Characterization of a rhodanese homologue from Haemonchus contortus and its immune-modulatory effects on goat immune cells in vitro

Abstract

Background: Modulation of the host immune response by nematode parasites has been widely reported. Rhodaneses (thiosulfate: cyanide sulfurtransferases) are present in a wide range of organisms, such as archaea, bacteria, fungi, plants and animals. Previously, it was reported that a rhodanese homologue could be bound by goat peripheral blood mononuclear cells (PBMCs) in vivo.

Methods: In the present study, we cloned and produced a recombinant rhodanese protein originating from Haemonchus contortus (rHCRD), a parasitic nematode of small ruminants. rHCRD was co-incubated with goat PBMCs to assess its immunomodulatory effects on proliferation, apoptosis and cytokine secretion.

Results: We verified that the natural HCRD protein localized predominantly to the bowel wall and body surface of the parasite. We further demonstrated that serum produced by goats artificially infected with H. contortus successfully recognized rHCRD, which bound to goat PBMCs. rHCRD suppressed proliferation of goat PBMCs stimulated by concanavalin A but did not induce apoptosis in goat PBMCs. The production of TNF-α and IFN-γ decreased significantly, whereas secretion of IL-10 and TGF-β1 increased, in goat PBMCs after exposure to rHCRD. rHCRD also inhibited phagocytosis by goat monocytes. Moreover, rHCRD downregulated the expression of major histocompatibility complex (MHC)-II on goat monocytes in a dose-dependent manner, but did not alter MHC-I expression.

Conclusions: These results propose a possible immunomodulatory target that may help illuminate the interactions between parasites and their hosts at the molecular level and reveal innovative protein species as candidate drug and vaccine targets.

Keywords: Haemonchus contortus; Immunomodulation; Peripheral blood mononuclear cell (PBMC); Rhodanese.

Fig. 1

Purification of recombinant rhodanese protein from Haemonchus contortus (rHCRD) and western blots. a Separation of purified rHCRD by SDS-PAGE using a 12% polyacrylamide gel and Coomassie brilliant blue R250 staining. b Western blots of rHCRD after purification. Antisera obtained from goats experimentally infected with H. contortus was used as the primary antibody to recognize the protein (Lane 1), whereas sera from uninfected goats was used as the control (Lane 2)

Fig. 2

Immunohistochemical localization of native rhodanese protein from Haemonchus contortus (HCRD) in frozen sections of H. contortus. Cy3-labelled goat anti-rat IgG (ab6953, Abcam) was used as the secondary antibody to detect HCRD protein by indirect immunofluorescence. DAPI was used to counter-stain sections in order to observe DNA

Fig. 3

Conjugation of recombinant rhodanese protein from Haemonchus contortus (rHCRD) and goat peripheral blood mononuclear cells (PBMCs). Goat PBMCs were treated with rHCRD (40 μg/ml) or untreated at 37 °C for 1 h. After fixing, the cells were incubated with rat anti-rHCRD antibody followed by Cy3-labelled goat anti-rat IgG (red), and DAPI (blue) staining was used to visualize the nuclei. A confocal laser scanning microscope was used to visualize the binding of rHCRD and goat PBMCs. The overlapping blue and red channels were merged. Tests were independently performed in triplicate

Fig. 4

Inhibitory effect of recombinant rhodanese protein from Haemonchus contortus (rHCRD) on goat peripheral blood mononuclear cell (PBMC) proliferation. ConA (10 μg/ml) was used to stimulate goat PBMCs for 72 h with or without a range of concentrations of rHCRD and His-tagged protein. CCK-8 incorporation was used to measure proliferation and the cell proliferation index was calculated based on the assumption that the absorbance at 450 nm of the blank group was 100%. Tests were independently performed in triplicate. (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

Recombinant rhodanese protein from Haemonchus contortus (rHCRD) did not induce apoptosis in goat peripheral blood mononuclear cells (PBMCs). PBMCs were cultured for 24 h with or without a range of concentrations of rHCRD and His-tagged protein. Propidium iodide (PI) and annexin V were used to stain the cells, which were subsequently analyzed by flow cytometry to quantify apoptotic cells. a Apoptotic cells (annexin V+/PI-) were plotted as a percentage of the total cell population. b Death of goat PBMCs after exposure to rHCRD is shown by a dot plot. Tests were independently performed in triplicate. (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 6

The cytokine profile of goat peripheral blood mononuclear cells (PBMCs) is modulated by recombinant rhodanese protein from Haemonchus contortus (rHCRD). Concanavalin A (ConA, 10 μg/ml) was used to stimulate goat PBMCs for 72 h with or without a series of concentrations of rHCRD and His-tagged protein. Enzyme-linked immunosorbent assays (ELISAs) were used to quantify cytokine secretion in the cell culture supernatant. a IL-2. b IL-4. c IL-17A. d IL-10. e TNF-α. f TGF-β1. g IFN-γ. Tests were independently performed in triplicate. (*P < 0.05, ** P< 0.01, ***P < 0.001)

Fig. 7

Inhibitory effect of recombinant rhodanese protein from Haemonchus contortus (rHCRD) on phagocytosis of goat monocytes. Cells were collected after rHCRD or his-tagged protein treatment for 48 h and incubated with FITC-dextran (1 mg/ml) for 1 h at 37 °C. The phagocytic activity of cells was analyzed on flow cytometry and calculated as mean fluorescence intensity (MFI). The data presented are results of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 8

Inhibitory effect of recombinant rhodanese protein from Haemonchus contortus (rHCRD) on the expression of MHC-II by goat monocytes. Cells were cultured in the presence of varies rHCRD concentrations and his-tagged protein or control buffer (PBS/DTT) for 24 h. The cells treated with LPS were used as positive control. MHC-II expression was analysed on flow cytometric analysis and calculated as the percentage of mean fluorescence intensity (MFI) of controls. Bars represent the MFI ± SD of controls. The data presented are results of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). a MHC-I. b MHC-II