Purification of Native Fasciola hepatica Fatty Acid-Binding Protein and Induction of Alternative Activation of Human Macrophages.

This chapter presents the different techniques to purify the native forms of Fasciola hepatica fatty acid-binding protein (Fh12) using size exclusion chromatography and isoelectric focusing (IEF). Also, it presents the procedure to study the immunological effect of the purified protein Fh12 using monocyte-derived macrophages (MDM) obtained from healthy human donors. For this purpose, I present the procedure to isolate and culture peripheral blood mononuclear cells (PBMCs) to generate alternatively activated macrophages (AAMΦ) by in vitro exposure to Fh12.

Possible Role for Toll-Like Receptors in Interaction of Fasciola hepatica Excretory-Secretory Products with Human Monocyte Cell Line.

This chapter presents a proteomic approach to purify and identify native excretory-secretory products (ESPs) in the range of >10-30 kDa proteins capable of interacting with toll-like receptors (TLRs). Here we present a protocol to fractionate the total ESPs using an ultrafiltration system to recover ESP proteins >10-30 kDa. The fraction of the proteins >10-30 kDa is purified by ion exchange chromatography (IEC) using a mono Q-column in a fast protein liquid chromatography system (FPLC) to separate its components based on charge. Finally, a screening system is presented using THP1-Blue CD14 cells to investigate whether TLRs could also be targeted by Fasciola hepatica ESPs and the interaction with TLR4 using HEK293 Blue-TLR4 cells.

Fasciola hepatica ESPs Could Indistinctly Activate or Block Multiple Toll-Like Receptors in a Human Monocyte Cell Line.

Fasciola hepatica is a parasitic helminth that induces Th2/Treg responses in its mammalian host. Some reports have suggested that ESPs achieve these polarized immune responses by delaying the activation of dendritic cells and macrophages during the early stages of innate immunity, a process that is mediated by TLR4. The present study aimed to investigate whether TLRs other than TLR4 could also be targeted by F. hepatica ESPs. To achieve this aim a screening system was optimized using THP1-Blue CD14 cells. ESPs were first separated based on their molecular weight and according their net charge by ion exchange chromatography (IEC). Results demonstrated that F. hepatica ESPs mainly cathepsin, serpin and endopin are capable of activating TLR2, TLR4, TLR8 and likely also TLR5 and TLR6. In contrast, fatty acid binding protein strongly suppressed the stimulation induced by various TLR-ligands. Further studies are needed to understand how these apparent contradictory effects of molecules of the same protein mix complement each other in the context of an active infection resulting in the polarized Th2-immune response that characterize F. hepatica infections.

Fasciola hepatica fatty acid binding protein inhibits TLR4 activation and suppresses the inflammatory cytokines induced by lipopolysaccharide in vitro and in vivo.

TLR4, the innate immunity receptor for bacterial endotoxins, plays a pivotal role in the induction of inflammatory responses. There is a need to develop molecules that block either activation through TLR4 or the downstream signaling pathways to inhibit the storm of inflammation typically elicited by bacterial LPS, which is a major cause of the high mortality associated with bacterial sepsis. We report in this article that a single i.p. injection of 15 µg fatty acid binding protein from Fasciola hepatica (Fh12) 1 h before exposure to LPS suppressed significantly the expression of serum inflammatory cytokines in a model of septic shock using C57BL/6 mice. Because macrophages are a good source of IL-12p70 and TNF-α, and are critical in driving adaptive immunity, we investigated the effect of Fh12 on the function of mouse bone marrow-derived macrophages (bmMΦs). Although Fh12 alone did not induce cytokine expression, it significantly suppressed the expression of IL-12, TNF-α, IL-6, and IL-1ß cytokines, as well as inducible NO synthase-2 in bmMΦs, and also impaired the phagocytic capacity of bmMΦs. Fh12 had a limited effect on the expression of inflammatory cytokines induced in response to other TLR ligands. One mechanism used by Fh12 to exert its anti-inflammatory effect is binding to the CD14 coreceptor. Moreover, it suppresses phosphorylation of ERK, p38, and JNK. The potent anti-inflammatory properties of Fh12 demonstrated in this study open doors to further studies directed at exploring the potential of this molecule as a new class of drug against septic shock or other inflammatory diseases.

Fasciola hepatica fatty acid binding protein induces the alternative activation of human macrophages.

The liver fluke Fasciola hepatica is a highly evolved parasite that uses sophisticated mechanisms to evade the host immune response. The immunosuppressive capabilities of the parasite have been associated with antigens secreted through the parasite's tegument, called excretory-secretory products (ESPs). Proteomic studies have identified the fatty acid binding protein (FABP) as one of molecules present in the parasite ESPs. Although FABP has been investigated for potential use in the development of vaccines against fascioliasis, its direct interaction with cells of immune system has not been studied. In this study, FABP was purified in native form from soluble extracts of F. hepatica adult flukes using a combination of molecular sieving chromatography and preparative isoelectric focusing. The immunological effect of the purified protein, termed Fh12, was assayed in vitro using monocyte-derived macrophages (MDM) obtained from healthy human donors. Results from the assay indicate that Fh12 produced a significantly increased arginase expression and activity and induced the expression of chitinase-3-like protein (CHI3L1). The assay also showed that Fh12 downregulated the production of nitric oxide (NO) and the expression of nitric oxide synthase (NOS2). This indicates that Fh12 induced the production of alternatively activated macrophages (AAMϕ). The results also demonstrated the ability of Fh12 to downregulate the secretion of the proinflammatory and inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and IL-1ßB, even after stimulation with lipopolysaccharide (LPS), as well as its ability to stimulate the overexpression of IL-10. These results suggest a potent anti-inflammatory role for Fh12, which could occur via targeting of Toll-like receptor 4 (TLR4).

Fasciola hepatica saposin-like protein-2-based ELISA for the serodiagnosis of chronic human fascioliasis.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed and evaluated for its diagnostic ability to detect human IgG antibodies against Fasciola hepatica saposin-like protein-2. The assay was compared with an indirect ELISA with excretory-secretory products (FhES) from adult F. hepatica. In an analysis of the sera of 37 patients infected with F. hepatica, 40 patients with other parasitic infections, and 50 healthy controls, the sensitivity of both ELISA assays was 100%. However, the FhSAP2-based ELISA was more specific (95.6%) than the FhES-ELISA (91.9%). These results demonstrated that FhSAP2 can be used in the serodiagnosis of chronic human fascioliasis with the additional advantage of being relatively cheap and easy to produce. Studies are in progress to evaluate this FhSAP2-ELISA assay in a large-scale prevalence surveys in endemic areas.