Type III interferon is a critical regulator of innate antifungal immunity

Abstract

Type III interferons (IFN-λs) are the most recently found members of the IFN cytokine family and engage IFNLR1 and IL10R2 receptor subunits to activate innate responses against viruses. We have identified IFN-λs as critical instructors of antifungal neutrophil responses. Using Aspergillus fumigatus (Af) as a model to study antifungal immune responses, we found that depletion of CCR2⁺ monocytes compromised the ability of neutrophils to control invasive fungal growth. Using an unbiased approach, we identified type I and III IFNs as critical regulators of the interplay between monocytes and neutrophils responding to Af We found that CCR2⁺ monocytes are an important early source of type I IFNs that prime optimal expression of IFN-λ. Type III IFNs act directly on neutrophils to activate their antifungal response, and mice with neutrophil-specific deletion of IFNLR1 succumb to invasive aspergillosis. Dysfunctional neutrophil responses in CCR2-depleted mice were rescued by adoptive transfer of pulmonary CCR2⁺ monocytes or by exogenous administration of IFN-α and IFN-λ. Thus, CCR2⁺ monocytes promote optimal activation of antifungal neutrophils by initiating a coordinated IFN response. We have identified type III IFNs as critical regulators of neutrophil activation and type I IFNs as early stimulators of IFN-λ expression.

Figure 1. Dysfunctional antifungal neutrophils have impaired expression of IFN-inducible genes

(A) Reactive oxygen species (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 MFI for 5 mice per group assayed by FACS as shown. (B)Af pulmonary fungal burden 48 hour after infection. (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 FC>2.5 in control neutrophils > 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 software. (E–J) Kinetics of IFN expression in the lung of mice at different times after Af infection. E–G shows gene expression as determined by qRT-PCR using TaqMan probes. Data shown is mean ± SEM of 5 mice per time point. H–J graphs show mean ± SEM of each cytokine as measured by ELISA in 5 mice per time point. (K–M) Pulmonary qRT-PCR of IFN gene expression at 3 (K) or 48 hours after Af infection (L–M). Throughout the figure, *p<0.05, **p<0.01 as calculated by Mann-Whitney non-parametric test of experimental group relative to control using Prism software.

Figure 2. Type I and type III IFN receptor signaling are both essential for protection against invasive aspergillosis

IFNAR^−/− (green lines or symbols), IFNLR1^−/− (blue lines or symbols), STAT1^−/− (orange line), double deficient IFNAR^−/−IFNLR1^−/− (purple line or symbols) and wild type 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–B) Kaplan-Meier survival plot for 10 mice per group analyzed in two independent experiments. (C–D) Representative images of Gomori ammoniacal silver-stained lung sections captured at 40X magnification. (E) Representative FACS plot of ROS detection in neutrophils derived from WT (dark gray histogram), IFNAR^−/−IFNLR1^−/−(purple) and p47^Phox−/− (light gray histogram) mice. (F) Mean fluorescence intensity 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 (F–G) Each symbol represents one mouse. Data shown is cumulative of three independent experiments. **p< 0.01, **** p<0.0001 values shown in F and G were calculated by Kruskall-Wallis non-parametric test for multiple comparisons for each knockout group compared to WT control. Statistical analysis was done with Prism software.

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

(A–B) IFNAR^−/− (green), IFNLR1^−/− (blue), double deficient IFNAR^−/−IFNLR1^−/− (DKO; purple), and wild type 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–G) CCR2⁺Ly6C⁺ monocytes (red bars), CD45⁺ cells depleted of monocytes (hatched bars), and CD45 pulmonary cells (gray bars) were isolated form 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 after overnight ex vivo culture (E and F). (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 bars) and in CCR2-depleted mice that were treated with 1 µg of IFN-α (green bars), 1 µg of IFN-λ (blue bars), or 1 µg of IFN-α and 1 µg of IFN-λ (purple bars). Responses in CCR2⁺ competent littermates (black bars) were used as positive controls. Data shown is mean ± SEM for four mice per group and is for one experiment representative of two. *p<0.05 as calculated by Mann-Whitney non-parametric test of experimental group relative to WT control mice using Prism software.

Figure 4. Type I and type III IFN receptor expression on hematopoietic cells is required for protection against invasive aspergillosis

(A–B) Lethally irradiated recipients were reconstituted with donor bone marrow for 8 weeks before infection with 8 × 10⁷ CEA10 Af conidia. (A) Kaplan-Meier survival plot for 8 mice per group analyzed in two independent experiments. (B) Representative images of Gomori ammoniacal silver-stained lung sections captured at 40X 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 prior to in vitro treatment with IFNs. (D–E) Neutrophils from bone marrow, spleen, or lung of WT mice (D) and IFNLR1^−/− mice (E) were FACS sorted >99% purity prior to in vitro treatment with IFNs and examined for responsiveness by Western Blot analysis of STAT1 phosphorylation. Neutrophils were sorted as Live CD45⁺CD11b⁺Ly6C^intLy6G⁺.

Figure 5. Human cells produce type I and III IFN 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–C) IFNLR1 expression as measured by MFI in each gated population in human peripheral blood (B) or bone marrow samples (C). (D–E) qRT-PCR of relative gene expression of IFN genes and type III IFN receptor in RNA isolated from human peripheral blood (E) or bone marrow samples (D). Data shown is mean ± SEM of 5 (B and E) or 4 (C and D) individual donors. **p<0.01 as calculated by paired t-test using Prism software.

Fig. 6. Mice with neutrophil-specific deletion of IFNLR1 or STAT1 succumb to invasive aspergillosis

Conditional gene targeting in neutrophils was achieved by crossing Ifnlr1^fl/fl and Stat1^fl/fl mice with MRP8^cre. Gene targeted animals and control mice were infected with 8 × 10⁷ CEA10 Af conidia. Control groups were WT B6 mice, Ifnlr1^fl/fl, Stat1^fl/fl, and MRP8^cre(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 40X 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 is cumulative of two independent experiments. (D) Pulmonary levels of TNF as measured by ELISA. (E–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 is cumulative of two independent experiments. Statistical analysis was done with Kruskall-Wallis, non-parametric test for multiple comparisons using Prism software. *p<0.05, ***p<0.001 for each labeled sample as compared to 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 post-infection. (A) Representative FACS plot of control, CCR2-depleted mice, and CCR2-depleted mice that received CD45.1⁺CCR2⁺monocytes. (B–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 in the lung (C). Each symbol represents one mouse. Data shown is cumulative of two independent experiments. (D–E) CCR2-depleted mice were left untreated or were 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, CCR2-depleted mice were treated 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 is 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 40X magnification. Data shown is cumulative of two-three independent experiments. Statistical analysis was done with Kruskall-Wallis, non-parametric test for multiple comparisons using Prism software. *p<0.05, ***p<0.001 for each labeled sample as compared to CCR2-depleted mice.