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Gene expression for Th1/Th2 cytokines (IL-4 and IFN-ɣ), regulatory cytokines (TGF-β and IL-10) and the transcriptional factor FoxP3 was analyzed in the liver and hepatic lymph nodes (HLN) from sheep immunized with partially protective and non-protective vaccine candidates and challenged with Fasciola hepatica. FoxP3 T cells were also evaluated by immunohistochemistry (IHQ). The most remarkable difference between the partially protected vaccinated (V1) group and the non-protected vaccinated (V2) group was a more severe expansion of FoxP3 T cells recorded by IHQ in both the liver and HLN of the V2 group as compared to the V1 group, whereas no differences were found between the V2 group and the infected control (IC) group. Similar results were recorded for FoxP3 gene expression although significant differences among V1 and V2 groups were only significant in the HLN, while FoxP3 gene expression was very similar in the V2 and IC groups both in the liver and HLN. No significant differences for the remaining cytokines were recorded between the V1 and V2 groups, but in the liver the V2 group shows significant increases of IFN-ɣ and IL-10 as compared to the uninfected control (UC) group whereas the V1 group did not. The lower expansion of FoxP3 T cells and lower increase of IFN-ɣ and IL-10 in the partially protected vaccinated group may be related with lower hepatic lesions and fluke burdens recorded in this group as compared to the other two infected groups. The most relevant change in regulatory cytokine gene expression was the significant increase of TGF-β in the liver of IC, V1 and V2 groups as compared to the UC group, which could be related to hepatic lesions. [ABSTRACT FROM AUTHOR]

The immunomodulatory capacity of F. hepatica antigens is probably one of the main reasons for the development of a driven non-protective Th2 immune response. In this study, we analysed the cellular response of hepatic lymph node cells and CD4+ T cells in terms of proliferative response, efficiency of antigen presentation and cytokine production, to F. hepatica-derived molecules, at early and late stages of the infection. Thirty-one sheep were allocated into five groups and were slaughtered at 16 dpi and 23 wpi. In order to analyse antigen-specific response, the following F. hepatica recombinant molecules were used: rFhCL1, rFhCL2, rFhCL3, rFhCB1, rFhCB2, rFhCB3, rFhStf-1, rFhStf-2, rFhStf-3 and rFhKT1. A cell proliferation assay using hepatic lymph node cells and an antigen presentation cell assay using CD4+ T cells were performed. At 16 dpi, all molecules but rFhStf-2 and rFhKT1 elicited a significant cell proliferative response on hepatic lymph node cells of infected animals. At both early and late stage of the infection, antigen presentation of rFhCB3 and rFhCL2 resulted in higher stimulation index of CD4+ T cells which was IL-2 mediated, although no statistically significant when compared to uninfected animals. Significant cytokine production (IL-4, IL-10 and IFN-γ) was conditioned by the antigen-specific cell stimulation. No CD4+ T cell exhaustion was detected in infected sheep at the chronic stage of the infection. This study addressed antigen-specific response to F. hepatica-derived molecules that are involved in key aspects of the parasite survival within the host. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The expression of T regulatory cells (Foxp3), regulatory (interleukin [IL]-10 and transforming growth factor beta [TGF-β]) and proinflammatory (tumor necrosis factor alpha [TNF-α] and interleukin [IL]-1β) cytokines was quantified using real time polymerase chain reaction (qRT-PCR) in the liver of sheep during early stages of infection with Fasciola hepatica (1, 3, 9, and 18 days post-infection [dpi]). Portal fibrosis was also evaluated by Masson's trichrome stain as well as the number of Foxp3+ cells by immunohistochemistry. Animals were divided into three groups: (a) group 1 was immunized with recombinant cathepsin L1 from F. hepatica (FhCL1) in Montanide adjuvant and infected; (b) group 2 was uniquely infected with F. hepatica; and (c) group 3 was the control group, unimmunized and uninfected. An overexpression of regulatory cytokines of groups 1 and 2 was found in all time points tested in comparison with group 3, particularly at 18 dpi. A significant increase of the number of Foxp3+ lymphocytes in groups 1 and 2 was found at 9 and 18 dpi relative to group 3. A progressive increase in portal fibrosis was found in groups 1 and 2 in comparison with group 3. In this regard, group 1 showed smaller areas of fibrosis than group 2. There was a significant positive correlation between Foxp3 and IL-10 expression (by immunohistochemistry and qRT-PCR) just as between portal fibrosis and TGF-β gene expression. The expression of proinflammatory cytokines increased gradually during the experience. These findings suggest the induction of a regulatory phenotype by the parasite that would allow its survival at early stages of the disease when it is more vulnerable. [ABSTRACT FROM AUTHOR]

The immunomodulatory capacity of F. hepatica antigens is probably one of the main reasons for the development of a driven non-protective Th2 immune response. In this study, we analysed the cellular response of hepatic lymph node cells and CD4 + T cells in terms of proliferative response, efficiency of antigen presentation and cytokine production, to F. hepatica-derived molecules, at early and late stages of the infection. Thirty-one sheep were allocated into five groups and were slaughtered at 16 dpi and 23 wpi. In order to analyse antigen-specific response, the following F. hepatica recombinant molecules were used: rFhCL1, rFhCL2, rFhCL3, rFhCB1, rFhCB2, rFhCB3, rFhStf-1, rFhStf-2, rFhStf-3 and rFhKT1. A cell proliferation assay using hepatic lymph node cells and an antigen presentation cell assay using CD4 + T cells were performed. At 16 dpi, all molecules but rFhStf-2 and rFhKT1 elicited a significant cell proliferative response on hepatic lymph node cells of infected animals. At both early and late stage of the infection, antigen presentation of rFhCB3 and rFhCL2 resulted in higher stimulation index of CD4 + T cells which was IL-2 mediated, although no statistically significant when compared to uninfected animals. Significant cytokine production (IL-4, IL-10 and IFN-γ) was conditioned by the antigen-specific cell stimulation. No CD4 + T cell exhaustion was detected in infected sheep at the chronic stage of the infection. This study addressed antigen-specific response to F. hepatica-derived molecules that are involved in key aspects of the parasite survival within the host.