Eosinophils subvert host resistance to an intracellular pathogen by instigating non-protective IL-4 in CCR2-/- mice

Abstract

Eosinophils contribute to type II immune responses in helminth infections and allergic diseases; however, their influence on intracellular pathogens is less clear. We previously reported that CCR2^-/- mice exposed to the intracellular fungal pathogen Histoplasma capsulatum exhibit dampened immunity caused by an early exaggerated interleukin (IL)-4 response. We sought to identify the cellular source promulgating IL-4 in infected mutant animals. Eosinophils were the principal instigators of non-protective IL-4 and depleting this granulocyte population improved fungal clearance in CCR2^-/- animals. The deleterious impact of eosinophilia on mycosis was also recapitulated in transgenic animals overexpressing eosinophils. Mechanistic examination of IL-4 induction revealed that phagocytosis of H. capsulatum via the pattern recognition receptor complement receptor (CR) 3 triggered the heightened IL-4 response in murine eosinophils. This phenomenon was conserved in human eosinophils; exposure of cells to the fungal pathogen elicited a robust IL-4 response. Thus, our findings elucidate a detrimental attribute of eosinophil biology in fungal infections that could potentially trigger a collapse in host defenses by instigating type II immunity.

Figure 1. Identification of IL-4⁺ cells in CCR2^−/− mice during H. capsulatum infection

(A) Quantitative analysis of IL4-eGFP⁺ cells in the lungs of WT and CCR2^−/− mice at day 3 p.i by flow cytometry. (B) Identification of IL4-eGFP⁺ eosinophils at day 3 p.i. Eosinophils defined as SSC^hi SiglecF⁺ FcεRI⁻ CD11b⁺. (C) Comparison of IL4-eGFP⁺ cells in the lungs of WT and CCR2^−/− mice prior to infection. (D) IL-4 expression in CCR2^−/− lung eosinophils at day 3 p.i. Cells were stimulated for 4 hours with 120 ng/ml phorbol 12-myristate 13-acetate (PMA)/ionomycin, the last 2 hours in the presence of Brefeldin A (BFA). (E and F) Analysis of leukocytes associated with innate IL-4 production in WT and CCR2^−/− (non-reporter) mice at day 3 p.i. All FACS plots are representative of 1 of 8 mice from two independent experiments. Graphical data are depicted as mean ± SEM, n=8 from 2 independent experiments.

Figure 2. Consequence of eosinophil depletion in CCR2^−/− mice

(A) FACS analysis of eosinophils in the lungs of CCR2^−/−.4get mice treated with anti-IL5 or isotype control antibody at day 3 p.i. (B, C, and D) FACS analysis of pulmonary IL4-eGFP⁺ cells in control IgG or anti-IL5-treated CCR2^−/−.4get mice at day 7 p.i. (E) Measurement of IL-4 protein concentration in lung homogenates of control IgG or anti-IL5-treated CCR2^−/− mice at day 7 p.i by ELISA. (F and G) qRT-PCR analysis of IL-4-dependent genes following eosinophil depletion (day 7 p.i). (H) Pulmonary fungal burden represented as log₁₀CFU in control IgG or anti-IL5-treated CCR2^−/− mice at day 7 p.i. All FACS plots are representative of 1 of 7–8 mice from two independent experiments. Graphical data are depicted as mean ± SEM, n=7–8 from 2 independent experiments.

Figure 3. H. capsulatum infection in IL-5 transgenic mice

(A) Eosinophil influx in lungs of WT and IL5-transgenic mice prior to infection, as analyzed by flow cytometry. FACS plots representative of 1 of 6 mice from two independent experiments. Fungal burden depicted in log₁₀CFU (B), and IL-4 protein concentration (C) in lungs of WT and IL-5 transgenic animals after 7 days of H. capsulatum challenge. Values are represented as mean ± SEM, n=7–8 from 2 independent experiments.

Figure 4. Phagocytosis of H. capsulatum by eosinophils results in IL-4 secretion

(A and B) FACS analysis of bone marrow derived eosinophils challenged with 1 or 5 MOI GFP⁺ H. capsulatum yeasts for 6 hour. Plots representative of 1 of 5 independent experiments. (C and D) Measurement of IL-4 protein concentration in culture supernatants of eosinophils infected with different MOI of H. capsulatum yeasts for varying time points as indicated. Values are mean ± SEM from at least 4 independent experiments. ** = p < 0.01 as compared to control, either time 0 for C or uninfected for D.

Figure 5. Phagocytosis of H. capsulatum yeasts by murine eosinophils

Z stacked images of an eosinophil infected with 5 MOI of H. capsulatum. The images were collected at 0.66μm intervals (1000X magnification). Representative image of 1 of 3 independent experiments. 5 μm bar is shown.

Figure 6. Eosinophil uptake of H. capsulatum is dependent on CR3

FACS analysis (A) and measurement of IL-4 protein in culture supernatants (B) of infected-bone marrow derived eosinophils pre-incubated with control IgG or CD18 blocking antibody (5μg/ml) for 90 minutes. Subsequently, cells were infected with 5 MOI GFP⁺ H. capsulatum yeasts for 6 hours. (C) FACS analysis of infected-bone marrow derived eosinophils pre-incubated with DMSO, Syk inhibitors Piceannatol (50μM) and BAY 61-3606 (1μM), PI3K inhibitor, LY294002 (2μM) or JNK inhibitor, SP600125 (10 μg/ml) for 90 minutes prior to infection. FACS plots are representative of 1 of 3 independent experiments. (D) % phagocytosis inhibition for each of the inhibitiors. (E) Measurement of IL-4 protein in culture supernatants of infected-bone marrow derived eosinophils pre-incubated with DMSO or Piceannatol. Graphical values are represented as mean ± SEM from 4–5 independent experiments.

Figure 7. Association of H. capsulatum with eosinophils in vivo

(A) FACS analysis of H. capsulatum-infected eosinophils in vivo. CCR2^−/− mice were infected with 1x10⁷ GFP⁺ H. capsulatum yeasts and GFP⁺ eosinophils in the lungs were analyzed by flow cytometry 3 days p.i. Plot representative of 1 of 6 mice from two independent experiments. (B) Microscopic analysis of GFP-Hc infected eosinophils from lungs of CCR2^−/− mice, 3 days after challenge and analyzed with an ImageStreamX® flow cytometer.

Figure 8. H. capsulatum infection and IL-4 response in human eosinophils

(A) FACS analysis of human eosinophils incubated without or with 5 MOI GFP⁺ H. capsulatum yeasts for 6 hours. (B) Infected human eosinophils analyzed with an ImageStreamX® flow cytometer. (C) FACS analysis of IL-4 MFI in infected and uninfected human eosinophils. Plots and images are representative of 1 of 3 independent experiments.