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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 depletes specifically neutrophils and leads to an increased number of eosinophils during the acute 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). Cell populations from lungs of mice were assessed by flow cytometry as described in the Materials and Methods section. Representative flow cytometry contour plots of pulmonary cells within the gated CD45+ subpopulations are shown as (a) eosinophils, (c) alveolar macrophages, (e), tissue macrophages, (g) DCs, (i) B cells, (k) CD4 T cells, (m) CD8 T, and (o) NK cells at 96 h after infection. Numbers inside the gates indicate the mean percentages of the gated subsets. Bar plots of the respective cell subpopulation per lung are shown (b, d, f, h, j, l, n, and p). 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. 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; ns: not significant.
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fig3: The anti-Ly6G mAb depletes specifically neutrophils and leads to an increased number of eosinophils during the acute 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). Cell populations from lungs of mice were assessed by flow cytometry as described in the Materials and Methods section. Representative flow cytometry contour plots of pulmonary cells within the gated CD45+ subpopulations are shown as (a) eosinophils, (c) alveolar macrophages, (e), tissue macrophages, (g) DCs, (i) B cells, (k) CD4 T cells, (m) CD8 T, and (o) NK cells at 96 h after infection. Numbers inside the gates indicate the mean percentages of the gated subsets. Bar plots of the respective cell subpopulation per lung are shown (b, d, f, h, j, l, n, and p). 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. 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; ns: not significant.

Mentions: In order to evaluate the specificity of the anti-Ly6G mAb, we determined other cell populations by flow cytometry (eosinophils, alveolar and tissue macrophages, DCs, B cells, CD4 and CD8 T cells, and NK cells) during these early periods. We did not observe any difference in those cell populations among any of the groups of mice at 48 h or 96 h after inoculation with PBS or P. brasiliensis yeast (Figures 3(c)–3(p)), with the exception of eosinophils, which were significantly more abundant in the neutrophil-depleted infected mice group at 96 h postinfection (Figures 3(a) and 3(b)). Nonetheless, P. brasiliensis infection reduced the number of alveolar (Figures 3(c)-3(d)) and tissue (Figures 3(e)-3(f)) macrophages as well as B cells (Figures 3(i)-3(j)) in the all postinfection periods evaluated; CD4 (Figures 3(k)-3(l)) and CD8 T (Figures 3(m)-3(n)) cells were also diminished in number at 48 h after infection, while DC (Figures 3(g)-3(h)) and NK (Figures 3(o)-3(p)) cells were increased in number at 96 h after challenge in those infected animals.


Depletion of Neutrophils Exacerbates the Early Inflammatory Immune Response in Lungs of Mice Infected with Paracoccidioides brasiliensis
The anti-Ly6G mAb depletes specifically neutrophils and leads to an increased number of eosinophils during the acute 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). Cell populations from lungs of mice were assessed by flow cytometry as described in the Materials and Methods section. Representative flow cytometry contour plots of pulmonary cells within the gated CD45+ subpopulations are shown as (a) eosinophils, (c) alveolar macrophages, (e), tissue macrophages, (g) DCs, (i) B cells, (k) CD4 T cells, (m) CD8 T, and (o) NK cells at 96 h after infection. Numbers inside the gates indicate the mean percentages of the gated subsets. Bar plots of the respective cell subpopulation per lung are shown (b, d, f, h, j, l, n, and p). 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. 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; ns: not significant.
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Related In: Results  -  Collection

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fig3: The anti-Ly6G mAb depletes specifically neutrophils and leads to an increased number of eosinophils during the acute 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). Cell populations from lungs of mice were assessed by flow cytometry as described in the Materials and Methods section. Representative flow cytometry contour plots of pulmonary cells within the gated CD45+ subpopulations are shown as (a) eosinophils, (c) alveolar macrophages, (e), tissue macrophages, (g) DCs, (i) B cells, (k) CD4 T cells, (m) CD8 T, and (o) NK cells at 96 h after infection. Numbers inside the gates indicate the mean percentages of the gated subsets. Bar plots of the respective cell subpopulation per lung are shown (b, d, f, h, j, l, n, and p). 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. 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; ns: not significant.
Mentions: In order to evaluate the specificity of the anti-Ly6G mAb, we determined other cell populations by flow cytometry (eosinophils, alveolar and tissue macrophages, DCs, B cells, CD4 and CD8 T cells, and NK cells) during these early periods. We did not observe any difference in those cell populations among any of the groups of mice at 48 h or 96 h after inoculation with PBS or P. brasiliensis yeast (Figures 3(c)–3(p)), with the exception of eosinophils, which were significantly more abundant in the neutrophil-depleted infected mice group at 96 h postinfection (Figures 3(a) and 3(b)). Nonetheless, P. brasiliensis infection reduced the number of alveolar (Figures 3(c)-3(d)) and tissue (Figures 3(e)-3(f)) macrophages as well as B cells (Figures 3(i)-3(j)) in the all postinfection periods evaluated; CD4 (Figures 3(k)-3(l)) and CD8 T (Figures 3(m)-3(n)) cells were also diminished in number at 48 h after infection, while DC (Figures 3(g)-3(h)) and NK (Figures 3(o)-3(p)) cells were increased in number at 96 h after challenge in those infected animals.

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