Escamilla A, Pérez-Caballero R, Zafra R, Bautista M J, Pacheco I L, Ruiz M T, Martínez-Cruz M S, Martínez-Moreno A, Molina-Hernández V, Pérez J
Department of Anatomy and Comparative Pathology, Faculty of Veterinary Medicine, University of Córdoba, Spain.
Department of Animal Health (Parasitology), Faculty of Veterinary Medicine, University of Córdoba, Spain.
Vet Parasitol. 2017 Apr 30;238:49-53. doi: 10.1016/j.vetpar.2017.03.015. Epub 2017 Mar 18.
Several immunomodulatory properties have been described in Fasciola hepatica infections. Apoptosis has been shown to be an effective mechanism to avoid the immune response in helminth infections. The aim of the present work was to study apoptosis in peritoneal leucocytes of sheep experimentally infected with F. hepatica during the early stages of infection. Five groups (n=5) of sheep were used. Groups 2-5 were orally infected with 200 metacercariae (mc) and sacrificed at 1, 3, 9 and 18days post-infection (dpi), respectively. Group 1 was used as the uninfected control (UC). Apoptosis was detected using three different methods 1) immunocytochemistry (ICC) with a polyclonal antibody anti-active caspase-3; 2) an annexin V flow cytometry assay using the Annexin V-FITC/propidium iodide (PI); and 3) transmission electron microscopy (TEM). The differential leucocyte count revealed that the majority of peritoneal granulocytes were eosinophils, which increased significantly at 9 and 18 dpi with respect to the uninfected controls. The ICC study revealed that the percentage of caspase-3 apoptotic peritoneal leucocytes increased significantly from 3 dpi onwards with respect to the uninfected controls. The flow cytometry annexin V assay detected a very significant (P<0.001) increase of apoptotic peritoneal macrophages, lymphocytes and granulocytes, which remained higher than in the UC until 18 dpi. Transmission electron microscopy studies also confirmed the presence of apoptosis in peritoneal eosinophils at 18 dpi. This is the first report of apoptosis induced by F. hepatica in the peritoneal leucocytes of sheep in vivo. The results of this work suggest the importance of apoptosis induction for the survival of the juvenile parasites in the peritoneal migratory stages of infection.
肝片吸虫感染具有多种免疫调节特性。凋亡已被证明是蠕虫感染中避免免疫反应的一种有效机制。本研究的目的是在感染早期，研究实验感染肝片吸虫的绵羊腹膜白细胞中的凋亡情况。使用了五组（每组n = 5）绵羊。第2 - 5组经口感染200个囊蚴，并分别在感染后1、3、9和18天处死。第1组用作未感染对照（UC）。使用三种不同方法检测凋亡：1）用抗活性半胱天冬酶-3多克隆抗体进行免疫细胞化学（ICC）；2）使用膜联蛋白V-FITC/碘化丙啶（PI）进行膜联蛋白V流式细胞术检测；3）透射电子显微镜（TEM）。白细胞分类计数显示，大多数腹膜粒细胞是嗜酸性粒细胞，与未感染对照相比，在感染后9天和18天显著增加。ICC研究显示，与未感染对照相比，从感染后3天起，半胱天冬酶-3凋亡腹膜白细胞的百分比显著增加。流式细胞术膜联蛋白V检测发现，凋亡的腹膜巨噬细胞、淋巴细胞和粒细胞非常显著（P<0.001）增加，直到感染后18天一直高于未感染对照。透射电子显微镜研究也证实了在感染后18天腹膜嗜酸性粒细胞中存在凋亡。这是肝片吸虫在绵羊体内诱导腹膜白细胞凋亡的首次报道。本研究结果表明，在感染的腹膜迁移阶段，诱导凋亡对幼虫寄生虫的存活具有重要意义。
