Fig 2: The effect of TLR2-neutralizing antibodies on HP-NAP-induced ROS production by ATRA-induced differentiated HL-60 cells and neutrophils. (A) TLR2-independent ROS production in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 4 × 106 cell/mL were pretreated with 10 µg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 µM HP-NAP, 1 µg/mL Pam3CSK4, 10 µg/mL LPS, or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 30 min. ROS production was measured as DHE-derived fluorescence by flow cytometry as described in Figure 2C. Representative histograms are shown in the left panel. Data in the right panel are expressed as the fold change as described in Figure 2C and as mean ± SD of three independent experiments. (B) TLR2-independent ROS production in neutrophils induced by HP-NAP. Neutrophils at a density of 2 × 106 cell/mL were pretreated with 10 µg/mL of anti-TLR2, anti-TLR4 or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 µM HP-NAP, 1 µg/mL Pam3CSK4, 10 µg/mL LPS or D-PBS, pH 7.2, as the unstimulated control for the indicated time. ROS production by neutrophils was determined by H2DCF-DA-derived derived fluorescence assay as described in Figure 2A. Data from neutrophils stimulated with HP-NAP for 2.5 h are shown as bar graph and expressed as mean ± SD of four independent experiments in duplicate. *: p value < 0.05, **: p value < 0.01, ***: p value < 0.001 as compared with the unstimulated cells. ##: p value < 0.01, ###: p value < 0.001 as compared with HP-NAP-stimulated cells in the isotype control group. n.s.: non-significant.

Fig 3: Specific interaction of HP-NAP with recombinant human TLR2 protein. (A) Binding of TLR2 to HP-NAP as determined by an ELISA-based solid-phase binding assay. The indicated amounts of recombinant human TLR2-10xHis and TLR4-10xHis proteins were incubated with 0.05 µg of HP-NAP coated on an ELISA plate. The binding of TLR2 and TLR4 to HP-NAP was detected by anti-His tag antibody or its isotype control antibody using the ELISA-based solid-phase binding assay as described in Materials and Methods. The results are presented as absorbance at OD450 nm. Data are expressed as mean ± SD of two independent experiments. (B) Specific binding of TLR2 to HP-NAP as determined by competitive binding with Pam3CSK4. The indicated amounts of Pam3CSK4 (Pam3) and BSA, as a negative control, were incubated with 0.625 µg of the recombinant human TLR2-10xHis protein at 4 °C overnight and then incubated with 0.05 µg of HP-NAP coated on an ELISA plate. The binding of TLR2 to HP-NAP was detected by the ELISA-based solid-phase binding assay as described in A. The results were presented as absorbance at OD450 nm. Data are expressed as mean ± SD of two independent experiments.

Fig 4: Contribution of pattern recognition receptors and cell wall components on the phagocytosis of Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa by human monocyte-derived Dendritic cells. In (A), Immune cells were preincubated with 200 µg mL-1 laminarin or 10 µg mL-1 of any of the following antibodies: anti-mannose receptor (MR), anti-CR3, anti-DCSIGN, anti-TLR2, or anti-TLR4. Then, cells were coincubated with Acridine Orange-labeled conidia or yeast-like cells at an immune cell-fungus ratio of 1:6, for 2 h at 37°C and 5% (v/v) CO2. Human cells were analyzed by flow cytometry, collecting 50,000 events, which were defined as a human cell interacting with at least one fluorescent fungal cell. All the interactions were performed in the presence of 5 µg mL-1 polymyxin B. No treatment refers to cells preincubated only with PBS. Results correspond to cells in the late stage of phagocytosis. For all cases, 100% corresponds to the system with no treatment, and the absolute values were similar to those shown in Figure 6 or (B) of this figure. CR3, complement receptor 3. *P < 0.05 when compared to the no-treatment condition of the same strain. †P< 0.05 when compared to conidia from the same species. In (B), Similar experiments as described in (A), but human cells were not preincubated with any blocking agent. Before coincubation with human monocyte-derived dendritic, conidia and yeast-like cells were inactivated by heat (HK) treated with endoglycosidase H (Endo-H), ß-eliminated to remove O-linked glycans, or both treated with endoglycosidase H and ß-elimination. No treatment refers to live cells without any treatment. *P < 0.05 when compared to the no-treatment condition of the same strain. Results are shown as mean ± standard deviation from data generated with samples from eight donors analyzed by duplicate.

Fig 5: Contribution of pattern recognition receptors and cell wall components on the phagocytosis of Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa by human monocyte-derived macrophages. In (A), Human monocyte-derived macrophages were preincubated with 200 µg mL-1 laminarin or 10 µg mL-1 of any of the following antibodies: anti-mannose receptor (MR), anti-CR3, anti-TLR2, or anti-TLR4. Then, cells were coincubated with Acridine Orange-labeled conidia or yeast-like cells at a macrophage-fungus ratio of 1:6, for 2 h at 37°C and 5% (v/v) CO2. Human cells were analyzed by flow cytometry, collecting 50,000 events, which were defined as a human cell interacting with at least one fungal cell. All the interactions were performed in the presence of 5 µg mL-1 polymyxin B. No treatment, cells preincubated with PBS. Results correspond to cells in the late stage of phagocytosis. For all cases, 100% corresponds to human cells preincubated with PBS, and the absolute values were similar to those shown in Figure 1 or (B) of this figure. CR3, complement receptor 3. *P < 0.05 when compared to the no-treatment condition of the same strain. †P< 0.05 when compared to conidia from the same species. In (B), Similar experiments as described in (A), but human cells were not preincubated with any blocking agent. Before coincubation with human monocyte-derived macrophages, conidia, and yeast-like cells were inactivated by heat (HK), treated with endoglycosidase H (Endo-H), ß-eliminated to remove O-linked glycans, or both treated with endoglycosidase H and ß-elimination. No treatment refers to live cells without any treatment. *P < 0.05 when compared to the no-treatment condition of the same strain. For both panels, data were shown as means ± SD from eight donors analyzed by duplicate.