response in the colon, with lower expression of inflammatory markers such as S100a8 and S100a9, but not in the jejunum. Similarly, the colonic expression of Cyp11a1 and Lrh1, involved in local steroidogenesis, was lower in CLP mice, regardless of genotype. WT but not KO mice showed an increased in of tissue non-specific alkaline phosphatase (TNAP) in liver, an enzyme involved in detoxification of phosphorylated molecules such as bacterial lipopolysaccharide, but lower expression of Tsc22d3, whose role is described to be protective against sepsis [5]. Inflammation markers were also augmented in the lung and kidney.

Conclusion

In the early stages of the CLP model of sepsis the colon and jejunum are inflamed, and epithelial deletion of the glucocorticoid receptor appears to modulate colonic inflammation, but not barrier function or systemic status.

References

1. Singer, M, et al., Jama, 2016. 315(8): p. 801-10.
2. Carrico, CJ, et al., Arch Surg, 1986. 121(2): p. 196-208.
3. Ocon, B, et al., Biochem Pharmacol, 2016. 116: p. 73-88.
4. Arredondo-Amador, M, et al., Br J Pharmacol, 2021. 178(12): p. 2482-2495.
5. Ballegeer, M, et al., Shock, 2019. 52(2): p. 208-214.

Introduction

Extracellular vesicles (EVs) play an essential role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. EVs are membrane-enclosed nanoparticles secreted by the majority of cells. They contain various types of biomolecules, including proteins, lipids, and non-coding RNAs such as miRNAs. It has been indicated that miRNAs found in EVs affect mRNA stability of recipient cells, thereby playing a crucial role in diseases progression. However, there is no clear consensus as to whether the packaging of miRNAs into EVs occurs randomly or selectively. Lipopolysaccharide (LPS) is an endotoxin of gram-negative bacteria known to initiate the response of immune system and was associated with pathogenesis of inflammatory bowel diseases and cancer. Fundamental mechanisms of molecular packaging into EVs and specificity remain unclear and need to be studied further.

Aims & Methods

The aim of this study was to assess the miRNA profile of extracellular vesicles compared to the miRNA profile of their host cells (colon adenocarcinoma and healthy colon cells), after stimulation with lipopolysaccharide.
Methods: Experiments were conducted using primary human colonic epithelial cells (HCoEpiC) and adenocarcinoma cells (Caco-2, HCT116) (5 passages per cell line). Cells were treated with bacterial LPS from Salmonella enterica (Serotype typhimurium) (Sigma-Aldrich) (10 µg/ml) 24 hours and after 24, 48, 72 hours their EVs were collected using polyethylene-glycol-based method. RNA was purified from cells and EVs using commercial kits. Small RNA sequencing was performed using HiSeq 2500 platform (Illumina, CA, USA). Bioinformatic analysis performed using R package and algorithms for small sequencing data (DEseq2 and etc.).

Results

After 24 hours LPS treatment, miR-146 and miR-451 were overexpressed in HCT116, Caco-2 and HCoEpic cells. The profile of deregulated miRNAs found in EVs of these cell lines was as follows: (i) miR-122-5p, miR-451a, miR-30b-5p, 381-3p, 99a-5p were upregulated and miR-20a-5p, miR-17-5p, miR-93-5p, miR-335-5p, miR-378c were downregulated in Caco-2 cell line produced EVs, (ii) miR-215-5p was downregulated in HcoEpic cell line produced EVs, and (iii) miRNAs expression was not deregulated in HCT116 cell line EVs. After 48- and 72-hours LPS treatment miRNA expression was deregulated only in HCOepic cell line: miR-451a, miR-147b-3p, miR-146a-5p, but not in the EVs secreted by this cell line. This research shows that host cells hold a sorting mechanism that guides specific miRNAs into exosomes. It is important to mention that we noticed that certain miRNAs sequences were found only in EVs of cells, but not in the cells, and some of them (even the most overexpressed) had not been packaged into EVs.

Conclusion

The miRNA profile of extracellular vesicles differs from miRNA profile of the host cells after stimulation with lipopolysaccharide.

References

Additional information. The study is a part of the MOL-CRC-SCREEN project that has received funding from European Regional Development Fund (project No. 01.2.2-LMT-K-718-03-0086) under grant agreement with the Research Council of Lithuania (LMTLT).

Introduction

The intestinal epithelium serves as a site for digestion and absorption of the nutrients and dietary factors, while forming the selective barrier to separate the body’s internal environment from the external luminal environment. The intestinal barrier is established by a combination of several mechanisms, including the mucous layer, antimicrobial peptides, and the intercellular tight junction (TJ)⁽¹⁾. The TJ structure is a multiprotein complex composed of both transmembrane proteins such as claudins and occludin, and cytosolic plaque proteins such as zonula occludens and cingulin, and regulates the paracellular passage of macromolecules, ions, and water. Our and other research groups have shown that the regulation of the intestinal TJ barrier is closely related to intestinal microbial metabolism^(2-4), however, the precise underlying mechanisms are still unclear.

Aims & Methods

The aim of this study was to investigate the role of intestinal microflora in the regulation of the colonic TJ barrier using mice and Caco-2 and MDCKII epithelial cells. In particular, we focused on the molecular mechanisms of n-butyrate-mediated expression of claudin-23. To investigate the effect of intestinal microflora on the TJ expression, RNA sequencing analysis was performed in the colonic tissues from germ-free (GF) and specific pathogen-free (SPF) mice. Claudin-23 expression was also examined in the colon of experimental colitis mice. Human intestinal Caco-2 cells were treated with various microbial metabolites and components including n-butyrate. The TJ barrier function was assessed by measuring the transepithelial resistance (TER) and FITC-dextran (FD) permeability. Claudin-23 expression and transcriptional activity were examined by qRT-PCR, immunoblot, immunofluorescence, and luciferase reporter analyses. The siRNA targeting SP1 and the selective inhibitor of AMPK were used in combination in some experiments. To investigate the role of claudin-23 in the epithelial barrier function, TER and FD permeability were measured in MDCKII epithelial cells stably expressing human claudin-23.

Results

Among several TJ molecules, the claudin-23 mRNA was lower in the colon of GF mice than that of the SPF mice. Claudin-23 mRNA expression was higher in the lower intestinal segments, such as ileum, cecum, and colon, compared with those in the duodenum and jejunum. Claudin-23 expression in the colitis mice was downregulated compared to the normal mice. n-butyrate, but not other microbial metabolites or components, upregulated the claudin-23 expression at both mRNA and protein levels in intestinal Caco-2 cells. N-butyrate enhanced the intestinal TJ barrier integrity, indicated by increased TER and decreased FD permeability. Luciferase reporter assay showed that n-butyrate increased the claudin-23 transcriptional activity. The SP1 siRNA and AMPK inhibitor reduced the n-butyrate-mediated claudin-23 expression and promoter activity. Exogenously expressed claudin-23 localized to the junctional region of MDCKII cells, but did not affect the TER and FD permeability.

Conclusion

Claudin-23 expression in the mouse colon is regulated by a microbial metabolite, n-butyrate. Our results suggest that the transcription factor, SP1, and the AMPK pathway are involved in its transcriptional regulation. Although the precise role of claudin-23 in the intestine is still unclear, the n-butyrate-mediated claudin-23 expression may contribute to the intestinal homeostasis.

References

(1) Suzuki T. Anim Sci J. 2020;91(1):e13357.
(2) Isayama K et al. Exp Cell Res. 2023;425(2):113528.
(3) Yokoo K et al. FASEB J. 2021;35(11):e21854.
(4) Tinh NTT et al. Foods. 2021;10(2):240.

Introduction

RGS proteins promote silencing of the GPCR responses, e.g. cannabinoid (CB) receptors. In our previous work we showed that RGS6 is highly expressed in the gut and RGS6-/- animals exhibit increased intestinal motility in physiological conditions¹. Activation of CB receptors cause several well-recognized effects in the GI tract, including smooth muscle relaxation, reduced motility and analgesia. However, little is known about how the effect of those compounds is modulated by RGS6.

Aims & Methods

The aim of this study was to elucidate the function of RGS6 in physiology and pathophysiology of the GI tract. METHODS: We used intestinal Caco-2 cells (WT and RGS6-/-) to measure the effect of CB agonist WIN 55,212-2 on the level of intracellular, second messenger cAMP and phosphorylation of extracellular signal-regulated kinase (ERK). Then in the in vivo conditions we used an RGS6-/- mice to evaluate the effect of this protein on intestinal motility (stress-induced hypermotility) in the course of DSS-induced inflammation in in naïve mice as well in the presence of WIN 55,212-2.

Results

WIN 55,212-2 (1 µM) caused significant reduction of both basal and forskolin-stimulated intracellular cAMP in WT and RGS6-/- Caco-2 cells. This effect was dose-dependent and was significantly stronger in the absence of RGS6. Phosphorylation of ERK was significantly increased in RGS-/- Caco-2 cells as compared to WT and stimulation with WIN 55,212-2 (1 µM) diminished this effect in the dose-dependent manner. Previously we observed a significant increase in RGS6 expression in the mouse colon samples during intestinal inflammation¹. However, we did not see any difference between anti-inflammatory effect of WIN 55,212-2 (1 mg/kg i.p.) in WT vs. RGS6-/- animals in the model of DSS-induced colitis. On the other hand, we observed that in RGS6-/- mice with colitis WIN 55,212-2 (1 mg/kg i.p.) alleviates stress-induced motility significantly stronger than in WT animals.

Conclusion

Serving as a central control point in GPCR signaling cascades, RGS proteins hold great promises as targets for the drug development. Here we present RGS6 protein as a novel candidate for drug targeting in the GI tract. Our study suggests that loss and perhaps also pharmacological blockade of RGS6 might be used to increase the potency of cannabinoids in the gut.

References

¹ M Swierczynski et al. Regulator of g protein signaling (RGS) protein 6 blunts the effect of cannabinoids on intestinal motility and visceral pain sensation, UEG week 2024, PP1069. Copenhagen, Denmark DOI: 10.1002/ueg2.12461