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Role of IL-17A on resolution of pulmonary C. neoformans infection.

Wozniak KL, Hardison SE, Kolls JK, Wormley FL - PLoS ONE (2011)

Bottom Line: Signaling through the IFN-γ receptor (R) was required for increased IL-17A production, however, a Th17-type cytokine profile was not observed.Neutrophils were found to be the predominant leukocytic source of IL-17A, rather than T cells, suggesting that the IL-17A produced was not part of a T cell-mediated Th17-type immune response.Also, depletion of IL-17A did not affect the local production of other cytokines.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The University of Texas at San Antonio, San Antonio, Texas, United States of America. Karen.Wozniak@utsa.edu

ABSTRACT
The current studies evaluated the role of interleukin (IL)-17A in the induction of protective immunity against pulmonary cryptococcosis in mice. Protection against pulmonary infection with C. neoformans strain H99γ was associated with increased IL-17A production. Signaling through the IFN-γ receptor (R) was required for increased IL-17A production, however, a Th17-type cytokine profile was not observed. Neutrophils were found to be the predominant leukocytic source of IL-17A, rather than T cells, suggesting that the IL-17A produced was not part of a T cell-mediated Th17-type immune response. Depletion of IL-17A in mice during pulmonary infection with C. neoformans strain H99γ resulted in an initial increase in pulmonary fungal burden, but had no effect on cryptococcal burden at later time points. Also, depletion of IL-17A did not affect the local production of other cytokines. IL-17RA⁻/⁻ mice infected with C. neoformans strain H99γ survived the primary infection as well as a secondary challenge with wild-type cryptococci. However, dissemination of the wild-type strain to the brain was noted in the surviving IL-17RA⁻/⁻ mice. Altogether, our results suggested that IL-17A may be important for optimal protective immune responsiveness during pulmonary C. neoformans infection, but protective Th1-type immune responses are sufficient for protection against cryptococcal infection.

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Lung neutrophils are the predominant leukocyte population expressing IL-17A during pulmonary infection with C. neoformans strain H99γ.BALB/c mice received an intranasal inoculum of 1×104 CFU of C. neoformans strain H99γ in 50 µl of sterile PBS (gray bars). Naïve Balb/c mice (white bars) are shown as controls. The lungs were excised at day 7 post-inoculation and a single cell suspension generated using enzymatic digestion. The leukocytes were stained with anti-mouse antibodies (CD45, 1A8 (Neut) CD4, CD8, F4/80 (Mac), CD11b/CD11c (DC), CD4/Fox3p (Treg), CD4/DX5 (NKT),γδ, B220 (B cell), SiglecF/CD11b (Eosinophil), FcεR1α/CD117/CD34 (Mast cell)), fixed, permeabilized, and incubated with anti-mouse antibodies specific for IL-17A and quantified by flow cytometry. Flow cytometry data are cumulative results of five independent experiments using pooled leukocytes from 5 mice per group per experiment. Results shown are the percentage of leukocytes expressing the indicated surface markers and IL-17A. Asterisks (*) indicate where significant differences were observed (P<0.0001) between naïve mice and mice infected with C. neoformans strain H99γ.
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pone-0017204-g002: Lung neutrophils are the predominant leukocyte population expressing IL-17A during pulmonary infection with C. neoformans strain H99γ.BALB/c mice received an intranasal inoculum of 1×104 CFU of C. neoformans strain H99γ in 50 µl of sterile PBS (gray bars). Naïve Balb/c mice (white bars) are shown as controls. The lungs were excised at day 7 post-inoculation and a single cell suspension generated using enzymatic digestion. The leukocytes were stained with anti-mouse antibodies (CD45, 1A8 (Neut) CD4, CD8, F4/80 (Mac), CD11b/CD11c (DC), CD4/Fox3p (Treg), CD4/DX5 (NKT),γδ, B220 (B cell), SiglecF/CD11b (Eosinophil), FcεR1α/CD117/CD34 (Mast cell)), fixed, permeabilized, and incubated with anti-mouse antibodies specific for IL-17A and quantified by flow cytometry. Flow cytometry data are cumulative results of five independent experiments using pooled leukocytes from 5 mice per group per experiment. Results shown are the percentage of leukocytes expressing the indicated surface markers and IL-17A. Asterisks (*) indicate where significant differences were observed (P<0.0001) between naïve mice and mice infected with C. neoformans strain H99γ.

Mentions: We sought to determine the leukocyte population that was the predominant source of IL-17A in the lungs of mice infected with C. neoformans strain H99γ. Total leukocytes were isolated from lung tissues of naïve mice and C. neoformans strain H99γ-infected mice on day 7 post-challenge, and the lymphocyte subpopulations characterized for intracellular IL-17A expression by flow cytometry. Leukocyte populations examined included CD4+ T cells, CD8+ T cells, γδ T cells, regulatory T cells, natural killer T cells, macrophages, dendritic cells, neutrophils, B cells, eosinophils, and mast cells. Figure 2 demonstrates that the majority of intracellular IL-17A expression in both naïve mice and mice infected with C. neoformans strain H99γ was observed in Ly6G+ neutrophils (detected with the 1A8 monoclonal antibody). We also observed that mice infected with C. neoformans strain H99γ had significantly higher intracellular IL-17A in total CD45+ cells, CD4+ T cells, and dendritic cells compared to naïve mice. Although neutrophils appear to be the predominant source of IL-17A, we cannot conclude that increased IL-17A production by other cell types in response to C. neoformans strain H99γ infection does not contribute to the observed phenotype. Representative flow cytometry plots are shown in Figure S1.


Role of IL-17A on resolution of pulmonary C. neoformans infection.

Wozniak KL, Hardison SE, Kolls JK, Wormley FL - PLoS ONE (2011)

Lung neutrophils are the predominant leukocyte population expressing IL-17A during pulmonary infection with C. neoformans strain H99γ.BALB/c mice received an intranasal inoculum of 1×104 CFU of C. neoformans strain H99γ in 50 µl of sterile PBS (gray bars). Naïve Balb/c mice (white bars) are shown as controls. The lungs were excised at day 7 post-inoculation and a single cell suspension generated using enzymatic digestion. The leukocytes were stained with anti-mouse antibodies (CD45, 1A8 (Neut) CD4, CD8, F4/80 (Mac), CD11b/CD11c (DC), CD4/Fox3p (Treg), CD4/DX5 (NKT),γδ, B220 (B cell), SiglecF/CD11b (Eosinophil), FcεR1α/CD117/CD34 (Mast cell)), fixed, permeabilized, and incubated with anti-mouse antibodies specific for IL-17A and quantified by flow cytometry. Flow cytometry data are cumulative results of five independent experiments using pooled leukocytes from 5 mice per group per experiment. Results shown are the percentage of leukocytes expressing the indicated surface markers and IL-17A. Asterisks (*) indicate where significant differences were observed (P<0.0001) between naïve mice and mice infected with C. neoformans strain H99γ.
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pone-0017204-g002: Lung neutrophils are the predominant leukocyte population expressing IL-17A during pulmonary infection with C. neoformans strain H99γ.BALB/c mice received an intranasal inoculum of 1×104 CFU of C. neoformans strain H99γ in 50 µl of sterile PBS (gray bars). Naïve Balb/c mice (white bars) are shown as controls. The lungs were excised at day 7 post-inoculation and a single cell suspension generated using enzymatic digestion. The leukocytes were stained with anti-mouse antibodies (CD45, 1A8 (Neut) CD4, CD8, F4/80 (Mac), CD11b/CD11c (DC), CD4/Fox3p (Treg), CD4/DX5 (NKT),γδ, B220 (B cell), SiglecF/CD11b (Eosinophil), FcεR1α/CD117/CD34 (Mast cell)), fixed, permeabilized, and incubated with anti-mouse antibodies specific for IL-17A and quantified by flow cytometry. Flow cytometry data are cumulative results of five independent experiments using pooled leukocytes from 5 mice per group per experiment. Results shown are the percentage of leukocytes expressing the indicated surface markers and IL-17A. Asterisks (*) indicate where significant differences were observed (P<0.0001) between naïve mice and mice infected with C. neoformans strain H99γ.
Mentions: We sought to determine the leukocyte population that was the predominant source of IL-17A in the lungs of mice infected with C. neoformans strain H99γ. Total leukocytes were isolated from lung tissues of naïve mice and C. neoformans strain H99γ-infected mice on day 7 post-challenge, and the lymphocyte subpopulations characterized for intracellular IL-17A expression by flow cytometry. Leukocyte populations examined included CD4+ T cells, CD8+ T cells, γδ T cells, regulatory T cells, natural killer T cells, macrophages, dendritic cells, neutrophils, B cells, eosinophils, and mast cells. Figure 2 demonstrates that the majority of intracellular IL-17A expression in both naïve mice and mice infected with C. neoformans strain H99γ was observed in Ly6G+ neutrophils (detected with the 1A8 monoclonal antibody). We also observed that mice infected with C. neoformans strain H99γ had significantly higher intracellular IL-17A in total CD45+ cells, CD4+ T cells, and dendritic cells compared to naïve mice. Although neutrophils appear to be the predominant source of IL-17A, we cannot conclude that increased IL-17A production by other cell types in response to C. neoformans strain H99γ infection does not contribute to the observed phenotype. Representative flow cytometry plots are shown in Figure S1.

Bottom Line: Signaling through the IFN-γ receptor (R) was required for increased IL-17A production, however, a Th17-type cytokine profile was not observed.Neutrophils were found to be the predominant leukocytic source of IL-17A, rather than T cells, suggesting that the IL-17A produced was not part of a T cell-mediated Th17-type immune response.Also, depletion of IL-17A did not affect the local production of other cytokines.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The University of Texas at San Antonio, San Antonio, Texas, United States of America. Karen.Wozniak@utsa.edu

ABSTRACT
The current studies evaluated the role of interleukin (IL)-17A in the induction of protective immunity against pulmonary cryptococcosis in mice. Protection against pulmonary infection with C. neoformans strain H99γ was associated with increased IL-17A production. Signaling through the IFN-γ receptor (R) was required for increased IL-17A production, however, a Th17-type cytokine profile was not observed. Neutrophils were found to be the predominant leukocytic source of IL-17A, rather than T cells, suggesting that the IL-17A produced was not part of a T cell-mediated Th17-type immune response. Depletion of IL-17A in mice during pulmonary infection with C. neoformans strain H99γ resulted in an initial increase in pulmonary fungal burden, but had no effect on cryptococcal burden at later time points. Also, depletion of IL-17A did not affect the local production of other cytokines. IL-17RA⁻/⁻ mice infected with C. neoformans strain H99γ survived the primary infection as well as a secondary challenge with wild-type cryptococci. However, dissemination of the wild-type strain to the brain was noted in the surviving IL-17RA⁻/⁻ mice. Altogether, our results suggested that IL-17A may be important for optimal protective immune responsiveness during pulmonary C. neoformans infection, but protective Th1-type immune responses are sufficient for protection against cryptococcal infection.

Show MeSH
Related in: MedlinePlus