Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood from healthy donors (HDs) by density gradient centrifugation at 500 g for 30 min on Ficoll lymphocyte separating solution (Dakewe Biotech) at room temperature. All donors gave written informed consent to participate in the study. CD14+ blood monocytes were purified from PBMCs by magnetic cell sorting using CD14 microbeads (Miltenyi Biotec, Germany) and used for generation of M1 and M2 macrophages following the protocol of Vogelpoel et al. (10). Monocytes were cultured for 6 days in RPMI 1640 (Hyclone) containing 10% fetal bovine serum (FBS, Biological Industries) supplemented with 20 ng/ml recombinant human M-CSF (Peprotech) for M2, or 500 U/ml recombinant human GM-CSF (Peprotech) for M1 macrophages. For M2, at day 3, half of the medium was replaced by new medium containing cytokines. At day 7, the medium was totally replaced in the presence of 20 ng/ml recombinant human IL-4 (Peprotech), respectively. Since the new guideline for in vitro polarization of M1 and M2 macrophages (1) was not followed in the present study, the differentiated M1 and M2 cells herein prepared are specified (GM-CSF)-M1 and (M-CSF)-M2 macrophages, respectively.

Anti-huLTF antibodies from RA patients (LTF-Abs) and LTF-ICs were prepared as previously described (17). Briefly, LTF-Abs were sequentially purified by affinity chromatography on LTF-S4B (prepared in our laboratory) and Protein A-S4B columns (Pierce) from six pooled plasma samples shown by ELISA to contain high levels of anti-LTF antibodies. The eluted IgG fractions were concentrated by centrifugation with buffer exchange to phosphate-buffered saline (PBS) (Amicon Ultra, Millipore) and were depleted of endotoxin by filtration through a polymyxin B column (Detoxigel). IgG concentrations were determined by optical density at 280 nm; IgG was aliquoted, and stored at −80°C. Preparation and characterization of a mouse mAbs against huLTF (M860), bovine serum albumin (BSA) (J1) or chicken ovalbumin (M562) in this laboratory have also been documented (24). For preparation of LTF-IC, human LTF (2 µg/ml, Sigma-Aldrich) and M860 (2 µg/ml, mAbs of LTF, purified with protein G antibody affinity chromatography, GE Healthcare Life Sciences) or LTF-Abs were mixed in a sterile tube with gentle rotation at 37°C for an hour. IC between LTF and M860 were separated from the uncoupled Ab and antigen using Sephadex Superfine G-75 column. The elutions of IC were pooled, desalted and concentrated. Endotoxin was removed by polymyxin B coupled beads repeatedly and the level of endotoxin in IC was below 1 EU/mg which was detected by Chromogenic LAL Endotoxin Assay Kit (Genscript). ICs between BSA (Sigma-Aldrich) and J1 were used as control and prepared similarly.

For phenotyping, cells were collected, washed with PBS, and the pellets were incubated for 30 min at 4°C with 50 µl APC-conjugated mouse anti-human CD14, CD163, CD16, or CD32, or PE-conjugated mouse anti-human CD86 or CD206, or FITC-conjugated mouse anti-human CD64, or APC-, PE-, FITC-conjugated isotype control Abs (Biolegend). After washes, the cells were subjected to analysis by flow cytometry Attune NxT (Life Technology).

In vitro differentiated macrophages were harvested by gentle pipetting and stimulated (3–5 × 10⁴ cells/well) with 30 µg/ml LTF, M860 (LTF-Abs), M860-IC (LTF-IC), or 100 ng/ml LPS (from Escherichia coli 0111:B4; Sigma-Aldrich) in 96-well plates (Nunc) for 18 h in a 5% CO₂ incubator at 37°C, then the supernatants were collected and stored at 4°C, until analysis by ELISA.

For PAMPs and inflammatory cytokine restimulation assays, LTF-IC-pretreated macrophages (5 × 10⁴ cells/well) were restimulated with different stimuli, including 10 ng/ml LPS, 1 µg/ml zymosan and 1 µg/ml curdlan, or with cytokines including 10 ng/ml IL-1β, 1,000 U/ml IFN-α/β, 2,000 U/ml IFN-γ, 100 ng/ml IL-6, 5 ng/ml IL-12, and the cell supernatants were collected. Cytokine levels were determined by TNFα ELISA kit.

The levels of TNFα, IL-1β, IL-6, IL-10, IL-17, IL-23, and IFN-γ in human macrophage culture supernatants were measured by using ELISA kits (from eBioscience) according to the manufacturer’s instructions. Standard curves were established using human recombinant cytokines provided in the kits.

Memory T cells were isolated from PBMCs of HDs using flow sorting (Arial III, BD Biosicences) stained with anti-CD45RO-PE (Biolegend) and anti-CD4-APC (MiltenyiBiotec). For in vitro activation of T cells, 2 × 10⁵ (M-CSF)-M2 macrophages were stimulated with 30 µg/ml LTF, M860, M860-IC or 100 ng/ml LPS, and then cocultured with 2 × 10⁵ allogeneic memory CD45RO⁺CD4⁺ T cells in presence of 2 µg/ml anti-IL-4 (R&D) and 10 µg/ml anti-IFN-γ (BD Pharmingen), costimulated with 100 ng/ml anti-CD3 antibodies (eBioscience) coated in the plates. Every 2 days, half of the medium was replaced by RPMI 1640 (Hyclone) containing 10% FBS and 20 U/ml recombinant human IL-2 (Peprotech). After 4 days, cells were transferred to 96-well flat-bottomed culture plates (Nunc).

For intracellular cytokine staining, T cells were restimulated by cell stimulation cocktail (including PMA, ionomycin, brefeldin A, and monensin, eBioscience) for 6 h. Cells were harvested and washed, fixed with fixation buffer (Biolegend) for 20 min at room temperature, washed again, permeabilized with Intracellular staining perm wash buffer (Biolegend) for 30 min. Cells were incubated with anti-IL-17-FITC (1:50; MiltenyiBiotec) and anti-IFN-γ-APC (1:50; MiltenyiBiotec) for 60 min at room temperature and analyzed by flow cytometry (Canto II, BD Biosicences). For cytokine analysis, resting T cells were restimulated with 1 µg/ml anti-CD3 and 1 µg/ml anti-CD28 (eBioscience). Supernatants were harvested after 24 h and stored at 4°C until analysis by ELISA.

For mRNA-level analysis, cells were lysed at the indicated time points, after which mRNA extraction was performed using Omega RNA Isolation Kit and cDNA synthesis using First Strand cDNA Synthesis Kit (Takara). Quantitative RT-PCR (StepOnePlus real-time PCR system; Applied Biosystems, Life Technology), was performed using SYBR green (Takara) and primer pairs as listed in Table 1. The mRNA levels were normalized to housekeeping gene expression [2^{Ct(housekeeping) − Ct(target)}], and folds were calculated compared with an unstimulated control sample.

Blocking antibodies were preincubated with (M-CSF)-M2 macrophages for 2 h in a 5% CO₂ incubator at 37°C, after which stimuli and culture medium were added resulting in a final antibody concentration of 5 µg/ml anti-CD16/32/64 and 2 µg/ml anti-CD14. Syk or TLR4 was inhibited by incubating M2 macrophages with titration of R406, Belnacasan VX-765 (both from Selleckchem) or CLI-095 (Invitrogen) for 2 h at 37°C before stimulation.

All experiments were repeated at least three times and the results are expressed as mean ± SD. Comparison of the data was performed using the Student’s t-test. Significance was defined as a P value of <0.05%.

This study was approved by the Ethics Committees of Soochow University Medical School, Suzhou, China. Written informed consent was obtained from all participants prior to inclusion in the study.

Human (GM-CSF)-M1 and (M-CSF)-M2 macrophages were generated in vitro by treating freshly purified human CD14⁺monocytes (Figure S1A in Supplementary Material) for 7 days with GM-CSF or M-CSF plus IL-4, respectively. The resultant cells displayed expected surface marker expression profiles of M1 (CD86 and CD64^high) or M2 (CD14, CD163, and CD16^high) phenotypes (Figure S1B in Supplementary Material). Q-PCR results confirmed mRNA transcription for the genes of ALDH1A2, CLEC5A, and INHBA in the (GM-CSF)-M1 and SLC40A1, FOLR2, and HMOX1in the (M-CSF)-M2 cells (Figure S1C in Supplementary Material), which is consistent with the characteristic gene expression profile of M1 and M2 macrophages. As expected, (GM-CSF)-M1 cells readily produced inflammatory cytokine TNFα, while (M-CSF)-M2 produced anti-inflammatory cytokine IL-10 in response to subsequent LPS stimulation (Figure S1D in Supplementary Material).

Based on our recent finding that LTF-IC was able to coligatie TLR4 and FcγRIIa on human monocytes/macrophages and induce a strong TNFα response (17), we wondered whether LTF-IC could also elicit inflammatory cytokine production by human (M-CSF)-M2 cells in vitro. In the experiment shown in Figure 1, (M-CSF)-M2 cells were stimulated with a mixture (1:1) of human LTF (hLTF) and LTF-specific IgG autoantibodies (affinity purified from RA sera) for 18 h followed by quantitation of TNFα, IL-1β, IL-6, and IL-10 in the culture supernatant. Clearly LTF-IC, but not anti-LTF IgG or hLTF alone, or huLTF plus control hIgG, was able to elicit production of TNFα, IL-1β, IL-6, and IL-10 by (M-CSF)-M2 cells (Figure 1A), while LTF-IC treatment had minimal effect on (GM-CSF)-M1 macrophages (Figure S2A in Supplementary Material). There was considerable individual variation in TNFα response of (M-CSF)-M2 cells generated from PBMCs of different donors, but the trend of all individual samples remained consistent (Figure 1B). Based on LTF-specific IgG Ab screening results of our earlier study on RA sera (17), serum samples from four patients with high titer anti-LTF autoantibodies were added to wells precoated with hLTF, the plate-bound ICs thus formed exhibited capability to induce TNFα production by (M-CSF)-M2 cells (Figure 1C).

Like autoantibody-containing LTF-ICs, complex between hLTF and mouse anti-hLTF mAb M860 (M860-IC) is also capable of eliciting proinflammatory cytokine, but not IL-10, production by human monocytes (17), which can be explained by the fact that mouse IgG1 binds human FcγRs with relatively high affinity (26, 27). In the present study, M860-IC strongly triggered (M-CSF)-M2 secrete of TNFα, IL-1β, IL-6, IL-10, and IL-23 (Figure 1D). In subsequent experiments M860 was employed as a replacement of human autoantibodies against hLTF. Dose curve of M860-IC-induced TNFα production by (M-CSF)-M2 cells, shown in Figure 1E, indicates that minimal concentration for LTF-IC to activate (M-CSF)-M2 cells was approximately 3 µg/ml. TNFα secretion by M860-IC-treated (M-CSF)-M2 cells was readily detectable after 4 h stimulation (Figure 1F). The above results are further confirmed by Q-PCR data showing strongly increased mRNA transcription of TNFA, IL1B, IL6, IL12A, IL12B, and IL23A genes in (M-CSF)-M2 macrophages after 4–6 h exposure to LTF-IC (Figure 2).

Lactoferrin IC treatment of (M-CSF)-M2 macrophages resulted in not only inflammatory cytokine secretion but also a switch from M2-specific (CD14, CD163, CD206, SLC40A1, FOLR2, and HMOX1) to M1-specific (CD86, ALDH1A2, CLEC5A, and INHBA) marker expression as evidenced by FACS and Q-PCR analysis (Figure 3). In addition to the phenotypical changes, LTF-IC-primed (M-CSF)-M2 macrophages acquired a significantly enhanced functional state in that they remained hyperactive to LPS stimulation for several days. As shown in Figure 4, LTF-IC-primed (M-CSF)-M2 macrophages (12 h stimulation followed by washes and a resting period of 24 h) vigorously responded to suboptimal concentration LPS by producing large amounts of TNFα, IL-1β, IL-6, and IL-23, albeit the ability to make IL-10 was not significantly affected (data not shown). By contrast, LPS-primed (M-CSF)-M2 became unresponsive to subsequent LPS stimulation. Although there was considerable individual variation in LPS responsiveness (TNFα production) by LTF-IC-primed (M-CSF)-M2 cells from different donors, the trend of all donors remained consistent (Figure 4C). Dose curve and time course studies (Figures 4D,E) indicate that a 4 h treatment with LTF-IC at a dose of 0.3 µg/ml was enough to make (M-CSF)-M2 macrophages significantly more responsive (LPS-induced TNFα secretion) than unprimed controls. More importantly, LTF-IC-primed (M-CSF)-M2 macrophages were high responders to not only LPS but also β-glucans (zymosan and curdlan) and IL-1β (Figure 4F). Though not as impressive, their responsiveness to IL-6 and IL-12, but not IFNs, was also significantly increased. Finally, the hyperactivity of LTF-IC-primed (M-CSF)-M2 macrophages lasted for up to 7 days in vitro (Figure 4G). It should be noted that LPS-induced TNFα production in (GM-CSF)-M1 macrophages was significantly down-regulated by LTF-IC (Figure S2B in Supplementary Material).

CD14/TLR4 is known to be the main surface receptor in monocytes for interaction with LTF, and LTF-IC activation of human monocytes was susceptible to suppression by heparin (blocker of LTF and receptor binding), blocking Abs against CD14 or CD32 as well as TLR4-specific inhibiting agents (17). Similarly, TNFα production by LTF-IC-treated (M-CSF)-M2 macrophages was significantly blocked by heparin, CLI095 (TLR4-specific chemical inhibitor), and mAbs against human CD14 or CD32, but not CD16 or CD64 (Figure 5A). Consistently, R406, a chemical inhibitor of CD32a signal transduction molecule Syk, dose dependently blocked LTF-IC-induced (M-CSF)-M2 activation (Figure 5A). Furthermore, LTF-IC-mediated decrease in surface expression of CD14, CD163, and CD206 molecules (Figure 5B) and suppression of M2 signature genes (SLC40A1, FOLR2, and HMOX1) (Figure 5C) in (M-CSF)-M2 cells were significantly reversed by heparin, CLI095, R406 or blocking mAbs against human CD14 or CD32. Taken together, both the CD14-TLR4 pathway and CD32-Syk axis play pivotal roles in LTF-IC-mediated (M-CSF)-M2 activation and subsequent conversion to an M1-like phenotype. Next we asked whether costimulation by unconjugated TLR agonist(s) and deposited IgG could also achieve similar M2 to M1 switch. As summarized in Table 2, combination of plate-coated IgG and soluble LTF was as effective as LTF-IC in eliciting IL-1β, IL-6, and TNFα secretion by (M-CSF)-M2 cells in vitro.

Macrophages are regarded as antigen-presenting cells capable of inducing CD4⁺ T helper (Th) cell activation and polarization through cytokines (e.g., IL-1β, IL-6, and TNFα) they produce (28–30). It has also been reported that IgG-opsonized bacteria were able to promote human Th17 response via synergy between TLRs (TLR2, 4, 5) and FcγRIIa in dendritic cells (31). It is therefore reasonable to question whether LTF-IC-primed M2 macrophages could facilitate Th17 cell activation and/or polarization in a similar fashion. In the experiment shown in Figure 6, CD4⁺ T cells, freshly purified from PBMC of HDs, were cultured together with LTF-IC-stimulated (M-CSF)-M2 macrophages for 96 h, followed by quantitation of IL-17 and IFNγ in the culture supernatant and intracellular staining for the same cytokines in CD4⁺ cells. Percentage of IL-17⁺Th cells in the LTF-IC-primed group was significantly higher than that of the controls as evidenced by intracellular staining (Figures 6A,B). Apparently CD4⁺ T cells strongly responded to LTF-IC-primed (M-CSF)-M2 cells, but not the controls, by producing large amounts of IL-17 and IFN-γ (Figures 6C,D).