PA-802 High dose vitamin D3 in vitro has no impact on neutrophil and monocyte antimicrobial functions

BackgroundVitamin D3 (vit.D3) plays an important role in immune responses, and deficiency has been linked to inflammation and higher susceptibility to infections. In vitro studies using pure cell cultures demonstrated vit.D3’s contribution to microbial killing capacity of macrophages. However, the effect on neutrophils is poorly described. We assessed the effect of high dose vit.D3 in vitro on activation and microbial killing capacities of neutrophils and monocytes under near-physiological conditions using fresh whole blood from adult healthy donors. MethodsWhole blood was exposed to Mycobacterium bovis Bacille Calmette-Guérin (BCG) and Lipopolysaccharide (LPS) post treatment with 100 nM vit.D3 for 2hr, 6hr, and 24hr. Cellular phenotyping by flow cytometry was performed to quantify expression of neutrophil (CD16bri14low) and monocyte (CD14bri16low) activation markers (CD11b, CD62L), bacterial phagocytic capacity, and reactive oxygen species (ROS) production. Interleukin 8 (IL-8) and myeloperoxidase serum levels were quantified using ELISA, and correlated with intracellular killing capacities by performing colony forming unit (CFU) analysis. ResultsVit. D3 had no significant direct effect on CD11b/62L expression, phagocytic capacity, ROS production, inflammatory marker expression (IL-8, MPO) and killing efficacy independent of the pre-treatment durations. This may reflect differences in vit.D3 concentrations used and kinetics of vit.D3 mediated response patterns in the cells studied when compared to previous reports. Although these results cannot be extrapolated onto in vivo conditions as vit.D3 effects under physiological conditions can be more complex, the whole blood assay proves a valuable tool to analyse host responses ex vivo in patient cohorts. This assay is employed in our ongoing EDCTP funded 96-week randomized placebo-controlled clinical trial (VITALITY) involving high-dose vit.D3 supplementation (20,000 IU/week) in HIV positive adolescents to assess effects on neutrophil and monocyte antimicrobial responses. ConclusionAn interplay of background effects of HIV and other comorbidities need to be considered as they may influence overall benefits of vit.D3 in this population.