IDDF2023-ABS-0188 Twist1-mediated FGFR2+ fibroblasts to FAP+ fibroblasts differentiation in the pathogenesis of intestinal fibrosis in crohn’s disease

Background

Intestinal fibrosis is a common complication of inflammatory bowel disease and is characterized by the excessive deposition of extracellular matrix (ECM). This study aims to systematically clarify the cellular origin of pathogenic ECM in intestinal fibrosis and explore the potential therapeutic targets.

Methods

A total of 32 patients with Crohn’s disease who underwent surgical treatment due to fibrotic obstruction were included in this study. The intestinal tissues collected from both fibrotic and normal sites were applied for single-cell sequencing, flow cytometry, immunofluorescence staining and biochemistry examinations. The intestinal fibrosis model was constructed with fibroblast-specific Twist1 knockout mice (Col1a2-Cre, Twist1 fllox/flox) treated with 2,4,6-trinitrobenzenesulfonic acid.

Results

The single-cell sequencing data showed that stromal cells are the main source of ECM but are highly heterogeneous. Compared to normal tissues, the proportion of FAP⁺ fibroblasts was significantly increased in fibrotic tissues (from 1.0-3.6% to 4.2-15.2%), while the proportion of FGFR2+ cells decreased significantly (from 39.6-60.1% to 16.5-38.6%). After separating the subsets of stromal cells by flow sorting, we found that the ECM-related genes, including COL1A1, ACTA2, DESMIN, TGF-β1 and SMAD3, were consistently over-expressed in FAP⁺ fibroblasts when compared to other subsets (all p< 0.05). An even higher expression of these genes was observed in FAP⁺ fibroblasts isolated from fibrotic tissues. From immunofluorescence staining, a large amount of collagen and laminin were distributed around FAP⁺ cells. Pseudotime analysis showed an abnormal trajectory of cell differentiation from FGFR2+ fibroblasts to FAP+ fibroblasts in the fibrotic tissues, and Twist1 was the most pronounced gene which differentially expressed during this process. After specifically knocking out Twist1 in the fibroblasts of the mice, we observed that the proportion of FAP⁺ fibroblasts was reduced, and the formation of fibrosis was inhibited compared to the control group (IDDF2023-ABS-0188 Figure 1. Twist1 mediated FGFR2 Fibroblasts to FAP Fibroblasts differentiation in the pathogenesis of intestinal fibrosis in Crohn’s disease).

Conclusions

The number of FAP+ fibroblasts increased significantly in the fibrotic intestine, resulting in a massive secretion of ECM. The transcription factor Twist1 plays a pivotal role in cell differentiation from FGFR2+ fibroblasts to FAP+ fibroblasts, which contributes to the pathogenesis of intestinal fibrosis.