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Vitamin D 2  (ergocalciferol) and vitamin D 3  (cholecalciferol) are fat-soluble secosteroid hormones obtained from plant and animal sources, respectively. Fish incorporates vitamin D 2  and D 3  through the diet. In mammals, vitamin D forms are involved in mineral metabolism, cell growth, tissue differentiation, and antibacterial immune response. Vitamin D is an essential nutrient in aquafeeds for finfish. However, the influence of vitamin D on fish cell immunity has not yet been explored. Here, we examined the effects of vitamin D 2  and vitamin D 3  on  Salmo salar  primary macrophage immune response to  A. salmonicida  subspecies  salmonicida  infection under  in vitro  conditions. We determined that high concentrations of vitamin D 2  (100,000 ng/ml) and D 3  (10,000 ng/ml) affect the growth of  A. salmonicida  and decrease the viability of  S. salar  primary macrophages. In addition, we determined that primary macrophages pre-treated with a biologically relevant concentration of vitamin D 3  for 24 h showed a decrease of  A. salmonicida  infection. In contrast, vitamin D 2  did not influence the antibacterial activity of the  S. salar  macrophages infected with  A. salmonicida . Vitamin D 2  and D 3  did not influence the expression of canonical genes related to innate immune response. On the other hand, we found that  A. salmonicida  up-regulated the expression of several canonical genes and suppressed the expression of  leukocyte-derived chemotaxin 2  ( lect-2 ) gene, involved in neutrophil recruitment. Primary macrophages pre-treated for 24 h with vitamin D 3  counteracted this immune suppression and up-regulated the transcription of  lect-2 . Our results suggest that vitamin D 3  affects  A. salmonicida  attachment to the  S. salar  primary macrophages, and as a consequence, the  A. salmonicida  invasion decreased. Moreover, our study shows that the positive effects of vitamin D 3  on fish cell immunity seem to be related to the  lect-2  innate immunity mechanisms. We did not identify positive effects of vitamin D 2  on fish cell immunity. In conclusion, we determined that the inactive form of vitamin D 3 , cholecalciferol, induced anti-bacterial innate immunity pathways in Atlantic salmon primary macrophages, suggesting that its utilization as a component of a healthy aquafeed diet in Atlantic salmon could enhance the immune response against  A. salmonicida .

frontiers in immunology

Effects of Vitamin D2 (Ergocalciferol) and D3 (Cholecalciferol) on Atlantic Salmon (Salmo salar) Primary Macrophage Immune Response to Aeromonas salmonicida subsp. salmonicida Infection