PLoS One. 2013 Oct 22;8(10):e78128.

M2 macrophages activate WNT signaling pathway in epithelial cells: relevance in ulcerative colitis.

Cosín-Roger J, Ortiz-Masiá D, Calatayud S, Hernández C, Alvarez A, Hinojosa J, Esplugues JV, Barrachina MD.

Departamento de Farmacología and CIBERehd, Facultad de Medicina, Universidad de Valencia, Valencia, Spain.



Macrophages, which exhibit great plasticity, are important components of the inflamed tissue and constitute an essential element of regenerative responses. Epithelial Wnt signalling is involved in mechanisms of proliferation and differentiation and expression of Wnt ligands by macrophages has been reported. We aim to determine whether the macrophage phenotype determines the expression of Wnt ligands, the influence of the macrophage phenotype in epithelial activation of Wnt signalling and the relevance of this pathway in ulcerative colitis. Human monocyte-derived macrophages and U937-derived macrophages were polarized towards M1 or M2 phenotypes and the expression of Wnt1 and Wnt3a was analyzed by qPCR. The effects of macrophages and the role of Wnt1 were analyzed on the expression of β-catenin, Tcf-4, c-Myc and markers of cell differentiation in a co-culture system with Caco-2 cells. Immunohistochemical staining of CD68, CD206, CD86, Wnt1, β-catenin and c-Myc were evaluated in the damaged and non-damaged mucosa of patients with UC. We also determined the mRNA expression of Lgr5 and c-Myc by qPCR and protein levels of β-catenin by western blot. Results show that M2, and no M1, activated the Wnt signaling pathway in co-culture epithelial cells through Wnt1 which impaired enterocyte differentiation. A significant increase in the number of CD206+ macrophages was observed in the damaged mucosa of chronic vs newly diagnosed patients. CD206 immunostaining co-localized with Wnt1 in the mucosa and these cells were associated with activation of canonical Wnt signalling pathway in epithelial cells and diminution of alkaline phosphatase activity. Our results show that M2 macrophages, and not M1, activate Wnt signalling pathways and decrease enterocyte differentiation in co-cultured epithelial cells. In the mucosa of UC patients, M2 macrophages increase with chronicity and are associated with activation of epithelial Wnt signalling and diminution in enterocyte differentiation.

PMID: 24167598



Inflammatory bowel disease (IBD) is a chronic disorder of the gastrointestinal tract characterized by disruption of the epithelial barrier function and inflammation. In last years, mucosal healing has been established as a key treatment goal in IBD that predicts sustained clinical remission and resection-free survival of patients. Regeneration of the mucosa depends on the coordinated regulation between proliferation and differentiation of the progenitor cells into epithelial cell lineages, a process that is mainly regulated by the Wnt signalling pathway.

Macrophages constitute one of the central components of inflamed tissue and are considered an essential element of regenerative responses. Several macrophage phenotypes have been described which differ substantially in terms of receptor expression, effector function and cytokine production. We aim to analyse the involvement of the macrophage phenotype in the activation of Wnt signalling pathways in intestinal epithelial cells and to evaluate its relevance in the mucosa of UC patients.

We demonstrate that M2 macrophages, but not non-polarized nor M1 macrophages, induce the expression of Wnt ligands (Wnt1 and Wnt3a) in monocyte-derived macrophages from both healthy donors and UC patients as well in macrophages from U937 cells. Furthermore, our results show that M2 macrophages increased, in epithelial cells in co-culture, the expression of nuclear b-catenin, the central component of the canonical Wnt pathway, and the expression of Lgr5 and c-Myc, two target genes of the Wnt signaling pathway. Moreover, in those epithelial cells the b-catenin bounded to Tcf4 was higher than epithelial cells co-cultured with non-polarized macrophages or M1 macrophages, reinforcing that M2 macrophages activate the Wnt pathway in intestinal epithelial cells.

The pathophysiological relevance of these findings was analysed in human samples from both newly diagnosed and chronic UC patients. We observed in both cases that the number of M1 and M2 macrophages was increased in damaged mucosa versus non-damaged mucosa; however, only the proportion of M2/CD68+ macrophages was significantly higher in damaged mucosa of chronic patients than in the mucosa of newly diagnosed, suggesting that the M2 phenotype increases with the chronicity of the disease. Furthermore, we also observed that M2 macrophages express Wnt1 in the mucosa of these patients and there was a significant a positive correlation between the number of M2 macrophages and the expression of b-catenin in damaged mucosa leading us to suggest that M2 macrophages mediate the activation of Wnt pathway in damaged mucosa of UC patients.

In summary, the present study demonstrates that M2, and not M1, macrophages activates the Wnt signalling pathway in co-cultured epithelial cells through the release of Wnt ligands (Fig 1). In the mucosa of UC patients our results reveal that M2 macrophages increase with the chronicity, act as a source of Wnt1 and they are associated with activation of Wnt signalling at the base of the crypts.

Dolores Barrachina-fig1Figure 1: M2 macrophages activate the Wnt signaling pathway in intestinal epithelial cells. In the intestinal mucosa, macrophages are strategically positioned near epithelial cells of the crypts. M2 macrophages express Wnt ligands (Wnt1 and Wnt3a) and activate the Wnt signaling pathway (nuclear b-catenin and Lgr5) in epithelial cells.


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