(a) expression of intracellular IL-17A in NIMP-R14⁺ bone marrow cells from C57BL/6, Rag2^−/−Il2rg^−/−, Il6^−/− and Il17a-GFP reporter mice recovered three days after receiving a subcutaneous injection of heat-killed, swollen Aspergillus fumigatus conidia (primed). (b) NIMP-R14⁺ neutrophils isolated from total bone marrow cells by density centrifugation showing purity of isolation (inset shows Wrights-Giemsa stained neutrophils). (c) Representative histogram showing percent IL-17A/GFP expressing NIMP-R14⁺ bone marrow neutrophils from naïve and primed Il17a-GFP mice. (d). Representative IL-17/GFP expressing cells from primed Il17a-GFP reporter mice (original magnification is ×400). (e) Serum IL-6 and IL-23 from naïve and primed C57BL/6 and Rag2^−/−Il2rg^−/− mice. (f) Cytokine production by splenocytes from naive C57BL/6 and Rag2^−/−Il2rg^−/− mice following 18h incubation with Aspergillus hyphal extract (AspHE) or unstimulated (US). (g) IL-17A gene expression in naïve C57BL/6 bone marrow neutrophils incubated with media alone (unstimulated, US) or after 1h incubation with supernatants from AspHE – stimulated splenocytes plus neutralizing antibodies to IL-1β, TGFβ, IL-6 or IL-23. (a-c): Representative scatter plots from four mice per group; (e, f): mean +/- SD of four mice per group, p<0.001 naïve and primed mice (e), and between unstimulated and AspHE – stimulated neutrophils (f). Each experiment was repeated twice with similar results.

(a) GFP-Rorc⁺ NIMP-R14⁺ cells in total bone marrow cells from Rorc^+/GFP , and Rorc^GFP/GFP mice (upper panels), and RORγt protein in NIMP-R14⁺ cells from C57BL/6 and Rorc^+/GFP mice (lower panels). (b) IL-6R, gp130, IL-23R, and IL-12Rβ1 expression in naïve and IL-6+IL-23 – stimulated bone marrow neutrophils from C57BL/6 mice. (c) Intracellular IL-17A and GFP–Rorc⁺ expression in IL-6+IL-23 – stimulated neutrophils (upper panels), and intracellular IL-17A and RORγt in bone marrow neutrophils from C57BL/6 and Rorc^+/GFP mice. (d) Representative Confocal images showing expression of RORγt and IL-17 (original magnification is ×400). (e) IL-17 gene expression in IL-6+IL-23 stimulated neutrophils from C57BL/6 and Rorc^+/GFP mice. 20μg of IL-6 and 2μg of IL-23 were used to stimulate all neutrophils. All data are representative of 4 mice per group and two repeat experiments.

Western blot of total cell lysates (a) and nuclear extracts (b) of isolated bone marrow neutrophils from naïve C57BL/6 mice after stimulation with supernatants from AspHE stimulated splenocytes containing IL-6+IL-23. (c) Nuclear extracts of neutrophils stimulated with 20μg recombinant murine (r)IL-6 and/or 2μg rIL-23. Blots were probed with antibody to RORγt, β-actin and the TATA box Binding Protein (TBP). (d) Representative Confocal images of intracellular RORγt in IL-6+IL-23 – stimulated C57BL/6 neutrophils and counterstained with DAPI. (e) Electrophoretic mobility shift assay (EMSA) analysis of IL-6+IL-23 stimulated bone marrow neutrophils from C57BL/6 mice. Nuclear extracts incubated with biotinylated oligonucleotide probes corresponding to the putative RORγt binding site of the mouse IL-17A promoter region. Extracts were also incubated with a biotinylated mutated (Mut) probe, with the biotinylated probe and competing cold probe, and with polyclonal anti- RORγt antibody (supershift assay). These experiments were repeated twice with similar results.

(a) Total cytokines in culture supernatants of peripheral blood mononuclear cells (PBMC) incubated 18h with AspHE (mean+/- SD). (b) IL-17 gene expression in neutrophils incubated 1h with PBMC supernatants (after stimulation with AspHE) plus neutralizing antibodies to IL-1β, TGF–β, IL-6 or IL-23. (c-e): IL-17 gene expression (c) total cellular IL-17 protein (d) and intracellular IL-17A (e) in IL-6+IL-23 – stimulated peripheral blood neutrophils from two healthy donors. (f) Intracellular IL-17 and RORγt, and cell surface IL-6R and IL-23R expression in unstimulated (upper panels) and IL-6+IL-23 - stimulated (lower panels) peripheral blood neutrophils from a single donor. (g) Confocal images of intracellular IL-17A and RORγt in neutrophils from a single donor. Neutrophils were stimulated with 20μg rIL-6 and 2μg rIL-23, and experiments were repeated using neutrophils from different donors.

IL-17RA and IL-17RC gene expression in bone marrow derived neutrophils from C57BL/6 mice (a), IL-17-/- mice (b), and human peripheral blood (c) after 1h incubation with IL-6+IL-23, and a further 1h with AspHE. (d) Cell surface IL-17RA and IL-17RC on IL-6+IL-23 stimulated human neutrophils. (e) Clec7A (Dectin-1) and Clec4n (murine Dectin-2) gene and cell surface expression in IL-6+IL-23 – stimulated C57BL/6 bone marrow neutrophils. (f) Clec6a (human Dectin-2) and Clec7A gene and cell surface expression in IL-6+IL-23 – stimulated human peripheral blood neutrophils. (g) IL-17RC expression in IL-6+IL-23 stimulated bone marrow neutrophils from C57BL/6, Clec7a^-/- and Clec4n^-/- mice after incubation with AspHE. Neutrophils from Il17rc^-/- mice were used as negative controls (CTRL). (h) IL-17RC and Clec4n gene expression in IL-6+IL-23 – stimulated bone marrow neutrophils from Rorc^+/GFP and Rorc^GFP/GFP mice. (i) IL-17RC and Dectin-2 cell surface expression in IL-6+IL-23 – stimulated bone marrow neutrophils from Rorc^GFP/GFP mice. (j) Intracellular IL-17A in IL-6+IL-23 – stimulated neutrophils from Clec7a^-/- and Clec4n^-/- mice. Data with mouse cells are representative of four mice, and experiments were repeated twice with similar results. Data with human cells are from a single donor, with a repeat experiment; results from additional donors are shown in .

IL-17RC and IL-17A gene expression (a) and intracellular IL-17A (b) in IL-6+IL-23 – stimulated neutrophils from Il17a^-/-, Il17rc^-/-, and Cybb^-/- mice (data are representative of three mice per group). (c, upper panel) ROS production (intracellular CFDA) in bone marrow neutrophils from naïve or primed C57BL/6 mice, and from naïve Il17a^-/-, Il17rc^-/-, and Cybb^-/- mice stimulated 3h with IL-6+IL-23 and then incubated 1h with growing Aspergillus hyphae +/- rmIL-17A or anti-IL-17RC. RFU: Relative fluorescent units; (lower panel) Fungal growth of dsRed expressing Aspergillus after 18h incubation with each neutrophil population. Fungal mass was measured by fluorimetry of dsRed, and represented as RFU. Mean +/- SD of three samples per group; controls are medium only (Ctrl) and naïve neutrophils (naïve). (d) ROS production and fungal mass of human peripheral blood neutrophils incubated with Aspergillus hyphae +/- rhIL-17A, anti-IL-17RC, or the ROS inhibitor DPI. (c-f) mean +/- SD of three wells per experimental condition from neutrophils pooled from three mice per group (c) or from a single donor (d). Data are representative of three repeat experiments.

Primed Il17–GFP reporter mice infected with RFP Aspergillus showing representative corneas 24h post-infection (a), total RFP in infected corneas assessed by image analysis (b) (data points represent individual corneas), total NIMP- R14⁺ neutrophils (c), and percent IL-17/GFP expressing neutrophils in the corneal stroma (d). (e-j) IL-6+IL-23 – stimulated bone marrow neutrophils from Il17–GFP, Il17a^-/-, Il17rc^-/-, and Cybb^-/- mice stimulated were injected intravenously into recipient Cd18-/- mice, which were then infected intrastromally with RFP Aspergillus. Representative corneas of Il17–GFP 24h post-infection (e), total RFP in infected corneas assessed by image analysis (f) (data points represent individual corneas), and total NIMP- R14⁺ neutrophils (g). Representative images of Aspergillus infected Cd18^-/- corneas in mice given Il17a^-/-, Il17rc^-/-, or Cybb^-/- neutrophils (h), total RFP Aspergillus (i), and total neutrophils (j). Experiments were repeated twice with similar results.