Male C57BL/6 (wild-type, WT), TLR2 knockout (TLR2^-/-), and TLR4 knockout (TLR4^-/-) mice of 6–8 weeks of age were used. They were acquired from the vivarium on the campus of the University of São Paulo at Ribeirão Preto, São Paulo, Brazil, and housed in the animal facility of the Molecular and Cellular Biology Department, Faculty of Medicine of Ribeirão Preto, University of São Paulo, under optimized hygienic conditions. Animal procedures were approved by the Ethical Committee for Ethics in Animal Research (CETEA) of the School of Medicine at Ribeirão Preto, University of São Paulo, under protocol number 20/2013-1.

The full-length prediction encoding ORF for paracoccin, cloned in vector pUC57 by Alegre et al. (2014), was used as a template for PCR with forward primer 5′-CTCGAGATGGCGTTTGAAAACCAGATTG-3′ and reverse primer 5′-GCGGCCG CCCAGCTGCTGGTGCTAAAGC-3′, containing the restriction sites of the XhoI and NotI enzymes, respectively. The reaction was carried out in 30 cycles (30 s at 94°C, 30 s at 57°C, and 60 s at 72°C). The purified PCR product was cloned into the pGEM-T vector (Promega, Fitchburg, WI, USA), and the insert was removed from the vector with the aforementioned restriction enzymes and ligated into the pGAPzαA vector (Invitrogen, Carlsbad, CA, USA). The pGAPzαA-PCN vector was obtained and sequenced to determine the ligation success and the correct sequence of the insert. This vector was then linearized with the restriction enzyme AvrII so as to be used for the transformation of the Pichia pastoris GS115 strain, as described by Maleki et al. (2010). In short, 10 μg of the purified (using Illustra kit plasmidPrep Mini Spin – GE Healthcare, Little Chalfont, UK) and linearized plasmid were electroporated into the yeast in 0.2 cm cuvettes at 1.5 kV (25 μF and 200 Ω), using Gene Pulser (Bio-Rad, Hercules, CA, USA).

The transformants obtained on the selective YPD medium containing Zeocin (100 μg/mL) were confirmed by PCR. A clone was grown in 300 mL YPD liquid medium at 30°C, 220 rpm, for 72 h, for paracoccin recombinant expression and secretion. The culture supernatant was collected, dialyzed against phosphate buffered saline (PBS; 10 mM, pH 7.2), and concentrated 10 times using centrifugal filtration devices with a 10,000-molecular weight cut-off (Millipore, Darmstadt, Germany).

For paracoccin purification, the prepared culture supernatant was centrifuged at 10000 × g, at 5°C, and applied to a chitin column, manufactured as described by dos Reis Almeida et al. (2010), and equilibrated with PBS. After rinsing with 20 column volumes of PBS, the material adsorbed to chitin was eluted with 0.1% acetic acid. The eluate was concentrated and dialyzed against PBS. The product obtained from chromatography was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and submitted to quality control by assessing its lectin and enzymatic activities. The final product was named p-rPCN.

A high affinity polystyrene microplate (Corning Costar, Badhoevedorp, The Netherlands) was coated with laminin (250 ng/well) in carbonate-bicarbonate buffer (0.1 M; pH 9.6) overnight, at 4°C. After rinsing with PBS-Tween 20 (PBS-T) and blocking with PBS-T with 3% gelatin (blocking buffer) for 1 h at 37°C, rPCN (200 ng) or blocking buffer (negative control) was added to the wells in triplicates, and the microplates were incubated at room temperature for 2 h. After rinsing with PBS-T, IgY anti-rPCN antibodies (diluted 1:250 in PBS-T containing 1% gelatin) were added to the wells and incubated for 1 h at 37°C. After rinsing, murine biotinylated IgG anti-IgY (diluted to 1:1000 in PBS-T containing 1% gelatin) was added and incubated for 1 h at 37°C. As the last step, streptavidin-peroxidase conjugate (1:250) was added to each well for 1 h at room temperature. Following rinsing, the reaction was revealed by using the tetramethylbenzidine substrate (Pierce, Thermo Fisher Scientific Inc., Waltham, MA, USA), and color development was detected at a wavelength of 450 nm in a microplate reader (Power Wave X – BioTek Instruments, Inc., Winooski, VT, USA).

The N-acetyl-β-D-glucosaminidase activity of yeast recombinant paracoccin was assayed as previously described in detail (dos Reis Almeida et al., 2010; Almeida et al., 2015). The substrate ρ-nitrophenyl-N-acetyl-β-D-glucosaminide (100 μL, 5 mM; Sigma–Aldrich, St Louis, MO, USA) was mixed with 350 μL of 0.1 M sodium acetate (pH 5.5) and 5 μg of p-rPCN. As a negative control, we used the vehicle medium. The reaction was incubated for 16–18 h at 37°C, with 1 mL of 0.5 M sodium carbonate added. The enzyme activity values were determined by using a microplate reader at 405 nm (Power Wave X, BioTek Instruments, Inc.).

Murine peritoneal macrophages were obtained from WT, TLR2^-/-, and TLR4^-/- mice after an intraperitoneal injection with 1.0 mL of sterile 3% sodium thioglycollate (Sigma–Aldrich). After 3 days, the peritoneal cavity was washed with 5.0 mL of cold sterile PBS and the cells were harvested and centrifuged at 300 × g for 10 min. The cell pellet was resuspended in Dulbecco’s modified Eagle’s medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific Inc.), and the cell counting was performed in a Neubauer chamber. The cells were plated in 48-well culture plates (1 × 10⁶ cells/mL) and incubated overnight at 37°C in a 5% CO₂ atmosphere. The non-adherent cells were removed by gentle rinsing with PBS, while the adherent macrophages were incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Elicited peritoneal macrophages were stimulated with p-rPCN (5.0 μg/mL). The positive controls were LPS (1 μg/mL) with IFN-γ (1 ng/mL) added for TLR2^-/- macrophages and Pam3CSK4 (100 ng/mL) for TLR4^-/- macrophages. The medium alone was used as a negative control. After variable incubation periods (specified in the legends), the culture supernatants were harvested and assessed for the concentration of mediators.

Nitric oxide concentration was inferred by measuring nitrite levels in the macrophage supernatants by using the Griess reagent system (Green et al., 1982). In short, 50 μL of the supernatants were distributed in 96-well microplates and incubated with 50 μL/well of Griess reagent (1.0% sulfanilamide, 0.1% naphthalenediamine dihydrochloride, and 2.5% H₃PO₄) at room temperature for 10 min. The absorbance at 550 nm was read using a Power Wave-X microplate reader (BioTek Instruments, Inc.). The absorbance was converted to micromolar (μM) NO on the basis of a standard curve, concomitantly generated by using known concentrations of NaNO₂.

Supernatants of stimulated macrophages were assessed for their levels of IL-12p40, IL-6, and TNF-α. The cytokines were detected by an enzyme-linked immunosorbent assay (ELISA) using an OptEIA kit (Pharmingen, San Diego, CA, USA), according to the manufacturer’s instructions. Standard curves allowed determining cytokine concentrations in pg/mL. The absorbance was read at 450 nm in the Power Wave-X microplate scanning spectrophotometer (BioTek Instruments, Inc.).

Macrophages (1 × 10⁶ cells/mL) from WT, TLR2^-/-, and TLR4^-/- mice were incubated with rPCN (5 μg/mL), LPS (1 μg/mL), Pam3CSK4 (100 ng/mL), IFN-γ (1 ng/mL) plus IL-12p40 (50 ng/mL) (M1 macrophage inducers), IL-10 plus IL-4 (50 ng/mL both) (M2 macrophage inducers), or with medium alone, for 6 h. RNA from macrophages was extracted using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. The total RNA was reverse-transcribed into cDNA by the ImProm-II Reverse Transcription System (Promega) using oligo(dT). Quantitative real-time PCR was performed using SYBR Green (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) on a 7500 Real-Time PCR System (Applied Biosystems). The relative expression of transcripts was quantified using the ΔΔCt method, and β-actin was used as endogenous control. The PCR primers used were: β-actin (S-CCTAAGGCCAACCGTGAAAA; AS-GAGGCATACAGGGACAGCACA), Ym1 (S-TCACAGGTCTGGCAATTCTTCTG; AS-ACTCCCTTCTATTGGCCTGTCC), Arginase-1 (S-GTTCCCAGATGTACCAGGATTC; AS-CGATGTCTTTGGCAGATATGC), FIZZ1 (S-CCTGAGATTCTGCCCCAGGAT; AS-TTCACTGGGACCATCAGCTGG), iNOS2 (S-CCGAAGCAAACATCACATTCA; AS-GGTCTAAAGGCTCCGGGCT), STAT1 (S-CACGCTGCCTATGATGTC; AS-CCTGGAGATTACGCTTGC), STAT3 (S-GGCACCTTGGATTGAGAG; AS-TGCTGATAGAGGACATTGG), SOCS1 (S-AGGATGGTAGCACGCAAC; AS-GAAGACGAGGACGAGGAG), and SOCS3 (S-AGGAGAGCGGATTCTACTG; AS-TCACACTGGATGCGTAGG).

The results are expressed as mean ± SEM. The statistical analysis was done using the GraphPad Prism software (GraphPad Software, San Diego, CA, USA). The homogeneous variance was analyzed, and the difference between means of groups was calculated by the analysis of variance (one-way) and Bonferroni’s test thereafter. Differences with p < 0.05 were considered statistically significant.

Paracoccin purified from Paracoccidioides brasiliensis has both lectin and enzymatic properties, which are mimicked by a recombinant form of the protein, expressed in Escherichia coli (b-rPCN) (Alegre et al., 2014). To be stricter concerning the required absence of LPS in our preparations, we expressed paracoccin in the P. pastoris strain GS115, transformed with the pGAPzαA vector and containing the synthetic transcript of this protein. The secreted protein was purified in a chitin column and called p-rPCN. The electrophoretic profile of the bound fraction showed that it consists of a single 27 kDa band (Figure 1A), which is the expected molecular mass for paracoccin. Our recombinant PCN exhibited the properties of the native protein, which was evident from several facts. First, the fact that its purification was founded on binding to chitin showed that p-rPCN retains the carbohydrate binding capacity of the native protein. Second, it could be detected by binding to laminin (Figure 1B), a property that depends on the lectin domain of paracoccin, as previously reported for PCN and b-rPCN. Finally, using the substrate ρ-nitrophenyl-N-acetyl-β-D-glucosaminide, we demonstrated that p-rPCN exhibits N-acetyl-β-D-glucosaminidase activity (Figure 1C). These results validated the use of p-rPCN in the present study aiming to detail the paracoccin effects exerted on macrophages.

We have previously demonstrated that native PCN and b-rPCN induce mouse peritoneal macrophages to release high levels of TNF-α and NO (Coltri et al., 2006; Alegre et al., 2014). Then, we examined whether p-rPCN could also stimulate mouse peritoneal macrophages to release inflammatory mediators. Indeed, p-rPCN induced the release of significant levels of NO, IL-12p40, and TNF-α in macrophages (Figures 2A–C, respectively) at 24, 48, and 72 h after the stimulus. The time-course production of IL-12 (Figure 2B) and TNF-α (Figure 2C) was close to that determined by the positive control and consisted of a mixture of LPS and IFN-γ. These findings demonstrate that p-rPCN induces macrophages to release proinflammatory cytokines.

To advance in the study of the effects of paracoccin on macrophage activation, using real-time PCR, we quantified the expression of M1 (iNOS2) and M2 (Arginase-1, Ym-1, and FIZZ1) polarization markers in response to p-rPCN. Peritoneal macrophages stimulated with p-rPCN showed a more than 200-fold increase in iNOS mRNA, attaining levels that were similar to those in macrophages stimulated by IFN-γ plus IL-12 (Figure 3A). The analysis of M2 markers revealed that the p-rPCN stimulus did not change Arginase-1, Ym-1, and FIZZ1 expression, whose levels remained close to those found in unstimulated cells and were significantly lower than those measured by the IL-10 plus IL-4 control stimulus (Figures 3B–D). In addition, the expression measurement of key signaling mediators associated with M1/M2 dichotomy showed that the levels of STAT1 and SOCS3 mRNA increased by 10- and 380-fold, respectively, in response to the p-rPCN stimulus (Figures 3E,F). On the other hand, this stimulus did not affect the expression of STAT3 and SOCS1 (Figures 3G,H). Furthermore, the ratio of the expression of SOCS3 and SOCS1 increased 15-fold in response to p-rPCN (Figure 3I). These results indicate that p-rPCN induces the classical activation of macrophages, with the involvement of STAT1 and SOCS3.

Given that recombinant paracoccin expressed in E. coli interacts with the N-glycans of TLR2 and TLR4 on antigen-presenting cells and promotes IL-12 production (Alegre-Maller et al., 2014), we examined the relevance of TLR2 and TLR4 in the production of inflammatory mediators induced by p-rPCN. We stimulated peritoneal macrophages obtained from WT, TLR2^-/-, or TLR4^-/- mice with p-rPCN for 48 h and quantified the levels of IL-12p40, TNF-α, and IL-6 in the cell supernatants. We found that the lack of TLR2 diminished the IL-12p40 production induced by p-rPCN compared to that of WT macrophages, while the TNF-α and IL-6 levels were unaffected. The absence of TLR4, in turn, abolished the p-rPCN-induced production of IL-12p40 and TNF-α (Figures 4A,B) and severely impaired the IL-6 release (Figure 4C). Interestingly, the cytokine production induced by the control agonists of either receptor (Pam3CSK4 for TLR2 and LPS plus IFN-γ for TLR4) was inhibited by the absence of the respective TLR, but less affected than the production promoted by p-rPCN. We conclude that TLR2 and TLR4 participate in the induction of proinflammatory cytokine production in macrophages stimulated by paracoccin; a more notable role is attributed to TLR4.

To expand the investigation of the roles of TLR2 and TLR4 on the effects of paracoccin on macrophages, we analyzed whether the M1 polarization of these cells promoted by p-rPCN could be affected by the absence of TLR2 or TLR4. Following the stimulation of peritoneal macrophages harvested from WT, TLR2^-/-, or TLR4^-/- mice, the cells were assessed for the expression of iNOS, Arginase-1, Ym-1, and FIZZ1. The iNOS expression stimulated by p-rPCN was not affected by the absence of TLR2, but was blocked in TLR4^-/- macrophages (Figure 5A). The expression of M2 polarization markers, which did not respond to the p-rPCN stimulus, remained in cells from TLR2^-/- or TLR4^-/- mice at levels as low as those verified in non-stimulated macrophages (medium) (Figures 5B–D). Therefore, we conclude that TLR4 is critical for the paracoccin-induced M1 polarization of macrophages.