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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

Depletion of neutrophils increases levels of chemokines and proinflammatory cytokines in lungs of mice infected with P. brasiliensis during the early stages of infection. BALB/c mice were challenged intranasally with 1.5 × 106 P. brasiliensis (Pb18) yeast cells and treated with an isotype control Ab or the anti-Ly6G mAb specific to neutrophils. Supernatants from lung homogenates of mice sacrificed at 96 h after infection were analyzed using a commercial kit and the Luminex System as described in Material and Methods. (a) chemokines (KC, LIX, MIP-2, MCP-1, MIP-1α, and MIP-1β) and (b) proinflammatory cytokines (IL-1α, IL-1β, IL-6, IL-15, IL-17, and TNF-α). Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). ∗P < 0.05 and ∗∗P < 0.01 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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fig4: Depletion of neutrophils increases levels of chemokines and proinflammatory cytokines in lungs of mice infected with P. brasiliensis during the early stages of infection. BALB/c mice were challenged intranasally with 1.5 × 106 P. brasiliensis (Pb18) yeast cells and treated with an isotype control Ab or the anti-Ly6G mAb specific to neutrophils. Supernatants from lung homogenates of mice sacrificed at 96 h after infection were analyzed using a commercial kit and the Luminex System as described in Material and Methods. (a) chemokines (KC, LIX, MIP-2, MCP-1, MIP-1α, and MIP-1β) and (b) proinflammatory cytokines (IL-1α, IL-1β, IL-6, IL-15, IL-17, and TNF-α). Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). ∗P < 0.05 and ∗∗P < 0.01 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: We also quantified chemokine and proinflammatory cytokine levels in the lungs of infected and noninfected mice treated with the mAb and sacrificed at 48 and 96 h after inoculation. We observed that several chemokines and proinflammatory cytokines showed significantly higher levels in mice infected and treated with the specific mAb when compared with infected-untreated or isotype-treated mice. The molecules that depicted significant increases in those mAb treated-mice at 96 h after infection were CXCL1 (KC), CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CXCL2 (MIP-2), CXCL5 (LIX), IL-1α, IL-6, G-CSF, M-CSF, and TNF-α (for all, P < 0.01) and IL-1β, IL-15, IL-10, and LIF (for all, P < 0.05). In contrast, IFN-γ (P < 0.01), IL-2, IL-17, and CCL5 (RANTES) (for all, P < 0.05) were significantly lower (Figures 4 and 5). Similar results were observed at 48 h after infection (data not shown).


Depletion of Neutrophils Exacerbates the Early Inflammatory Immune Response in Lungs of Mice Infected with Paracoccidioides brasiliensis
Depletion of neutrophils increases levels of chemokines and proinflammatory cytokines in lungs of mice infected with P. brasiliensis during the early stages of infection. BALB/c mice were challenged intranasally with 1.5 × 106 P. brasiliensis (Pb18) yeast cells and treated with an isotype control Ab or the anti-Ly6G mAb specific to neutrophils. Supernatants from lung homogenates of mice sacrificed at 96 h after infection were analyzed using a commercial kit and the Luminex System as described in Material and Methods. (a) chemokines (KC, LIX, MIP-2, MCP-1, MIP-1α, and MIP-1β) and (b) proinflammatory cytokines (IL-1α, IL-1β, IL-6, IL-15, IL-17, and TNF-α). Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). ∗P < 0.05 and ∗∗P < 0.01 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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fig4: Depletion of neutrophils increases levels of chemokines and proinflammatory cytokines in lungs of mice infected with P. brasiliensis during the early stages of infection. BALB/c mice were challenged intranasally with 1.5 × 106 P. brasiliensis (Pb18) yeast cells and treated with an isotype control Ab or the anti-Ly6G mAb specific to neutrophils. Supernatants from lung homogenates of mice sacrificed at 96 h after infection were analyzed using a commercial kit and the Luminex System as described in Material and Methods. (a) chemokines (KC, LIX, MIP-2, MCP-1, MIP-1α, and MIP-1β) and (b) proinflammatory cytokines (IL-1α, IL-1β, IL-6, IL-15, IL-17, and TNF-α). Data shown represent median and IQR (n = 4-5 mice/group; representative of two independent experiments). ∗P < 0.05 and ∗∗P < 0.01 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: We also quantified chemokine and proinflammatory cytokine levels in the lungs of infected and noninfected mice treated with the mAb and sacrificed at 48 and 96 h after inoculation. We observed that several chemokines and proinflammatory cytokines showed significantly higher levels in mice infected and treated with the specific mAb when compared with infected-untreated or isotype-treated mice. The molecules that depicted significant increases in those mAb treated-mice at 96 h after infection were CXCL1 (KC), CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CXCL2 (MIP-2), CXCL5 (LIX), IL-1α, IL-6, G-CSF, M-CSF, and TNF-α (for all, P < 0.01) and IL-1β, IL-15, IL-10, and LIF (for all, P < 0.05). In contrast, IFN-γ (P < 0.01), IL-2, IL-17, and CCL5 (RANTES) (for all, P < 0.05) were significantly lower (Figures 4 and 5). Similar results were observed at 48 h after infection (data not shown).

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