10.6084/m9.figshare.4270094.v1 Ethier C. Ethier C. Yu Y. Yu Y. Cameron L. Cameron L. Lacy P. Lacy P. Davoine F. Davoine F. Supplementary Material for: Calcitriol Reduces Eosinophil Necrosis Which Leads to the Diminished Release of Cytotoxic Granules Karger Publishers 2016 Eosinophils Apoptosis Inflammation Cytotoxicity 2016-11-30 15:37:41 Figure https://karger.figshare.com/articles/figure/Supplementary_Material_for_Calcitriol_Reduces_Eosinophil_Necrosis_Which_Leads_to_the_Diminished_Release_of_Cytotoxic_Granules/4270094 <p><b><i>Background:</i></b> Asthma severity and eosinophilia correlate with a deficiency in vitamin D and its active metabolite calcitriol. Calcitriol modulates numerous leukocyte functions, but its effect on eosinophils is not fully understood. We postulated that calcitriol exerts a direct effect on eosinophil biology by modulating cell survival. <b><i>Methods:</i></b> Purified peripheral blood eosinophils from atopic donors were incubated in the presence of calcitriol for up to 14 days with or without IL-5. The effect of calcitriol on eosinophil viability was measured using the annexin-V/propidium iodide flow cytometry assay. We also examined the release of eosinophil peroxidase (EPX) in media using a flow cytometry assay with anti-EPX antibodies, and the enzymatic activity of EPX was measured by an OPD-based colorimetric assay. <b><i>Results:</i></b> We observed that calcitriol sustained cell viability in eosinophils with a concurrent reduction of necrotic cells. This effect was amplified by the addition of IL-5. In parallel, we observed that a physiological dose of calcitriol (10 nM) significantly reduced eosinophil necrosis and cytolytic release of EPX in media when coincubated with IL-5. <b><i>Conclusion:</i></b> These results suggest that calcitriol may exert a direct effect on eosinophils by reducing necrosis and the cytolytic release of inflammatory mediators like EPX.</p>