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.This work was supported by EU grants (H2020-635408-PARAGONE) and the Spanish Ministry of Science grant AGL2015-67023-C2-1-R. TM receives funding from the Scottish Government.Accepted manuscriptVeterinari

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.This work was supported by EU grants (FPVII-265862-PARAVAC, H2020-635408-PARAGONE) and the Spanish Ministry of Science grant AGL2009-08726. TEM studies were carried out by the Central Services for Research of the University of Córdoba (SCAI)Veterinari

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 18 days 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.This work was supported by EU grants (H2020-635408-PARAGONE) and the Spanish Ministry of Science grant AGL2015-67023-C2-1-R. The TEM studies were carried out by the Central Research Services (SCAI) of the University of CórdobaAccepted manuscriptVeterinari

Comparative dynamics of peritoneal cell immunophenotypes in sheep during the early and late stages of the infection with Fasciola hepatica by flow cytometric analysis

Background: The peritoneal cell populations (PCP) are thought to play a crucial role during the early immune response in Fasciola hepatica infection while newly excysted juveniles (NEJ) are migrating in the peritoneal cavity (PC) towards the liver. In this study, we aimed to determine the immunophenotypes of the PCP and to analyse the dynamics of the recruitment of the PCP during the early and late stage of the infection in sheep infected with F. hepatica. Methods: Thirty-seven sheep were divided into three groups: Group 1 (n = 20) and 2 (n = 10) were challenged with F. hepatica, Group 3 (n = 7) was not infected and remained as uninfected control (UC). After the slaughtering, peritoneal lavages were carried out to isolate peritoneal cell populations at 1, 3, 9 and 18 days post-infection (dpi) for Group 1 and at 14 weeks post-infection (wpi) for Group 2 and 3. Flow cytometry was conducted to assess the dynamics of peritoneal cavity cell populations. Results: TCD4 cells showed a significant decrease at 1 and 18 dpi when compared to UC; no statistical differences were detected for TCD8 and WC1+ γδ during the early stage of the infection with respect to the UC. CD14 cells exhibited a decreasing trend, with a significant decrease at 9 and 18 dpi when compared to the UC. The dynamics of MHCII and CD83 cells showed a similar increasing pattern from 3 to 18 dpi. During the chronic stage, both TCD4 and TCD8 cells showed no significant differences when compared to the UC, although a slight but statistically significant higher level of WC1+ γδ cells was observed. A lower percentage of antigen-presenting cells (APCs) was detected with respect to the UC. Conclusions: The recruitment of the lymphocytes subsets did not show a significant increase during the course of the infection and only WC1+ γδ cells displayed a significant increase at the chronic stage. For the CD14, a decreasing trend was observed during the early stage, which was statistically significant at the chronic stage of the infection. Peritoneal CD83 and MHCII cells developed an increasing trend during the early stage of infection, and showed a significant decrease at the late stage of the infection.This study was funded by the European Union Grant H2020-635408- PARAGONE and by National Grant AGL2015-67023-C2-1-R. RPC was supported by an FPU grant of the Spanish Ministry of Education, Culture and Sport. Funding bodies were neither involved in the design of the study nor in analysis and interpretation of the dataVeterinari

Th1/Th2 balance in the liver and hepatic lymph nodes of vaccinated and unvaccinated sheep during acute stages of infection with Fasciola hepatica

The expression of IFNγ and IL4 was quantified using q-PCR in the liver and hepatic lymph nodes (HLN) of sheep during early stages of infection with Fasciola hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.This work was supported by EU grants (FPVII-265862-PARAVAC, H2020-635408-PARAGONE) and National grant (AGL2015-67023-C2-1-R). We thank Prof. John P. Dalton, Queen’s University Belfast, Northern Ireland, for providing recombinant FhCL1. Sequencing analysis and quality analysis of RNA were carried out by the Central Services for Research of the University of CórdobaVeterinari