The following reagents were purchased for recombinant protein expression: Escherichia coli (E. coli) BL21 (DE3) plysS cells (Merck KGaA), pMD 19-T vectors (Takara Biotechnology Co., Ltd.), pET-His vectors (Wuhan Miaoling Bioscience & Technology Co., Ltd.), and primer STAR HS DNA polymerase (Takara Biotechnology Co., Ltd.). Af RNASET2 cDNAs were synthesized by Nanjing GenScript Biotech Corp. Lysozyme (Sangon Biotech Co., Ltd.) and blot membranes (nitrocellulose and polyvinylidene fluoride; Merck KGaA) were used for pre-purification cell lysis and electrophoresis analysis, respectively.

A total of 18 female BALB/c mice (6 weeks of age; 20-22 g) were purchased from Guangdong Medical Laboratory Animal Center, and housed in a specific pathogen free facility with six mice per cage under a stable temperature (24±1°C) and humidity (55±10%). Mice were kept in open polypropylene cages with clean chip bedding under a 12-h light/dark cycle with free access to a standard rodent diet. The animals were acclimatized to the laboratory for at least 1 week prior to the start of the experiments. The health status of experimental mice was monitored twice daily and humane endpoints were used to determine if mice met the criteria for euthanasia prior to the study end point, namely weight loss exceeding 10-15% of the body weight, lethargy, inability to stand, or anorexia. All surgical procedures were performed under 1.5-2% isoflurane anesthesia. At the end of the experiments, animals were humanely sacrificed using CO₂ asphyxiation. The flow rate of CO₂ displaced ~50% of the chamber volume per minute for euthanasia. The gas flow was maintained for at least 1 min following clinical death, and death was evaluated monitoring the lack of movement or visible breathing for 5 min. Both the breeder of the animals and the assessor of the results are blinded. All studies involving mice were performed according to protocols approved by the Animal Ethical and Welfare Committee of the School of Medicine of Shenzhen University. All efforts were made to minimize discomfort and suffering of the animals.

The RAW264.7 murine macrophages were purchased from Guangzhou Cellcook Biotechnology Co., Ltd., and cultured in complete DMEM with 10% FBS and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin), at 37°C in a humidified incubator with 5% CO₂.

The analysis of the amino acid sequence alignment was performed to investigate the sequence conservation of Af RNASET2 gene in the ribonuclease T2 family. GenBank (https://www.ncbi.nlm.nih.gov/genbank) was used to download the amino acid sequences of Af RNASET2 [GenBank ID, XP754496.2; based on the Af AF293 genome (Genome ID, 18)], Sj CP1412 (GenBank ID, AY570741.2), Sm ω-1 (GenBank ID, DQ013207.1), and Clonorchis sinensis (Cs) RNASET2 (GenBank ID, GAA50115.1) were imported into DNAMAN software (version 8.0; Lynnon Biosoft) for alignment. The sequences were saved in FASTA format for multiple alignment analysis. The homologous alignment was calculated according to the ratio of the number of conserved amino acids to total number of amino acids.

The Af RNASET2 cDNA was synthesized following codon optimization and submitted to the National Center for Biotechnology Information (NCBI) database. The Af RNASET2 gene was amplified using PCR with Primer STAR HS DNA polymerase and the aforementioned cDNA template. The following PCR primers were used: Forward, 5′-AGA TGG ATC CAT GAA ATT CAA CAT AAC TAT CGC-3′ and reverse 5′-AGC GAT AAG CTT ATG TAC ATG TTA ATT CTT TTT CA-3′; and the following thermocycling conditions: Initial denaturation at 94°C for 30 sec, and 30 cycles of 55°C for 30 sec, and 72°C for 50 sec. The PCR product, confirmed by Sanger DNA sequencing (GenScript Biotech Corporation), was subcloned into a pET-His vector with BamHI and HindIII restriction enzymes. The recombinant expression vector pET-His-Af RNASET2 was confirmed by double enzyme digestion with BamHI/HindIII. Following double enzyme digestion with BamHI/HindIII for 2 h at 37°C, the products were analyzed using a 1% agarose gel.

The recombinant plasmid pET-His-Af RNASET2 was then transformed into E. coli BL21 (DE3) plysS cells. The transformed E. coli were grown overnight in Luria-Bertani medium containing 100 mg/l ampicillin at 37°C, and induction was performed using isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. Following cultivation and an additional 3 h at 37°C, E. coli cells were harvested by centrifugation at 9,600 x g for 5 min at 4°C. Following mixing with protein extraction buffer (20 mM Tris HCl, 150 mM NaCl and 1 mg/ml lysozyme), harvested cells were sonicated (50 kHz; 3 sec bursts, 5 sec inter-burst interval for a total of 20 min) in an ice bath, and then centrifuged at 9,600 x g for 20 min at 4°C. Subsequently, 10% SDS-PAGE was performed to analyze the status of recombinant protein expression. The recombinant protein in the soluble fraction was purified with a Ni-NTA column (cat. no. 17040303) and gel filter (Hi Load Superdex 16/600; cat. no. 28-9893-33) (both from GE Healthcare). Specifically, rAf RNASET2 was expressed in the inclusion bodies, which were dissolved in 6 M urea with 25 mM β-mercaptoethanol. Denatured proteins were refolded and diluted 10-fold with 0.5 M L-arginine (pH 8.0) (19). Arginine buffer was added to the denatured protein solution slowly (flow rate of 0.5 ml/min) and incubated overnight at 4°C. For renaturation, rAf RNASET2 was dialyzed with PBS at 4°C for 24 h via a dialysis membrane (Sangon Biotech Co., Ltd.) (19). Following renaturation, protein concentrations were determined using the Bradford method (Sangon Biotech Co., Ltd). The rAf RNASET2 protein endotoxin was removed using an endotoxin removal kit following the manufacturer's instructions and quantitation was performed using an endotoxin assay kit, which revealed that the levels were reduced to <0.25 endotoxin units/ml (both Genscript Biotech Corporation).

Yeast RNA (Thermo Fisher Scientific, Inc.) was dissolved in reaction buffer (50 mmol/l Tris HCl, 50 mmol/l NaCl, pH 7.0) to prepare a stock solution (1 mg/ml). The rAf RNASET2 (1 µg) aliquots were mixed thoroughly with 50 µg yeast RNA at 37°C for 20, 40, 60, 80, and 100 min. In addition, a 30-min pretreatment with an RNase inhibitor (RNasin; Thermo Fisher Scientific, Inc.) served as a positive control for confirmation of digestion by RNASET2. Ribonuclease activity was observed using the analysis of enzymolysis products in 1% agarose electrophoresis gels, which was stained with Gelview dye (BioTeke Corporation). Bands were visualized on a UV transilluminator (Analytik Jena US LLC).

RAW264.7 macrophages (1×10⁶) were incubated with the indicated concentrations (20 or 40 µg/ml) of rAf RNASET2 (pretreated with RNasin) in PBS at 37°C under 5% CO₂. After 24 h of co-culture, the cells and supernatant were collected and centrifuged at 300-400 × g for 5 min at 4°C. The cells were collected and incubated with the labeled antibodies (FITC-CD16/32 (cat. no. 11-0161-81; 1:500; BioLegend, Inc.) or FITC-CD206 (cat. no. MA5-16870; 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) for M2 or M1 macrophage detection, respectively, and APC-F4/80 (cat. no. 17-4801-80; 1:500; BioLegend, Inc.) in PBS containing 1% bovine serum albumin (Amresco LLC) for flow cytometry. All incubations were performed at room temperature for 30 min in the dark and subsequently, the cells were washed with PBS. Macrophage surface markers were detected using a Cytoflex flow cytometer and analyzed using the CytExpert software (version 2.0) (both Beckman Coulter, Inc.). IL-10 levels were measured in the culture supernatant of RAW264.7 cells stimulated by incubation with rAf RNASET2 (20 µg/ml) alone or in the presence of MAPK signal inhibitors (U0126, 20 µM; SP600125, 20 µM and SB203580, 1 µM) for 24 h. The cells were centrifuged at 300-400 x g for 5 min at 4°C and subsequently analyzed using ELISA kits according to the manufacturer's instructions (cat. no. HZ-IL-10-Mu; Shanghai and Shanghai Zhen Biotech Co., Ltd.).

RAW264.7 macrophages were co-cultured with rAf RNASET2 (20 µg/ml), inactive rAf RNASET2 (pretreated with RNasin (2 U/µl)), and PBS and LPS controls at 37°C with 5% CO₂ for 24 h, and with rAf RNASET2 (20 µg/ml) alone or in the presence of MAPK signal inhibitors (U0126, 20 µM; SP600125, 20 µM and SB203580, 1 µM) for 24 h, under normal cell culture conditions. RNA was extracted after the cells were centrifuged at 300-400 x g for 5 min at 4°C, using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was subsequently reverse transcribed into cDNA using a TIANScript II RT kit (Tiangen Biotech, Co., Ltd.) according to the manufacturer's protocol: 42°C for 50 min, followed by 95°C for 5 min then cooled on the ice at 4°C for 10 min. RT-qPCR analysis was performed with SYBRGreen (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the primers listed in Table I.

For the qPCR, 5 µl 2X SYBRGreen PCR buffer, 0.2 µl forward and reverse primers (both 10 µmol), and 5 ng template were used to a total volume of 10 µl with sterile water. The thermocycling conditions used were as follows: Initial denaturation 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The real-time PCR was performed on a qTOWER 2.2 system (Analytik Jena US LLC). All qPCRs were performed in triplicate with 18S rRNA gene as a reference housekeeping gene. Relative expression was calculated using the 2−^ΔΔCq method (20).

RAW264.7 macrophages were co-cultured with rAf RNASET2 (20 µg/ml), inactive rAf RNASET2 (pretreated with RNasin (2 U/µl), and PBS and LPS controls at 37°C in a humidified incubator with 5% CO₂ for 24 h. The cells were centrifuged at 300-400 × g for 5 min at 4°C, and subsequently lysed in RIPA buffer (Beyotime Insitute of Biotechnology) with a protease-inhibitor cocktail (MedChemExpress LLC). The protein concentration was determined using the Bradford assay (Sangon Biotech Co., Ltd.) and the protein samples were subsequently diluted to 1 mg/ml, then separated (20 µg/lane) using 10 or 12% SDS-PAGE, following which the proteins were transferred onto PVDF membranes. The membranes were blocked overnight at 4°C with 5% skimmed milk in TBS +0.05% Tween-20. The following primary antibodies were used: Phosphorylated (p)-ERK1/2 (cat. no. 4370; Cell Signaling Technology Inc.), ERK1/2 (cat. no. 46951; Cell Signaling Technology Inc.), p-p38 (cat. no. 4511; Cell Signaling Technology Inc.), p-38 (cat. no. 8690; Cell Signaling Technology Inc.) p-JNK (cat. no. 4668; Cell Signaling Technology Inc.), JNK (cat. no. 9252; Cell Signaling Technology Inc.), IL-10 (cat. no. ab33471; Abcam), induced nitric oxide synthase (iNOS; cat. no. ab213987; Abcam), ARG1 (cat. no. ab124917; Abcam), and cyclooxygenase 2 (COX2; ab179800; Abcam) (all 1:1,000) were used. The primary antibody for the loading control, GAPDH (cat. no. sc-365062; Cell Signaling Technology, Inc.) was used at a dilution of 1:2,000. After washing with PBS, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. nos. sc-2004 and sc-2005) (both 1:2,000 and Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Proteins were visualized using an enhanced chemiluminescence kit (Dalian Meilun Biology Technology Co., Ltd.) according to the manufacturer's protocol.

Purified rAf RNASET2 was mixed with Imject Aluminum adjuvant (Thermo Fisher Scientific, Inc.) to form a water-in-oil adjuvant antigen. Mice were randomly divided into immunized, adjuvant control, and PBS control groups (n=6 per group), and injected intraperitoneally with 100 µg rAf RNASET2 and 2 mg of aluminum adjuvant (Af RNASET2 + adjuvant), 2 mg of aluminum adjuvant alone (adjuvant), or with PBS only (control) once a week for 3 weeks. A total of 7 days following the third immunization injection, about 50 µl orbital venous blood samples were collected and the sera were isolated following centrifugation at 3,000 x g for 20 min at 4°C. The levels of IL-4, IL-13, IL-10, and interferon-γ (INF-γ) in mouse sera were detected using cytokine ELISA kits (IL-4, cat. no. HZ-IL-4-Mu; IL-10, cat. no. HZ-IL-10-Mu; IL-13, cat. no. HZ-IL-13-Mu and INF-γ, cat. no. HZ-INF-γ-Mu) (all Shanghai and Shanghai Zhen Biotech Co., Ltd.) according to the manufacturer's instructions.

Quantitative data are presented as mean ± SDs. Analyses were performed using Prism 7 software (GraphPad Software, Inc.). Student's t-tests or one-way ANOVA followed by Dunnett's post hoc test were performed, to detect statistical significance between groups. P<0.05 was considered to indicate a statistically significant difference.

The amino acid sequence of Af RNASET2 (GenBank XP_754496.2) was obtained based on the Af AF293 genome (Genome ID 18) in the NCBI database. The Af RNASET2 gene encodes 401 amino acids with a 15-amino-acid N-terminal secretory signal; the mature RNASET2 protein is 382 amino acids long. As shown in Fig. 1, a homology comparison based on amino acid sequence alignment revealed that Af RNASET2 was 11.84, 13.20, and 12.17% homologous with Sj CP1412, Sm ω-1, and Cs RNASET2, respectively. Notably, Af RNASET2 was predicted to contain two conserved amino acid sequence (CAS) domains (⁸³WTIHGLWP⁸⁹ and ¹⁴⁰WEHEWNKHG¹⁴⁸), namely CAS-1 and CAS-2, and a conserved pair of cysteine residues (cysteine¹⁰⁰ and cysteine¹⁵⁸) characteristic of T2 RNase-glycosylation sites were also found.

To obtain the rAf RNASET2 protein, cDNA encoding mature Af RNASET2 (glutamicacid 26-arginine 407 region), without its signaling peptide (methionine 1-phenylalanine 25 domain) was synthesized, submitted to the NCBI (GenBank MN593022), and subcloned to a pET-His prokaryotic expression vector for expression and purification. cDNA from Af RNASET2 were generated using PCR amplification and subcloned into pET-His vectors using BamHI/HindIII restriction enzymes. The band at 1,200 bp was consistent with the length of the Af RNASET2 gene (Fig. 2A). The pET-His-Af RNASET2 plasmid was confirmed using DNA sequencing. Subsequently, the pET-His-Af RNASET2 plasmid was transformed into E. coli BL21(DE3) plysS cells for expression and purification. SDS-PAGE revealed that rAf RNASET2 (~45 kDa) was expressed in the sediment following IPTG induction (Fig. 2B; Lane 3). A portion of the rAf RNASET2 protein sample was purified using Ni-NTA-resin and renatured into soluble proteins by gradient dialysis with urea (Fig. 2C). Purified soluble rAf RNASET2 lysed yeast RNA in a time-dependent manner and RNA digestion activity of rAf RNASET2 was effective with ribonuclease A (Fig. 2D) and could be inhibited with the ribonuclease inhibitor RNasin (Fig. 2E).

The levels of the Th2 response-related cytokines IL-4 (P>0.01; Fig. 3A), IL-10 (P<0.01; Fig. 3B), and IL-13 (P<0.01; Fig. 3C) were significantly increased in rAf RNASET2-immunized mouse sera, collected 7 days following the third immunization compared with that in PBS control groups. In addition, there was no change in the expression of the Th1 response-related cytokine, INF-γ, between Af RNASET2+ adjuvant and the control group (P>0.05; Fig. 3D). This indicates that rAf RNASET2 enhanced Th2 responses in vivo.

The flow cytometry results revealed that in vitro stimulation of RAW264.7 macrophages with purified rAf RNASET2 significantly increased the expression level of the M2 surface marker CD206 (Fig. 4B), Notably, CD206 expression was increased from 3.00 to 21.73% following treatment with 40 µg/ml rAf RNASET2 (P<0.01). However, there was no significant change in the level of the M1 surface marker CD16/32 (P<0.01; Fig. 4A).

The RT-qPCR results revealed that RAW264.7 cells treated with rAf RNASET2 for 24 h had significantly increased expression levels of M2-related genes, including Arg1 (P<0.01; Fig. 5A), Il-10 (P<0.01; Fig. 5C), and Il-13 (P<0.01; Fig. 5D), without a significant change in the expression level of the M2 marker iNOS (P>0.05; Fig. 5B). Western blot analysis revealed marked increases in ARG1 and IL-10 protein levels, with no changes in iNOS and M1-related gene COX2 levels, in RAW264.7 cells treated with rAf RNASET2 (Fig. 5E). LPS upregulated the protein expression level of COX2 in RAW264.7 cells. An ELISA also revealed that the levels of IL-10 in the culture supernatant of RAW264.7 cells stimulated by rAf RNASET2 were also increased (Fig. 6B). These results indicate that rAf RNASET2 promoted M2 macrophage polarization in RAW264.7 cells.

Conversely, in a parallel experiment conducted with RNasin-inactivated rAf RNASET2, the aforementioned M2-like changes were not observed, as evidenced by no significant changes in the mRNA levels of ARG1, IL-10 and IL-13 (P>0.01 vs. control group; Fig. 5). These results indicated that high rAf RNASET2 enzyme activity was required for the M2 polarization/Th2 response of macrophages to rAf RNASET2.

Western blot analysis revealed that active, but not inactive, rAf RNASET2 upregulated the expression levels of p-ERK1/2, p-p38, and p-JNK in RAW264.7 cells (Fig. 6A). In addition, as IL-10 is important for Th2 responses (21), the transcription and protein level of IL-10 was subsequently detected to reflect the effect of MAPK inhibitors on the rAf RNASET2-induced M2 macrophage polarization. RT-qPCR revealed that MAPK inhibitors (U0126, SP600125 and SB203580 for ERK, JNK and p38, respectively) significantly reduced the rAf RNASET2-induced increases in the protein expression levels of IL-10 in RAW264.7 cells (P<0.01; Fig. 6B). In addition, ELISA revealed that rAf RNASET2-induced IL-10 secretion in the cell supernatant was similarly decreased by MAPK inhibitors (P<0.01; Fig. 6C).