Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection

Abstract

Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein that possesses antimicrobial activity primarily because of its ability to chelate zinc and manganese. In the current study, we showed that neutrophils from calprotectin-deficient S100A9(-/-) mice have an impaired ability to inhibit Aspergillus fumigatus hyphal growth in vitro and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9(-/-) mice by injecting recombinant calprotectin. Furthermore, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both zinc and manganese binding are necessary for calprotectin's antihyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatus ∆zafA mutant, which demonstrates deficient zinc transport, exhibits impaired growth in infected corneas and following incubation with neutrophils or calprotectin in vitro as compared with wild-type. Collectively, these studies demonstrate a novel stage-specific susceptibility of A. fumigatus to zinc and manganese chelation by neutrophil-derived calprotectin.

Figure 1. Effect of neutrophil calprotectin on A. fumigatus corneal infection

A. C57BL/6 or S100A9^−/− mice were infected intrastromally with 50,000 A. fumigatus dsRed conidia (A-D) and corneas were imaged at 24 and 48h pi. B. Fungal burden measured by dsRed fluorimetry at 24 and 48h pi. C. CFU from whole eye homogenates measured at 48h pi. D. Neutrophil number in infected corneas measured as total NIMP-R14+ cells in corneal cell suspensions at 24 and 48h pi by flow cytometry. E. PASH and IHC for neutrophils (NIMP-R14), S100A8 and S100A9 in adjacent corneal sections from A. fumigatus infected C57BL/6 mice at 24 and 48h pi (400X). F. S100A8 and S100A9 expression by ELISA in naïve or A. fumigatus infected corneas of C57BL/6 or CD18^−/− mice. G. NIMP-R14 staining in S100A8 and S100A9-positive cells from A. fumigatus infected corneas of C57BL/6 mice at 24h pi. H-I. S100A9^−/− mice were infected intrastromally with 50,000 A. fumigatus dsRed conidia and PBS or recombinant calprotectin (1 µg) was injected subconjunctivally at the time of infection. H. Representative images of A. fumigatus dsRed in infected corneas at 24 hrs pi. I. Fungal burden measured by dsRed fluorimetry at 24h pi. Experiments were repeated three times with 3–5 mice per group, A Mann-Whitney U test was used to determine significance. All data show mean +/− SEM. See also Figure S1.

Figure 2. Effect of neutrophil calprotectin on A. fumigatus hyphal growth in vitro

Peritoneal neutrophils or neutrophil lysates from C57BL/6 or S100A9^−/− mice were incubated 16h with A. fumigatus hyphae (A-B), and hyphal growth was assessed by calcofluor staining A. Representative images of hyphae (200X); B-C. Quantification of hyphal growth in the presence of neutrophils or neutrophil lysates. % Fungal mass was calculated by fluorimetry of calcofluor and shown as percent of total hyphae when grown in media alone (experimental/control × 100). Representative In vitro experiments with 3–6 technical replicates per condition, significance was determined by student’s t-test. All data show mean +/− SEM. See also Figure S1.

Figure 3. Zn and Mn binding contribute to calprotectin Anti Aspergillus activity

A. fumigatus hyphae were incubated +/− recombinant calprotectin for 16h and fungal growth was assessed by calcofluor staining (A-D). % Fungal mass was calculated by fluorimetry of calcofluor and shown as percent of total hyphae when grown in media alone (experimental/control × 100). A. Representative images of hyphae (200X) in media +/− 25 µg/ml calprotectin B. Quantification of hyphael growth + 50 µg/ml calprotectin +/− increasing ZnSO₄ +/− 1 or 5 µM MnSO₄C-D. Quantification of hyphal growth of hyphae incubated with wild-type calprotectin (CP) vs. CPΔMn calprotectin (lacks Mn binding motif; note difference in the y axis scale) (C) or CP vs. or CPΔMn/Zn calprotectin (lacks Mn and Zn binding motifs). E-FA. fumigatus hyphae were incubated with 2 × 10⁵ human neutrophils in RPMI +/− 5 µM ZnSO₄ or MnSO₄ and fungal growth was assessed by calcofluor staining. E. % Fungal mass was calculated by fluorimetry of calcofluor and shown as percent of total hyphae when grown in media alone (experimental/control × 100). F. Representative images of hyphae (200X) in media +/− neutrophils, ZnSO₄ or MnSO_4. Representative In vitro experiments with 3–6 technical replicates per condition, significance was determined by student’s t-test. Human neutrophils were tested from three separate donors.

Figure 4. ZafA mediated zinc uptake in A. fumigatus virulence and susceptibility to calprotectin

WT or ΔzafAA. fumigatus hyphae were incubated with peritoneal neutrophils from C57BL/6 or S100A9−/− mice or recombinant calprotectin for 16h (A-B). Fungal growth was assessed by calcofluor staining and % Fungal mass was calculated by fluorimetry of calcofluor and shown as percent of total hyphae when grown in media alone (experimental/control × 100). A. Growth of hyphae incubated with C57BL/6 or S100A9^−/− neutrophils (0.5 × 10⁵/well). B. Growth of hyphae incubated with increasing doses of CP. Representative In vitro experiments with 3–6 technical replicates per condition, significance was determined by student’s t-test. C57BL/6 mice were infected intrastromally with 50,000 WT or ΔzafA A. fumigatus conidia (C-D). C. Fungal growth assessed in infected corneal sections by GMS stain (hyphae appear black) at 48h pi (400X). D. CFU of WT vs. ΔzafA from whole eye homogenates measured at 48h pi. E-F. Neutrophil infiltration in C57BL/6 mice infected with WT or ΔzafAA. fumigatus assessed by IHC for NIMP-R14 in corneal sections and quantified using Metamorph (400x). In vivo experiment was repeated three times with at least 3 mice per group. Significance was assess by Mann-Whitney U test. All data show mean +/− SEM. See also Figure S2.

Figure 5. Conidia killing by neutrophils is independent of calprotectin

A. Representative dot plots of peritoneal C57BL/6 or S100A9^−/− neutrophils incubated with FLARE (dsRed+ AF633+) conidia 8h. B. Uptake and viability of FLARE conidia incubated with C57BL/6 or S100A9−/− neutrophils in vitro for 8 hrs. Representative In vitro experiments with 3–6 technical replicates per condition, significance was determined by student’s t-test. C57BL/6 or S100A9^−/− bone marrow chimeras were challenged intratracheally with 3 × 10⁷ FLARE conidia (C-E). C. Representative dot plots of neutrophils from lung and BALF analyzed for dsRed and AF633 fluorescence by flow cytometry. The tan gates indicate bystander neutrophils, and the red (R1) and blue (R2) gates indicate neutrophils that contain live or killed conidia, respectively. D. Uptake and viability of FLARE conidia in lung or BALF neutrophils 12h pi. E. Total neutrophils in lung or BALF 12h pi in C57BL/6 or S100A9^−/− bone marrow chimeras. In vivo experiments were repeated twice with at least 3 mice per group. Significance was determined by Mann-Whitney U test. All data show mean +/− SEM. See also Figure S3.