Flores-Villalva Susana, Remot Aude, Carreras Florence, Winter Nathalie, Gordon Stephen V, Meade Kieran G
School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland; CENID Fisiología, INIFAP, Querétaro, Mexico.
INRAE, Université de Tours, ISP, F-37380 Nouzilly, France.
Vet Immunol Immunopathol. 2023 Feb;256:110536. doi: 10.1016/j.vetimm.2022.110536. Epub 2022 Dec 16.
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)D 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)D for 2 h, and then infected with M. bovis BCG. Results showed that 1,25(OH)D 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)D 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)D 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)D showed an enhanced microbicidal activity against both BCG and a virulent strain of M. bovis. Lastly, data showed that 1,25(OH)D 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)D improves antimycobacterial killing in bovine PBLs via the synergistic activity of monocytes and granulocytes and enhanced activation of innate immunity.
人们越来越认识到维生素D对健康以及抵抗包括结核病在内的传染病具有有益作用。然而，研究主要集中在小鼠和人类物种上，关于牛的功能数据有限。因此，本研究的目的是使用功能测定和基因表达谱分析相结合的方法，评估维生素D代谢物1,25(OH)D对牛外周血白细胞（PBL）在感染牛分枝杆菌卡介苗（BCG）时的杀菌活性和免疫调节作用。对25(OH)D循环水平较低的荷斯坦 - 弗里生公牛犊的血液用1,25(OH)D刺激2小时，然后感染牛分枝杆菌卡介苗。结果表明，补充1,25(OH)D可使卡介苗的杀灭率平均显著提高16%，尽管杀灭率在1%至38%之间有所不同。然后在1,25(OH)D孵育之前对PBL进行连续细胞亚群清除，并像之前一样感染卡介苗，以分析主要细胞类型对分枝杆菌生长控制的贡献。使用特异性抗体以及单核细胞、粒细胞、CD3 +、CD4 +和CD8 + T淋巴细胞的磁性细胞分离或密度梯度离心来捕获每个细胞亚群。结果表明，粒细胞的清除对卡介苗生长影响最大，导致细菌菌落显著增加。相比之下，单独或联合清除CD4或CD8 T细胞（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)D显著增加了PBL、单核细胞清除样本中的细菌杀灭率，并且在粒细胞清除亚群中也观察到了类似趋势。此外，对用1,25(OH)D处理的分选中性粒细胞的特异性分析表明，其对卡介苗和强毒牛分枝杆菌菌株的杀菌活性增强。最后，数据表明1,25(OH)D刺激增加了活性氧（ROS）的产生以及编码宿主防御肽（HDP）和病原体识别受体（PRR）的基因表达，这些因素在抗分枝杆菌的杀菌活性中起重要作用。总之，维生素D代谢物1,25(OH)D通过单核细胞和粒细胞的协同活性以及增强先天免疫激活，改善了牛PBL中的抗分枝杆菌杀灭作用。
