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P027. Vitamin D potentiates the immunosuppressive effect of anti-TNF induced regulatory macrophages

A. Levin1, M. Wildenberg1, C. Vos2, J. Brandse3, J. De Bruyn3, G. D'Haens3, G. van den Brink3, 1AMC, Tytgat institute, Amsterdam, Netherlands, 2LUMC, Gastroenterology, Leiden, Netherlands, 3AMC, Gastroenterology, Amsterdam, Netherlands

Background

We have previously shown that anti-TNF-alfa antibodies induce a distinct population of macrophages with immune regulatory and wound healing properties. An environmental factor that has been shown to induce tolerogenicity in dendritic cells is Vitamin D. Interestingly, epidemiological studies have shown that Crohn's disease patients have a high prevalence of vitamin D deficiency. These results incited us to investigate the role of Vitamin D in anti-TNF induced macrophages. The aim of this study was to see if the Vitamin D receptor pathway is involved in the induction of anti-TNF induced macrophages and if Vitamin D can potentiate immunosuppressive effect of these macrophages.

Methods

PBMC's were isolated from peripheral blood of healthy donors. Mixed lymphocyte reactions were established by co-culturing PBMC's of two healthy individual donors in a 1:1 ratio. Cultures were treated with anti-TNF to induce anti-TNF induced macrophages. Inflammatory M1 type macrophages were generated by culturing of monocytes in the presence of IFN-gamma. Gene expression of anti-TNF compared to M1 macrophages was determined by microarray followed by pathway analysis.

To determine the effect of Vitamin D on the immunosuppressive effect of anti-TNF induced regulatory macrophages, cell culture experiments were performed in the presence or absence of 1,25-dihydroxyvitamin D. Macrophages were isolated with CD14 microbeads and co-cultured with activated T-cells from a third donor. Proliferation was measured by 3H thymidine incorporation.

Results

Anti-TNF induced macrophages displayed an increased expression of a number of components of the vitamin D receptor pathway, including the vitamin D receptor (VDR) and osteopontin (OPN). Addition of 1,25-dihydroxyvitamin D to the cultures did not result in enhanced numbers of regulatory macrophages. However, macrophages generated in the presence of 1,25-dihydroxyvitamin D did show an increased inhibition of T-cell proliferation, indicating increased immunosuppressive function.

Conclusion

Anti-TNF induced macrophages show an increased activation of the vitamin D receptor pathway. Furthermore the immunosuppressive properties of wound healing macrophages induced by anti-TNF-alfa can be potentiated by 1,25-dihydroxyvitamin D.