Abstract Background Intestinal fibrosis is a severe complication of Crohn’s disease (CD), resulting from accumulation of extracellular matrix (ECM) and tissue remodelling. This process can lead to strictures and bowel obstruction, requiring surgical resection in up to 50% of patients. Fibrotic alterations are largely refractory to current anti-inflammatory therapies. Precision-cut intestinal slices (PCIS) are viable tissue sections, containing intestinal epithelial and lamina propria cells, as well as smooth muscle cells of the lamina muscularis mucosae. Previous studies have demonstrated that PCIS preserve the native immune microenvironment of CD patients ex vivo. The aim of this study was to investigate pro-fibrotic responses in intestinal tissue from patients with and without Crohn’s disease. Methods PCIS were prepared from ileum or colon resections of patients with and without CD, and cultured ex vivo for 24 h. Tissue viability was assessed by LDH release assay and baseline gene expression by RNA sequencing. To induce relevant pro-fibrotic responses in non-CD PCIS, TGF-β and/or TNF-α were applied at three different concentrations (10-100 ng/ml). IL-6, MMP-9, MMP-7, ProCollagen1a1, PRO-C3 and C3M were measured in the supernatant by ELISA. Results PCIS maintained typical intestinal morphology and viability during culture. Compared to non-CD tissues, CD tissues showed upregulation of several extracellular matrix genes, including ELN, COL5A3 and CDH6, ex vivo. Overrepresentation analysis of deregulated genes indicated upregulation of pathways associated with tissue remodelling in CD PCIS. Furthermore, tissue slices from CD patients displayed significantly higher levels of C3M and elevated (non-significant) levels of PRO-C3, indicating increased type III collagen turnover in CD tissue ex vivo. TGF-β induced MMP-7 secretion in non-CD tissues in a dose-dependent manner. This was further increased by the addition of TNF-α. In contrast, induction of MMP-9 and IL-6 was primarily driven by TNF-α alone. Neither stimulation had a clear effect on ProCol1a1 levels in the supernatant at the tested timepoint. Conclusion Our data show that CD PCIS exhibit signs of fibrotic remodelling and secrete clinically relevant collagen biomarkers ex vivo. The increased turnover of type III collagen in CD tissue is consistent with previous studies that have linked C3M/PRO-C3 to disease activity in Crohn’s disease patients. The induction of pro-fibrotic responses in non-CD tissue suggests that MMP-7 is a relevant marker that is upregulated independently of inflammation. From a technical point of view, increasing incubation times and integrating deeper tissue layers would be beneficial. Conflict of interest: Mrs. Schröder, Valerie: No conflict of interest Grieger, Klaudia: No conflict of interest Schmidt, Kevin: No conflict of interest Dehmel, Susann: No conflict of interest Pehrsson, Martin: Employed at Nordic Bioscience A/S Kulik, Ulf: No conflict of interest Nickelsen, Swantje: No conflict of interest Gundert, Benjamin: No conflict of interest Aselmann, Heiko: No conflict of interest Mortensen, Joachim: Employee at Nordic Bioscience, who have developed the C3M (patent ref. 9359633 US) and PRO-C3 (patent ref. 9726674 US) assays Braun, Armin: No conflict of interest Hesse, Christina: No conflict of interest Sewald, Katherina: No conflict of interest
Beneke et al. (Thu,) studied this question.