Global brain and grey matter volume loss occurred within the first year after a CIS; brain volume loss predicted conversion to MS.
Background Fibrosis constitute the main complications associated to Crohn′s disease (CD). NOTCH signalling has been implicated in lung, kidney, liver and cardiac fibrosis. Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their microenvironment. The aim of the present study is to analyze the role of NOTCH ligands derived from macrophages in the complications of CD. Methods The aim of the present study is to analyze the role of NOTCH ligands derived from macrophages in the complications of CD. We have analyzed: the protein expression of NOTCH ligands and receptors in CD patients with fistulizing (B3) and stenting pattern (B2), the protein expression of NOTCH ligands in macrophages treated with the main cytokines present in CD patients (IFNγ-, IL10-, IL4, TNFα-U937 treated cells), the protein expression of HES1 and fibrosis markers in DLL4-HT29 and DLL3-HT29 treated cells. Results are expressed as fold induction (mean±SEM). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test or t-tet. Results The expression of DLL4 and NOTCH4 were significantly higher in intestinal samples from B3 CD patients (3,2 ± 0,6 N=4* and 3,8 ± 0,6 N=8*, respectively) than in B2 patients (1,6 ± 0,2 N=4 and 1,7 ± 0,3 N=8, respectively) and controls (1,0 ± 0,1N=3 and 1,0 ± 0,1 N=8, respectively). IFNγ-U937 treated cells increased significantly the protein expression of DLL3 and DLL4 (1,6 ± 0,09 N=6* and 1,3 ± 0,1 N=7*, respectively) respect vehicle; IL4 increased significantly the expression of DLL4 (1,4 ± 0,1 N=7*) and TNFα increased significantly the expression of DLL3 (1,4 ± 0,1 N=5*), respect vehicle. DLL4-HT29 treated cells increased significantly fibrosis markers (VIMENTIN: 1,7 ± 0,1 N=3*; SNAIL: 2,2 ± 0,2 N=3*) and HES1 (1,4 ± 0,06 N=3*), respect vehicle (1,0 ± 0,07 N=6; 1,0 ± 0,05 N=6; and 1,0 ± 0,05 N=6, respectively). DLL3-HT29 treated cells only produced a reduction in the protein expression of ECADHERIN (0,4 ± 0,1 N=3*), respect vehicle (1,0 ± 0,09 N=6). Conclusion Macrophages may act as a source of NOTCH ligands who could act as fibrosis mediators in CD patients with a fistulizing (B3) behavior. The microenvironment rich in IFNγ could activate the fibrosis process in epithelial cells by favoring the expression of DLL4 and DLL3 in macrophages. DLL4 mainly activates transcription factors involved in mesenchymal epithelial transition in colonic epithelial cells (SNAIL), while DLL3 seems to have a more relevant role in cell-cell junction modification (ECADHERIN).
Background Macrophages contribute to fibrosis by releasing different mediators and the pattern of secretion may vary depending on the surrounding environment. We previously described that the mRNA expression of IFNγ was significantly higher in intestinal samples from CD patients. Methods The aim of the present study is to analyze the role of IFNγ-treated macrophages in epithelial mesenchymal transition (EMT) through the WNT pathway. The mRNA and protein expression of IFNγ in surgical resections from Crohn′s disease. U937 were differentiated to macrophages and then treated with IFNγ (10 ng/ml) for 4 days, the mRNA expression of WNT2b, WNT6 and TGFβ were determined by RT-PCR and protein. IFNγ-U937 were coculture with HT29 cells for 3 days and the expression of EMT markers, βCATENIN and WNT2b in HT29 cells were analyzed by WB. In some cases, HT29 cells were treated with the inhibitor of the WNT-pathway, XAV939 (1 μM), or were transfected with vectors-targeting human FZD4 (miFzd4). Results are expressed as mean±SEM. Statistical analysis was performed by ANOVA + Newman-Keuls or unpaired t-test. Results The mRNA and protein expression of IFNγ were significantly higher in intestinal samples from B2 CD patients (11.4±1.6 fold induction and 70,0 ± 2,3 pg/mg, respectively) and B3 CD patients (14.2±1.8 fold induction and 80,4 ± 6,8 pg/mg, respectively) than in controls (1,0±0,1 fold induction and 18,9 ± 0,3 pg/mg, respectively). U937 cells treated with IFNγ increased significantly the protein expression of WNT2b (1,3 ± 0,07 N=13* vs vehicle) but not the expression of WNT6 or TGFβ. IFNγ-U937 co-cultured with HT29 increased significantly the protein expression of EMT markers, βCATENIN and WNT2b in HT29 cells (VIMENTIN: 2,7 ± 0,3 N=6* vs vehicle-HT29; SNAIL1: 1,5 ± 0,1 N=5* vs vehicle-HT29; βCATENIN: 1,4 ± 0,09 N=5* vs vehicle-HT29; WNT2b: 2,0 ± 0,3 N=8* vs vehicle-HT29). Treatment HT29 cells with XAV939 (VIMENTIN: 1,4 ± 0,4 N=6 vs vehicle-HT29 XAV; SNAIL1: 1,0 ± 0,1 N=5 vs vehicle-HT29 XAV; βCATENIN: 0,8 ± 0,1 N=5 vs vehicle-HT29 XAV) or with miFzd4 (VIMENTIN: 0,3 ± 0,07 N=12* vs IFNγ-HT29 mock; SNAIL1: 0,5 ± 0,06 N=12* vs IFNγ-HT29 mock; βCATENIN: 0,6 ± 0,04 N=12* vs IFNγ-HT29 mock; FZD4: 0,5 ± 0,04 N=12* vs IFNγ-HT29 mock) partially reversed the increases produced by IFNγ-U937 in HT29 cells. Conclusion IFNγ-macrophages may stimulate the expression of WNT ligands in an autocrine and paracrine manner. The expression of WNT ligands at the epithelial level could favor mesenchymal epithelial transition through WNT2b ligand and FZD4 receptor.
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