State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, Gansu, China.
Department of Parasitology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Bahawalpur, 63100, Punjab, Pakistan.
Parasit Vectors. 2021 May 22;14(1):276. doi: 10.1186/s13071-021-04784-5.
The liver fluke Fasciola gigantica secretes excretory-secretory proteins during infection to mediate its interaction with the host. In this study, we investigated the immunomodulatory effects of a recombinant tegumental calcium-binding EF-hand protein 4 of F. gigantica (rFg-CaBP4) on goat monocytes.
The rFg-CaBP4 protein was induced and purified by affinity chromatography. The immunogenic reaction of rFg-CaBP4 against specific antibodies was detected through western blot analysis. The binding of rFg-CaBP4 on surface of goat monocytes was visualized by immunofluorescence assay. The localization of CaBP4 within adult fluke structure was detected by immunohistochemical analysis. The cytokine transcription levels in response to rFg-CaBP4 were examined using ABI 7500 real-time PCR system. The expression of the major histocompatibility complex (MHC) class-II molecule (MHC-II) in response to rFg-CaBP4 protein was analyzed using Flow cytometry.
The isopropyl-ß-D-thiogalactopyranoside-induced rFg-CaBP4 protein reacted with rat sera containing anti-rFg-CaBP4 polyclonal antibodies in a western blot analysis. The adhesion of rFg-CaBP4 to monocytes was visualized by immunofluorescence and laser scanning confocal microscopy. Immunohistochemical analysis localized native CaBP4 to the oral sucker, pharynx, genital pore, acetabulum and tegument of adult F. gigantica. Co-incubation of rFg-CaBP4 with concanavalin A-stimulated monocytes increased the transcription levels of interleukin (IL)-2, IL-4, interferon gamma and transforming growth factor-β. However, a reduction in the expression of IL-10 and no change in the expression of tumor necrosis factor-α were detected. Additionally, rFg-CaBP4-treated monocytes exhibited a marked increase in the expression of the major histocompatibility complex (MHC) class-II molecule (MHC-II) and a decrease in MHC-I expression, in a dose-dependent manner.
These findings provide additional evidence that calcium-binding EF-hand proteins play roles in host-parasite interaction. Further characterization of the immunomodulatory role of rFg-CaBP4 should expand our understanding of the strategies used by F. gigantica to evade the host immune responses.
肝片形吸虫 Fasciola gigantica 在感染过程中分泌排泄分泌蛋白，以介导其与宿主的相互作用。在这项研究中，我们研究了重组肝片形吸虫表皮钙结合 EF 手蛋白 4（rFg-CaBP4）对山羊单核细胞的免疫调节作用。
通过亲和层析诱导和纯化 rFg-CaBP4 蛋白。通过 Western blot 分析检测 rFg-CaBP4 对特异性抗体的免疫反应。通过免疫荧光分析观察 rFg-CaBP4 在山羊单核细胞表面的结合。通过免疫组织化学分析检测 CaBP4 在成虫结构内的定位。使用 ABI 7500 实时 PCR 系统检测 rFg-CaBP4 诱导的细胞因子转录水平。使用流式细胞术分析 rFg-CaBP4 蛋白诱导的主要组织相容性复合体（MHC）II 类分子（MHC-II）的表达。
异丙基-β-D-硫代半乳糖吡喃糖苷诱导的 rFg-CaBP4 蛋白在 Western blot 分析中与含有抗 rFg-CaBP4 多克隆抗体的大鼠血清反应。通过免疫荧光和激光扫描共聚焦显微镜观察 rFg-CaBP4 与单核细胞的粘附。免疫组织化学分析将天然 CaBP4 定位到成虫的口吸盘、咽、生殖孔、髋臼和表皮。rFg-CaBP4 与刀豆球蛋白 A 刺激的单核细胞共孵育可增加白细胞介素（IL）-2、IL-4、干扰素γ和转化生长因子-β的转录水平。然而，检测到 IL-10 的表达减少和肿瘤坏死因子-α的表达没有变化。此外，rFg-CaBP4 处理的单核细胞以剂量依赖性方式表现出主要组织相容性复合体（MHC）II 类分子（MHC-II）表达的显著增加和 MHC-I 表达的降低。
这些发现提供了额外的证据，表明钙结合 EF 手蛋白在宿主-寄生虫相互作用中发挥作用。进一步表征 rFg-CaBP4 的免疫调节作用应扩大我们对肝片形吸虫逃避宿主免疫反应所使用策略的理解。
