Introduction

Intestinal fibrosis is a late Crohn's disease (CD) complication characterized by poor treatment efficacy and prognosis^[1]. Mesenchymal stem cells (MSCs) have the characteristics of multidirectional differentiation and immune regulation and are expected to become a new means of CD treatment^[2]. Human dental-pulp MSC (hDP-MSC), originating from the neural crest, is easy to obtain and exhibits a strong immune-modulating ability^[3].

Aims & Methods

We aimed in this study to demonstrate the roles of hDP-MSC in intestinal fibrosis. We established a TNBS-induced mouse chronic intestinal fibrosis model and observed the effect of hDP-MSC on the mouse model, with human umbilical cord MSC (hUC-MSC) as a positive control. To track the location of MSC in vivo, MSCs tagged with indocyanine green were injected into the tail vein of the mouse, and fluorescence was analyzed with in vivo imaging system. Besides, we performed Hematoxylin-Eosin staining, Masson staining, western blotting, and ELISA to determine the expression level of pro-fibrotic markers and inflammatory cytokines. Furthermore, to determine the possible mechanism of MSC in intestinal fibrosis, transcriptome sequencing of intestinal tissues of all four groups was applied to analyze pathway changes in the MSC treatment group.

Results

Both intravenous injections of hDP-MSC and hUC-MSC attenuated body weight loss, reduced colon length shortening, lowered disease activity index (DAI), and macroscopic fibrosis score of TNBS modeling mice, with hDP-MSC more statistically significant. Western blotting showed that the expression level of Fibronectin, Collagen I, and α-SMA was increased in the TNBS modeling group, but was decreased in the MSC treatment group. ELISA showed that hDP-MSC significantly decreased the levels of proinflammatory cytokine IL-1β, IL-6, and TNF-α. For transcriptome sequencing, the top ten differentially expressed gene (DEG) of the hDP-MSC versus TNBS modeling group was MMP3, Pla2g5, Rfx2, Sh3rf3, H4c4, Slc34a2, Cldn4, Hbegf, Ibhba, Myo18b. KEGG pathway enrichment of all DEGs showed that the TGF-β signaling pathway was inhibited in the hDP-MSC group, while the IFN-γ signaling pathway was activated.

Conclusion

Collectively, our findings identify that hDP-MSC ameliorates intestinal fibrosis through balancing pro-and anti-fibrotic signaling pathways, which might act as a novel therapeutic approach for the treatment of intestinal fibrosis in Crohn’s disease.

References

[1] Wang Y, Huang B, Jin T, et al. Intestinal fibrosis in inflammatory bowel disease and the prospects of mesenchymal stem cell therapy [J]. Front Immunol, 2022, 13(835005. DOI:10.3389/fimmu.2022.835005
[2] Lian L, Huang Q, Zhang L, et al. Anti-fibrogenic potential of mesenchymal stromal cells in treating fibrosis in crohn's disease [J]. Dig Dis Sci, 2018, 63(7): 1821-1834. DOI:10.1007/s10620-018-5082-8
[3] Stanko P, Altanerova U, Jakubechova J, et al. Dental mesenchymal stem/stromal cells and their exosomes [J]. Stem Cells Int, 2018, 2018(8973613. DOI:10.1155/2018/8973613

Introduction

Inflammatory Bowel Diseases (IBD) are chronic immune-mediated disorders of the gastrointestinal tract. Fibrosis is a frequent manifestation in IBD driven by inflammatory cytokines. During fibrosis, extracellular matrix proteins accumulate in the tissue, causing the disruption of the epithelial barrier and stenosis, ultimately causing malabsorption and intestinal motility issues. This can eventually lead to further serious complications, strictures, fistulas, or ileus. No drugs are available targeting fibrosis, in these cases, only surgical intervention can be used. A defined primary in vitro cell culture model is not available for intestinal fibrosis modeling.

Aims & Methods

Therefore, we aimed to establish and characterize human primary fibroblast cell culture from intestinal biopsies of control and IBD patients.
Human primary fibroblast cell cultures were generated from colonic biopsy of patients with IBD (N=4) and control (N=4). All downstream analyses were applied in passage number 3. For the phenotype observation, immunofluorescent staining was used. Targeted proteins were COL1A1, TGF-ß1, PAI-1, α-SMA, and PHH3. Proteome Profiler Human Cytokine Array Kit was used for the secreted cytokine pattern observation. For the gene expression analysis, RNA was isolated, and the qRT-PCR technique was utilized for the following target genes: COL1A1, ACTA2, TGF-ß1, FN1, PAI-1, H3C4. Scratch assay was used to examine wound closure, we monitored the closing for 48 hours taking photographs throughout. We evaluated the collected data and performed statistical analysis.

Results

We can successfully generate and maintain human colon primary fibroblast cell cultures from colonic biopsies of healthy and Crohn’s disease (CD) origins. Morphological differences were observed between control and diseased patient samples. In the protein examinations, the fluorescent intensity of fibrotic markers (COL1A1, TGF-ß1, PAI-1) and the mitosis marker PHH3 were significantly higher in the CD samples, indicating that the cells maintained fibrotic phenotype and the diseased fibroblasts had a higher proliferation rate. The myofibroblast marker, α-SMA, didn’t show a significant difference. In the cytokine pattern analysis, 22 cytokines showed a twofold increase or decreased by half between the two groups. HGF and DPPIV showed higher quantities in the CD-originated fibroblast. These cytokines are connected to the induction of fibrosis. However, the ANG, BDNF, PTX-3, VEGF, and VCAM-1 were higher in the control samples. Interestingly, no significant differences were noted between the two groups in the targeted genes examined by qRT-PCR measurements. We observed the closure of the artificial wound was faster and more effective in the Crohn’s disease culture compared to the control.

Conclusion

In conclusion, we can generate primary human intestinal fibroblast cell cultures and maintain them to passage number 3, approximately a month long. Fibrotic markers, secreted cytokines and morphological differences distinguish the diseased cultures from the healthy subjects; therefore, it could be useful for in vitro fibrosis modeling in the future. On the other hand, the gene expression analysis doesn’t discriminate between the two groups at passage number 3. To continue with our work, we will observe the fibrotic properties in earlier passage numbers.

Introduction

MCL1 immunostaining has been shown in fibroblasts of the fibrotic ileum of CD patients¹ suggesting that senescence of these cells may be a mechanism of fibrosis perpetuation. We aimed to analyse here the expression of senescence markers in murine intestinal fibrosis and to determine the effects, on this process, of galacto-oligosaccharide-functionalized Mesoporous Silica Nanoparticles (NPs) loaded with an anti-MCL1 drug, S63845 (MSN-S63845).

Aims & Methods

MCL1 and β-Galactosidase immunohistochemistry (IHQ) were performed in colons of mice underwent two DSS cycles, obtained from a previous samples collection. C57Bl/6 mice (20–25 g) were drinking DSS at 2% during 2 cycles (a cycle consists in 7 days drinking water with 2% DSS, followed by ten days drinking water). After the first cycle, mice were administered intraperitoneally, 5 days a week, with MSN-S63845 (8 mg/kg), empty MSN, vehicle or S63845 (8 mg/kg), until the end of the experiment. Body weight was monitored daily, and mice were sacrificed 34 days after the beginning of the experiment. A sample from the colon was paraffin embedded for both histological analysis (collagen deposition was quantified by the QuPath software in Syrius Red- stained slides) and MPO IHQ. Other sample was used for RT-PCR studies. The number of CD45, CD3 and Ly6G positive cells in blood was analysed by FACS. Unless other mention, results are expressed as mean±sem.

Results

Both MCL1 and β-galactosidase immunostaining were detected in leukocytes and fibroblasts of the colon of chronic DSS-treated mice. The effects of blocking MCL1, selectively in β-gal positive cells (MSN-S63845), or non-selectively (S63845) did not alter changes in body weight associated to DSS, but significantly increased the colon length (cm) of mice receiving MSN-S63845 (5.9±0.2, P=0.002^**) or S63845 (6.2±0.3, P=0,0402^*) compared with the respective vehicles (5.1±0.1 or 5.2±0.2, respectively). Treatment of MSN-S63845 compared with empty MSN induced a significant diminution in: a) the thickness of the submucosa (32.4±2.9 vs 51.6±5.9, P = 0,024^*), b) the number of MPO positive cells (67±15.7 vs 171.2±15.4, P=0.001^**) c) the mRNA expression of inflammatory cytokines il17 (0.08 (0.02-0.5) vs 1 (0.3-1.6) P=0.03^#), cxcl15 (0.4 (0.2-0.6) vs 0.8 (0.5-3.5) P=0.07) and cxcl5 (0.1 (0.1-1.2) vs 0.5 (0.5-4.2) P=0.048^#) and profibrotic markers, tgfβ1 (0.5±0.04 vs 1.03±0.1, P=0.003^**). In contrast, treatment with S63845 failed to significantly modify any of these parameters. Mice receiving both MSN-S63845 or S63845 exhibited a similar number of CD45, CD3 and Ly6G positive cells in blood than those receiving the respective vehicles.
^#Mann-Whitnney; *t-test

Conclusion

The selective blockade of MCL1 in β-galactosidase positive cells of murine inflamed intestine reduces fibrosis development.

References

1. Seco-Cervera M et al Biochim Biophys Acta Mol Basis Dis. 2024 Feb;1870(2):166966BBA

Introduction

Over 50% of Crohn’s disease (CD) patients develop intestinal fibrosis, often resulting in strictures. Although anti-inflammatory therapies have improved disease management, fibrotic complications continue to represent a major unmet clinical challenge. Emerging evidence suggests that the gut microbiota may contribute to fibrosis independently of inflammation¹, yet the microbial signatures associated with different stages of fibrosis remain poorly defined.

Aims & Methods

We performed a meta-analysis of RNA-seq data using the IBD TaMMA framework² on fibrotic CD and healthy tissue. Mucosal fibroblasts, epithelial, and endothelial cells were isolated via FACS and profiled transcriptomically. Epithelial-derived organoids and co-culture systems were used to model host–microbe interactions. Fibroblasts stimulated with Roseomonas mucosa lysates (10 µg/mL) were assessed for fibrosis markers (α-SMA, vimentin, FAP) by immunofluorescence and qRT-PCR (ACTA2, PDPN, FAP).

Results

Gene ontology analysis revealed enrichment of antimicrobial immune responses during fibrosis progression. Microbial profiling indicated a consistent enrichment of Roseomonas mucosa in CD samples across fibroblasts, epithelial, endothelial, and immune cell compartments. Notably, fibroblasts stimulated with R. mucosa lysates showed increased expression of fibrosis markers compared to controls.

Conclusion

These findings suggest that Roseomonas mucosa or its components may influence the transcriptional programming of mucosal cells and contribute to fibrosis development in CD. Ongoing spatial transcriptomics and RNA-scope analyses aim to define the spatial and temporal dynamics of microbial–host interactions in fibrotic tissue, advancing our understanding of microbiota-driven fibrosis.

References

1.  Watanabe, D. & Kamada, N. Contribution of the gut microbiota to intestinal fibrosis in crohn’s disease. Front Med (Lausanne) 9, 826240 (2022).
2. Massimino, L. et al. The Inflammatory Bowel Disease Transcriptome and Metatranscriptome Meta-Analysis (IBD TaMMA) framework. Nat. Comput. Sci. 1, 511–515 (2021).

Introduction

Emerging evidence implicates Notch signaling in intestinal mucosal barrier preservation, though its mechanistic basis remains undefined.

Aims & Methods

This study investigates the molecular interplay between Notch activation and tight junction (TJ) regulation during inflammatory injury. In vivo experiments, the dextran sulfate sodium (DSS)-induced colitis mice were used and LY411,575, a γ-secretase inhibitor, was orally administered for Notch signaling inhibition. In vitro experiments, we modulated Notch signaling by either activating or inhibiting it through the overexpression of Hes1 or LY411,575, respectively. The tight junction (TJ) and relevant myosin light chain kinase (MLCK) pathway were systematically investigated. To elucidate the mechanism underlying Notch activation, we conducted a comprehensive analysis of ligands and receptors associated with the Notch pathway in Caco-2 cells.

Results

Compared to DSS-colitis mice, inhibition of Notch activation by LY411,575 led to severe exacerbation of colitis, accompanied by TJ deterioration and concurrent activation of MLCK pathway. In vitro experiments confirmed the Notch signaling's regulatory effect on TJ function, with MLCK inhibitor ML-7 effectively mitigating the TJ dysfunction induced by LY411,575. Additionally, TNF-α-treated Caco-2 cells exhibited significantly increased expression of Jag1, Notch1, and Hes1. Silencing Jag1 using siRNA resulted in reduced NICD1 and Hes1 expression.

Conclusion

Activation of Jag1/Notch1/Hes1 signaling pathway protects the intestinal mucosal barrier from inflammatory injury by abrogating MLCK-dependent TJ dysregulation. Therapeutic Notch activation may represent a novel strategy for mucosal protection in IBD pathogenesis.

Disclosure

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Introduction

Inflammatory Bowel Disease (IBD), including Crohn’s Disease (CD), is characterized by chronic intestinal inflammation and complex host-microbiome interactions. In advanced cases of inflammation in CD patients, the terminal ileum is commonly affected, often becoming obstructed and requiring surgical removal. Approximately 70% of CD patients undergo surgical intervention, and inflammation frequently recurs even after ileocecal resection. Unfortunately, it is still unclear why recurrence happens in some patients and not in others. Increasing evidence suggests that the gut microbiome may play a crucial role in modulating inflammation and influencing disease course, yet spatial insights into host-microbe interactions in CD-affected ileum remain limited.

Aims & Methods

This project aims to spatially characterize the host and microbial transcriptomes in the terminal ileum of CD patients in remission versus recurrence following ileocecal resection, and to explore how human cell responses to host-microbiome interactions may relate to therapeutic outcomes. We collected 24 FFPE ileum samples from CD patients post-resection, including 8 remission and 16 recurrence cases. Spatial transcriptomics was performed using the 10x Genomics Xenium platform with the human colon pre-designed panel (322 genes) and a custom add-on of 100 genes. The custom panel included markers for ileum-specific cell types (32 genes), barrier function (8 genes), redox response (19 genes), and inflammation (30 genes). Additionally, we designed 11 custom bacterial probes in collaboration with 10x Genomics to enable the detection of microbial transcripts.

Results

We successfully performed spatial transcriptomic preprocessing on all 24 samples, with each yielding approximately 200,000–300,000 cells. Samples were annotated using the cell2location pipeline, and we were able to successfully identify the major cell type categories (Epithelial, Mesenchymal, Myeloid, Neuronal, Plasma Cells, Red blood cells, and T cells). The custom bacterial probes enabled detection of microbial transcripts, which were predominantly localized around the lumen and were clearly distinguishable from human tissue regions. Furthermore, we observed differences in bacterial transcript abundance across samples, which could help explain the diversified clinical outcome.

Conclusion

This preliminary analysis demonstrates the feasibility of spatially profiling host and microbial transcripts in FFPE ileum samples from CD patients. Our findings support future investigations into how host-microbiome interactions may be associated with CD progression and recurrence.