Involvement of the Dectin-1 Receptor upon the Effector Mechanisms of Human Phagocytic Cells against Paracoccidioides brasiliensis

Abstract

Paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America, occurs after inhalation of mycelial components of Paracoccidioides spp. When the fungus reaches the lungs and interacts with the alveolar macrophages and other cells, phagocytic cells such as neutrophils and monocytes are immediately recruited to the injured site. The interaction between surface molecules of pathogens and homologous receptors, present on the surface membrane of phagocytes, modulates the innate immune cell activation. Studies have shown the importance of fungal recognition by the Dectin-1 receptor, which can induce a series of cellular protective responses against fungi. The objective of the present study was to evaluate Dectin-1 receptor expression and the effector mechanisms of human monocytes and neutrophils activated or not with different cytokines, such as IFN-γ, TNF-α, and GM-CSF, followed by the challenge with Paracoccidioides brasiliensis (P. brasiliensis or Pb265). Therefore, analysis of Dectin-1 receptor expression was done by flow cytometry whereas the effector mechanisms were evaluated by fungal recovery by colony-forming unit (CFU) counting and hydrogen peroxide (H₂O₂) production. Our results showed that, after treatment with IFN-γ, TNF-α, and GM-CSF and challenge with Pb265, cells, especially monocytes, demonstrated an increase in Dectin-1 expression. Both types of cells treated with the cytokines exhibited a decreased fungal recovery and, conversely, an increased production of H₂O₂. However, when cultures were treated with an anti-Dectin-1 monoclonal antibody, to block the P. brasiliensis binding, a decrease in H₂O₂ production and an increase in fungal recovery were detected. This effect was observed in all cultures treated with the specific monoclonal antibody. These results show the involvement of the Dectin-1 receptor in fungal recognition and its consequent participation in the induction of the killing mechanisms against P. brasiliensis.

Figure 1

Dectin-1 receptor expression (MFI) of monocytes (MO) (a) and neutrophils (PMN) (b) treated or not with IFN-γ, TNF-α, and GM-CSF for 18 hours followed by the Pb265 challenge or not for 4 hours. Data are expressed as the mean of 8 healthy volunteer donors tested. ^∗Statistical significance between groups is indicated (^∗p < 0.05 × MO control).

Figure 2

Histogram of a representative experiment for Dectin-1 receptor blockage for monocytes (a) and neutrophils (b), showing MFI of monocytes or neutrophil cultures (blue line) or monocytes or neutrophils treated with 3.0 μg/mL (orange line) of anti-Dectin-1 monoclonal antibody. The red line represents MFI of the isotypic control.

Figure 3

Percentage of fungal recovery by colony-forming unit (CFU) analysis of monocyte (MO) cultures treated or not with IFN-γ (a), TNF-α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged with Pb265 for 4 hours. Data are expressed as the mean of 8 healthy volunteer donors tested. Statistical significance between groups is indicated.

Figure 4

Percentage of fungal recovery by colony-forming unit (CFU) analysis of neutrophil (PMN) cultures treated or not with IFN-γ (a), TNF-α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged with Pb265 for 4 hours. Data are expressed as the mean of 8 healthy volunteer donors tested. Statistical significance between groups is indicated.

Figure 5

Hydrogen peroxide (H₂O₂) production by monocytes (MO) treated or not with IFN-γ (a), TNF-α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged or not with Pb265 for 4 hours. Box-and-whisker plot showing data distribution of 8 healthy volunteer donors. Statistical significance between groups is indicated.

Figure 6

Hydrogen peroxide (H₂O₂) production by neutrophils (PMN) treated or not with IFN-γ (a), TNF-α (b), and GM-CSF (c) for 18 hours in the presence or absence of the anti-Dectin-1 monoclonal antibody (AD) and challenged or not with Pb265 for 4 hours. Box-and-whisker plot showing data distribution of 8 healthy volunteer donors. Statistical significance between groups is indicated.