Rachele Riganò

Echinococcus granulosus Antigen B Impairs Human Dendritic Cell Differentiation and Polarizes Immature Dendritic Cell Maturation towards a Th2 Cell Response

ABSTRACT Despite inducing a strong host cellular and humoral immune response, the helminth Echinococcus granulosus is a highly successful parasite that develops, progresses, and ultimately causes chronic disease. Although surgery remains the preferred therapeutic option, pharmacological research now envisages antihelminthic strategies. To understand the mechanisms that E. granulosus uses to escape host immunosurveillance and promote chronic infection, we investigated how two hydatid cyst components, purified antigen B (AgB) and sheep hydatid fluid (SHF), act on host dendritic cell (DC) differentiation from monocyte precursors and how they influence maturation of DC that have already differentiated. We evaluated the immunomodulatory potential of these antigens by performing immunochemical and cytofluorimetric analyses of monocyte-derived DCs from healthy human donors. During monocyte differentiation, AgB and SHF downmodulated CD1a expression and upregulated CD86 expression. Compared with immature DCs differentiated in medium alone (iDCs), AgB- and SHF-differentiated cells stimulated with lipopolysaccharide included a significantly lower percentage of CD83+ cells (P < 10−4) and had weaker costimulatory molecule expression. When stimulated with AgB and SHF, iDCs matured and primed lymphocytes towards the Th2 response typical of E. granulosus infection. SHF and particularly AgB reduced the production of interleukin-12p70 (IL-12p70) and tumor necrosis factor alpha in lipopolysaccharide-stimulated iDCs. Anti-IL-10 antibodies increased the levels of IL-12p70 secretion in AgB- and SHF-matured DCs. AgB and SHF induced interleukin-1 receptor-associated kinase phosphorylation and activated nuclear factor-κB, suggesting that Toll-like receptors could participate in E. granulosus-stimulated DC maturation. These results suggest that E. granulosus escapes host immunosurveillance in two ways: by interfering with monocyte differentiation and by modulating DC maturation.

Immunological markers indicating the effectiveness of pharmacological treatment in human hydatid disease

The relation of interferon‐gamma (IFN‐γ), IL‐4, IL‐10 production and specific IgE, total IgG, IgG subclass expression to the effectiveness of pharmacological treatment in human hydatid disease (Echinococcus granulosus infection) was evaluated in 27 hydatid patients divided into four clinical groups according to their response to albendazole/mebendazole therapy (full, partial, low and non‐responders). After parasite antigen stimulation, peripheral blood mononuclear cells (PBMC) from full responders produced significantly more IFN‐γ (P= 0·038), significantly less IL‐4 (P= 0·001) and less IL‐10 than PBMC from non‐responders. PBMC from partial and low responders produced intermediate cytokine concentrations. ELISA determining immunoglobulin production showed that sera from all non‐responders had IgE and IgG4 antibodies, both regulated by IL‐4. In contrast to IgG4, IgE decreased rapidly in full responders. Full responders also showed the highest percentage of IgG3 reactions. Qualitative analysis of total IgG responses in hydatid patients’ sera determined by immunoblotting showed that binding profiles to hydatid cyst fluid antigen differed in the four groups of treated patients. Non‐responders had the highest percentage of reactions to all subunits of antigens 5 and B, and full responders had the highest percentage of reactions to antigen 5 alone. The high IFN‐γ production associated with a lack of IL‐4 and low IL‐10 production in the full responders, and vice versa the high IL‐4 and IL‐10 production associated with lack of or low IFN‐γ production in the non‐responders implies Th1 cell activation in protective immunity and Th2 cell activation in susceptibility to hydatid disease. IgE may be a useful marker of therapeutic success in hydatid patients with pretreatment specific IgE antibodies. IgG subclass responses and differential immunoglobulin subclass binding pattern to hydatid antigens may also be useful in the immunosurveillance of hydatid disease.

An update on immunodiagnosis of cystic echinococcosis.

Immunological parameters are increasingly investigated as possible markers for the development of cystic echinococcosis. Among the newer immunologic tests for assessing the host-parasite relationship, assay of immunoglobulin isotypes with the use of distinct parasite antigens and detection of Th1/Th2 cytokine expression are an interesting new approach. The findings upon which we have constructed our immunological hypothesis of the host-parasite relationship are: (1) immunoglobulin isotype profiles differ in patients with distinct clinical outcomes of the disease; in particular, antigen B is the antigen of choice to detect specific IgG4, which is the immunoglobulin isotype most clearly associated with the progression of the disease; (2) the isolation and characterisation of recombinant parasite proteins that behave as molecular markers of allergic reactions associated with cystic echinococcosis; (3) Th1/Th2 cell activation is involved in the clinical outcome of Echinococcus granulosus infection and, in particular Th2 response, is associated with susceptibility to the disease, whereas a Th1 response is associated with protective immunity.

Echinococcus granulosus‐specific T‐cell lines derived from patients at various clinical stages of cystic echinococcosis

To investigate the role of T lymphocytes in the immune response to Echinococcus granulosus, using sheep hydatid fluid (SHF) and antigen B (AgB), we generated T‐cell lines from patients with active, transitional and inactive hydatid cysts. We established 16 T‐cell lines, eight specific to SHF and eight specific to AgB. At surface phenotyping 88–98% of cells displayed the helper/inducer CD4 antigen. In all patients, at all clinical stages of hydatid cyst disease, T‐cell stimulation with SHF and AgB invariably amplified a large number of almost identical Vβ subfamily fragments. Irrespective of antigen‐specificity, the two cell lines from the patient with an inactive cyst had a Th1 profile, because they exclusively expressed and produced IFN‐γ. Conversely, the T‐cell lines derived from the seven patients with active and transitional hydatid cysts had mixed Th1/Th2 and Th0 clones. The functional characteristics of the 16 T‐cell lines differed markedly in the various clinical stages of cystic echinococcosis, thus providing new in vitro evidence that Th1 lymphocytes contribute decisively to the inactive stage of hydatid disease, Th2 lymphocytes in the active and transitional stages. The parasite‐specific T‐cell lines, especially the two Th1 lines from the patient with an inactive cyst, may help identify Th1 protective epitopes on SHF and AgB.

Immunological responses to antigen B from Echinococcus granulosus cyst fluid in hydatid patients

The immunological reactivity of Echinococcus granulosus antigen B was evaluated in 30 hydatid patients. Antigen B was purified from sheep hydatid cyst fluid by electroelution from a non‐reducing SDS‐PAGE gel (AgB). In ELISA and immunoblotting (IB), determining antibody production in sera from patients with hydatid disease and with other parasitic infections, purified AgB showed higher specificity than a partially purified antigen named pH5PPT (100% vs 83% in pH5PPT‐ELISA and 58% in pH5PPT‐IB). AgB‐IB achieved higher sensitivity than AgB‐ELISA (80% vs 63%). All AgB‐IB positive sera recognized the 12 kDa subunit. Qualitative AgB‐IB assessment of IgG isotype responses identified IgG4 as the predominant isotype (87%). The other isotypes showed a lower percentage of positive reactions: IgG1, 33%; IgG2, 21%; and IgG3, 17%. PBMC proliferative assay revealed a cellular response to AgB in 100% of patients’ PBMC. These findings confirm antigen B, especially its smallest subunit, as a good diagnostic molecule.

In vitro production of cytokines by peripheral blood mononuclear cells from hydatid patients

The role of cytokines in human hydatidosJs (Echinococcus granulosus infection) was evaluated in immunoassays determining production of IL‐4, IL‐10 and interferon‐gamma (IFN‐γ) in peripheral blood mononuclear cell (PBMC) cultures from 30 hydatid patients and 14 uninfected controls. In ceil cultures from hydatid patients parasite and non‐parasite antigen stimulation significantly increased IL‐4 production (P·0·005). Spontaneous and milogen‐driven IL‐4 production was similar in patients and controls. IL‐10 and IFN‐γ production did not differ statistically in the two groups, even though some hydatid patients produced these cytokines in large amounts. Notably, antigen‐driven IFN‐γ concentrations were invariably higher in patients than in uninfected controls. Data analysis showed a relationship between IgE and IgG4 responses and parasite‐driven cytokine production. High IgE and IgG4 responders produced high IL‐4 and IL‐10 concentrations. High IgE responders showed decreased IFN‐γ production, but high IgG4 responders had IFN‐γ levels slightly higher than those of low responders. Cytokine response patterns did not relate to the clinical stage of disease. The significantly increased IL‐4 and the high IL‐10 concentrations found in PBMC from many hydatid patients in this study are consistent with Th2 cell activation in human hydatidosis. The presence of antigen‐driven IFN‐γ production in patients with E. granulosus infection implies concurrent intervention of the Th1 or Th0 cell subset.

Immunomodulatory mechanisms during Echinococcus granulosus infection

The pathologic events that ensue after humans ingest the eggs of Echinococcus granulosus and continue while cystic echinococcosis develops, provide an excellent example illustrating the evasive strategies helminth parasites use to develop, progress and cause chronic disease. The hydatid cyst secretes and exposes numerous immunomodulatory molecules to the hosts immune system. By characterizing these molecules we can understand the mechanisms that E. granulosus uses for increasing the efficiency and persistency of infection in the host. These molecules modulate both the innate and adaptive arms of the immune response and appear to target cellular and humoral responses. In this review, we discuss recent advances in the immunobiology of host-E. granulosus interactions that provide intriguing insights into the complex interplay between host and parasite that ultimately facilitates parasite survival.

Molecular cross-talk in host-parasite relationships: the intriguing immunomodulatory role of Echinococcus antigen B in cystic echinococcosis.

Cystic echinococcosis (CE), a zoonosis caused by the development of Echinococcus granulosus tapeworm larvae in the internal organs of ungulates and humans, continues to pose a major public health burden in underdeveloped and industrialised areas worldwide. Research designed to improve parasitic disease control and find out more about parasite biology has already identified a number of E. granulosus antigenic molecules. The major E. granulosus immunomodulant antigen isolated from hydatid fluid is antigen B, a 120 kDa polymeric lipoprotein consisting of various 8 kDa subunits. By inhibiting elastase activity and neutrophil chemotaxis and eliciting a non-protective Th2 cell response, antigen B helps the parasite evade the human response. In this review, we briefly discuss current information on the molecular characteristics and immunomodulatory properties of E. granulosus antigen B. Besides focusing on findings that provide intriguing insights into the complex interplay between host and parasite, we suggest how this information could extend the current therapeutic options in inflammatory diseases.

Molecular and immunological characterization of the C-terminal region of a new Echinococcus granulosus Heat Shock Protein 70

By screening an expression library of Echinococcus granulosus with IgE from sera of patients with cystic echinococcosis (CE) and allergic reactions, we isolated the C‐terminal region of a new heat shock protein (HSP)70 of E. granulosus. The protein, named Eg2HSP70, has close homology with the C‐terminal region of Dermatophagoides farinae and human HSP70. We investigated the humoral and cell‐mediated immune responses to this antigen in patients with CE grouped according to the presence of allergic reactions. Immunoblotting detected total IgG, IgG4 and IgE specific to Eg2HSP70 (83% of sera contained IgG, 31% IgG4 and 57% IgE). No significant difference was found in immunoglobulin percentages according to the presence of allergic reactions. Immunoblotting inhibition showed that no IgG or IgE specific to Eg2HSP70 cross‐reacted with D. farinae or human HSP70. Eg2HSP70‐stimulated PBMC from 26 patients produced significantly greater amounts of TNF‐α, IFN‐γ, and IL‐10 than unstimulated cultures in all patients, irrespective of the presence of allergic reactions (P < 0·05). They also produced significantly greater amounts of IL‐4 than unstimulated cultures exclusively in patients with allergic reactions (P < 0·05). These findings show that Eg2HSP70 is a new antigenic molecule inducing both B and T cell responses.

Long-term serological evaluation of patients with cystic echinococcosis treated with benzimidazole carbamates

Seeking better immunological markers indicating the long‐term outcome of cystic echinococcosis (CE) after chemotherapy we studied 23 patients receiving albendazole, clinically followed for 8 years, and grouped ultrasonographically according to therapeutic outcome. Antibody responses against a partially purified fraction of hydatid fluid (HFF) and antigen B (AgB) were evaluated by indirect haemagglutination (IHA), ELISA and immunoblotting (IB). Although IHA titres varied over the course of treatment, differences in mean antibody titres to HFF between groups were significant only at 4 years (P = 0·031). IgG isotype expression remained unchanged during follow‐up whereas IgE expression decreased in patients with cured or stable disease. AgB disclosed higher IgG4 expression (P < 10–4; P = 0·025) and lower IgG1 expression than HFF (P < 10–4; P = 0·022). IHA antibody titres were higher in patients with progressive than in those with cured or stable disease, even in those with the same cyst type. ELISA isotype profiles differed between groups, particularly for type CE 3, 4 and 5 cysts: higher serum IgG1 and IgG3, lower IgG4 and IgE in patients with cured or stable disease. Although combined serological testing provides scarce information on the long‐term outcome of CE after chemotherapy it may be useful for reviewing in a retrospective study the outcome of a cyst and for assessing the host‐parasite relationship.