Fasciola gigantica tegumental calcium-binding EF-hand protein 4 exerts immunomodulatory effects on goat monocytes

Abstract

Background: The liver fluke Fasciola gigantica secretes excretory-secretory proteins during infection to mediate its interaction with the host. In this study, we investigated the immunomodulatory effects of a recombinant tegumental calcium-binding EF-hand protein 4 of F. gigantica (rFg-CaBP4) on goat monocytes.

Methods: The rFg-CaBP4 protein was induced and purified by affinity chromatography. The immunogenic reaction of rFg-CaBP4 against specific antibodies was detected through western blot analysis. The binding of rFg-CaBP4 on surface of goat monocytes was visualized by immunofluorescence assay. The localization of CaBP4 within adult fluke structure was detected by immunohistochemical analysis. The cytokine transcription levels in response to rFg-CaBP4 were examined using ABI 7500 real-time PCR system. The expression of the major histocompatibility complex (MHC) class-II molecule (MHC-II) in response to rFg-CaBP4 protein was analyzed using Flow cytometry.

Results: The isopropyl-ß-D-thiogalactopyranoside-induced rFg-CaBP4 protein reacted with rat sera containing anti-rFg-CaBP4 polyclonal antibodies in a western blot analysis. The adhesion of rFg-CaBP4 to monocytes was visualized by immunofluorescence and laser scanning confocal microscopy. Immunohistochemical analysis localized native CaBP4 to the oral sucker, pharynx, genital pore, acetabulum and tegument of adult F. gigantica. Co-incubation of rFg-CaBP4 with concanavalin A-stimulated monocytes increased the transcription levels of interleukin (IL)-2, IL-4, interferon gamma and transforming growth factor-β. However, a reduction in the expression of IL-10 and no change in the expression of tumor necrosis factor-α were detected. Additionally, rFg-CaBP4-treated monocytes exhibited a marked increase in the expression of the major histocompatibility complex (MHC) class-II molecule (MHC-II) and a decrease in MHC-I expression, in a dose-dependent manner.

Conclusions: These findings provide additional evidence that calcium-binding EF-hand proteins play roles in host-parasite interaction. Further characterization of the immunomodulatory role of rFg-CaBP4 should expand our understanding of the strategies used by F. gigantica to evade the host immune responses.

Keywords: Fasciola gigantica; Host-parasite interactions; Immune responses; Monocytes; Tegumental calcium-binding EF-hand protein 4.

Fig. 1a–c

Fasciola gigantica tegumental calcium-binding EF-hand protein (CaBP) 4 (Fg-CaBP4) sequence and phylogenetic analysis. a The predicted protein homology and secondary structures with the best template found in the Phyre2 search tool, and BLAST search against the National Center for Biotechnology Information (NCBI) non-redundant protein sequence highlighting the two major domains, EF-hand and dynein-light chain, in the Fg-CaBP4 protein. b The predicted 3-dimensional model of Fg-CaBP4 shows 55% similarity to Fasciola hepatica CaBP2 protein (Protein Data Bank: p6422). c A neighbor joining phylogenetic tree showing genetic relatedness between Fg-CaBP4 and CaBPs from other helminth parasites in the NCBI database

Fig. 2

The relative expression of CaBP4 in F. gigantica life cycle stages. The messenger RNA (mRNA) transcriptional patterns of CaBP4 in eggs, juvenile [28 and 42 days post-infection (dpi)] and adult (98 dpi) flukes were examined by real-time polymerase chain reaction. The transcription value of CaBP4 in the eggs was normalized to 1.0. The relative changes in the gene transcription ratio were normalized to the transcription of a reference gene and calculated by the 2^−ΔΔCt method. The data are presented as mean ± SD from three independent experiments. Data were analyzed by one-way ANOVA. ns Non-significant; ***P < 0.001, ****P < 0.0001

Fig. 3a, b

Western blotting was performed to confirm the expression of recombinant Fg-CaBP4 (rFg-CaBP4) protein [molecular weight protein ladder (M)]. a The recombinant protein expression before isopropyl-ß-D-thiogalactopyranoside induction (0 h), protein expression at different times (2, 4, 6 h) and the purified protein resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (P). b The purified protein was transferred to a polyvinylidene difluoride membrane which was probed with rat sera containing anti-rFg-CaBP4 antibodies (1) or incubated with normal rat sera (2)

Fig. 4

Immunolocalization of CaBP4 within adult F. gigantica flukes. The CaBP4 protein was detected in the tegument and inner lining of the oral sucker and ventral sucker (Acetabulum). Red denotes the target protein stained with cyanine dye 3 (Cy3)-conjugated secondary antibody. Blue denotes nuclei stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI). No red fluorescence was detected in the control panel. Merge Combined DAPI and Cy3, BF bright fluorescence. Scale bars 250 μm

Fig. 5

Binding of rFg-CaBP4 to the surface of goat monocytes. Goat monocytes were treated with rFg-CaBP4 (20 μg/ml) or left untreated for 60 min at 37 °C. All cells were fixed and incubated with rat anti-rFg-CaBP4 antibody followed by Cy3-labeled goat anti-rat IgG (red). The cell nuclei were counter labeled with DAPI (blue). The binding of rFg-CaBP4 to the surface of goat monocytes was observed as red staining with a confocal laser scanning microscopy. Scale bars 10 μm. For abbreviations, see Figs. 3 and  4

Fig. 6

Effect of rFg-CaBP4 on goat monocyte cytokine production. Cells were stimulated with concanavalin A (ConA) (10 µg/ml) and incubated with rFg-CaBP4 for 26 h. Then, expression of the mRNAs of interleukin (IL)-2, IL-4, IL-10, IFN-γ, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was quantified by reverse transcription-polymerase chain reaction. The data are presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001, ns [compared with the untreated (Control) group]

Fig. 7

rFg-CaBP4-mediated differential expression of major histocompatibility complex (MHC) on the surface of goat monocytes. Monocytes were cultured in the presence of the indicated concentrations of rFg-CaBP4 and control buffer for 24 h. MHC class-I (MHC-I) and MHC-II expressions were measured by flow cytometric analysis and calculated as the percentage of mean fluorescence intensity (MFI). The data are presented as the MFI ± SD from three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns (compared to untreated control)