Introduction

Fibrosis is a complication commonly present in Crohn’s disease (CD) patients with a structuring (B2) or penetrating (B3) behaviour, with no available treatment. This process is characterized by an excessive extracellular matrix deposition, mainly associated with a dysregulated function of myofibroblasts.

Aims & Methods

We analyse here, the expression of markers of senescence in human small intestinal fibroblasts (HSIF). HSIF (P10760, Innoprot, Spain) were treated during 48h with 20ng/ml of TNFα, 2ng/ml of IL-1β, 5ng/ml of TGFβ1, and 100ng/ml of PDGF. In some cases, fibroblasts were treated with 20nM of siRNA to ETS1 gene (siETS1) or negative control (NC). Gene expression profiles were analysed by RT-qPCR.

Results

Treatment of fibroblasts with IL-1β, PDGF, and TNF-α significantly increased the mRNA expression of different senescence-associated secretory phenotype (SASP) factors (IL-1α, IL-1β, IL-8, and Serpine1). In addition IL-1β also increase the expression of metalloprotease ADAM12 and the chitinase CHI3L1 and the transcription factor related to senescence ETS proto-oncogene 1 (ETS1) and the antiapoptotic factor MCL1, all compared with vehicle treatment. Furthermore, treatment with PDGF significantly increased the mRNA expression of MCL1, ETS1, P21 and P16. Treatment with TGFβ1 increase extracellular matrix components COL1A1 and COMP and the senescence related factor ETS1 and decrease the expression of IL1B. Treatment with TNFα increase the expression of ETS1 but decrease the expression of ADAM12. Finally, a cellular model of depletion of ETS1 treated with IL1β showed that deficiency of this protein can reduce levels of IL1B, IL1A, and CHI3L1 gene expression that were upregulated with IL-1β treatment. Also, this depletion increases the induction of SERPINE1 and ADAM12 observed after treatment with IL-1β.

Conclusion

In human primary intestinal fibroblasts, treatment with IL1-β, PDGF, and TNF-α increased the expression of senescence associated secretory phenotype while treatment with PDGF increased the expression of markers involved in cell cycle arrest. The induction of ETS1 by both treatments and the involvement of this transcription factor regulating gene expression could suggest that ETS1 functions as a double-edged sword inducing inflammation but also repressing the expression of ECM remodelers in intestinal fibroblasts.

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

Fibrosis constitute an important complication of CD. MicroRNAs (miRNAs), which are small RNA molecules that regulate gene expression, have been shown to participate in the molecular interactions of both inflammation and fibrosis. Lower levels of miR-378a-3p have been reported associated to murine liver fibrosis [1] and we analyze here the relevance of miR-378a-3p on intestinal fibrosis.

Aims & Methods

B57BL/6 mice were intravenously injected with 2,5mg/kg of negative control (NC) or mm-miR-378a-3p mimic, twice a week and received vehicle or Dextran Sulfate Sodium (DSS) for 2 cycles (7 days drinking DSS 2% in water solution followed by 10 days drinking water). Body weight and DAI score was obtained every day and the colon was collected after sacrifice. Sirius and hematoxylin-eosin dyes were employed to determine the fibrosis and structural state in 5µm slides of intestinal tissue. Gene expression and miRNA profiles were analyzed by RT-qPCR. Human small intestinal fibroblasts (HSIF; P10760, Innoprot, Spain) were transfected with 20nM of NC or hsa-miR-378a-3p mimic during 24h.

Results

No significant changes in body weight, DAI score, and colon length were detected between mice receiving NC and those receiving mimic, all along the two DSS cycles. Colon of mice treated with DSS, exhibited a significant diminution in the mRNA expression of mm-miR-378a-3p compared with naïve samples. In DSS-treated mice, the iv administration of the mimic compared with the NC: a) significantly increased levels of miR-378a-3p in the colon; b) increased the number of neutrophils, and heightened changes in the glandular epithelia and architectural distortion C) decreased the number of lymphoid follicles; d) slightly increased collagen deposition, as analyzed by sirius red and e) significantly increased the mRNA expression of Tgfb1, Il1b, and Mmp2. Treatment of human intestinal fibroblasts with hsa-miR-378a-3p mimic did not significant modify the mRNA expression of markers of fibrosis but it significantly increased the mRNA expression of two antiapoptotic molecules BCL2 and MCL1.

Conclusion

Levels of mm-miR-378a-3p are diminished in a murine model of intestinal fibrosis; The exogenous administration of miR-378a-3p, increased the acute inflammatory response and the expression of fibrosis markers in murine fibrotic colon and the expression of antiapoptotic molecules in human intestinal fibroblasts.

References

1. Hyun, J.; Wang, S.; Kim, J.; Rao, K.M.; Park, S.Y.; Chung, I.; Ha, C.S.; Kim, S.W.; Yun, Y.H.; Jung, Y. MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression. Nat Commun 2016, 7, 10993, doi:10.1038/ncomms10993.

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

The Notch signaling pathway and senescence are both complex processes that play important roles in a wide range of biological processes, including tissue repair and regeneration, as well as the development of age-related diseases. Studies have suggested that the Notch pathway may be involved in the regulation of senescence and in the development of fibrosis. Senescence is a normal physiological process that plays a role in tissue repair and regeneration, however the accumulation of senescent cells has been implicated in the development of fibrosis. Fibrosis represent the main complications related to Crohn´s disease (CD). Previous studies carried out by our group have shown a high expression of NOTCH pathway in CD samples (Rodriguez-Antequera, et al., 2019).

Aims & Methods

The general aim of the present study is to determine the possible potential of Notch pathway as a therapeutic target in intestinal fibrosis associated with Crohn's disease. Specifically, we pretend: to analyze the localization of the receptors of NOTCH3/4 and the expression of NOTCH markers in the intestinal tissue of patients with complicated CD, and to study the relevance of the Notch pathway in the senescence of intestinal fibroblasts. We have analyzed in intestinal samples from CD patients with complicated lesions: the localization of NOTCH3/4 receptors by IH, the protein expression of senescence/apoptotic markers (BCL2, P16, MCL1 and P53) and NOTCH markers (NOTCH3/4, DLL3/4 and HES1) by WB. We correlated the expression of NOTCH pathway markers with senescence markers of each patient. We carry out in vitro studies and analyze: the protein expression of HES1 (effector Notch pathway) and the protein expression of senescence/apoptotic proteins in HSIF fibroblasts treated with DLL4 or DLL3. Results are expressed as fold induction (mean±SEM). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test or by unpaired Student’s t test.

Results

NOTCH4 is found more specifically in the crypts of the mucosa, as well as in cells of the lamina propria. NOTCH3 was located preferentially in muscular areas -muscularis mucosa, endothelium and muscularis externa. The expression of DLL4/3, NOTCH4 and HES1 were significantly higher in the affected tissue compared to the ileal samples of control patients. BCL2, MCL1, P16 and P53 showed significantly elevated levels in the affected CD tissue, compared to the ileal sample of control patients. P16, the main marker of senescence, correlates positively and significantly with DLL3/4, NOTCH3 and HES1 in intestinal tissue (Table). DLL3, and not DLL4, produced in intestinal fibroblasts a significant increase in the protein expression levels of BCL2 and P53, compared to the vehicle.

Table. Protein correlation of NOTCH markers with senescence/apoptotic markers in intestinal mucosa. The table show the Pearson r. The results of applying the Spearman’s correlation analysis were considered significant at P < 0.05*, P < 0.01** and P < 0.001***.

Conclusion

NOTCH pathway is involved in the regulation of key cellular functions and processes essential for the pathogenesis of intestinal fibrosis in CD patients. DLL3 seems to have a relevant role in activation of senescence in fibroblasts.

References

M Rodriguez-Antequera, J Cosin-Roger, D Macias-Ceja, P Salvador, L Gisbert-Ferrándiz, S Coll, J Manyé, R Alós, F Navarro-Vicente, S Calatayud, M D Barrachina, D Ortiz-Masia, P041 Differences in NOTCH signalling between stricturing and penetrating behaviour in Crohn’s disease, Journal of Crohn's and Colitis, Volume 13, Issue Supplement_1, March 2019, Page S107, https://doi.org/10.1093/ecco-jcc/jjy222.165

Disclosure

Nothing to disclose

Introduction

The Notch pathway may selectively mediate fibrogenic processes, such as epithelial-mesenchymal transition. The dysregulation of EMT is linked to numerous pathologies, including fibrotic diseases¹. Previous studies by our group have demonstrated elevated expression of NOTCH3, NOTCH4 receptors, and the DLL4 ligand in the intestines of patients with complicated Crohn's disease (CD)². This study aims to explore the potential of the Notch pathway as a therapeutic target in intestinal fibrosis associated with CD.

Aims & Methods

We analyzed ileal samples from CD patients with complicated lesions to assess the localization of NOTCH3/4 receptors using immunohistochemistry (IHC) and performed RT-qPCR and Western blot (WB) analysis. In vitro studies utilized DLL4-coated plates (1 µg/ml), with or without the γ-Secretase Inhibitor DAPT (10 μL/ml), to evaluate protein and mRNA expression of HES1 and EMT markers (VIMENTIN, SNAI, and E-CADHERIN) in two primary epithelial cell lines: small intestine (HIEC6) and colon (CoEpi) cells over 48 hours. Data, presented as fold induction (mean ± SEM) relative to water-treated cells or control tissues, were analyzed using parametric or non-parametric ANOVA (Kruskal-Wallis) based on normality testing (*P < 0.05 or **P < 0.01).

Results

RT-qPCR analysis revealed significantly increased mRNA levels of NOTCH3 and NOTCH4 receptors. WB analysis showed elevated levels of NOTCH3 (235±87**, compared to control tissues 100±8) but no significant change in NOTCH4 (87±13 vs. control 100±7). Cellular localization studies showed NOTCH3 in endothelial and smooth muscle cells, lamina propria cells, and lower intensity in the epithelium, whereas NOTCH4 was predominantly found in lamina propria cells. In vitro assays indicated that DLL4 significantly altered mRNA and protein expression of HES1 and EMT markers (VIMENTIN and SNAIL) in both epithelial cell lines. Co-treatment with DAPT significantly reversed some EMT markers induced by DLL4 (VIMENTIN and SNAIL).

Conclusion

NOTCH3 appears to play a role in the fibrogenic processes in CD, with elevated expression in affected tissues. The activation of EMT markers by DLL4 suggests that Notch signaling plays a crucial role in driving fibrosis and tissue remodeling in CD, positioning it as a potential therapeutic target.

References

1. Macias-Ceja DC, Mendoza-Ballesteros MT, Ortega-Albiach M, Barrachina MD, Ortiz-Masià D. Role of the epithelial barrier in intestinal fibrosis associated with inflammatory bowel disease: relevance of the epithelial-to mesenchymal transition. Front Cell Dev Biol. 2023 Sep 26;11:1258843. doi: 10.3389/fcell.2023.1258843. PMID: 37822869; PMCID: PMC10562728.
2. S Marti-Chafer, E Jimenez-Mallen, T Mendoza, D C Macias-Ceja, C Bauset, M Seco-Cervera, S Calatayud, J Cosin-Roger, M D Barrachina, D Ortiz-Masiá, P038 Notch pathway in fibrosis: a new anti-fibrotic therapy in Crohn's disease?, Journal of Crohn's and Colitis, Volume 17, Issue Supplement_1, February 2023, Page i205, https://doi.org/10.1093/ecco-jcc/jjac190.016

Introduction

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease (CD), is a chronic disorder characterized by recurrent intestinal inflammation and complications. Although there has been significant research on bacterial dysbiosis in IBD, the role of the gut mycobiome has been largely overlooked, especially in surgical tissue samples.

Aims & Methods

We aim to characterize the intestinal mycobiome in surgical resections from IBD patients.
Intestinal fungal communities in surgical resections obtained from 20 UC and 30 CD patients, with non-IBD colonic and ileal resections as controls respectively, were analyzed. Fungal DNA was extracted and the ITS region was amplified and sequenced. This data was processed by Dada2 software. Statistical analyses were performed in R, using Adonis tests for multivariate analysis. Rarefaction curves, richness and diversity indexes were calculated and compared by Wilcoxon. Constrained correspondence analysis (CCA) was used for visualization, and ANCOMBC2 for taxon abundance comparisons. P-values were adjusted using FDR method. Spearman's correlation was applied to correlate mycobiome data with microbiota sequencing. Data were expressed as mean ± SEM and compared using t-tests, one-way ANOVA, or Kruskal-Wallis tests, with significance set at p < 0.05.

Results

Results revealed modest changes in fungal diversity and richness in UC resections compared with control-UC samples. However, CD exhibited significant mycobiome alterations at genus level, including an increased abundance of Malassezia (log2FC=-1.4), specifically Malassezia globosa (log2FC=-9,4) at species level, alongside a reduction in Yarrowia lipolytica (log2FC=8.2) compared to the control-CD samples. Regarding fungal correlations, in surgical samples from UC patients, the majority of the correlations were positive while any negative correlation was statistically significant. On the other hand, CD patients showed few negative and weaker positive correlations. Correlation analyses further suggested complex interkingdom interactions between fungal and bacterial communities. In UC patients, Dothideomycetes unclassified stands out with the majority of significant correlations, whereas in CD patients most of the significant negative correlations found in control-CD disappeared while new significant correlations appeared.
Additionally, mycobiome differences between complicated CD-phenotypes were analysed and Cryptococcus pseudolongus and Cladosporium unclassified were significantly more abundant in B3 samples than in B2, whereas Dothideomycetes unclassified was more abundant in B2 than in B3. Moreover, distinct fungal signatures capable of discriminating between them were identified by analyzing ROC curves and Dothideomycetes unclassified presented an AUC value of 0,823 and Cladosporium unclassified with an AUC of 0,737.

Conclusion

Intestinal mycobiome has been characterized for the first time in surgical resections from both UC and CD patients, observing statistical differences in the genera Malassezia and Yarrowia in CD patients. Additionally, several associations between fungal and bacterial species were identified, highlighting the direct interaction between the bacteriome and mycobiome. Notably, complicated-CD phenotypes exhibit a distinct mycobiome pointing Dothideomycetes unclassified and Cladosporium unclassified as potential discriminators between these phenotypes.