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-1β 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.

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-1βB, 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).

Recently, we reported that a nativeFasciola hepaticafatty acid binding protein (FABP) termed Fh12 is a powerful anti-inflammatory protein capable of suppressing the LPS-induced expression of inflammatory markersin vivoandin vitro. Because the purification of a protein in native form is, in many situations not cost-beneficial and unsuitable for industrial grade scale-up, this study accomplished the task of optimizing the expression and purification of a recombinant form of FABP (Fh15). Additionally, we ascertained whether this molecule could exhibit a similar suppressive effect on TLR-stimulation and inflammatory cytokine expression from macrophages than those previously demonstrated for the native molecule. Results demonstrated that Fh15 suppresses the expression of IL-1β and TNFα in murine macrophages and THP1 Blue CD14 cells. Additionally, Fh15 suppress the LPS-induced TLR4 stimulation. This effect was not impaired by a thermal denaturing process or blocked by the presence of anti-Fh12 antibodies. Fh15 also suppressed the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti-inflammatory drug in preclinical studies in the near future.

Tegument protein extract fromFasciola hepaticaadult flukes (FhTA) was obtained and assessed for its potential as a diagnostic agent for the serological detection of human fascioliasis using an indirect enzyme-linked immunosorbent assay (ELISA). In an analysis of sera from 45 patients infected withF. hepatica, sera from 41 patients with other parasitic infections, and sera from 33 healthy controls, the FhTA-ELISA showed sensitivity, specificity, and accuracy of 91.1%, 97.3%, and 95%, respectively. Specific IgG1 and IgG4 were the antibody isotypes mainly detected in sera from patients with fascioliasis. Polypeptides of 52, 38, 24 to 26, and 12 to 14 kDa were identified by Western blotting as the most immunoreactive components of the FhTA. A proteomic approach led us to identify enolase, aldolase, glutathioneS-transferase, and fatty acid binding protein as the major immunoreactive components of the FhTA.