Microscopical Techniques to Analyze the Hepatic and Peritoneal Changes Caused by Fasciola hepatica Infection.

The helminth parasite Fasciola hepatica modulates the host immune response at early stages of infection (Rodríguez et al., PLoS Negl Trop Dis 9:e0004234, 2015; Vukman et al., J Immunol 190:2873-2879, 2013). Nevertheless, little is known about the cell composition of the peritoneal fluid at these early stages of infection.In this chapter, we describe a method to perform peritoneal lavages and to recover peritoneal fluid from sheep experimentally infected and noninfected with F. hepatica at early stages of infection. In addition, with the aim to characterize the peritoneal fluid immune cell phenotype, we describe a procedure to obtain the total leukocyte count, the differential leukocyte count and the preparation and storage of peritoneal fluid smears, together with the application of an immunocytochemical technique and an automatic method to count the immunoreactive cells. Finally, the present protocol describes the evaluation of the gross and the histopathological lesions together with the immunohistochemical analysis of the hepatic tissue.

Study of peritoneal macrophage immunophenotype in sheep experimentally infected with Fasciola hepatica.

During Fasciola hepatica infection, the parasite has the capability to modulate the host immune response towards a non-protector Th2 type instead of Th1. This type of immune response is closely related to the alternative activation of macrophages (M2 profile) as has been shown in vivo in murine models. In this study, an experiment was carried out in order to evaluate the expression of CD68, CD14, CD206 and iNOS in cells present in the peritoneal fluid of sheep during early stages of infection with F. hepatica (1, 3, 9 and 18 days post-infection, dpi) by immunocytochemistry. To the authors' knowledge, this is the first report that studies the in vivo immunophenotype of macrophages from the peritoneal fluid of sheep infected with F. hepatica. Throughout the experiments the absolute number of leucocytes progressively increased, reaching its highest value at 18 dpi, mainly due to the increase of eosinophils. This immunocytochemical study had two purposes: 1) CD68 expression was assessed with Hansel counterstaining, to optimally identify peritoneal macrophages, eosinophils and lymphocytes; 2) expression of CD14, CD206 and iNOS was evaluated to identify alternative or classical pathways of macrophage activation. The results showed a significant increase in CD14 from day 3 dpi compared with the non-infected group. CD206 expression at all time-points showed a significant and dramatic increase in comparison with the uninfected group. On the other hand, iNOS expression showed little variation, and was significantly decreased at 18 dpi in comparison with the uninfected group. These results suggest that F. hepatica induces an alternative activation of peritoneal macrophages of sheep from the first day post-infection, which may facilitate parasite survival. This is the first report describing M2 activation of peritoneal macrophages in ruminants infected with F. hepatica.

Apoptosis of peritoneal leucocytes during early stages of Fasciola hepatica infections in sheep.

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.

Distribution of Foxp3+ T cells in the liver and hepatic lymph nodes of goats and sheep experimentally infected with Fasciola hepatica.

Foxp3 regulatory T cells (Tregs) are now considered to play a key role in modulation of immune responses during parasitic helminth infections. Immunomodulation is a key factor in Fasciola hepatica infection; however, the distribution and role of Foxp3+ Tregs cells have not been investigated in F. hepatica infected ruminants. The aim of this study was to evaluate the presence of Foxp3+ Tregs in the liver and hepatic lymph nodes from experimentally infected sheep and goats during acute and chronic stages of infection. Three groups of goats (n=6) and three groups of sheep (n=6) were used in this study. Goats in groups 1-2 and sheep in groups 4-5 were orally infected with metacercarie of ovine origin. Groups 1 and 4 were killed during the acute stage of the infection, at nine days post infection (dpi); groups 2 and 5 were killed during the chronic stage, at 15 and19 weeks post infection respectively (wpi). Groups 3 (goats) and 6 (sheep) were left as uninfected controls. Fluke burdens and liver damage were assessed and the avidin-biotin-complex method was used for the immunohistochemical study. At nine dpi in acute hepatic lesions, the number of both Foxp3+ and CD3+ T lymphocytes increased significantly in goats and sheep. In the chronic stages of infection (15-19wpi), the number of Foxp3+ and CD3+ T lymphocytes were also significantly increased with respect to control livers, particularly in portal spaces with severely enlarged bile ducts (response to adult flukes) while the increase was lower in granulomas, chronic tracts and smaller portal spaces (response to tissue damage). Foxp3+ Tregs were increased in the cortex of hepatic lymph nodes of sheep (chronic infection) and goats (acute and chronic infection). The estimated proportion of T cells which were Foxp3+ was significantly increased in the large bile ducts and hepatic lymph node cortex of chronically infected goats but not sheep. This first report of the expansion of Foxp3+ Tregs in acute and chronic hepatic lesions in ruminants suggests that these cells may be involved in both parasite survival and modulation of hepatic damage. Future studies should be focused on the investigation of parasite molecules and cytokines involved in this process.

Fasciola hepatica induces eosinophil apoptosis in the migratory and biliary stages of infection in sheep.

The aim of the present work was to evaluate the number of apoptotic eosinophils in the livers of sheep experimentally infected with Fasciola hepatica during the migratory and biliary stages of infection. Four groups (n=5) of sheep were used; groups 1-3 were orally infected with 200 metacercariae (mc) and sacrificed at 8 and 28 days post-infection (dpi), and 17 weeks post-infection (wpi), respectively. Group 4 was used as an uninfected control. Apoptosis was detected using immunohistochemistry with a polyclonal antibody against anti-active caspase-3, and transmission electron microscopy (TEM). Eosinophils were identified using the Hansel stain in serial sections for caspase-3, and by ultrastructural features using TEM. At 8 and 28 dpi, numerous caspase-3(+) eosinophils were mainly found at the periphery of acute hepatic necrotic foci. The percentage of caspase -3(+) apoptotic eosinophils in the periphery of necrotic foci was high (46.1-53.9) at 8 and 28 dpi, respectively, and decreased in granulomas found at 28 dpi (6%). Transmission electron microscopy confirmed the presence of apoptotic eosinophils in hepatic lesions at 8 and 28 dpi. At 17 wpi, apoptotic eosinophils were detected in the infiltrate surrounding some enlarged bile ducts containing adult flukes. This is the first report of apoptosis induced by F. hepatica in sheep and the first study reporting apoptosis in eosinophils in hepatic inflammatory infiltrates in vivo. The high number of apoptotic eosinophils in acute necrotic tracts during the migratory and biliary stages of infection suggests that eosinophil apoptosis may play a role in F. hepatica survival during different stages of infection.