Background: Defective bacterial clearance by macrophages is believed to play an important role in Crohn's disease (CD). Phenotypes of macrophages include inflammatory M1 and anti-inflammatory M2. Vitamin D has been shown to reduce colitis severity but the mechanisms remain unclear. The effect of Vitamin D on M1 and M2 macrophages in IBD has not been investigated. The aim was to identify possible differences in M1 and M2 macrophages between CD patients and controls and to determine the effect of 1,25 vit D on macrophage function and phenotype markers.
Methods: PBMC were isolated from peripheral blood of 45 CD patients and 33 controls not taking vitamin D supplements. REB approval and patient consent was obtained. Monocytes were isolated using CD14 microbeads. M1 and M2 type macrophages were generated by culturing of monocytes in the presence of GM-CSF and M-CSF, respectively. Cytokines were determined by ELISA following stimulation with 100 ng/ml LPS. Phagocytosis was determined by measuring the uptake of FITC-latex beads using flow cytometry. Chemotaxis assays were performed in transwell plates. Expression of M1 and M2 markers was determined by qPCR.
Results: No difference in chemotaxis or phagocytosis was observed in CD macrophages compared to controls. M2 displayed greater phagocytic activity than M1 (94.6% vs 80.3%). Phagocytosis was not altered after treatment with 1,25D. M1 migrated in slightly higher numbers toward CCL2 and fMLP compared to M2. Vit D slightly increased migration of both types toward fMLP (+18–26%). LPS-induced production of TNFα, IL-12p40 and IL-10 was comparable between macrophages in CD and controls. M1 produced higher amounts of IL-12p40 and TNFα compared to M2 (p<0.0005 and p<0.05, respectively) whereas IL-10 production was greater in M2 (1566 vs 152 pg/ml, p<0.005). Preincubation with 1,25D greatly decreased IL-12p40 production by M1 (-64.9%, p<0.0005) as well as that by M2 (-100%, p<0.05). 1,25D also decreased TNFα production by M1 (-49.6%, p<0.05) and IL-10 by M2 (-28.2%, p<0.05). M2 macrophages preferentially express F13A1, PTGS2, CD163, CXCL10, CD14 and MMP2, whereas TGFβ, CCL1 and CYP27B1 expression was higher in M1. Marker expression was similar between CD and control macrophages. M1 and M2 markers were not differentially modulated by vit D.
Conclusions: Peripheral blood derived macrophage chemotaxis and phagocytosis in CD is similar to those from controls. The main effect of 1,25D was to markedly decrease pro-inflammatory cytokine production from M1 macrophages. However, 1,25D did not modulate macrophage polarization to the anti-inflammatory M2 phenotype.
This study was supported by a grant from the Dairy Farmers of Canada.