A role for tumor necrosis factor and bacterial antigens in the pathogenesis of Crohn's disease-associated fistulae.

BACKGROUND: Intestinal fistulae represent a severe complication of Crohn's disease (CD). The authors have demonstrated that epithelial-to-mesenchymal transition plays a pivotal role in their pathogenesis. High levels of interleukin-13 and tumor necrosis factor (TNF) are detected in myofibroblast-like transitional cells covering the fistula tracts. Here, a functional role was investigated for the transcription factor Ets-1, TNF, and the bacterial wall component (muramyl dipeptide [MDP]) in the pathogenesis of CD-associated fistulae. METHODS: Perianal fistulae from CD patients were analyzed by immunohistochemistry. Primary colonic lamina propria fibroblasts (CLPFs) were isolated from CD patients with or without fistulizing disease. Messenger RNA (mRNA) levels were assessed by real-time polymerase chain reaction in CLPF or HT29 intestinal epithelial cells (IECs) grown as monolayers or spheroids. RESULTS: Strong expression of Ets-1 transcription factor was demonstrated in transitional cell covering the fistula tracts by immunohistochemistry. TNF induced mRNA expression of ETS-1 and ß6-integrin in HT29 IEC and in CLPF from fistulizing CD patients. These effects could be fully blocked by administration of anti-TNF antibodies. In HT29 cells, TNF further induced mRNA levels of TNF and transforming growth factor beta by treatment for 24 hours. In fistula CLPF derived from CD patients, TNF induced expression of ß6-integrin, TNF, and transforming growth factor beta. Of note, the bacterial wall component, MDP, induced mRNA levels of ETS-1, transforming growth factor beta, interleukin-13, SNAIL1, and ß6-integrin in HT29 IEC monolayers and fistula CLPF by treatment for 24 hours. CONCLUSIONS: TNF and MDP induce the expression of factors associated with epithelial-to-mesenchymal transition and invasion in IEC and fistula CLPF. Our findings indicate that TNF and MDP might synergize in the pathogenesis of CD-associated fistulae.

The role for dickkopf-homolog-1 in the pathogenesis of Crohn's disease-associated fistulae.

BACKGROUND: One of the most challenging conditions in Crohn's disease (CD) patients is the treatment of perianal fistulae. We have recently shown that epithelial-to-mesenchymal transition (EMT) plays a crucial role during CD-fistulae development. Dickkopf-homolog 1 (DKK-1) is known to play a key role during EMT. Here, we investigated a role for DKK-1 in the pathogenesis of CD-associated fistulae. METHODS: Dkk-1 protein expression in CD-fistula specimens were investigated by immunohistochemistry. Colonic lamina propria fibroblasts (CLPF) were obtained from either non-IBD control patients or patients with fistulizing CD. HT-29 intestinal epithelial cells (IEC) were either grown as monolayers or spheroids. Cells were treated with either TNF-α, TGF-ß or IL-13. Knock-down of DKK-1 or ß-Catenin was induced in HT-29-IEC by siRNA technique. mRNA expression was determined by real-time-PCR. RESULTS: Dkk-1 protein was specifically expressed in transitional cells lining the fistula tracts. TGF-ß induced DKK-1 mRNA expression in HT-29-IEC, but decreased it in fistula CLPF. On a functional level, DKK-1 knock-down prevented TGF-ß-induced IL-13 mRNA expression in HT-29-IEC. Further, loss of ß-Catenin was accompanied by reduced levels of DKK-1 and, again, IL-13 in IEC in response to TGF-ß. In turn, treatment of HT-29-IEC as well as fistula CLPF with IL-13 resulted in decreased levels of DKK-1 mRNA. Treatment with TNF-α or the bacterial wall component, muramyl-dipeptide, decreased DKK-1 mRNA levels in HT-29-IEC, but enhanced it in fistula CLPF. DISCUSSION: We demonstrate that DKK-1 is strongly expressed in cells lining the CD-fistula tracts and regulates factors involved in EMT initiation. These data provide evidence for a role of DKK-1 in the pathogenesis of CD-associated perianal fistulae.