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

Abstract

Vitamin D₂ (ergocalciferol) and vitamin D₃ (cholecalciferol) are fat-soluble secosteroid hormones obtained from plant and animal sources, respectively. Fish incorporates vitamin D₂ and D₃ 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 D₂ and vitamin D₃ on Salmo salar primary macrophage immune response to A. salmonicida subspecies salmonicida infection under in vitro conditions. We determined that high concentrations of vitamin D₂ (100,000 ng/ml) and D₃ (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 D₃ for 24 h showed a decrease of A. salmonicida infection. In contrast, vitamin D₂ did not influence the antibacterial activity of the S. salar macrophages infected with A. salmonicida. Vitamin D₂ and D₃ 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 D₃ counteracted this immune suppression and up-regulated the transcription of lect-2. Our results suggest that vitamin D₃ 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 D₃ on fish cell immunity seem to be related to the lect-2 innate immunity mechanisms. We did not identify positive effects of vitamin D₂ on fish cell immunity. In conclusion, we determined that the inactive form of vitamin D₃, 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.

Keywords: Aeromonas salmonicida; Atlantic salmon; Gram-negative; innate immunity; primary macrophages; vitamin D2; vitamin D3.

Figure 1

Aeromonas salmonicida subsp. salmonicida growth curve in TSB media supplemented with (A) 10, 100, 1,000, 10,000, and 100,000 ng ml⁻¹ of vitamin D₂ and (B) 10, 100, 1,000, and 10,000 ng ml⁻¹of vitamin D₃. Growth was determined by reading O.D. 600 nm at different time points until 48 h. Each value is the mean ± S.E.M (n = 3). Symbols (*, +) indicate differences between each group at different time points of measure, p < 0.05.

Figure 2

Atlantic salmon primary macrophages treated with vitamin D₂ or D₃. (A) Live cells and (B) percentage of viability of primary macrophages treated with 10, 100, 1,000, 10,000, and 100,000 of vitamin D₂, were measured after 24 and 48 h of treatment. (C) Live cells and (D) percentage of viability of primary macrophages treated with 10, 100, 1,000, and 10,000 ng ml⁻¹ of vitamin D₃ were measured after 24 and 48 h of treatment. Each value represents the mean ± S.E.M (n = 6). Lower case letters (a, b) show differences between treatments after 24 h. Upper case letters (A,B,C) show differences between treatments after 48 h. Asterisks (*) represent significant differences between treatments (*p < 0.05, **p < 0.01).

Figure 3

Gentamicin exclusion assay in Atlantic salmon primary macrophages pre-treated for 24 h with either control or 100 ng ml⁻¹ of vitamin D₂ or vitamin D₃, and then infected with Aeromonas salmonicida subsp. salmonicida. Live cells of primary macrophages pre-treated with vitamin D₂ (A), or vitamin D₃ (E); percentage of viability of primary macrophages pre-treated with vitamin D₂ (B), or vitamin D₃ (F), Colony forming unit (CFU) of A. salmonicida in Atlantic salmon primary macrophages pre-treated with vitamin D₂ (C), or vitamin D₃ (G); and percentage of attachment and invasion of A. salmonicida in Atlantic salmon primary macrophages pre-treated with vitamin D₂ (D), or vitamin D₃ (H), were measured 1, 2, 3, and 4 h post-infection. Initial A. salmonicida inoculum calculated in TSA Congo red plates are shown above the CFU figures. Each value represents the mean ± S.E.M (n = 6). Asterisks (*) represent significant differences between treatments on each time-point (**p < 0.01, ***p < 0.001). Lower case letters (a, b) show differences in the control at different time points post-infection. Upper case letters (A, B) show differences in vitamin D₂ or D₃ pre-treated cells in different time points post-infection.

Figure 4

Gene expression of (A,F) Interleukin 1b (il-1b), (B,G) Interleukin 8 (il-8), (C,H) Tumor necrosis factor alpha (tnf-α), (D,I) Leukocyte-derived chemotaxin 2 (lect-2), and (E,J) soluble toll-like receptor 5 (stlr5) in Atlantic salmon primary macrophages isolated from head kidney pre-treated 24 h with either the control, vitamin D₂ (100 and 1,000 ng ml⁻¹) or vitamin D₃ (100 and 1,000 ng ml⁻¹), and then inoculated with PBS (control) or infected with live (J223) or exposed to formalin-killed A. salmonicida for 3 h. Relative expression was calculated using the 2^(−ΔΔCt) method and Log₂ converted using β-actin and 60S ribosomal protein L32 (rpl32) as internal reference genes. Each value is the mean ± S.E.M (n = 6). Different letters represent significant differences between treatments, p < 0.05.