Each sample was mixed with p-nitrophenyl phosphate solution (Sigma-Aldrich). Thirty minutes later, absorbance at 405nm was measured. Protein content was quantified using Bradford Assay (Bio-Rad Laboratories, Madrid, Spain). sellekchem Statistical analysis Data were expressed as mean��SEM and compared by analysis of variance (one way-ANOVA) with a Newman-Keuls post hoc correction for multiple comparisons or an unpaired Student��s t test where appropriate. A P value of <0.05 was considered to be statistically significant. The clinical correlations were analyzed using Spearman��s correlation coefficient. Ethical Considerations The study was approved by the Institutional Review Board of The Hospital of Manises (Valencia, Spain). Written informed consent was obtained from all patients.
Results M2 macrophages induce the expression Wnt ligands Human monocyte-derived macrophages from healthy donors and UC patients and macrophages derived from U937 cells (Figure S1) were polarized to the M1 or M2 phenotype and we analysed the expression of Wnt1 and Wnt3a in these cells. In all cases, the mRNA expression of the canonical Wnt ligands, Wnt1 and Wnt3a, was significantly increased in M2 cells when compared with the expression observed in both M1- and non-polarized macrophages (Figure 1). Figure 1 The expression of Wnt ligands is selectively induced by M2 macrophages. M2 macrophages activate the Wnt signalling pathway in epithelial cells To determine the influence of the Wnt ligands expression by macrophages on epithelial cells, we established a co-culture system in which macrophages and epithelial cells were in close proximity and shared the culture medium.
As shown in Figure 2A, M2 macrophages induced a significant increase in nuclear accumulation of ��-catenin in Caco-2 cells compared with that induced by M1 or non-polarized macrophages while non-significant differences were observed in total or cytoplasmic ��-catenin in Caco-2 cells co-cultured with different macrophage phenotypes. Analysis of ��-catenin expression in Tcf-4 immunoprecipitates revealed an increase of the amount of ��-catenin attached to Tcf-4 in those Caco-2 cells that had been co-cultured with M2 macrophages when compared with the other experimental groups (Figure 2B).
Furthermore, results also show an increase in the mRNA levels of Lgr5 and c-Myc, two target genes of the Wnt route, in Caco-2 cells that had been co-cultured with M2 macrophages while no Carfilzomib significant induction of these genes was observed in neither cells co-cultured with non-polarized nor M1 macrophages (Figure 2C). Figure 2 M2 macrophages activate the Wnt signalling pathway in epithelial cells. The role of Wnt1 released from M2 macrophages on the epithelial activation of Wnt pathway was determined using a miRNA approach to selectively knockdown Wnt1 by transient transfection in M2 cells (Figure 2D).