Center of Scientifc Technology of Ningxia Medical University, Yinchuan, China.
Ningxia Key Laboratory of Prevention and Treatment of Common Infectious Diseases, Yinchuan, China.
BMC Vet Res. 2022 May 5;18(1):165. doi: 10.1186/s12917-022-03268-7.
Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune indicators, especially T-lymphocyte parameters, is necessary for research on sheep diseases and vaccines, to better understand the immune response to bacteria and viruses for reducing the use of antibiotics and improving the welfare of sheep. We randomly selected 36 sheep of similar ages to analyze cell-related immune indicators in peripheral blood mononuclear cells (PBMCs). The proportions of CD4 and CD8 T cells in PBMCs were detected by flow cytometry. We used Concanavalin A (Con A) and Phorbol-12-myristate-13-acetate (PMA)/Ionomycin to stimulate PBMCs, and measured the expression of IFN-γ, IL-4, and IL-17A using enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISpot). Simultaneously, PMA/Ionomycin/brefeldin A (BFA) was added to PBMCs, then the expression of IFN-γ, IL-4, and IL-17A was detected by flow cytometry after 4 h of culturing. In addition, we observed the proliferation of PBMCs stimulated with Con A for 3, 4, and 5 days.
The proportions of CD4 T lymphocytes (18.70 ± 4.21%) and CD8 T lymphocytes (8.70 ± 3.65%) were generally consistent among individuals, with a CD4/CD8 ratio of 2.40 ± 0.79. PBMCs produced high levels of IFN-γ, IL-4, and IL-17A after stimulation with PMA/Ionomycin and Con A. Furthermore, PMA/Ionomycin stimulation of PBMC yielded significantly higher cytokine levels than Con A stimulation. Flow cytometry showed that the level of IFN-γ (51.49 ± 11.54%) in CD8 T lymphocytes was significantly (p < 0.001) higher than that in CD4 T lymphocytes (14.29 ± 3.26%); IL-4 (16.13 ± 6.81%) in CD4 T lymphocytes was significantly (p < 0.001) higher than that in CD8 T lymphocytes (1.84 ± 1.33%), There was no difference in IL-17A between CD4 (2.83 ± 0.98%) and CD8 T lymphocytes (1.34 ± 0.67%). The proliferation of total lymphocytes, CD4 T lymphocytes, and CD8 T lymphocytes continued to increase between days 3 and 5; however, there were no significant differences in proliferation between the cell types during the stimulation period.
Evaluating primary sheep immune indicators, especially T lymphocytes, is significant for studying cellular immunity. This study provided valuable data and theoretical support for assessing the immune response of sheep to pathogens and improving sheep welfare.
绵羊是世界范围内重要的家畜物种，也是畜牧业和兽医研究中必不可少的大型动物模型。了解基本的免疫指标，尤其是 T 淋巴细胞参数，对于研究绵羊疾病和疫苗至关重要，这有助于更好地了解对细菌和病毒的免疫反应，以减少抗生素的使用并提高绵羊的福利。我们随机选择了 36 只年龄相近的绵羊，分析外周血单个核细胞（PBMC）中的细胞相关免疫指标。通过流式细胞术检测 PBMC 中 CD4 和 CD8 T 细胞的比例。我们使用刀豆蛋白 A（Con A）和佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）/离子霉素刺激 PBMC，并使用酶联免疫吸附试验（ELISA）和酶联免疫斑点试验（ELISpot）测量 IFN-γ、IL-4 和 IL-17A 的表达。同时，向 PBMC 中加入 PMA/离子霉素/布雷菲德菌素 A（BFA），然后在培养 4 小时后通过流式细胞术检测 IFN-γ、IL-4 和 IL-17A 的表达。此外，我们观察了 Con A 刺激 3、4 和 5 天后 PBMC 的增殖情况。
CD4 T 淋巴细胞（18.70±4.21%）和 CD8 T 淋巴细胞（8.70±3.65%）在个体之间的比例大致一致，CD4/CD8 比值为 2.40±0.79。PBMC 在 PMA/离子霉素和 Con A 刺激后产生高水平的 IFN-γ、IL-4 和 IL-17A。此外，PMA/离子霉素刺激 PBMC 产生的细胞因子水平明显高于 Con A 刺激。流式细胞术显示，CD8 T 淋巴细胞中的 IFN-γ（51.49±11.54%）水平明显（p<0.001）高于 CD4 T 淋巴细胞（14.29±3.26%）；CD4 T 淋巴细胞中的 IL-4（16.13±6.81%）明显（p<0.001）高于 CD8 T 淋巴细胞（1.84±1.33%）；CD4（2.83±0.98%）和 CD8 T 淋巴细胞（1.34±0.67%）之间的 IL-17A 无差异。总淋巴细胞、CD4 T 淋巴细胞和 CD8 T 淋巴细胞的增殖在第 3 天至第 5 天持续增加；然而，在刺激期间细胞类型之间的增殖没有显著差异。
评估绵羊的初级免疫指标，尤其是 T 淋巴细胞，对于研究细胞免疫具有重要意义。本研究为评估绵羊对病原体的免疫反应和提高绵羊福利提供了有价值的数据和理论支持。
