Figure 1.
Dkk-1 is strongly expressed in TCs lining the fistula tract of CD patients.
Immunohistochemical stainings of healthy control subjects (Co, A), fistula tissue derived from CD-patients (fistCD, B), CD-patients with active disease (aCD, C), patients with an active ulcerative colitis (aUC, D), CD patients in remission (remCD, E) and colitis patients in remission (remUC, F) were analyzed. The representative section of the fistula associated to CD highlights the presence of Dkk-1 in TCs lining the fistula tract (red arrow), cells covering the fistula tract (black arrows), whereas surrounding cells show almost no staining signal (white arrows). Asterisks indicate the fistula tract. Pictures are acquired with a 10-fold magnification.
Figure 2.
TGF-β induces mRNA expression of DKK-1 in human IEC, but reduces it in control and CD-derived fistula fibroblasts.
DKK-1 mRNA expression is demonstrated in (A) HT-29 cells treated with TGF-β (50 ng/ml) for 24 h (n = 3), (B) HT-29 spheroids treated with TGF-β for 1, 3, 5 or 7 days, fibroblasts of (C) non-IBD-control patients (n = 3) and (D) CD-associated fistulae (n = 3), both treated with TGF-β for 24 h. Asterisks indicate significant differences versus the respective control. * = p<0.05, ** = p<0.01.
Figure 3.
Knock-down of DKK-1 prevents TGF-β induced up-regulation of IL-13.
(A) DKK-1 expression in control-transfected cells and after DKK-1-siRNA transfection with or without TGF-β (50 ng/ml) treatment (n = 5). (B) IL-13 (n = 4), (C) β6-Integrin (n = 5) and (D) SLUG mRNA expression (n = 4) in HT-29-IEC in control-transfected and transfected cells with or without application of TGF-β for 24 h. Asterisks indicate significant differences versus the respective control whereas # indicates significant differences versus TGF-β treatment and ° indicates significance in comparison to non-treated siRNA transfected cells. */# = p<0.05, **/## = p<0.01, ***/###/°°° = p<0.001.
Figure 4.
IL-13 levels are decreased in β-Catenin knock-down-cells compared to β-Catenin-competent cells.
(A) mRNA expression and (B) protein levels of β-Catenin (n = 2), mRNA levels of (C) DKK-1, (D) IL-13 and (E)TCF-4 in control and β-Catenin-knock-down HT-29 IEC stimulated with or without TGF-β (50 ng/ml) for 24 h (n = 5 each experiment).Asterisks indicate significant difference versus the no stimulated control cells whereas # indicates significant differences versus TGF-β treated control cells. */# = p<0.05, **/## = p<0.01, *** = p<0.001.
Figure 5.
IL-13-induced decrease of DKK-1 mRNA expression is reversed by an anti-IL-13 antibody in IEC and in fibroblasts.
(A) DKK-1 mRNA expression in HT-29 cells stimulated with IL-13 (100 ng/ml) for 30 min, 24 h and 72 h (n = 3). (B) Effect on DKK-1 levels in HT-29-IEC treated for 24 h alone with IL-13, an anti-IL-13 antibody (100 µg/ml) or a combination of both (n = 12). (C) Expression pattern of DKK-1 in HT-29-spheroids exposed to IL-13 for 1, 5 and 7 days (n = 3). (D) DKK-1 mRNA expression in intestinal control fibroblasts (n = 5) and (E) in CD- patients with a fistulizing course (n = 4). Asterisks indicate significant differences versus the respective control whereas # indicates significant differences versus IL-13 treatment. */# = p<0.05, ## = p<0.01, ***/### = p<0.001.
Figure 6.
TNF-α inhibits DKK-1 mRNA expression in human IEC.
(A) DKK-1 mRNA expression in response to TNF-α (50 ng/ml) and/or an anti-TNF-α antibody (24 µg/ml) for 24 h in HT-29-IEC (n = 3 for each experiment). (B) Control CLPF from healthy individuals (n = 3 for each experiment) and (C) CD CLPF (n = 4 for each experiment) in response to TNF-α (50 ng/ml) and/or an anti-TNF-α antibody (24 µg/ml) for 24 h. Asterisks indicate the difference versus the respective untreated control whereas # indicates significant differences versus TNF-α-treated cells. * = p<0.05, **/## = p<0.01, *** = p<0.001.
Figure 7.
DKK-1 expression is decreased in IEC when MDP is present.
DKK-1 mRNA expression in (A) HT-29-IEC (n = 3), (B) in control CLPF (n = 3) and (C) in CD CLPF (n = 4) in response to MDP (100 ng/ml) or LPS (100 ng/ml) for an exposition of 24 h. Asterisks indicate the difference versus the respective untreated control. ** = p<0.01.
Figure 8.
DKK-1 regulates TGF-β induced IL-13 secretion in CD-associated perianal fistulae.
Under chronic inflammatory conditions, TNF-α, and also IL-13, are present at high levels and exert inhibitory effects on DKK-1 mRNA expression in IEC. TNF-α further stimulates TGF-β secretion that promotes DKK-1 mRNA expression in IEC. The effects of the cytokine and the growth factor on DKK-1 are mediated via β-catenin/TCF. On a functional level, TGF-β induces IL-13 secretion via a β-catenin/DKK-1-dependent pathway. These mechanisms through synergism between TGF-β and IL-13 finally lead to an enhanced expression of genes associated with EMT and therefore to the formation of perianal fistulae associated to CD.