Background. Intestinal fibrosis is a complication of inflammatory bowel disease (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC). Chronic inflammation causes excessive deposition of extracellular matrix by activated fibroblasts and subsequent stenosis with many patients necessitating surgery. The pro-fibrotic cytokine TGF-β1 activates intestinal fibroblasts and stimulates epithelial cells to undergo to epithelial-to-mesenchymal transition (EMT). A comprehensive understanding of gut fibrosis pathogenesis in IBD remains uncertain hampering the development of effective therapeutic strategies.
Here, we aimed at inducing a fibrotic response in Patient-Derived Organoids (PDOs) issued from colonic mucosal biopsies of pediatric IBD patients and controls.
Methods. Samples were taken during endoscopy from colon uninflamed areas of CD/UC patients and age-matched controls at the Department of Maternal Infantile and Urological Sciences, Sapienza University of Rome. PDOs were generated from intestinal stem cells contained in crypts isolated from biopsies. The inflammation-mediated fibrosis was induced by exposing PDOs to the pro-inflammatory cytokine TNFα (100ng/ml) for one day, followed by treatment with TNFα (100ng/mL) and TGFβ1 (10 ng/ml) for three days.
The fibrotic response was proven by analyzing the levels of inflammatory and fibrotic markers by RT-PCR and immunofluorescence. Transcriptomic changes were assessed by RNA-Sequencing analysis.
Results. Treatment with TNF-α and TGF-β1 of intestinal PDOs from CD, UC and control patients caused morphological changes towards a mesenchymal-like phenotype as well as up-regulation inflammatory (IL-1β), mesenchymal (SLUG), and fibrotic markers (SERPINE1 and COL4A1). The mesenchymal markers Vimentin and Fibronectin were greatly increased in PDOs from CD, UC and control patients upon fibrotic stimuli as assessed by immunofluorescence. Transcriptomic profiling highlighted that in all PDOs, regardless of the source (control, CD or UC), genes regulated by the co-exposure to TNF-α and TGF-β1 are mainly involved in fibrosis-related processes such as cell migration, cell adhesion, cytoskeleton reorganization and in inflammation (Fig. 1). Finally, we demonstrated that CD-PDOs display a specific response to fibrosis compared to both control and UC- PDOs, as assessed by the expression of a subset of 46 genes (Fig 2).
Conclusions. We show that intestinal CD, UC and control PDOs exposed to both TNF-α and TGF-β1 develop fibrosis, therefore representing a new tool to study inflammation-mediated intestinal fibrosis. Moreover, our data suggest that fibrotic CD-PDOs display a specific gene expression signature compared to UC and control PDOs. Further analysis will highlight whether the CD-specific fibrotic signature may explain the high incidence of fibrosis in CD patients.

<b>Figure 1.</b> Intestinal PDO from CD, UC and control patients were treated with TNF-α and TGF-β1 and total RNA Seq was performed. Top-10 Gene Ontology (GO) terms enriched in differentially expressed genes between untreated and TNF-α and TGF-β1 treated PDOs are depicted. <i>Red arrows </i>indicate GO terms shared among the three groups .

Figure 1. Intestinal PDO from CD, UC and control patients were treated with TNF-α and TGF-β1 and total RNA Seq was performed. Top-10 Gene Ontology (GO) terms enriched in differentially expressed genes between untreated and TNF-α and TGF-β1 treated PDOs are depicted. Red arrows indicate GO terms shared among the three groups .

<b>Figure 2. </b>Heatmap depicting the 46 genes that are differentially expressed between CD-PDOs treated with TNF-α and TGF-β1 and control-PDOs treated with TNF-α and TGF-β1. This gene expression signature differentiates fibrotic CD-PDOs from both fibrotic control and UC PDOs.

Figure 2. Heatmap depicting the 46 genes that are differentially expressed between CD-PDOs treated with TNF-α and TGF-β1 and control-PDOs treated with TNF-α and TGF-β1. This gene expression signature differentiates fibrotic CD-PDOs from both fibrotic control and UC PDOs.

Salvatore Cucchiara

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