Research ArticleFUNGAL INFECTION

Type III interferon is a critical regulator of innate antifungal immunity

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Science Immunology  06 Oct 2017:
Vol. 2, Issue 16, eaan5357
DOI: 10.1126/sciimmunol.aan5357
  • Fig. 1 Dysfunctional antifungal neutrophils have impaired expression of IFN-inducible genes.

    (A) ROS production by airway-infiltrating neutrophils isolated from CCR2-depleted mice (red histogram) or control littermates (gray histogram) 48 hours after infection. Graph shows mean ± SEM of ROS mean fluorescence intensity (MFI) for five mice per group assayed by FACS. FITC, fluorescein isothiocyanate. (B) Af pulmonary fungal burden 48 hours after infection. CFU, colony-forming units. (C) Differential gene expression as assessed by RNA-seq of pulmonary neutrophils isolated from uninfected controls (naïve) or mice infected with Af for 48 hours. Heat map depicts the top 100 genes expressed at a fold change of >2.5 in control neutrophils more than in neutrophils from CCR2-depleted mice. (D) Venn diagram of differentially expressed genes. Predicted upstream regulators of the 231 genes as determined with Ingenuity Pathway Analysis (IPA) software. (E to J) Kinetics of IFN expression in the lung of mice at different times after Af infection. (E to G) Gene expression as determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using TaqMan probes. Data are means ± SEM of five mice per time point. Graphs of (H) to (J) show mean ± SEM of each cytokine as measured by ELISA in five mice per time point. (K to M) Pulmonary qRT-PCR of IFN gene expression at 3 hours (K) or 48 hours (L and M) after Af infection. *P < 0.05; **P < 0.01; ***P < 0.001, calculated by Mann-Whitney nonparametric test or Kruskal-Wallis test using Prism software.

  • Fig. 2 Both type I and III IFNR signaling are essential for protection against IA.

    IFNAR−/− (green lines or symbols), IFNLR1−/− (blue lines or symbols), STAT1−/− (orange line), double-deficient IFNAR−/−IFNLR1−/− (purple line or symbols), and WT control (black lines or symbols) B6 (A) and WT Balb/c (B) animals were challenged with 8 × 107 CEA10 Af conidia and monitored for survival. (A and B) Kaplan-Meier survival plot for 10 mice per group analyzed in two independent experiments. (C and D) Representative images of Gomori ammoniacal silver–stained lung sections captured at ×40 magnification. (E) Representative FACS plot of ROS detection in neutrophils derived from WT (dark gray histogram), IFNAR−/−IFNLR1−/− (purple histogram), and p47Phox−/− (light gray histogram) mice. (F) MFI of ROS generation by airway-infiltrating neutrophils at 48 hours after Af infection as analyzed in (E). (G) Pulmonary fungal burden at 48 hours after Af infection. Each symbol in (F) and (G) represents one mouse. Data shown are cumulative of three independent experiments. **P < 0.01; ***P < 0.001; ****P < 0.0001, calculated by Kruskal-Wallis nonparametric test for multiple comparisons for each knockout group compared with WT control. Statistical analysis was done with Prism software.

  • Fig. 3 CCR2+ monocytes are a relevant source of type I IFN in response to Af.

    (A and B) IFNAR−/− (green), IFNLR1−/− (blue), double-deficient IFNAR−/−IFNLR1−/− (DKO; purple), and WT control (black) B6 animals were challenged with 4 × 107 CEA10 Af conidia and examined for type I IFN expression at 12 hours after infection (A) or type III IFN at 48 hours after infection (B) by ELISA. (C to G) CCR2+Ly6C+ monocytes (red bars), CD45+ cells depleted of monocytes (hatched bars), and CD45 pulmonary cells (gray bars) were isolated from the lung of CCR2-GFP reporter mice at 3 hours (C, E, and G) or 48 hours (D and F) after infection with Af. Pulmonary, CCR2+Ly6C+ monocytes were also isolated from naïve CCR2-GFP reporter mice (white bars). All samples were examined for IFN transcription (C, D, and G) or secretion of IFN proteins (E and F) after overnight ex vivo culture. (H) Endogenous transcription of type III IFN was examined by qRT-PCR in RNA samples isolated from the lungs of CCR2-depleted mice that were left untreated (red) and in CCR2-depleted mice that were treated with 1 μg of IFN-α (green), 1 μg of IFN-λ (blue), or 1 μg each of IFN-α and IFN-λ (purple). Responses in CCR2+ competent littermates (black) were used as positive controls. DT, diphtheria toxin. Data are means ± SEM for four mice per group and for one experiment representative of two. *P < 0.05; **P < 0.01; ****P < 0.0001, calculated by Mann-Whitney nonparametric test of experimental group relative to WT control mice using Prism software.

  • Fig. 4 Type I and III IFNR expression on hematopoietic cells is required for protection against IA.

    (A and B) Lethally irradiated recipients were reconstituted with donor bone marrow (BM) for 8 weeks before infection with 8 × 107 CEA10 Af conidia. (A) Kaplan-Meier survival plot for eight mice per group analyzed in two independent experiments. (B) Representative images of Gomori ammoniacal silver–stained lung sections captured at ×40 magnification. (C) Western blot analysis of STAT1 phosphorylation at 15 min after treatment with 100 ng of recombinant murine IFN-α2 or mIFN-λ2 as indicated. Bone marrow neutrophils from each experimental group were FACS-sorted (>99% purity) before in vitro treatment with IFNs. (D and E) Neutrophils from the bone marrow, spleen, or lung of WT mice (D) and IFNLR1−/− mice (E) were FACS-sorted (>99% purity) before in vitro treatment with IFNs and examined for responsiveness by Western blot analysis of STAT1 phosphorylation. Neutrophils were sorted as Live CD45+CD11b+Ly6CintLy6G+.

  • Fig. 5 Human cells produce type I and III IFNs upon Af stimulation.

    (A) Representative FACS plot of IFNLR1 expression in gated neutrophils or lymphocytes in human peripheral blood cells without treatment or 6 hours after culture with Af. (B and C) IFNLR1 expression as measured by MFI in each gated population in human peripheral blood (B) or bone marrow (C) samples. (D and E) qRT-PCR of relative gene expression of IFN genes and type III IFNR in RNA isolated from human peripheral blood (E) or bone marrow (D) samples. Data are means ± SEM of five (B and E) or four (C and D) individual donors. **P < 0.01, calculated by paired t test using Prism software.

  • Fig. 6 Mice with neutrophil-specific deletion of IFNLR1 or STAT1 succumb to IA.

    Conditional gene targeting in neutrophils was achieved by crossing Ifnlr1fl/fl and Stat1fl/fl mice with MRP8cre. Gene-targeted animals and control mice were infected with 8 × 107 CEA10 Af conidia. Control groups were WT B6 mice, Ifnlr1fl/fl, Stat1fl/fl, and MRP8cre. (A) Kaplan-Meier survival plot for 10 mice per group analyzed in two independent experiments. (B) Representative images of Gomori ammoniacal silver–stained lung sections captured at ×40 magnification. (C) Neutrophil recruitment to the lung of infected mice at 48 hours after infection with 4 × 107 CEA10 Af conidia. Each symbol represents one mouse. Data are cumulative of two independent experiments. (D) Pulmonary levels of TNF as measured by ELISA. (E and F) ROS generation by airway-infiltrating neutrophils (E) and pulmonary fungal burden (F) 48 hours after Af infection. Each symbol represents one mouse. Data shown are cumulative of two independent experiments. Statistical analysis was done with Kruskal-Wallis nonparametric test for multiple comparisons using Prism software. **P < 0.01; ***P < 0.001 for each labeled sample as compared with WT mice.

  • Fig. 7 Neutrophil antifungal response in CCR2-depleted mice is rescued by adoptive transfer of CCR2+ monocytes or by treatment with recombinant IFNs.

    CD45.2+CCR2-depleted mice were infected with Af and either left untreated or treated by the adoptive transfer of CD45.1+CCR2-GFP+ monocytes at 3 hours after infection. Monocytes (~850,000) were isolated from the lung of Af-infected CD45.1+CCR2-GFP+ donor mice 3 hours after infection. (A) Representative FACS plot of control, CCR2-depleted mice, and CCR2-depleted mice that received CD45.1+CCR2+ monocytes. (B and C) WT control (black symbols), CCR2-depleted (red symbols), or CCR2-depleted mice adoptively transferred with CCR2+ monocytes (blue symbols) were examined at 48 hours after infection for ROS generation by neutrophils (B) or Af fungal burden (C) in the lung. Each symbol represents one mouse. Data shown are cumulative of two independent experiments. (D and E) CCR2-depleted mice were left untreated or treated with recombinant IFN-α2 and/or IFN-λ3 as indicated. Mice were treated with 1 μg of individual cytokines, except for the double-treated, which received 500 ng of IFN-α2 and 500 ng of IFN-λ3. To recapitulate the kinetics of IFN expression in control mice, we treated CCR2-depleted mice shortly after infection with type I IFN followed by type III IFN. Mice were treated with IFNs the same day of infection and then every other day. (D) Analysis of ROS generation by airway-infiltrating neutrophils isolated from controls (WT littermates treated with DT) or from untreated and treated CCR2-depleted mice. (E) Pulmonary fungal burden 48 hours after infection. Each symbol represents one mouse. Data shown are for one experiment representative of two independent ones. (F) Kaplan-Meier survival plot of CCR2-depleted mice treated with each IFN and mock-treated controls. (G) Representative images of Gomori ammoniacal silver–stained lung sections captured at ×40 magnification. Data shown are cumulative of two to three independent experiments. *P < 0.05; ***P < 0.001; ****P < 0.0001 for each labeled sample as compared with CCR2-depleted mice, calculated by Mann-Whitney or Kruskal-Wallis nonparametric statistical tests using Prism software.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/16/eaan5357/DC1

    Materials and Methods

    Fig. S1. Lymphoid-deficient and IFN-γ–deficient mice control fungal infection normally.

    Fig. S2. Global transcriptional response of antifungal neutrophils.

    Fig. S3. Inflammatory responses in IFNR−/− mice 48 hours after infection.

    Fig. S4. Analysis of p47Phox−/− mice susceptibility to Af infection.

    Fig. S5. Validation of hIFNLR1 antibody.

    Fig. S6. Neutrophil-specific gene excision is efficiently targeted in MRP8Cre mice.

    Fig. S7. Type III IFN transcription in CCR2-depleted mice is rescued by adoptive transfer of CCR2+ monocytes.

    Table S1. Exact P values of asterisks shown in the figures.

    Excel sheet with differentially expressed gene lists

    Excel sheet with source data for figures shown

  • Supplementary Materials

    Supplementary Material for:

    Type III interferon is a critical regulator of innate antifungal immunity

    Vanessa Espinosa, Orchi Dutta, Constance McElrath, Peicheng Du, Yun-Juan Chang, Bryan Cicciarelli, Amy Pitler, Ian Whitehead, Joshua J. Obar, Joan E. Durbin, Sergei V. Kotenko, Amariliz Rivera*

    *Corresponding author. Email: riveraam{at}njms.rutgers.edu

    Published 6 October 2017, Sci. Immunol. 2, eaan5357 (2017)
    DOI: 10.1126/sciimmunol.aan5357

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Lymphoid-deficient and IFN-γ–deficient mice control fungal infection normally.
    • Fig. S2. Global transcriptional response of antifungal neutrophils.
    • Fig. S3. Inflammatory responses in IFNR−/− mice 48 hours after infection.
    • Fig. S4. Analysis of p47Phox−/− mice susceptibility to Aƒ infection.
    • Fig. S5. Validation of hIFNLR1 antibody.
    • Fig. S6. Neutrophil-specific gene excision is efficiently targeted in MRP8Cre mice.
    • Fig. S7. Type III IFN transcription in CCR2-depleted mice is rescued by adoptive transfer of CCR2+ monocytes.
    • Table S1. Exact P values of asterisks shown in the figures.

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    Other Supplementary Material for this manuscript includes the following:

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