Cellular Crosstalk in Peripheral Blood is Critical for the Vitamin D Mediated Antimycobacterial Immune Response to BCG in Cattle.
Susana Flores-Villalva1,2,3, Stephen V. Gordon2 and Kieran G. Meade1,2.
1Animal & Bioscience Research Department, Animal & Grassland Research and Innovation Centre, Teagasc, Grange.
2UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
3CENID Fisiología, INIFAP. Querétaro, México.

Bovine tuberculosis is a significant economic burden for the global livestock industry, and it has a major impact on animal and human health in low- and mid-income countries. Thus, bovine tuberculosis control is a key example of the One Health approach that requires us to take a holistic view of human and animal health. As a further One Health link, the beneficial role of vitamin D for health and resistance against tuberculosis in humans is well established. However, although the metabolism of vitamin D in humans and cattle is similar, its role in susceptibility to bovine tuberculosis remains to be assessed. Therefore, the objective of this project is to assess the role of the vitamin D metabolite 1,25(OH)2D3 on the bovine innate immune response to Mycobacterium bovis using the vaccine strain BCG as a model. Blood from Holstein-Friesian bull calves (n=7) with low circulating levels of vitamin D (18.89 ± 2.027 ng/ml) was stimulated with 10 nM of 1,25(OH)2D3 for 2 hours, and then challenged with (1x106 CFU) BCG. Results showed that 1,25(OH)2D3 supplementation significantly reduced the growth of BCG by 0.48 log in comparison to 0.29 log in the non-supplemented peripheral blood. Then, to analyse the contribution of major cell types to mycobacterial growth control, depletion of monocytes, granulocytes, CD3+, CD4+, and CD8+ T lymphocytes was subsequently performed. Monocytes and lymphocytes were depleted from peripheral blood leukocytes (PBLs) by magnetic cell separation (MACS) and granulocyte depletion was performed by density gradient centrifugation. Each cell subset was stimulated with 1,25(OH)2D3 and challenged with BCG as before. Bacterial growth was assessed after 2 and 24 hours of infection, to analyse phagocytosis and killing of BCG. We found that the depletion of granulocytes had the greatest impact on BCG growth, leading to a significant enhancement of growth. Our results agree with previous reports in human where the depletion of CD4+ or CD8+ T cells individually or together (CD3+) had no impact on mycobacterial control. In concordance with our previous results, data showed that vitamin D stimulation significantly decreased BCG growth in PBLs but not when depleting any of the specific major cell subsets. Therefore, crosstalk between cells appears critical for the growth control promoted by 1,25(OH)2D3 in cattle. Finally, we observed that vitamin D significantly upregulated the gene expression of β-defensins (eDEFB4A, PAM defensin primer), CCL5, NOS2, IL-1β, IL17, DEFB10, thereby boosting the antimicrobial innate response in cattle. In conclusion, vitamin D metabolite 1,25(OH)2D3 improves antimycobacterial killing of bovine cells via the synergistic activity of peripheral blood cell populations and enhanced activation of innate immunity.

Scholarship to SFV is funded by CONACYT & INIFAP Mexico.

Susana Flores Villalva MSc

University College Dublin

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