Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, 110866, China.
The Research Unit for Pathogenic Mechanisms of Zoonotic Parasites, Chinese Academy of Medical Sciences, 120 Dongling Road, Shenyang, 110866, China.
Parasit Vectors. 2019 Nov 11;12(1):534. doi: 10.1186/s13071-019-3788-x.
Malaria, one of the largest health burdens worldwide, is caused by Plasmodium spp. infection. Upon infection, the host's immune system begins to clear the parasites. However, Plasmodium species have evolved to escape the host's immune clearance. T-cell immunoglobulin and mucin domain 3 (Tim-3), a surface molecule on most immune cells, is often referred to as an exhaustion marker. Galectin (Gal)-9 is a Tim-3 ligand and the T helper (Th) 1 cell response is inhibited when Gal-9 binds to Tim-3. In the present study, dynamic expression of Tim-3 on key populations of lymphocytes during infection periods of Plasmodium berghei and its significance in disease resistance and pathogenesis were explored.
Tim-3 expression on critical lymphocyte populations and the proportion of these cells, as well as the levels of cytokines in the sera of infected mice, were detected by flow cytometry. Further, in vitro anti-Tim-3 assay using an anti-Tim-3 antibody and in vivo Tim-3-Gal-9 signaling blockade assays using α-lactose (an antagonist of Gal-9) were conducted. An Annexin V Apoptosis Detection Kit with propidium iodide was used to detect apoptosis. In addition, proteins associated with apoptosis in lung and spleen tissues were confirmed by Western blotting assays.
Increased Tim-3 expression on splenic CD8 and splenic CD4, and circulatory CD4 T cells was associated with a reduction in the proportion of these cells. Furthermore, the levels of interleukin (IL)-2, IL-4, IL-6, IL-22, and interferon (IFN)-γ, but not that of tumor necrosis factor alpha (TNF-α), IL-10, and IL-9, increased to their highest levels at day 4 post-infection and decreased thereafter. Blocking Tim-3 signaling in vitro inhibited lymphocyte apoptosis. Tim-3-Gal-9 signaling blockade in vivo did not protect the mice, but induced the expression of the immunosuppressive molecule, T cell immunoreceptor with Ig and ITIM domains (TIGIT), in Plasmodium berghei ANKA-infected mice.
Tim-3 on lymphocytes negatively regulates cell-mediated immunity against Plasmodium infection, and blocking Tim-3-galectin 9 signaling using α-lactose did not significantly protect the mice; however, it induced the compensatory expression of TIGIT. Further investigations are required to identify whether combined blockade of Tim-3 and TIGIT signaling could achieve a better protective effect.
疟疾是全球最大的健康负担之一，由疟原虫感染引起。感染后，宿主的免疫系统开始清除寄生虫。然而，疟原虫已经进化到可以逃避宿主的免疫清除。T 细胞免疫球蛋白和粘蛋白结构域 3（Tim-3）是大多数免疫细胞表面的分子，通常被称为衰竭标记物。半乳糖凝集素（Gal）-9 是 Tim-3 的配体，当 Gal-9 与 Tim-3 结合时，辅助性 T 细胞（Th）1 细胞反应受到抑制。本研究探索了伯氏疟原虫感染期间关键淋巴细胞群上 Tim-3 的动态表达及其在抗病性和发病机制中的意义。
通过流式细胞术检测感染期小鼠关键淋巴细胞群及其细胞比例上的 Tim-3 表达以及血清细胞因子水平。此外，通过抗 Tim-3 抗体进行体外抗 Tim-3 测定，并用α-乳糖（Gal-9 的拮抗剂）进行体内 Tim-3-Gal-9 信号阻断测定。用碘化丙啶结合的 Annexin V 凋亡检测试剂盒检测凋亡。此外，通过 Western blot 测定法证实肺和脾组织中与凋亡相关的蛋白质。
脾 CD8 和脾 CD4 以及循环 CD4 T 细胞上的 Tim-3 表达增加与这些细胞比例的降低有关。此外，白细胞介素（IL）-2、IL-4、IL-6、IL-22 和干扰素（IFN）-γ的水平增加到感染后第 4 天的最高水平，然后下降，但肿瘤坏死因子-α（TNF-α）、IL-10 和 IL-9 的水平没有增加。体外阻断 Tim-3 信号抑制淋巴细胞凋亡。体内阻断 Tim-3-Gal-9 信号不仅没有保护小鼠，反而在伯氏疟原虫 ANKA 感染的小鼠中诱导了免疫抑制分子 T 细胞免疫受体 Ig 和 ITIM 结构域（TIGIT）的表达。
淋巴细胞上的 Tim-3 负调节针对疟原虫感染的细胞介导免疫，用α-乳糖阻断 Tim-3-半乳糖凝集素 9 信号不会显著保护小鼠；然而，它诱导了 TIGIT 的代偿性表达。需要进一步研究以确定是否联合阻断 Tim-3 和 TIGIT 信号可以达到更好的保护效果。
