Fasciola hepatica Fatty Acid Binding Protein Induces the Alternative Activation of Human Macrophages

Abstract

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 (AAM). The results also demonstrated the ability of Fh12 to downregulate the secretion of the proinflammatory and inflammatory cytokines tumor necrosis factor alpha (TNF-␣), interleukin-12 (IL-12), and IL-1␤B, 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 hepaticaFatty Acid Binding Protein Inhibits TLR4 Activation and Suppresses the Inflammatory Cytokines Induced by Lipopolysaccharide In Vitro and In Vivo

“…After the purification process, fractions containing a polypeptide band of around 12kDa with isoelectric points of 5.1 to 7.25 were confirmed as FABP1 by mass spectrometry and its purity verified by 12.5% SDS-PAGE as recently reported (29). Fh12 gave endotoxin levels lower than 0.1 EU/ml, which is similar to background levels and to complete RPMI medium, so the purified Fh12 protein was considered to be endotoxin free.…”

“…We previously demonstrated that Fh12 exerts a strong immunomodulatory effect on human macrophage-derived monocytes (29). Our results here demonstrate that in the presence of Fh12 alone, bmMΦs do not express pro-inflammatory or inflammatory cytokines, which suggests that Fh12 does not activate any TLR on macrophages.…”

“…Native Fh12 was purified from FhWE using a previously optimized protocol (29) that involves an initial ultracentrifugation step at 30,000 × g , followed by gel filtration chromatography with Sephadex G-50 (XK 26/100 column) and two consecutive preparative isoelectric focusing (IEF). Each aliquot from the last IEF separation was subjected to 12.5% SDS-PAGE and the proteins were visualized by silver staining or coomassie blue staining.…”

“…Each aliquot from the last IEF separation was subjected to 12.5% SDS-PAGE and the proteins were visualized by silver staining or coomassie blue staining. Fractions that exhibited a single polypeptide band of around 12kDa were manually excised from the gel and their identity as FABP confirmed by MALDI and MS/MS as previously described (29, 30). Fractions were pooled and termed Fh12.…”

“…bmMΦs were seeded into 24-well plates (Nunc) at 10 6 /ml in complete RPMI 1640 medium and then treated with a pre-determined concentration of Fh12 (5μg/ml) (29) for 30 min prior to being exposed to heat-killed Listeria monocytogenes (HKLM, 10 8 cells/ml); Poly(I:C) (100μg/ml); LPS (100 ng/ml); flagellin (1μg/ml); imiquimod (10μg/ml); or thiazoloquinolone (CL075, 10μg/ml). Control cells were treated with PBS, Fh12 or TLRs-ligands alone.…”