The effects of vitamin D on the cellular responses, molecular immunity, and mycobacterial killing in cattle

The positive effect of vitamin D (vit D) on health and resistance against infectious diseases, particularly tuberculosis (TB) is well recognized. However, research has predominantly focused on murine and human species and functional data in bovines is limited. The aims of this project were: chapter 2&3) To investigate the effect of 1,25(OH)2D3 on the microbicidal and immunoregulatory activities in peripheral blood leukocytes (PBL) and on neutrophils. Chapter 4) To develop a model to drive divergent 25(OH)D circulating levels in dairy calves, and Chapter 5) To investigate the influence of differential 25(OH)D circulating levels on the microbicidal and immunoregulatory activities of PBL following and ex-vivo BCG challenge. Results from chapter 2 showed that 1,25(OH)2D3 supplementation significantly increased BCG killing on PBL to 65.7% compared to 49.1% in the untreated blood. Serial cell subset depletion showed that depletion of granulocytes had the greatest impact on BCG growth and lead to a significant enhancement of bacterial colonies. Significantly enhanced bacterial killing was observed in CD14neg PBLs treated with 1,25(OH)2D3 and a similar trend was observed in Granneg subsets (P = 0.06). In contrast, depletion of CD4+ or CD8+ T cells individually or in combination (CD3+) had no impact on mycobacterial control. Data also showed that 1,25(OH)2D3 stimulation increased reactive oxygen species (ROS) production and decreased overall BCG growth in PBLs. 1,25(OH)2D3 increased the expression of a cluster of genes including DEFB7, TAP, ELANE, CCL2, CXCL10, IFNB, IL33 and PKR implicated in activation of the innate immune response to mycobacteria. Results from chapter 3 confirmed the microbicidal effects of 1,25(OH)2D3 on neutrophils showing an enhanced killing against BCG and M. bovis. Then, in chapter 4 calves were supplemented with vit D3 from birth to 7 months age. Two control groups (Ctl-In, Ctl-Out) received a diet containing 6,000 IU/Kg of vit D3 in milk replacer and 2,000 IU/Kg in rations, and two groups (VitD-In, VitD-Out) were fed with 10,000 IU/Kg of vit D3 in milk replacer and 4,000 IU/Kg in rations. After weaning, Ctl-Out and VitD-Out groups were moved outdoors, whereas Ctl-In and VitD-In groups were kept indoors. Results showed a high incidence of vitamin D deficiency (VDD) at birth with mean 25(OH)D of 7.64 ±3.206. Significant elevated 25(OH)D concentrations were observed after weaning, with the maximal 25(OH)D level achieved in VitD-Out reaching 60.86±7.318. Greatest divergence in haematology profile was observed between Ctl-In and VitD-In groups, with Ctl-In calves showing an elevated count of neutrophils, eosinophils, and basophils associated with reduced 25(OH)D concentrations. Neither IL-8 nor ROS production in serum were significantly different between calves. In chapter 5 the assessment of the microbicidal activity and immunoregulatory effect to an ex-vivo BCG challenge was performed on Ctl-In and VitD-In groups. Results showed higher bacterial killing in the VitD-In group (48.0%) in contrast to Ctl-In group (29.5%). Besides, increased production of ROS and NO was detected in VitD-In calves. Whereas serum concentrations of IL-1ß and IL-8 were significantly lower. Likewise, a significant downregulation of a cluster of genes including IL1B, IL1R1, TNFA, CXCL1, CXCL2, CXCL5, CXCL8 was detected in VitD-in calves relative to Ctl-In animals. Collectively, results showed that 1,25(OH)2D3 improved mycobacterial killing in bovine PBLs via the synergistic activity of monocytes and granulocytes and enhanced activation of innate immunity. Evidence indicates that vit D3 supplementation boosts antimicrobial and innate immune responses but modulates excessive inflammation which may be detrimental for the outcome to infection. Finally, results identified neutrophils as one of the principal targets of vit D modulatory effects