Recently, we reported that a native Fasciola hepatica fatty acid binding protein (FABP) termed Fh12 is a powerful anti-inflammatory protein capable of suppressing the LPS-induced expression of inflammatory markers in vivo and in 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.

Due to the unsatisfactory performance of parasitological diagnosis of human fascioliasis; the use of immunodiagnosis based on the detection of anti-Fasciola antibodies is traditionally used as a diagnostic alternative using total or purified parasite excretory-secretory products (ESPs). Glutathione S-transferase (GST) protein, one of the F. hepatica ESP components, possesses well-known roles in the detoxification of xenobiotic and endogenously derived toxins within the host bile environment. GST has shown to be a good target for vaccine or drug development against fascioliasis. The current study aimed to evaluate the potential of GST protein purified from a soluble crude extract of adult flukes as an antigen for serodiagnosis of chronic human fascioliasis by indirect ELISA. The study included a panel of 116 serum samples collected from individuals with confirmed fascioliasis, individuals carrying heterologous parasitic infections and healthy subjects. The parasitological examination was used as gold standard and a previously optimized ESP-ELISA was used to compare the performance of the GST-ELISA method. Results demonstrated that GST-ELISA is 94.3% sensitive, 80.2% specific and exhibits a moderate positive correlation (r = 0.555) and substantial agreement (k = 0.786) with the results obtained with the ESP-ELISA method. Moreover, because no sera from patients with early F. hepatica infection were available, GST-ELISA was then tested with sera from rabbits experimentally infected with F. hepatica metacercariae. The assay was able to detect anti-Fasciola antibodies as early as the 3 week of infection (p < 0.0001) with peaks at 4 and 10 week post-infection.

Parasitic helminths and helminth-derived molecules have demonstrated to possess powerful anti-inflammatory properties and confirmed therapeutic effects on inflammatory diseases. The helminth Fasciola hepatica has been reported to suppress specific Th1 specific immune responses induced by concurrent bacterial infections, thus demonstrating its anti-inflammatory ability in vivo. In this study, we demonstrate that native F. hepatica glutathione S-transferase (nFhGST), a major parasite excretory-secretory antigen, majorly comprised of Mu-class GST isoforms, significantly suppresses the LPS-induced TNFα and IL1β of mouse bone-marrow derived macrophages in vitro and the pro-inflammatory cytokine/chemokine storm within C57BL/6 mice exposed to lethal doses of LPS increasing their survival rate by more than 85%. Using THP1-Blue CD14 cells, a human monocyte cell line, we also demonstrate that nFhGST suppresses NF-κB activation in response to multiple TLR-ligands, including whole bacteria clinical isolates and this suppression was found to be dose-dependent and independent of the timing of exposure. Moreover, the suppressive effect of nFhGST on NF-κB activation was shown to be independent of enzyme activity or secondary structure of protein. As part of its anti-inflammatory effect nFhGST target multiple proteins of the canonic and non-canonic NF-κB signaling pathway as well as also JAK/STAT pathway. Overall, our results demonstrate the potent anti-inflammatory properties of nFhGST and its therapeutic potential as an anti-inflammatory agent.

Cell free protein synthesis has become a powerful method for the high-throughput production of proteins that are difficult to express in living cells. The protein SAP2 of Fasciola hepatica (FhSAP2), which has demonstrated to be both, an excellent vaccine candidate against experimental fascioliasis and a good antigen for serodiagnosis of human chronic fascioliasis, is a typical example of a molecule that is difficult to produce. This is mainly due to its tendency to get over-expressed in inclusion bodies by prokaryotes. FhSAP2 expressed in an Escherichia coli-based expression system is poorly glycosylated, insoluble and often undergoes improper folding leading it to reduced immunogenicity. In this work, FhSAP2 was expressed in vitro using the eukaryote cell free system, TNT T7 Quick coupled transcription/translation, that has been designed for the expression of PCR-generated DNA templates. FhSAP2 was expressed in micro-volumes and purified by an affinity chromatography method, which gave a protein yield of 500 µg/ml as determined by bicinchoninic acid assay method. Circular dichroism, Western blotting and enzyme-linked immunosorbent assay analysis were used to confirm the secondary structure, purity and integrity of protein. Results demonstrate that FhSAP2 can be expressed in a cell-free system retaining its main conformational and antigenic properties. The protein purified could be used in immunization experiments and immunodiagnostic techniques.

Fasciola hepatica, a world-wide parasitic helminth, is a well-known master of immunomodulation. The survival of this parasite in the mammalian host is highly dependent on its ability to avoid immune responses of the host for which the parasite employs excretory-secretory products (ESPs). Glutathione S-transferase (GST), one of the proteins in the repertoire of parasite ESPs, is an enzyme recognized for its anti-oxidant properties. GST has been studied as a vaccine candidate against animal fascioliasis, but its role in the immunomodulation has been poorly studied. In a previous study, we demonstrated that GST alone incites no activation of the nuclear factor NF-κB, yet it can suppress NF-κB when THP1-CD14 cells are exposed to GST 30 minutes before stimulation with TLR ligands. In this study, we investigate whether GST could exert a similar suppressive effect on NF-κB activation in THP1-CD14 cells when added several hours after TLR-ligand stimulation and if GST is able to suppress the expression of inflammatory cytokines in bone marrow derived macrophages (bmMØs) from naïve mice induced by LPS. Finally, we performed in vivo experiments to investigate whether one 200ug intraperitoneal (IP) injection with GST could increase the survival rate of C57BL/6 mice exposed to a lethal dose of LPS. Our results demonstrate that 10ug of GST on THP1-CD14 cells up to 3 hours after TLR-ligand stimulation significantly suppressed (p≤0.01) NF-kB activation. GST also suppressed the expression of TNF-α and IL-1β cytokines (p&lt;0.05) in bmMØs in a dose dependent manner. Importantly, a single IP injection of GST boosted the survival rate of mice by 80% against lethal doses of LPS, suggesting GST is an anti-inflammatory molecule.