人们越来越认识到维生素D对健康和抵抗传染病的有益作用。包括肺结核。然而,研究主要集中在鼠和人类物种上,牛的功能数据有限。因此,这项研究的目的是使用功能测定和基因表达谱分析的组合,评估维生素D代谢物1,25(OH)2D3对牛外周血白细胞(PBL)的杀菌活性和免疫调节作用。用1,25(OH)2D3刺激25(OH)D循环水平低的荷斯坦-弗里斯牛的血液2小时,然后感染了牛分枝杆菌卡介苗.结果表明,补充1,25(OH)2D3可显著提高卡介苗的平均死亡率16%,虽然反应在1%到38%之间变化。然后在1,25(OH)2D3孵育之前对PBL进行系列细胞亚群的消耗,并如前所述感染BCG,以分析主要细胞类型对分枝杆菌生长控制的贡献。特异性抗体和磁性细胞分离或单核细胞密度梯度离心,粒细胞,CD3+,CD4+,和CD8+T淋巴细胞用于捕获每个细胞亚群。结果表明,粒细胞耗竭对BCG生长的影响最大,导致细菌菌落的显著增加。相比之下,单独消耗CD4+或CD8+T细胞,或组合(CD3+),对分枝杆菌生长控制没有影响。与我们之前的数据一致,1,25(OH)2D3显着增加PBL中的细菌杀灭率,在单核细胞耗尽的样本中,在粒细胞耗竭亚群中观察到类似的趋势。此外,用1,25(OH)2D3处理的分选的嗜中性粒细胞的特定分析显示,对BCG和牛分枝杆菌的强毒株的杀微生物活性均增强。最后,数据显示,1,25(OH)2D3刺激增加了活性氧(ROS)的产生和编码宿主防御肽(HDP)和病原体识别受体(PRR)的基因的表达,在对分枝杆菌的杀微生物活性中起重要作用的因素。总之,维生素D代谢产物1,25(OH)2D3通过单核细胞和粒细胞的协同活性和增强的先天免疫激活来改善牛PBLs中的抗分枝杆菌杀伤。
A growing appreciation is emerging of the beneficial role of vitamin D for health and resistance against infectious diseases, including tuberculosis. However, research has predominantly focused on murine and human species and functional data in bovines is limited. Therefore, the objective of this study was to assess the microbicidal activity and immunoregulatory effect of the vitamin D metabolite 1,25(OH)2D3 on bovine peripheral blood leukocytes (PBL) in response to Mycobacterium bovis BCG (BCG) infection using a combination of functional assays and gene expression profiling. Blood from Holstein-Friesian bull calves with low circulating levels of 25(OH)D was stimulated with 1,25(OH)2D3 for 2 h, and then infected with M. bovis BCG. Results showed that 1,25(OH)2D3 supplementation significantly increased BCG killing by on average 16 %, although responses varied between 1 % and 38 % killing. Serial cell subset depletion was then performed on PBL prior to 1,25(OH)2D3 incubation and BCG infected as before to analyse the contribution of major cell types to mycobacterial growth control. Specific antibodies and either magnetic cell separation or density gradient centrifugation of monocytes, granulocytes, CD3+, CD4+, and CD8+ T lymphocytes were used to capture each cell subset. Results showed that depletion of granulocytes had the greatest impact on BCG growth, leading to a significant enhancement of bacterial colonies. In contrast, depletion of CD4+ or CD8+ T cells individually, or in combination (CD3+), had no impact on mycobacterial growth control. In agreement with our previous data, 1,25(OH)2D3 significantly increased bacterial killing in PBL, in monocyte depleted samples, and a similar trend was observed in the granulocyte depleted subset. In addition, specific analysis of sorted neutrophils treated with 1,25(OH)2D3 showed an enhanced microbicidal activity against both BCG and a virulent strain of M. bovis. Lastly, data showed that 1,25(OH)2D3 stimulation increased reactive oxygen species (ROS) production and the expression of genes encoding host defence peptides (HDP) and pathogen recognition receptors (PRRs), factors that play an important role in the microbicidal activity against mycobacteria. In conclusion, the vitamin D metabolite 1,25(OH)2D3 improves antimycobacterial killing in bovine PBLs via the synergistic activity of monocytes and granulocytes and enhanced activation of innate immunity.