Limits...
Depletion of Neutrophils Exacerbates the Early Inflammatory Immune Response in Lungs of Mice Infected with Paracoccidioides brasiliensis

View Article: PubMed Central - PubMed

ABSTRACT

Neutrophils predominate during the acute phase of the Paracoccidioides brasiliensis infection. Herein, we determined the role of the neutrophil during the early stages of experimental pulmonary paracoccidioidomycosis using a monoclonal antibody (mAb) specific for neutrophils. Male BALB/c mice were inoculated intranasally with 1.5 × 106 or 2 × 106 P. brasiliensis yeast cells. The mAb was administered 24 h before infection, followed by doses every 48 h until mice were sacrificed. Survival time was evaluated and mice were sacrificed at 48 h and 96 h after inoculation to assess cellularity, fungal load, cytokine/chemokine levels, and histopathological analysis. Neutrophils from mAb-treated mice were efficiently depleted (99.04%). Eighty percent of the mice treated with the mAb and infected with 1.5 × 106 yeast cells died during the first two weeks after infection. When mice were treated and infected with 2 × 106 yeast cells, 100% of them succumbed by the first week after infection. During the acute inflammatory response significant increases in numbers of eosinophils, fungal load and levels of proinflammatory cytokines/chemokines were observed in the mAb-treated mice. We also confirmed that neutrophils are an important source of IFN-γ and IL-17. These results indicate that neutrophils are essential for protection as well as being important for regulating the early inflammatory immune response in experimental pulmonary paracoccidioidomycosis.

No MeSH data available.


Related in: MedlinePlus

The anti-Ly6G mAb efficiently depleted neutrophils demonstrating their essential role during the early stages of P. brasiliensis infection. BALB/c mice were intranasally inoculated with PBS or 1.5 × 106 P. brasiliensis (Pb18) yeast cells, treated with an isotype control Ab or the anti-Ly6G specific mAb against neutrophils, and analyzed during the acute phase of P. brasiliensis infection (48 h and 96 h after challenge). Neutrophils were assessed by flow cytometry as described in the Materials and Methods section, which were identified as CD45+/CD11b+/Gr1+. (a) Representative flow cytometry contour plots of neutrophils within the gated CD45+ subpopulations are shown. Numbers inside the gates indicate the mean percentages of the gated subsets. (b) Bar plots of the neutrophils per lung. Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). ∗P < 0.05 comparing infected-untreated mice versus control mice or comparing infected-anti-Ly6G mAb-treated mice versus infected-untreated mice. (c) BALB/c mice were challenged intranasally with 1.5 × 106 or 2 × 106 P. brasiliensis yeast cells, treated as previously described, and evaluated over 12 weeks. Results are representative of two independent experiments (n = 10-11 mice/group). A statistically significant difference between the survival plots of the anti-Ly6G and the isotype or untreated mice was determined (P < 0.001). (d) Comparison of fungal burden in the lungs of BALB/c mice infected intranasally with 1.5 × 106 P. brasiliensis yeast cells, treated or not as previously described, and sacrificed at 48 h and 96 h after challenge. Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). A statistically significant increase in fungal burden was observed in the lungs of infected mice treated with the anti-Ly6G mAb (∗∗P < 0.01) compared to the infected-untreated mice and the infected-isotype control Ab treated mice. PBS, control mice; PBS + Iso, control mice treated with isotype control Ab (clone: 2A3); PBS + antineutrophils, control mice treated with anti-Ly6G mAb (clone: 1A8); Pb18, infected-untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab; Pb18 + antineutrophils, infected mice treated with anti-Ly6G mAb.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5015031&req=5

fig1: The anti-Ly6G mAb efficiently depleted neutrophils demonstrating their essential role during the early stages of P. brasiliensis infection. BALB/c mice were intranasally inoculated with PBS or 1.5 × 106 P. brasiliensis (Pb18) yeast cells, treated with an isotype control Ab or the anti-Ly6G specific mAb against neutrophils, and analyzed during the acute phase of P. brasiliensis infection (48 h and 96 h after challenge). Neutrophils were assessed by flow cytometry as described in the Materials and Methods section, which were identified as CD45+/CD11b+/Gr1+. (a) Representative flow cytometry contour plots of neutrophils within the gated CD45+ subpopulations are shown. Numbers inside the gates indicate the mean percentages of the gated subsets. (b) Bar plots of the neutrophils per lung. Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). ∗P < 0.05 comparing infected-untreated mice versus control mice or comparing infected-anti-Ly6G mAb-treated mice versus infected-untreated mice. (c) BALB/c mice were challenged intranasally with 1.5 × 106 or 2 × 106 P. brasiliensis yeast cells, treated as previously described, and evaluated over 12 weeks. Results are representative of two independent experiments (n = 10-11 mice/group). A statistically significant difference between the survival plots of the anti-Ly6G and the isotype or untreated mice was determined (P < 0.001). (d) Comparison of fungal burden in the lungs of BALB/c mice infected intranasally with 1.5 × 106 P. brasiliensis yeast cells, treated or not as previously described, and sacrificed at 48 h and 96 h after challenge. Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). A statistically significant increase in fungal burden was observed in the lungs of infected mice treated with the anti-Ly6G mAb (∗∗P < 0.01) compared to the infected-untreated mice and the infected-isotype control Ab treated mice. PBS, control mice; PBS + Iso, control mice treated with isotype control Ab (clone: 2A3); PBS + antineutrophils, control mice treated with anti-Ly6G mAb (clone: 1A8); Pb18, infected-untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab; Pb18 + antineutrophils, infected mice treated with anti-Ly6G mAb.

Mentions: In order to investigate the efficiency of the mAb anti-Ly6G (clone 1A8) to deplete specifically neutrophils in BALB/c mice inoculated with PBS or 1.5 × 106 P. brasiliensis yeast cells, a flow cytometry assay was carried out for lung homogenates at 48 h and 96 h after inoculation. The anti-Ly6G mAb depleted only neutrophils with an efficiency of 99.04% in P. brasiliensis-infected mice when compared to infected-untreated mice (Figure 1(a)). Untreated and isotype control-treated infected mice showed equivalent numbers of neutrophils, characteristic of this model at the early phase of infection (Figures 1(a) and 1(b)).


Depletion of Neutrophils Exacerbates the Early Inflammatory Immune Response in Lungs of Mice Infected with Paracoccidioides brasiliensis
The anti-Ly6G mAb efficiently depleted neutrophils demonstrating their essential role during the early stages of P. brasiliensis infection. BALB/c mice were intranasally inoculated with PBS or 1.5 × 106 P. brasiliensis (Pb18) yeast cells, treated with an isotype control Ab or the anti-Ly6G specific mAb against neutrophils, and analyzed during the acute phase of P. brasiliensis infection (48 h and 96 h after challenge). Neutrophils were assessed by flow cytometry as described in the Materials and Methods section, which were identified as CD45+/CD11b+/Gr1+. (a) Representative flow cytometry contour plots of neutrophils within the gated CD45+ subpopulations are shown. Numbers inside the gates indicate the mean percentages of the gated subsets. (b) Bar plots of the neutrophils per lung. Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). ∗P < 0.05 comparing infected-untreated mice versus control mice or comparing infected-anti-Ly6G mAb-treated mice versus infected-untreated mice. (c) BALB/c mice were challenged intranasally with 1.5 × 106 or 2 × 106 P. brasiliensis yeast cells, treated as previously described, and evaluated over 12 weeks. Results are representative of two independent experiments (n = 10-11 mice/group). A statistically significant difference between the survival plots of the anti-Ly6G and the isotype or untreated mice was determined (P < 0.001). (d) Comparison of fungal burden in the lungs of BALB/c mice infected intranasally with 1.5 × 106 P. brasiliensis yeast cells, treated or not as previously described, and sacrificed at 48 h and 96 h after challenge. Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). A statistically significant increase in fungal burden was observed in the lungs of infected mice treated with the anti-Ly6G mAb (∗∗P < 0.01) compared to the infected-untreated mice and the infected-isotype control Ab treated mice. PBS, control mice; PBS + Iso, control mice treated with isotype control Ab (clone: 2A3); PBS + antineutrophils, control mice treated with anti-Ly6G mAb (clone: 1A8); Pb18, infected-untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab; Pb18 + antineutrophils, infected mice treated with anti-Ly6G mAb.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5015031&req=5

fig1: The anti-Ly6G mAb efficiently depleted neutrophils demonstrating their essential role during the early stages of P. brasiliensis infection. BALB/c mice were intranasally inoculated with PBS or 1.5 × 106 P. brasiliensis (Pb18) yeast cells, treated with an isotype control Ab or the anti-Ly6G specific mAb against neutrophils, and analyzed during the acute phase of P. brasiliensis infection (48 h and 96 h after challenge). Neutrophils were assessed by flow cytometry as described in the Materials and Methods section, which were identified as CD45+/CD11b+/Gr1+. (a) Representative flow cytometry contour plots of neutrophils within the gated CD45+ subpopulations are shown. Numbers inside the gates indicate the mean percentages of the gated subsets. (b) Bar plots of the neutrophils per lung. Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). ∗P < 0.05 comparing infected-untreated mice versus control mice or comparing infected-anti-Ly6G mAb-treated mice versus infected-untreated mice. (c) BALB/c mice were challenged intranasally with 1.5 × 106 or 2 × 106 P. brasiliensis yeast cells, treated as previously described, and evaluated over 12 weeks. Results are representative of two independent experiments (n = 10-11 mice/group). A statistically significant difference between the survival plots of the anti-Ly6G and the isotype or untreated mice was determined (P < 0.001). (d) Comparison of fungal burden in the lungs of BALB/c mice infected intranasally with 1.5 × 106 P. brasiliensis yeast cells, treated or not as previously described, and sacrificed at 48 h and 96 h after challenge. Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). A statistically significant increase in fungal burden was observed in the lungs of infected mice treated with the anti-Ly6G mAb (∗∗P < 0.01) compared to the infected-untreated mice and the infected-isotype control Ab treated mice. PBS, control mice; PBS + Iso, control mice treated with isotype control Ab (clone: 2A3); PBS + antineutrophils, control mice treated with anti-Ly6G mAb (clone: 1A8); Pb18, infected-untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab; Pb18 + antineutrophils, infected mice treated with anti-Ly6G mAb.
Mentions: In order to investigate the efficiency of the mAb anti-Ly6G (clone 1A8) to deplete specifically neutrophils in BALB/c mice inoculated with PBS or 1.5 × 106 P. brasiliensis yeast cells, a flow cytometry assay was carried out for lung homogenates at 48 h and 96 h after inoculation. The anti-Ly6G mAb depleted only neutrophils with an efficiency of 99.04% in P. brasiliensis-infected mice when compared to infected-untreated mice (Figure 1(a)). Untreated and isotype control-treated infected mice showed equivalent numbers of neutrophils, characteristic of this model at the early phase of infection (Figures 1(a) and 1(b)).

View Article: PubMed Central - PubMed

ABSTRACT

Neutrophils predominate during the acute phase of the Paracoccidioides brasiliensis infection. Herein, we determined the role of the neutrophil during the early stages of experimental pulmonary paracoccidioidomycosis using a monoclonal antibody (mAb) specific for neutrophils. Male BALB/c mice were inoculated intranasally with 1.5 &times; 106 or 2 &times; 106 P. brasiliensis yeast cells. The mAb was administered 24&thinsp;h before infection, followed by doses every 48&thinsp;h until mice were sacrificed. Survival time was evaluated and mice were sacrificed at 48&thinsp;h and 96&thinsp;h after inoculation to assess cellularity, fungal load, cytokine/chemokine levels, and histopathological analysis. Neutrophils from mAb-treated mice were efficiently depleted (99.04%). Eighty percent of the mice treated with the mAb and infected with 1.5 &times; 106 yeast cells died during the first two weeks after infection. When mice were treated and infected with 2 &times; 106 yeast cells, 100% of them succumbed by the first week after infection. During the acute inflammatory response significant increases in numbers of eosinophils, fungal load and levels of proinflammatory cytokines/chemokines were observed in the mAb-treated mice. We also confirmed that neutrophils are an important source of IFN-&gamma; and IL-17. These results indicate that neutrophils are essential for protection as well as being important for regulating the early inflammatory immune response in experimental pulmonary paracoccidioidomycosis.

No MeSH data available.


Related in: MedlinePlus