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P051 Macrophages as a source of Notch Ligands in Crohn's disease: implications in fibrosis

Jorge, M.A.(1);Barrachina, M.D.(1);Bauset, C.(1);Gisbert-Ferrándiz, L.(1);Cosin-Roger, J.(2);Coll, S.(1);Calatayud, S.(1);López-Serrano, A.(3);Ortiz-Masiá, D.(4);

(1)Universitat de Valencia, Facultad de Medicina, Valencia, Spain;(2)Hospital Doctor Peset, Fisabio, Valencia, Spain;(3)Hospital Doctort Peset, Servicio Digestivo, Valencia, Spain;(4)Universidad de Valencia, Facultad de Medicina, Valencia, Spain

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

We have analyzed: the mRNA expression of cytokines and Notch ligands in CD patients with fistulizing and stenting pattern, the mRNA and protein expression of macrophage markers and Notch ligands in macrophages treated with the main cytokines present in CD patients (IFNγ-, IL10-, IL4, TNFα-U937 treated cells), the mRNA expression of fibrosis markers of DLL4-HT29  treated cells. Results are expressed as fold induction (mean±SEM, n≥4). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test. Correlations were analyzed with the Spearman coefficient.

Results

The mRNA expression of IFNγ, TNFα, IL4, IL6 and IL10 was significantly higher in intestinal samples from B2- and B3-CD patients (11.4±1.6, 6.9±1.3, 16.4±3.9, 4.1±0.4, and 5.0±1.0 respectively for B2, and 14.2±1.8, 9.6±2.6, 21.5±4.1, 6.3±1.0 and 9.2±1.7 respectively for B3) than in controls (1.0±0.09, 1.1±0.2, 1.2±0.1, 1.1±0.1 and 1.0±0.1 respectively). The mRNA expression of DLL3 and DLL4 was significantly higher in intestinal samples from B2 (6.1±1.3 and 9.3±2.4, respectively) than in B3 CD patients (4.4±1.0 and 1.8±0.7, respectively) and in controls (0.9±0.2 and 0.7±1.2 respectively). IFNγ-U937 treated cells increased significantly the mRNA expression of DLL3 and DLL4 (2,9±0,6 N=5 N=4 and 3,7±1,1 N=4, respectively) respect vehicle;  IL1β increased significantly the expression of DLL4 (1,8±0,01 N=4) and TNFα increased significantly the expression of DLL3 (7,1±2,2 N=4), respect vehicle. DLL4-HT29 treated cells increased significantly fibrosis markers (VIMENTIN (4,6±0,6 N=6), α-SMA (20,9±8,2 N=6), SNAIL1 (1,8±0,4 N=6), SNAIL2 (8,3±1,3 N=6), ZEB1 (8,2±4,3 N=6) and ZEB2 (4,3±0,2 N=6), respect vehicle.

Conclusion

Macrophages may act as a source of Notch ligands who could act as fibrosis mediators in CD patients with a stenting (B2) behavior. The microenvironment rich in IFNγ could activate the fibrosis process in epithelial cells by favoring the expression of DLL4 in macrophages.

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