M2 MACROPHAGES, THE MAIN PHENOTYPE PRESENT IN CHRONIC PATIENTS WITH ULCERATIVE COLITIS, STIMULATE CELLULAR PROLIFERATION THROUGH WNT PATHWAY

J Cosín-Roger1, D Ortiz-Masiá1, J Hinojosa2, MD Barrachina1. 1Facultaty of Medicine (University of Valencia), Pharmacology and CIBERehd 46010, Spain, 2Hospital of Manises, Digestive 46940, Spain

Introduction: Epithelial barrier function is impaired in ulcerative colitis (UC). The release of mediators from inflammatory cells may modulate the ability of epithelial cells to regenerate damaged areas. Macrophages play a central role in inflammation and their plasticity allows them to differentiate in different phenotypes in response to micro-environmental triggers. Long-standing UC has been associated with a high risk of developing colonic adenocarcinoma. The Wnt/beta-catenin pathway is crucial in cancer development and progression, but its role in UC remains to be determined.

Objective: To analyze the activation pattern of macrophages in colon of patients with UC, to determine the relevance of different activation pattern on epithelial cell proliferation and to evaluate the role of the wnt-signaling pathway in the effects observed.

Patients and Methods: Both damaged and non-damaged mucosa from patients with UC was obtained. Paraffin embedded tissues were used for histological analysis (score 1-4) and expression of ki67 (cellular proliferation, score 1-4), CD86+ macrophages (M1 phenotype) and CD206+ macrophages (M2 phenotype) were analyzed by immunohistochemistry. Quantification was performed counting positive cells in a total area of 0,152mm2 using an inverted microscope.

Macrophages derived from U937 cells were differentiated to M1 phenotype (LPS 0,1ng/ml + IFN-γ 20ng/ml, during 24h) or M2 phenotype (IL-4 20ng/ml, during 72h) and gene expression of Wnt3a and Wnt1 was analyzed by real-time RT-PCR. After differentiation, M1 or M2 macrophages were co-cultured with Caco cells and epithelial cell proliferation was analyzed at 24h. Some Caco cells were treated with the inhibitor of Wnt pathway (XAV939, 1μM). Data were expressed as mean± SEM and performed in groups of n≥4.

Results: Number of CD206+ macrophages (8.2±2.2) was higher than CD86+ macrophages (0.2±0.1) in non-damaged mucosa of patients with UC. These numbers were significantly increased (18.8±1.7 and 1.0±0.2, respectively) in damaged mucosa.

Number of Ki67+ cells was higher in damaged (2.8±0.3) than in non-damaged mucosa (1.3±0.3). A positive correlation was observed between: a) mucosal damage and cellular proliferation (rSpearman=0.8676,p<0.0001,n=15); b) CD206+ macrophages and cellular proliferation (rSpearman=0.7841,p=0.0015,n=13) and c) CD206+ and histological damage (rSpearman=0.7366,p=0.0097,n=13). However, a correlation was not observed between CD86+ macrophages and cellular proliferation (rSpearman=0.4317,p=0.1408,n=13) or between CD86+ macrophages and histological damage (rSpearman=0.4396,p=0.1157,n=14).

The mRNA expression of Wnt1 and Wnt3a (fold induction vs non-differentiated macrophages) was significantly increased in M2 macrophages (1.7±0.1 and 5.6±1.8, respectively) while it was not significantly modified in M1 macrophages (0.8±0.2 and 1.3±0.5, respectively). In the co-culture system, non-differentiated macrophages increased epithelial proliferation in 167±16% while M1 macrophages did it in 183±20% (% at=24h vs t=0h). Proliferation induced by M2 macrophages was significantly higher (217±11%) that those observed in non-differentiated and M1 macrophages. This effect was prevented by pretreatment of cells with XAV939.

Conclusion: M2 macrophages increased the expression of wnt ligands and the proliferation of epithelial cells. The prevalence of M2 macrophages in the mucosa of patients with ulcerative colitis and its correlation with histological damage and cellular proliferation could promote a colon cancer in patients with a long-standing ulcerative colitis and it should be considered as a target for new therapeutic approaches.