Table 1.
Primers sequences.
Fig 1.
The anti-Ly6G mAb efficiently depleted neutrophils during chronic stage of PCM.
BALB/c mice were intranasally inoculated with PBS or 1.5x106 P. brasiliensis (Pb18) yeast cells, treated with an isotype control Ab or the anti-Ly6G specific mAb against neutrophils during the chronic phase of P. brasiliensis infection (4 or 8 week post-challenge). Neutrophils were assessed by flow cytometry as described in the Materials and Methods section. 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 + anti-neutrophils, control mice treated with anti-Ly6G mAb (clone: 1A8); Pb18, infected-untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab; Pb18 + anti-neutrophils, infected mice treated with anti-Ly6G mAb.
Fig 2.
Depletion of neutrophils is associated with a decreased number of CD4 T cells, B cells, eosinophils, DCs and MDSc during the chronic stages of P. brasiliensis infection.
BALB/c mice were inoculated with PBS or 1.5x106 P. brasiliensis (Pb18) yeast cells and treated with an isotype control Ab or anti-Ly6G mAb at 4 weeks (A, B, C, D) or 8 weeks (E, F, G, H) post-challenge and analyzed at 12 weeks post-infection. Cell populations from lungs of mice were assessed by flow cytometry. CD4 T cells (A, E), B cells (B, F) DCs (C, G), myeloid suppressor cells (D) and eosinophils (H) were identified as described in the Materials and Methods section. 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 + anti-neutrophils, control mice treated with anti-Ly6G mAb (clone: 1A8); Pb18, infected-untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab; Pb18 + anti-neutrophils, infected mice treated with anti-Ly6G mAb.
Fig 3.
Effect of neutrophil depletion on chemokine levels in lungs during the chronic stage of P. brasiliensis infection.
BALB/c mice were infected with 1.5x106 P. brasiliensis (Pb18) yeast cells and treated with an isotype control Ab or anti-Ly6G mAb at 4 weeks post-challenge and analyzed at 12 weeks post-infection. Chemokines levels from lungs of mice were assessed by Luminex 200 system as described in the Materials and Methods section. A) Chemokines associated with the recruitment of granulocyte cells (neutrophils, eosinophils and basophils); B) chemokines associated with the recruitment of mononuclear cells (lymphocytes, monocyte/macrophage and dendritic cells). Data shown represent median and IQR (n = 4–5 mice/group; representative of two independent experiments). *, P<0.05, **, P<0.01 and ***, P<0.001 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 + anti-neutrophils, control mice treated with anti-Ly6G mAb (clone: 1A8); Pb18, infected-untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab; Pb18 + anti-neutrophils, infected mice treated with anti-Ly6G mAb.
Fig 4.
Effect of neutrophils depletion on cytokine levels in lungs during the chronic stage of P. brasiliensis infection.
BALB/c mice were infected with 1.5x106 P. brasiliensis (Pb18) yeast cells and treated with an isotype control Ab or anti-Ly6G mAb at 4 weeks post-challenge and analyzed at 12 weeks post-infection. Cytokines levels associated with inflammatory response from lungs of mice were measured by Luminex 200 system as described in the Materials and Methods section. Data shown represent median and IQR (n = 4–5 mice/group; representative of two independent experiments). *, P<0.05, **, P<0.01 and ***, P<0.001 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 + anti-neutrophils, control mice treated with anti-Ly6G mAb (clone: 1A8); Pb18, infected-untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab; Pb18 + anti-neutrophils, infected mice treated with anti-Ly6G mAb.
Fig 5.
Neutrophil depletion is associated with decreased intracellular cytokine levels and changes in transcription factor mRNA expression related to T helper subsets during the chronic stages of P. brasiliensis infection.
BALB/c mice infected with 1.5x106 P. brasiliensis yeast cells and treated with an isotype control Ab or anti-Ly6G mAb at 4 weeks post-infected and analyzed at 12 weeks post-infection. Flow cytometry was performed to identify the following lymphocytes subsets: A) Th1 (LT CD4+IFNγ+), B) Th2 (LT CD4+IL-4+), C) Th17 (LT CD4+IL-17+), D) Th22 (LT CD4+IL-22+), and E) Treg cells (LT CD4+CD25+ FoxP3+). Data shown represent median and IQR (n = 4–5 mice/group; representative of two independent experiments). Relative quantification of the mRNA expression for T-bet (F), GATA-3 (G), RORc (H), Ahr (I), FoxP3 (J), iNOS2 (K) and Arginase-1 (L) were measured in lungs of mice. Results are expressed as mean ± SEM (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 + anti-neutrophils, control mice treated with anti-Ly6G mAb (clone: 1A8); Pb18, infected-untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab; Pb18 + anti-neutrophils, infected mice treated with anti-Ly6G mAb.
Fig 6.
Depletion of neutrophils is associated wit a reduction on the fungal burden in lungs, liver, and spleen from mice infected with P. brasiliensis at the chronic stages of infection.
Fungal load measurement was performed in lung, liver and spleen of mice at 12 weeks post-challenge with 1.5x106 P. brasiliensis (Pb18) yeast cells and treated with an isotype control Ab or anti-Ly6G mAb at 4 weeks (A, B, C) or 8 weeks (D, E, F) post-challenge. Data shown represent median and IQR (n = 4–5 mice/group; representative of two independent experiments). A statistically significant reduction in fungal burden was observed in the lungs of mice infected and treated with the anti-Ly6G mAb (*, P<0.05) compared to the infected untreated mice and the infected-isotype control Ab treated mice. Pb18, infected, untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab (clone: 2A3); Pb18 + anti-neutrophils, infected mice treated with anti-Ly6G mAb (clone: 1A8).
Fig 7.
Depletion of neutrophils is associated with a reduction in both granulomatous inflammatory response and fibrosis in lungs from mice challenged with P. brasiliensis.
Microphotographs are representative of lungs from uninfected (A, B, C), infected and untreated mice (D, E, F) or infected and treated with an isotype control Ab (G, H, I) or an anti-Ly6G mAb specific to neutrophils (J, K, L) at 12 weeks post-challenge and obtained from groups of 4–5 mice. Lungs were fixed, embedded in paraffin, cut and stained using H&E staining to determine lung inflammatory response (A, D, G, J), methenamine silver staining (B, E, H, K) to identify P. brasiliensis’ yeast cells and Masson’s trichrome (C, F, I, L) to identify and differentiate collagen fibers as described in the Materials and Methods section. Arrowheads indicate collagen fibers (in white). Quantitative analysis of total injured lung area (M) and size of granuloma (N) were assessed by ImageJ software as described in the Materials and Methods section. These results are representative of two independent experiments. Magnification 10X.
Fig 8.
Neutrophils depletion is associated with a decrease of collagen levels and changes on mRNA level expression of Colagen-1 and Colagen-3 in lungs of mice infected with P. brasiliensis yeast cells.
BALB/c mice were infected with 1.5x106 P. brasiliensis (Pb18) yeast cells and treated with an isotype control Ab or anti-Ly6G mAb at 4 weeks post-challenge and analyzed at 12 weeks post-infection. Total collagen (A) and relative quantification of the mRNA expression for Col-1α2 (B) and Col-3α1 (C) were performed in lungs of mice (n = 4–5 mice/group; representative of two independent experiments). Results are expressed as median ± IQR. *, 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 + anti-neutrophils, control mice treated with anti-Ly6G mAb (clone: 1A8); Pb18, infected, untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab; Pb18 + anti-neutrophils, infected mice treated with anti-Ly6G mAb.
Fig 9.
Reduction of the granulomatous inflammatory response in lungs of neutrophil-depleted mice challenged with P. brasiliensis during the chronic course of infection is associated with a decrease of expression of TGF-β1 and MMP-8 and overexpression of TGF-β3, MMP-12 and MMP-14.
BALB/c mice were infected with 1.5x106 P. brasiliensis (Pb18) yeast cells and treated with an isotype control Ab or anti-Ly6G mAb at 4 weeks post-challenge and analyzed at 12 weeks post-infection. Relative quantification of the mRNA expression was performed in lungs of mice for the following genes: A) TGF-β1, TGF-β3 and TGF-βRc2; B) MMP-1, MMP-8, MMP-12, MMP-13, MMP-14 and MMP-15; and C) TIMP-1 and TIMP-2 (n = 4–5 mice/group; representative of two independent experiments). Results are expressed as median ± IQR. *, 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 + anti-neutrophils, control mice treated with anti-Ly6G mAb (clone: 1A8); Pb18, infected, untreated mice; Pb18 + Iso, infected mice treated with isotype control Ab; Pb18 + anti-neutrophils, infected mice treated with anti-Ly6G mAb.