Abstract
Fasciola hepatica has been shown to have a high capacity for immunomodulation of the host response, making the development of protective vaccines extremely difficult. One of these immunomodulation mechanisms is the impairment of dendritic cells (DC) maturation and, therefore, suppression of antigenic presentation. The aim of this study was to evaluate the pathological changes as well as the characterization of two antigen presenting cells, DC (CD1b, CD83 and MHC-II positive) and follicular dendritic cells (FDC) (CNA.42, S100 and CD83 positive) by immunohistochemistry in the hepatic lymph nodes (HLN) and livers of sheep during the early stages of infection with F. hepatica [9 and 18 days post-infection (dpi)], compared with an uninfected group (UC) as a control. The results revealed a marked hyperplasia of HLN germinal centres at 9 and, in particular, 18 dpi, with respect to the UC group, with coincidental increased expression of CNA.42 in FDC of lymphoid follicles and CD1b in the DC of paracortical areas at 18 dpi. However, the expression of MHC-II and CD83 decreased at 9 and, particularly, at 18 dpi in HLN compared with that in the UC group. Since both markers are related to active presentation of antigens by DC and FDC, the results of the present study suggest that, despite the marked hyperplasia of HLN and increase in DC and FDC numbers during early stages of infection, the DC and FDC antigenic presentation capacity, as suggested by the expression of the markers MHC-II and CD83, is suppressed by the parasite. This suppression was not observed in the liver, probably because of the low number of DC. This is the first study of the immunophenotype of DCs and FDC in sheep infected with F. hepatica.

Introduction
Fasciolosis, caused by F. hepatica, results in major economic losses to the agricultural sector [1]. The parasite infects a wide range of domestic animals, including cattle, sheep and goats, and the disease has been recognized as an important zoonosis in Africa, Asia, Europe, America and Oceania [2, 3]. Control of the disease has traditionally been based on the use of anthelmintic drugs such as triclabendazole. Nevertheless, resistance to this and other drugs, together with public concern about the presence of drug metabolites in foodstuff, is increasing in numerous countries [4]. Because of this, there is increasing interest in the development of an immunological method of control for the disease [5, 6]. Despite major efforts during the last two decades, the search for an effective vaccine to control fasciolosis has been slow due to the different mechanisms used by F. hepatica to modulate the host immune response, which have rendered attempted therapies ineffective in killing the parasite [7,8,9]. One of these mechanisms is the impairment of the maturation of dendritic cells (DC), which facilitate both parasite survival and modulation of the immune response [10,11,12].

DC are specialized antigen-presenting cells that can be found in the paracortical area of the lymph nodes where they promote the activation of naive T lymphocytes. On the other hand, follicular dendritic cells (FDC) are cells of stromal origin located in the central region of primary follicles and in the light zone of germinal centres of secondary and tertiary lymphoid organs. They play a crucial role in B-cell activation and have the unique capacity to bind and retain native antigen in B-cell follicles for long periods of time [13, 14]. In previous papers, it has been reported that F. hepatica is able to downregulate the Th1 immune response and upregulate the Th2 response at early stages of infection in sheep [15] and mice [16], as well as in chronic stages in cattle [17]. This imbalance towards a Th2 immune profile is mediated through regulatory cytokines and cells, such as DC, that modulate and/or suppress inflammatory responses. It has been determined that different antigenic preparations of this parasite, such as total extract, F. hepatica tegumental antigen (FhTeg) and excretory-secretory products (ESPs), decrease the activation state of DC in mice [11, 12, 18, 19], and F. gigantica ESPs induce the modulation of buffalo DC [20]. More specifically, it has been reported that FhTeg induces DC modulation, resulting in a lack of T cell Th1 cytokine response and proliferation [21]. In addition, the glycan products produced by F. hepatica participate in the modulation of DC maturation and mediate the production of IL-10 and IL-4 during infection, inducing a Th2/regulatory-polarized immune response [22,23,24,25]. F. hepatica can also induce the development of phenotypes that are characterized by a decreased production of pro-inflammatory cytokines, as well as the expression of general markers that are characteristic of an M2 macrophage phenotype and have been shown to promote the differentiation of Th2 and Treg cells [26,27,28]. Moreover, it has been reported that F. hepatica cathepsin L1 (FhCL1), glutathione transferase (FhGST) and Kunitz-type molecule induce a modulatory effect on DC, which leads to the suppression of the adaptive immune response, including Th1- and/or Th17-related responses [10, 29]. To date, the effect of F. hepatica infection on FDC has not been evaluated.

On the other hand, it has been shown that recombinant forms of FhCL1 and FhGST can partially activate DC [10]. Moreover, a mucin-like peptide from F. hepatica (Fhmuc) induces parasite-specific adaptive immunity with increased levels of IFN-γ and specific IgG antibodies [30]. In a recent study, it was also reported that the interactions of DC with F. hepatica cathepsin L3 (FhCL3) confer a unique expression pattern of the cytokines IFN-γ and IL-13, which may be protective in this parasitosis or in other helminth infections [31].

Most studies investigating the modulation of DC by F. hepatica have been carried out in murine models, with only one study in buffaloes [20]. Therefore, to date, there is little available information on F. hepatica-induced modulation of DC in ruminants. The aim of the present study was to evaluate the expression of different markers of DC (CD1b, CD83 and MHC-II) and of FDC (CNA.42, S100 and CD83) in the HLNs and liver of sheep during the early stages of infection with F. hepatica. These markers have been selected because they have been used as markers of DC, FDC and antigen-presenting cells in formalin-fixed peripheral lymph nodes in sheep [13, 32]. This is the first study to evaluate the immunophenotype of DC and FDC present within the HLNs and livers of sheep experimentally infected with F. hepatica in the early stages of infection.