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P070 The influence of vitamin D on M1 and M2 macrophages in patients with Crohn’s disease

E. Seidman*, S. Dionne

Research Institute of the McGill University Hospital, Gastroenterology, Montreal, Canada

Background

Macrophages play an important role in IBD possessing pro- and anti-inflammatory activities, depending on their phenotype. M1 polarised macrophages contribute to DSS colitis, whereas M2 macrophages attenuate colitis severity. Vitamin D is a potent modulator of both innate and adaptive immunity, but its effect on M1 and M2 macrophages in IBD has not been investigated. The aim of this study was to M1 and M2 macrophage function in CD and to determine the effect of 1,25D on monocyte-derived macrophages in CD patients.

Methods

PBMC were isolated from peripheral blood of 28 CD patients. Monocytes were isolated by negative selection or CD14 microbeads. Inflammatory M1 type macrophages were generated by culturing of monocytes in the presence of GM-CSF, whereas M-CSF with or without IL-4 was used to produce anti-inflammatory M2 macrophages. Cytokines were determined by ELISA following stimulation with 100 ng/ml LPS. Phagocytosis was determined by measuring uptake of FITC-latex beads using flow cytometry. Expression of M1 and M2 markers was determined by qPCR.

Results

CD patients’ M1 macrophages produced much more IL-12p40 compared with M2 macrophages (4 130 pg/ml vs 84.2 pg/ml production, p < 0.0005). There was a trend towards higher TNF-α in M1 macrophages compared with M2 macrophages (p = 0.069). CCL22 levels were very low in M2 cells generated with M-CSF alone, but were produced by M1 macrophages, as well as M2 macrophages generated in the presence of IL-4. M2 macrophages produced significantly higher amounts of IL-10 than M1 macrophages did (1 638 vs 152 pg/ml, p < 0.005). Preincubation with 1,25D greatly decreased IL-12p40 production by M1 macrophages (-68.2%, p < 0.0005), as well as that by M2 macrophages (-100%, p < 0.05). In addition,1,25D also inhibited TNF-α production by M1 macrophages (-36.0%, p < 0.05). IL-10 levels were decreased 32% by 1,25D (p < 0.05). CCL22 production was increased by 1,25D (+165%, p < 0.05). M2 macrophages displayed greater phagocytic activity compared with M1 macrophages (94.6% vs 81.1%). Phagocytosis was minimally enhanced when M1 macrophages were treated with 1,25D (84.4%). 1,25D decreased expression of M1 markers CCL7 and CD80 by 28-41% but had no effect on M2 markers CX3CR1 and CD206.

Conclusion

M1 macrophages from CD patients displayed inflammatory activity/profiles with elevated production of the pro-inflammatory cytokines IL-12p40 and TNF-α, whereas enhanced anti-inflammatory IL-10 production characterised M2 macrophages. Moreover, 1,25D significantly inhibited pro-inflammatory cytokine production from M1 macrophages while increasing IL-10 production; 1,25D decreased M1 markers, enhanced phagocytosis, and CCL22 release, a chemokine that can promote tolerance.