Supplementary Material for: Vitamin D₃ Treatment Decreases Frequencies of CD16-Positive and TNF-α-Secreting Monocytes in Asthmatic Patients

Background: Previously, we demonstrated that glucocorticoid (GC) treatment of asthmatic patients resulted in decreasing frequencies of monocyte subsets expressing CD16 and capable of releasing TNF-α. Here, we wished to analyze whether the active form of vitamin D, i.e. vitamin D₃, referred to as 1α,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] can exert GC-like proapoptotic effects on CD16-positive monocytes and thus decrease the proinflammatory potential of these cells. Finally, we set out to investigate whether the addition of 1,25-(OH)₂D₃ would facilitate the use of lower doses of GC without decreasing their anti-inflammatory properties. Methods: Peripheral blood mononuclear cells collected from healthy individuals and asthmatic patients were cultured with 1,25-(OH)₂D₃ and/or varying doses of GC in the presence or absence of caspase inhibition. The cells were either directly stained for extracellular markers or prestimulated with lipopolysaccharide for the assessment of intracellular cytokine production and then analyzed by flow cytometry. Results: We found that 1,25-(OH)₂D₃ alone (and in combination with GC) decreased the frequency of CD14++CD16+ and CD14+CD16++ monocytes from asthmatic patients and significantly diminished TNF-α production by the monocytes. With regard to the CD14+CD16++ subset, the monocyte-depleting effects of 1,25-(OH)₂D₃ were abrogated in the presence of pan-caspase inhibitor, suggesting a proapoptotic mechanism of 1,25-(OH)₂D₃ action. Interestingly, we found that a combined treatment of 1,25-(OH)₂D₃ and GC allowed for a 5-fold reduction of the GC dose while maintaining their anti-inflammatory effects. Conclusions: This study has revealed novel immunomodulatory properties of 1,25-(OH)₂D₃ directed against monocyte subsets capable of TNF-α production. In addition, our data suggest that the introduction of 1,25-(OH)₂D₃ to anti-inflammatory therapy would possibly allow for the use of lower doses of GC.