Parasites, animals, and cells
The H. contortus strain Nanjing 2005 was acquired from Nanjing in Jiangsu province, China. Helminth-free goats aged 3–6 months were used to maintain the strain through continuous passage . Experimental animals were challenged with stage 3 larvae (L3), which were isolated from the feces of goats given monospecific infections, cultured at 26 °C and preserved in water at 4 °C at a larval concentration of 2500 per ml.
Native mongrel male goats aged 3–6 months were acquired from a herd maintained for study and education at Nanjing Agricultural University and kept in indoor pens containing six goats per pen. The goats were provided with cured hay and whole grain maize and allowed to drink water freely. Levamisole at a dose of 8 mg per kg body weight was given P.O. every 14 days to expel naturally-occurring strongylids. After 14 days, helminth eggs were collected from goat feces under a light microscope using standard parasitology techniques. Goats confirmed to be free from nematode infections were utilized to conduct the following experiments. Goats were observed every day throughout the study to ensure their health.
Sprague-Dawley rats weighing approximately 150 g were purchased from the Experimental Animal Center of Jiangsu, China (qualified certificate: SCXK 2008-0004), maintained in a sterile environment and supplied with sterile food and water.
The standard Ficoll-Hypaque (GE Healthcare, Little Chalfont, UK) gradient centrifugation method was used to separate PBMCs from whole blood with added heparin, and the obtained cells were washed twice with phosphate buffered saline (PBS) . The cell density was then adjusted to 1×106 cells/ml and cells were cultured in RPMI 1640 (GIBCO, Grand Island, New York, USA) containing 100 mg/ml streptomycin (GIBCO), 100 U/ml penicillin (GIBCO) and 10% heat-inactivated fetal calf serum (GIBCO) in a humidified cell chamber at 37 °C and 5% CO2. Trypan blue dye was used to determine cell viability, which was >95% for all relevant experiments.
To obtain goat monocytes, a 6-well flat-bottom tissue culture plate (Corning, New York, USA) was used to culture goat PBMCs with RPMI 1640 culture medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO), 100 U/ml penicillin and 100 mg/ml streptomycin (GIBCO). Plates were incubated at 37 °C in a humidified atmosphere with 5% CO2 for 1 h. Non-adherent cells were drained by washing twice with PBS. The cells (monocytes) stuck to the bottom of the plate were collected and adjusted to a density of 1×106 cells/ml, and viability was confirmed to be >95% in all preparations using a trypan blue exclusion test.
Cloning of HCRD and bioinformatics analyses
Based on the sequence of the open reading frame (ORF) of the rhodanese-like gene retrieved from the online database (GenBank accession number: CDJ82729.1), the following primers were designed to specifically amplify the gene using reverse transcription-polymerase chain reaction (RT-PCR): forward primer, 5′-ACGGATCCATGATGTGTCCACCTCCA-3′; and reverse primer, 5′-GCAAGCTTGGAGAACTGTAACTGCCT-3′. The underlined sequences indicate BamHI and HindIII restriction endonuclease sites. The RT-PCR products were ligated with pMD19-T vector (Takara, Dalian, China) to produce pMD-rhodanese. Fragments of rhodanese were then cleaved from the pMD-rhodanese plasmid using BamHI and HindIII before being subcloned into the relevant location of the pET32a vector (Invitrogen, Carlsbad, CA, USA). Sequencing analysis was used to confirm proper plasmid construction.
Expression and purification of rHCRD in Escherichia coli
To express the recombinant fusion protein, isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM was used to induce Escherichia coli BL-21 cells (DE3) cultured in Luria-Bertini (LB) medium containing 100 μg/ml ampicillin at 37 °C for 6 h. A His•Bind® 128 Resin Chromatography kit (Novagen, Madison, WI, USA) was used to purify the histidine (His)-tagged fusion protein from the precipitated bacterial lysate according to the manufacturer’s instructions. The refolded protein was purified in renaturation buffer (20 mM Tris-Cl, 500 mM NaCl, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, pH 8.0) containing different concentrations of urea (0, 2, 4, 6 or 8 M) and dialyzed using PBS (pH 7.4). The empty pET32a vector was used to produce a control His-tagged protein, which was expressed and purified using an identical procedure to that used to produce the rhodanese-His-tagged fusion protein. Subsequently, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie bright blue staining were used to assess the purity of rHCRD after purification, and the Bradford method was used to quantify protein samples. Detoxi-Gel Affinity Pak prepacked columns (Thermo Fisher Scientific, Waltham, MA, USA) were used to deplete lipopolysaccharide from the obtained rHCRD protein, and the concentration of the recombinant protein samples was adjusted to 1 mg/ml before performing limulus amebocyte lysate (LAL) assays. A Pyrosate® Kit (Cape Cod Inc., East Falmouth, MA, USA) was used for LAL gel clot assays to measure endotoxin units (EU) in protein samples. Only samples with endotoxin levels lower than 1 EU/mg recombinant protein were used in further experiments.
Generation of polyclonal antibodies
Five goats were used to produce antisera against H. contortus, which was used for western blots. A total of 5000 L3 larvae with infective activity was administered P.O. to goats who were maintained in a helminth-free environment. After 30 days, antiserum samples were collected and preserved at -70 °C for further use.
In order to generate polyclonal antibodies against rHCRD, a mixture of Freund’s complete adjuvant and 0.3 mg purified rHCRD was used to inoculate Sprague-Dawley rats by subcutaneous injection at a series of sites, based on the protocol proposed by Wang et al. . The rats received an initial immunization followed by four booster immunizations of the same dose at intervals of 14 days. Ten days after the final immunization, antiserum, which contained specific antibodies against rHCRD, was collected, and its reactivity was determined using an enzyme-linked immunosorbent assay (ELISA).
Western blot analysis
Protein samples containing 20 μg purified rHCRD were isolated by 12% SDS-PAGE, and the protein bands in the gel were transferred onto Hybond-C Extra nitrocellulose membranes (Amersham Biosciences, London, UK). The membranes were immersed in blocking buffer containing 5% skim milk and Tris-buffered saline (TBS) for 1 h under ambient conditions to block non-specific binding sites. Subsequently, TBS containing 0.1% Tween-20 (TBST) was used to wash the membranes five times for 5 min each. The primary antibody (i.e., antiserum obtained from goats experimentally infected with H. contortus) was diluted 1:100 in TBST and used to incubate the membranes for 1 h at 37 °C. The membranes were then washed five times with TBST and treated with horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (Sigma, St. Louis, MO, USA) diluted 1:2000 in TBST for 1 h at 37 °C. Finally, a fresh preparation of diaminobenzidine (DAB, Sigma), which acted as a chromogenic reagent, was added for 5 min to visualize the immune reaction.
Localization of HCRD by immunohistochemistry
Adult nematodes were immersed in TISSUE-TeK® O.C.T. compound (SAKURA, Torrance, CA, USA), washed with PBS and immersed in PBS containing 0.2% glutaraldehyde and 4% formaldehyde for 1.5 h. The nematodes were then flash frozen in liquid nitrogen and stored at -20 °C for further use. Cryostat sections with a thickness of 10 μm were washed with PBS and immersed in PBS containing 10% normal goat serum in order to block non-specific binding sites. Subsequently, serum from rats immunized with rHCRD diluted to 1:100 was used to incubate separate sections for 1 h at 37 °C. Serum from normal rats was used as the control. The sections were then washed three times with PBS for 15 min and treated with Cy3 goat anti-rat IgG (ab6953, Abcam, Cambridge, MA, USA) for 1 h. Finally, DAPI (Beyotime, Haimen, Jiangsu, China) and PBS were used to stain and wash the sections, respectively, and Anti-Fade Fluoromount solution (Beyotime) was applied to prevent fading as the samples were observed under a fluorescent microscope.
Binding of rHCRD to goat PBMCs
Goat PBMCs were isolated immediately prior to experiments as described above, inoculated into 24-well plates and cultured with 40 μg/ml rHCRD on glass cover slides for 1 h at 37 °C. Untreated cells were used as controls. Then, 0.1 M PBS was used to wash the slides, and 4% paraformaldehyde was used to fix the slides under ambient conditions for 30 min. Subsequently, PBS containing 5% normal goat serum was used to incubate the slides to block non-specific binding sites, and rat polyclonal antibody against rHCRD diluted 1:100 in PBS containing 5% normal goat serum was used to incubate the slides overnight at 4 °C. Cy3 goat anti-rat IgG (ab6953, Abcam) diluted 1:400 in PBS containing 5% normal goat serum was used to incubate the slides at 37 °C for 1 h before counterstaining with DAPI (Beyotime). The nucleus and rHCRD were indicated by blue and red, respectively, under scanning confocal laser microscopy (LSM710, Zeiss, Jena, Germany) with an oil immersion lens. Images were acquired by selecting the blue and red color channels corresponding to DAPI and Cy3, respectively. The fluorescent microscope settings used for observing control samples were identical to those used for rHCRD-treated cells. Unstained controls were used to detect background staining and auto-fluorescence of the protein and cells. ZEN software (Zeiss) was used to perform synergistic combinations and for photo acquisition. Observations were independently collected from three individual samples.
Cell proliferation assays
Concanavalin A (ConA, 10 μg/ml) was used to activate goat PBMCs at the same time as they were incubated with a range of concentrations of rHCRD at 37 °C and 5% CO2 for 72 h. Cell Counting Kit-8 assay reagent (Beyotime) was added into each well of a 96-well plate and the OD450 was measured using a microplate reader (Thermo Fisher Scientific) after incubation for 4 h. The OD450 of control cells in the blank group was set as 100%. The following equation was used to calculate the proliferation index of the cells: OD450 sample/OD450 control.
Flow cytometry was used to analyze cell apoptosis, as previously described . Briefly, a range of concentrations of rHCRD was used to culture goat PBMCs. Control cells received no rHCRD treatment. Subsequently, the cells were stained with annexin V and propidium iodide (PI, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.
Detection of cytokine secretion
To assess cytokine secretion, 10 μg/ml of ConA with or without rHCRD was used to stimulate goat PBMCs for 72 h. After collecting the supernatant, cytokines were measured using ELISAs. Commercial goat ELISA kits (Mlbio, Shanghai, China) were used to determine the concentrations of IL-2, IL-4, IL-10, IL-17A, TNF-α, IFN-γ and TGF-β1 in the supernatant of samples. Data obtained from three individual experiments were used to perform the analyses.
The phagocytic ability of goat monocytes in response to rHCRD was determined by FITC-dextran internalization followed by flow cytometry analysis (BD Biosciences). Monocytes were treated with rHCRD for two days and then incubated with FITC-dextran (1 mg/ml in RPMI1640) for 1 h at 37 °C. Cells incubated with an equal concentration of FITC-Dextran were used as the baseline for monocyte phagocytosis. Finally, cells were washed twice to eliminate excess FITC-dextran and results were analyzed using FlowJo 7.6 software (Tree Star, Ashland, OR, USA) with the median fluorescence intensity (MFI) values of the control set to 100%.
Analysis of major histocompatibility complex molecule expression
The purified monocytes (0.5×106 cells/ml) were poured into 24-well culture plates containing complete RPMI 1640 and different concentrations of rHCRD or equal volumes of control buffer for 24 h at 37 °C. Afterward, monocytes were marked with monoclonal major histocompatibility complex (MHC)-I (MCA2189A647) and MHC-II (MCA2226F) antibodies (AbD Serotec, BioRad Laboratories, CA, USA). The results were expressed as the percentage of MFI and analyzed on a FACS Calibur cytometer (BD Biosciences).
The data are represented as average ± standard deviation. Analysis of variance was used to calculate significant differences and Student’s t-tests were used to evaluate parametric samples (GraphPad Prism, San Diego, CA, USA).