5. Prep1: a new player in the development of IBD-associated fibrosis
S. D'Alessio1, A. Gandelli1, C. Correale1, E. Longobardi2, F. Blasi2, S. Danese1, 1Istituto Clinico Humanitas, Rozzano, 2IFOM-IEO Campus, Italy
Intestinal fibrosis is a common complication in Crohn's disease (CD) leading to stricture formation. Distinct cell types are involved in intestinal fibrosis, such as resident mesenchymal cells (fibroblasts, myofibroblasts and smooth muscle cells), whose activation is regulated by several soluble factors. The lack of efficient anti-fibrotic drugs is partly due to the fact that the main and specific cellular and molecular pathways leading to fibrosis remain to be identified. Prep1, a homeodomain transcription factor, has been found to be involved in inflammation through regulation of CCL2 and IL-10 expression. We hypothesized that Prep1 may be involved in the pathogenesis of IBD-associated fibrosis.
Prep1 expression and localization were evaluated in surgical specimens from normal individuals and patients with CD. Intramural gene transfer by injection of adenovector into the mouse rectal wall of healthy and colitic mice was established. Mice subjected or not to weekly intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) for 50 days, received an adenovector encoding murine Prep1 (AdPrep1) or control virus via intramural injection. We also evaluated the inhibitory effect of small interfering RNA (siRNA) targeting Prep1 in vivo on mouse intestinal fibrosis and in vitro on collagen deposition, proliferation and migration of myofibroblasts isolated from CD patients.
Prep1 protein levels were increased within the muscularis mucosa and muscularis propria of inflamed CD tissues in comparison with not inflamed specimens, particularly when fibrosis was evident. Co-staining with alfa-SMA and desmin confirmed specific Prep1 overexpression in myofibroblasts and smooth muscle cells. In vivo, intramural injection of AdPrep1 caused significant elevation of tissue Prep1 protein levels, transmural inflammatory cell infiltration, and transmural fibrosis in mice colon, in comparison with the control virus. Moreover, administration of siRNA targeting Prep1 remarkably reduced collagen deposition in colitic mice and significantly improved TNBS-induced chronic colitis both clinically and histologically. In vitro, Prep1 inhibition reduced collagen deposition, growth rate and migration of IBD-isolated myofibroblasts, in comparison with siRNA scramble-treated cells.
Our findings demonstrate for the first time that Prep1 can induce fibrosis in the gut and that may be a potential target for therapeutic intervention to target intestinal fibrosis associated with CD.