The THP-1 cells were acquired from the cell bank of Shanghai Institute of Biochemistry and Biology (Chinese Academy of Sciences, Shanghai, China). RPMI-1640 medium and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA, USA). LPS (O111:B4), PMA, Ficoll-Paque Plus, and commercial anti-Siglec-9 antibody were obtained from Sigma-Aldrich (St. Louis, MO, USA), Abs specific to GAPDH, total p38, phosphorylated JNK1/2, p38, p65, and IRF-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). His-trap Lambda Fab Select column was obtained from GE Healthcare (Piscataway, NJ, USA). Anthrax chimeric Fab antibody was prepared in our lab.

THP-1 cells were cultured in RPMI-1640 supplied with 10% FBS, 1% penicillin, and streptomycin in a 5%-CO₂ humidified incubator at 37°C. The THP-1 cells were stimulated with 10 nM PMA for 48 h, then THP-1-derived macrophages were differentiated. The PBMC-derived macrophages were cultured and differentiated as previously reported (24). Briefly, human PBMCs were separated by centrifugation on Ficoll-Paque Plus and purified by CD14-positive cells isolation kit (Miltenyi Biotec, CA, USA). The purified cells were differentiated in complete RPMI-1640 supplied with M-CSF (10 ng/ml) (BD Biosciences, CA, USA) for 6 days. Donor blood samples were randomly collected in the Jiangsu Province Blood Center. The study was approved by Ethical Committee of Anhui Medical University Affiliated with Bayi Clinical College and all participants signed an informed consent form when they filled the questionnaire.

A human naive Fab phage library for Siglec-9 selection was generated as previously described (25). Before the first round panning, the phage library was titrated and 1 × 10¹³ phage clones were collected for panning.

Single phage clone from the E. coli XL1-Blue infected by the seventh round of eluted phage was randomly picked up and grown in 1 ml super broth (SB) medium containing 100 µg/ml ampicillin and 1% glucose. VCSM13 helper phage (1 × 10⁹) was added to each vial. Then, 50 µl of supernatant from each vial was added to each well of 96-well plate, which was pre-coated with 100 ng extracellular domain of Siglec-9 antigen. After incubation for 2 hours and washing for three times, 50 µl of horseradish-peroxidase (HRP)-conjugated anti-M13 antibody solution was added to each well. Finally, the highest absorbance of the positive clones was selected for further evaluation.

Total RNA was extracted from positive cells by the TRIzol reagent (Invitrogen, CA, USA) and converted to complementary DNA (cDNA) by the Prime Script RT Master Mix kit (TaKaRa, Shiga, Japan) according to the manufacturer’s protocol. The variable regions of the heavy (V_H) chain and the light (V_L) chain were amplified with primers shown in Table S1 in Supplementary Material. The conserved regions of the heavy (C_H1) and light (C_L) chains were provided by the Barbas laboratory (Scripps Research Institute, USA). The V_H and V_L were amplified and cloned into pMD-18T vector, respectively. The heavy chain Fd and the light chain L were amplified with HF1, HR2, and LL1, LR2, respectively. The Fd and L were cloned into pETDuet-1 at NcoI/HindIII and EcoRV/XhoI, respectively.

The expression of hS9-Fab03 in E. coli BL21 was detected by Western blotting as previously described (26). The concentrations of proteins were determined by bicinchoninic acid protein assay kit (Thermo, Waltham, MA, USA). The proteins from bacteria lysate were dissociated by 12% SDS-PAGE and then transferred onto nitrocellulose membrane (Bio-Rad, CA, USA). The membrane was blocked by 5% non-fat milk in TBST for 1 h at room temperature, which was then incubated with HRP-conjugated goat anti-human antibody (Fab specific) (Santa Cruz Biotechnology, CA, USA). The THP-1-derived macrophages (1 × 10⁶ cells/well) were pretreated with the hS9-Fab03 (5 µg/ml) for 2 h and stimulated with LPS (1 µg/ml) for 0, 15, 30, and 60 min. The phosphorylation level of NF-κB/MAPKs/IRF-3 signaling pathway were detected and analyzed.

The THP-1-derived macrophages were treated with the hS9-Fab03 for 2 h; the cells were then incubated with goat anti-human IgG (Fab specific)-FITC antibody (Sigma-Aldrich, MO, USA) at 37°C for 1 h. The cells were analyzed by LSRII flow cytometer (BD Biosciences, CA, USA).

TNF-α, IL-1β, IL-6, IL-10, and IL-8 in supernatants were determined with ELISA kits (R&D Systems, MN, USA).

We optimized the coupling condition based on the isoelectric point of the Siglec-9 protein, and sodium acetate was chosen as the dilution buffer for Biacore X100 SPR system (GE, Sweden). The Siglec-9 protein was diluted to 30 µg/ml and coupled to the CM5 chip. The coupling level was set at 1,500 RU, the injection time was set to 180 s, the dissociation time was set to 15 min, and 50 mM Gly-HCl (pH = 1.7) was used as the regeneration buffer. Finally, the Siglec-9 protein was treated with serial concentrations of hS9-Fab03 to detected the antibodies’ binding affinity.

The total RNA was extracted using RNAfast200 (Feijie, Shanghai, China) following the manufacturer’s instructions. A quantity of 0.5 µg total RNA was used in a 10-µl reverse-transcription reaction using the PrimeScript RT Master Mix kit, then the cDNA was diluted into 40 µl as template for real time Q-PCR. Q-PCR analysis was performed using LightCycler (Roche Diagnostic, IN, USA). The primers used for human GAPDH, TNF-α, IL-6, IL-1β, IL-8, IL-10, and IFN-β were as described previously (27). Data were normalized by the level of GAPDH expression in each sample.

The significance of differences was analyzed by Student’s t-test and Mean ± SD was calculated, with a value of p < 0.05 considered to be statistically significant.

Generally, with seven rounds of affinity panning, 50 single phages were selected and analyzed by phage ELISA. While the positive and negative ratio was exceeded 4, the phage was considered positive. Ten positive phages were possessed, amplified, and confirmed by sequencing. After examining the sequence in VBASE2 database, the light chain was classified as lambda chain. Consequently, we amplified the V_L and C_L of selected positive clones with universe primers, which were designed by our lab as previously reported (28). The length of V_H and C_H1 were close to 400 bp (Figure 1A). Similarly, the length of V_L and C_L were approximately 400 and 350 bp, respectively (Figure 1B). Then, the heavy chain Fd and light chain L were spliced by overlap extension-polymerase chain reaction (29), which were approaching to 800 and 750 bp, respectively. Finally, the Fd and L fragments were inserted into the prokaryotic expression plasmid pETDuet-1 sequentially affirmed by digestion and sequencing (Figure 1C). Therefore, the prokaryotic expression vector of human anti-Siglec-9 Fab fragments (pETDuet-hSiglec-9 Fab03) had been constructed successfully.

The vector of pETDuet-hSiglec-9 Fab03 was transformed into E. coli BL21 and induced with 1 mmol/l IPTG overnight at 16 or 37°C, the recombinant E. coli was preferred to express more Fab fragments (data not shown). Then the lysate of the recombinant E. coli was examined by SDS-PAGE gel with Coomassie brilliant blue staining, the bands of Fd and L were overlapped due to their similar DNA length (Figure 1D). To determine whether the Fab fragments were expressed in the inclusion body, western blotting was utilized. We observed that the recombinant proteins were primarily secreted in the component of ultrasonic supernatant but not that of sediment (Figure 1E). After purification, the Fab antibodies were presented at 27 kDa (Figures 1F,G). Compared with SDS-PAGE electrophoresis, Native-PAGE could reduce the possibility of protein denaturalization. As shown in Figures 1H,I, results showed that two obvious bands were at 27 and 55 kDa, respectively.

Considered to light chain of the recombinant protein was lambda chain; the purification of the human anti-Siglec-9 Fab fragments (hS9-Fab03) was achieved by His-trap Lambda Fab Select column. The purification efficiency of target proteins was above 95% with the concentration of hS9-Fab03 up to 1.0 mg/ml (Figures 1J,K). Collectively, the hS9-Fab03 was successfully expressed at high concentration to explore its affinity and immune activity.

To examine whether the hS9-Fab03 could specifically bind to Siglec-9 antigen, the ELISA assay was carried out with different concentration of hS9-Fab03, and the commercial anti-Siglec-9 antibody was used as positive control. Next, the concentration of hS9-Fab03 was diluted from 0.5 to 0.07 mg/ml. The absorbance value at 450 nm was from 1.174 to 0.216 (Figure 2A). Finally, the ELISA results showed that the hS9-Fab03 could particularly bind to Siglec-9 antigen in a dose-dependent manner, which was as good as the commercial antibody.

Aiming to characterize the binding capacity of hS9-Fab03, we further calculated the affinity constant using the following formula: affinity constant (KD) = dissociation constant (Kd) ÷ binding constant (Ka) (30). Results from the Biacore X100 SPR analysis demonstrated that the affinity constant of hS9-Fab03 was 6.58 × 10⁻⁷ (Figure 2B). Taken together, these results indicated that hS9-Fab03 could effectively bind to Siglec-9 protein.

The binding ability of hS9-Fab03 was further analyzed with Flow cytometry in Siglec-9-positive THP-1-derived macrophages. Our results revealed that the population of hS9-Fab03-treated THP-1 cells was divided from unbinding cells, whereas no apparent difference was detected in blank and FITC antibody-treated cells (Figure 2C). This implied that hS9-Fab03 could effectively bind to Siglec-9 expressed on the cells’ surface.

Human macrophages have been widely used for studying TLRs signaling (31). To investigate the role of Siglec-9 during LPS-initiated inflammatory responses, we determined the efficient pre-incubated time and optimized concentrations of hS9-Fab03 and LPS. When the concentration of hS9-Fab03 range from 0.1 to 5 µg/ml, the inhibition efficiency of IFN-β and TNF-α mRNA levels were increased in a concentration-dependent manner (Figures S1A,B in Supplementary Material). Then, when the concentration of hS9-Fab03 was at 5 µg/ml, the pre-incubated time of hS9-Fab03 was ranging from 30 min to 4 h, the inhibition efficiency of IFN-β and TNF-α mRNA levels was better at 2 h (Figures S1C,D in Supplementary Material). Furthermore, treatment with 5 µg/ml of hS9-Fab03 reduced LPS-initiated inflammatory responses by approximately 85% of IFN-β expression and by approximately 70% of TNF-α expression than without antibody treatment (Figures S1E,F in Supplementary Material). Finally, we conducted the following experiments with pre-incubation time at 2 h and the concentration of hS9-Fab03 and LPS at 5 and 1 µg/ml, respectively.

Next, we investigated whether hS9-Fab03 ligation could regulate the expression of pro-inflammatory cytokines in human macrophages. The THP-1-differentiated macrophages were pretreated with the hS9-Fab03 and stimulated with LPS for different time. The mRNAs were collected to evaluate the expression of pro-inflammatory cytokines. We found that hS9-Fab03 could obviously inhibit LPS-initiated production of pro-inflammatory cytokines, such as TNF-α, IL-6, and IFN-β, no effect on the expression of IL-1β, IL-8, and IL-10 in THP-1-differentiated macrophages (Figures 3 and 4). Similarly, we observed that pre-incubation of hS9-Fab03 could also suppress the TNF-α, IL-6, IL-1β, IL-8, and IFN-β expression of pro-inflammatory in human PBMC-derived macrophages stimulated with LPS, but slightly increased IL-10 production in an early time point compared to group with LPS only (Figures 5 and 6). We also incubated different concentrations of hS9-Fab03 macrophages and found that hS9-Fab03 could effectively block LPS-induced TNF-α and IFN-β expression in a dose-dependent manner (Figures S1A,B in Supplementary Material). Thus, the results implied that the hS9-Fab03 could inhibit LPS-induced pro-inflammatory cytokines production in human macrophages.

To investigate the inhibitory effects of the hS9-Fab03 on TLR4 signaling transduction induced by LPS ligation, we detected the phosphorylation levels of NF-κB, MAPKs, and IRF-3 signaling pathways. As shown in Figure 7, LPS ligation significantly increased the phosphorylation level of these proteins in THP-1-differentiated macrophages, but the phosphorylation level of p65, p38, JNK1/2, and IRF-3 was suppressed when pretreated with 5 µg/ml of the hS9-Fab03. Thus, these results demonstrated that the hS9-Fab03 could suppress LPS-triggered TLR4 signaling transduction.