2.1 Ethical statement
The treatments of animals in our research conformed with the guidelines of the Animal Welfare Board of Nanjing Agricultural University, China. The protocols were approved by the Animal Ethics Committee of Nanjing Agricultural University, China. Science and Technology Agency of Jiangsu Province authorized the trials (Approval ID: SYXK (SU) 2010-0005).
2.2 Animals and cells
Wistar rats (female, body weight 180-220 g) were obtained from the Experimental Animal Center of Jiangsu, China (SCXK 2018-004), and fed in the Animal Experimental Laboratory of Nanjing Agricultural University under aseptic conditions.
5-7-month-old goats were reared in ventilated cages under clean and hygienic conditions to prevent accidental nematode infections. They were also provided with hay, water and alfalfa pellets at all times. During the study, no nematode eggs were found in goat feces. As described previously, peripheral blood mononuclear cells (PBMCs) from goats were isolated and cultured using peripheral venous blood samples (10mL per goat) obtained by venipuncture [16].
2.3 Cloning genes encoding HcL6 and sequence analysis
Total RNA was extracted from H. contortus adult worms using Trizol reagent (Invitrogen, New York, USA) [17]. The RNA (OD260/280 = 1.99, 1421.26 ng/μL) was transcripted by a cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s instructions. The genes encoding HcL6 were amplified by reverse transcriptase PCR (RT-PCR) using specific primers (Table 1) based on the sequences of HcL6 (EBI No. CDL93639.1). The PCR products were ligated into a prokaryotic expression vector (pET-28a (+)) (Novagen, USA) to generate recombinant HcL6, followed by endonuclease cleavage and sequence analysis (SnapGene 4.3.7, USA; Additional file 1).
2.4 Expression and purification of recombinant HcL6 protein
The expression of recombinant HcL6 protein was performed as described [18]. In brief, the recombinant plasmid pET-28a-HcL6 was transfected into E.coli BL21 (DE3) and incubated in Luria-Bertini medium with kanamycin at 37 ℃ until OD600 range from 0.6 to 0.8. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to induce protein expression. The his-tag fused recombinant proteins were purified with His TrapTM FF kit (GE Healthcare, USA) following the manufacturer’s protocol. ToxinEraserTM Endotoxin Removal kit (Genscript biotech company, Nanjing, Jiangsu, China) was used to remove endotoxin from the sample. Afterwards, 0.22 m filters were used to filter the rHcL6. A BCA method was used to determine the protein concentration (776.09 μg/mL) [19] and 12% SDS-PAGE gels were used to confirm the size and purity of the rHcL6 protein.
2.5 Preparation of polyclonal antibodies
Wistar rats were given 300 g of rHcL6 protein blended with Freund's complete adjuvant (FCA, diluted 1:1, Sigma, USA) to generate polyclonal antibodies. After the primary immunization, rats were boosted 4 times with the same dose of rHcL6 protein with Freund’s incomplete adjuvant (FIA, dilution to 1:1, Sigma, United States) at one-week intervals. In rat sera, the specific IgG titer (1:223) was determined by indirect enzyme-linked immunoabsorbent assay (ELISA) as previously described [20]. A negative control will be collected from rats before vaccination. All sera were stored at -70 °C.
2.6 Western blot analysis
rHcL6 proteins were resolved on 12% SDS-PAGE gels and electro-transferred onto nitrocellulose membranes. The membranes were incubated in 5% skimmed milk in TRIS-buffered saline containing 0.1% Tween-20 (TBST) at 37℃ for 1 h to block the non-specific binding sites. The blots of rHcL6 samples were treated with rat anti-rHcL6 serum (1:300 in TBST) or normal rat serum (control) overnight at 4℃. After the membranes were washed five times in TBST, horseradish peroxidase (HRP)-conjugated rabbit anti-rat IgG secondary antibody (Sigma-Aldrich) in TBST (1:5000) were incubated with the membranes for 1 h at 37℃. Finally, immunoreactions were detected by DAB kit (Sigma-Aldrich) for 3–5min color development.
2.7 The binding of HcL6 protein with Th9 cells in vitro
Total Th9 cells in goat PBMCs were sorted by the flow-sorting cytometry method as described elsewhere [15]. Briefly, PBMCs were cultured in RPMI-1640 media containing rHcL6 for 2 h at 37 °C in a humidified 5% CO2 incubator. Following that, phorbol myristate acetate (PMA, Sigma-Aldrich, MO, USA) and ionomycin (Sigma-Aldrich, MO, USA) were added to cells containing Brefeldin A Solution (BFA; BD Biosciences, San Jose, CA, USA) for another 4-6 hours before intracellular dyeing was performed [5]. After which, cells were obtained and incubated with fluorescein FITC-labeled anti-CD2, Alexa Fluor 488 dye-tagged CD4 antibodies (BD Biosciences, Becton, USA), PE-cy5-conjugated anti-IL-9 (GenScript, New Jersey, USA) and PE-conjugated anti-IL-10 antibodies (BD, Pharmingen, USA). The labeled cells were suspended in 500 µL PBS and analyzed with BD FACS ARIA II SORP (BD Biosciences) [21]. The Th9 cell population was represented by the CD2+CD4+IL-9+IL-10+ phenotype.
The collected Th9 cells were subjected to three washes using PBS (pH 7.4). Subsequently, Tide Quencher™ 3WS acid, Tide Quencher™ 2WS acid, and Tide Quencher™ 5WS acid (AAT Bioquest, CA, USA) were introduced to quench fluorescence. This process was conducted over 5 hours until no residual fluorescence was detectable. After several additional washes, the cell suspensions were loaded onto adhesion microscope slides and allowed to settle for 15-20 minutes before the supernatant was discarded. The Th9 cells were fixed using 4% paraformaldehyde (PA) for 15 minutes at room temperature. This step was followed by incubation with 5% bovine serum albumin (BSA) in PBS, fortified with 0.1% Tween-20 (PBST), at 37℃ for 30 minutes. The slides were then incubated overnight at 4℃ with rat anti-rHcL6 IgG (1:300) or normal rat IgG (serving as a control). Following three washes with PBST, Cy3-labeled goat anti-rat IgG (1:500) was applied to the slides and incubated for one hour at 37°C. Three additional PBST washes preceded the nuclear staining with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Sigma-Aldrich) for five minutes at room temperature. An anti-fade medium (Sigma-Aldrich) was utilized to prevent fluorescence quenching of the samples prior to microscopic examination. The final stage involved imaging the slides using an LSM780 laser scanning confocal microscope (Zeiss, Jena, Germany) at 100× magnification. Digital images were processed and analyzed using the ZEN 2012 software (Zeiss).
2.8 Detection of IL-9 transcriptions
The transcription of IL-9 was detected by real-time PCR. PBMCs (1 × 106 cells/mL) were incubated with various concentrations of rHcL6 (5, 10, 20, 40, 60 μg/mL) in vitro in 24-well culture plates supplied with RPMI-1640 medium for 24 h at 37 ℃. Subsequently, RNA was extracted from these cells using Total RNA Kit (Omega, USA) according to the manufacturer’s instructions. The mRNA transcription of IL-9 was assessed as described elsewhere [15]. Specific primers for the β-actin gene (endogenous reference) and target gene IL-9 (F: 5’-GATGCGGCTGATTGTTT-3’; R: 5’-CTCGTGCTCACTGTGGAGT-3’) were used and the relative transcription levels of IL-9 were normalized on β-actin based on the 2-∆∆Ct method [5]. Real-time PCR was performed and the data were recorded by ABI 7500 application (Applied Biosystems, USA).
2.9 IL-9 concentration assays
ELISA was used to examine the effects of rHcL6 on IL-9 secretion. PBMCs (1 × 106 cells/mL) were stimulated with lipopolysaccharide (LPS, 100 ng/mL). 2 h following stimulation, 2 mL of cells (1 × 106 cells/mL) were added to each well of a 6-well plate. Then cells were incubated with different concentrations of rHcL6 to check whether the protein could stimulate IL-9 secretion or not. All the samples were incubated at 37 ℃ and 5 % CO2 conditions. After 24 h incubations, the supernatants were collected by centrifugation. The IL-9 concentration in the supernatants was measured by ELISA. Different concentrations of IL-9 proteins (300pg/mL, 150pg/mL, 75pg/mL, 37.5pg/mL, 18.75pg/mL, 9.38pg/mL and 4.69pg/mL) and 100 μL of each cell supernatant were placed into a 96-well plate containing IL-9 monoclonal antibody at 37℃ for 90 minutes. Removing the supernatant, rat anti-IL-9 IgG polyclonal antibody coupled with biotin (1:200) were added to each well at 37℃ for 1 h. After being washed three times with TBST, the avidin-biotin-peroxidase complex was added at 37℃ for 30 mins. After three washes with TBST, TMB was added for 15-20 mins in the dark, then ELISA stop solution was added to finish the reaction. The optical density at 450 nm was measured using a microplate reader (Bio-Rad, Hercules, California, USA). Three independent experiments were performed in the test with three technical replicates for each group.
2.10 Detection of Th9 cells differentiation
To estimate the effects of rHcL6 on Th9 cells, PBMCs were incubated with various concentrations of rHcL6 (5, 10, 20, 40, and 60 μg/mL) in vitro in 12-well culture plates supplied with RPMI-1640 medium for 12 h at 37 ℃ with 5% CO2. Therefore, the method is the same as in 2.7.
2.11 Detection of key genes in Th9 immune response signaling pathways
This study investigated the transcription of key genes in Th9 immune response signaling pathways such as STAT1, STAT5, GTAT3, SMAD, NFAT, IRF4, PU.1, STAT6 and NF-ĸB by real-time PCR. PBMCs (1 × 106 cells/mL) were incubated with 20 μg/mL rHcL6 in vitro in 24-well culture plates supplied with RPMI-1640 medium for 24 h at 37 ℃. Subsequently, the method is the same as in 2.8. Therefore, the primers of these nine key genes were listed in Table 2.
2.12 Knocking down of STAT6/NF-κB/PU.1 gene by RNA interference
Three single sequence siRNA duplex (RiBo Bio, Guangzhou, MA) of STAT6/NF-ĸB/PU.1 siRNA were procured (Table 3). Transfection of these siRNAs was accomplished using the RNAiMAX Transfection Reagent (Thermo Fisher, MA, USA). Following transfection, the culture medium was replaced with fresh medium, and after 48 hours, the cells were ready for treatment. A dose-response study was initially conducted to assess the impact of 0nM, 10nM, 20 nM, 40nM, and 80nM of STAT6/NF-κB/PU.1 siRNA on the transcription level of STAT6/NF-κB/PU.1. Following this, transfected cells with 20nM of STAT6, 20nM of NF-κB, and 40nM of PU.1 were used to examine the expression of IL-9 and key genes within the Th9 immune response signaling pathways.
2.13 Statistical study
Statistical analyses were carried out using GraphPad Prism 7.0 (GraphPad Prism, USA). All data are presented as mean ± SD. Differences between groups were evaluated by one-way ANOVA followed by Tukey’s post-hoc test. The thresholds for statistical significance were set at *p < 0.05, **p < 0.01, and ***p < 0.001. Flow cytometry data were analyzed employing Flow Jo software (Version 10, USA).