(a) Human peripheral neutrophils stimulated with C. albicans with or without 3% plasma. Extracellular DNA stained for NET release with Sytox 4 h post stimulation. (b) Quantification of NET release by human peripheral neutrophils stimulated with a hgc1Δ yeast-locked C. albicans mutant (yeast) or pre-formed WT C. albicans hyphae without plasma. Percentage (%) Sytox positive events over total number of neutrophils. (c) Histone H3 (17 kDa) degradation (arrow) of neutrophils stimulated with WT hyphae or hgc1Δ yeast for the indicated times without plasma and assessed by immunoblotting. (d) NET release after direct stimulation of human peripheral neutrophils with C. albicans hgc1Δ yeast or separated by a transwell that allows contact but prevents phagocytosis. (e) Quantification of (d). Percentage (%) Sytox positive events over total number of neutrophils. (f) NET release after stimulation of human peripheral neutrophils with intact or fragmented WT C. albicans hyphae. (g) Quantification of (f). % Sytox positive events over total number of neutrophils. (a-g) US, unstimulated. Multiplicity of infection (MOI) = 10. Scale bars = 50 μm. Statistics by one-way ANOVA, followed by Tukey’s multiple comparison post test: * p<0.01, ** p<0.001, *** p<0.0001. Data are representative of at least three independent experiments.

(a) NET release after direct stimulation of human peripheral neutrophils with E. coli DH5α or K. pneumoniae KP52145 (MOI = 10) or separated by a transwell that allows contact but prevents phagocytosis. Extracellular DNA stained for NET release with Sytox 4 h post stimulation. US, unstimulated. Scale bars = 50 μm. (b) Quantification of (a). % NETs released over total number of neutrophils. Statistics by one-way ANOVA, followed by Sidak’s multiple comparison post test: NS p>0.5, * p<0.001, ** p<0.0001. Data are representative of three independent experiments. (c) NET release (Sytox, green) 4 h after stimulation of human peripheral neutrophils with BCG-dsRed. Scale bars: 50 μm. Necrotic neutrophils responding to single BCG and small aggregates (i) and NETing neutrophils responding to large BCG aggregates (ii). (d) Quantitation of NET release in (c) depicting the two neutrophil distributions (i, ii). Percentage (%) Sytox positive events over total number of neutrophils. Statistics by one-way ANOVA, followed by Tukey’s multiple comparison post test: ** p<0.0001. Data are representative of two independent experiments. (e) Time-lapse video microscopy stills of BCG-dsRed single bacteria (asterisks) and large aggregates (arrows) (red) incubated with human neutrophils in the presence of Sytox (green, extracellular DNA). Scale bar: 150 μm

(a) NET release in the lungs of WT (C57BL/6) mice infected intratracheally with 1×10⁵ c.f.u. WT C. albicans or a hgc1Δ yeast-locked mutant and assessed 24 h post infection by immunofluorescence microscopy for citrullinated histone H3 (Cit-H3, red), MPO (green) and DNA (DAPI, blue). White arrows depict areas of NET release. Lower panel: magnification detail in upper panel. Scale bars = 20 μm. (b) Weight of WT (C57BL/6) (n=6) and MPO deficient (n=5) mice infected with 1×10⁴ c.f.u. WT C. albicans or a hgc1Δ yeast-locked mutant. Weight normalized to staring weight at d0. Statistics by two-way ANOVA, followed by Sidak’s multiple comparison post test: NS p>0.5, ** p<0.0001. (c) C. albicans load in the lung and spleen 6 days post infection (n=3). Statistics by two-way ANOVA, followed by Tukey’s multiple comparison post test: NS p>0.5, * p<0.01. Data are representative of two independent experiments.

(a) Human peripheral neutrophils stimulated with WT hyphae or hgc1Δ yeast-locked mutant C. albicans. NE (red) and MPO (green) localization were assessed after 1 h by immunofluorescence via confocal microscopy. An optical section spanning the center of the cell is shown. Arrows indicate the nucleus. Asterisks indicate NE colocalisation with MPO. Scale bars = 5 μm. Data are representative of two independent experiments. (b) Quantification of (a). Nuclear NE over total NE per cell from multiple confocal sections of 15-20 cells per condition. Statistics by two-way ANOVA, followed by Sidak’s multiple comparison post test * p<0.0001. (c) Human peripheral neutrophils stimulated with hgc1Δ yeast-locked C. albicans. NE (yellow), CD63 (magenta), p40 (cyan) and DNA (DAPI, blue) localization after 1 h. Scale bar = 5 μm. (d, e) Human peripheral neutrophils stimulated with hgc1Δ yeast-locked C. albicans (red). CD63 (green) (d) or p47 (green) (e) and DNA (DAPI, blue) localization after 1 h. Scale bar = 5 μm. Data are representative of three independent experiments (b–e).

(a) NET release by human peripheral neutrophils untreated or treated with bafilomycin A1 (Baf, 1 μM) or nocodazole (Noc, 2.5 μM) and stimulated with hgc1Δ yeast-locked C. albicans (MOI = 10). (b) Production of reactive oxygen species by human peripheral neutrophils untreated or treated with bafilomycin A1 (Baf) or nocodazole (Noc) and stimulated with hgc1Δ yeast-locked C. albicans (MOI = 10). (c) NET release by neutrophils pre-incubated with hgc1Δ yeast-locked C. albicans at an MOI of 0, 20, 40 and 80 for 1 h and stimulated with C. albicans hyphae at an MOI of 10. (d) NET release by neutrophils pre-incubated with 0.1 μm polystyrene beads 1 h and stimulated with C. albicans hyphae at an MOI of 10. (a, c, d) Statistics by two-way ANOVA, followed by Sidak’s multiple comparison post test: * p<0.01, ** p<0.0001. Data are representative of at least three independent experiments. UT, untreated; LU, luminescence units.

(a) Phagocytosis of hgc1Δ yeast-locked C. albicans (MOI 40) by untreated neutrophils (left panel) or treated with anti-dectin-1 blocking antibody (right panel). The number of phagocytosed yeast particles per cell was assessed over 2 h by live microscopy. A trend line was fitted to the data from 10 individual neutrophils. (b) NET release by untreated peripheral human neutrophils (left panel) or treated with anti-dectin-1 blocking antibody (right panel) stimulated with hgc1Δ yeast-locked C. albicans or WT hyphae (MOI = 10). Assessment of NET release 4 h post stimulation. % NETs released over total number of neutrophils. Statistics by one-way ANOVA, followed by Sidak’s multiple comparison post test: * p<0.0001 (c) Time-lapse microscopy of live human peripheral neutrophils treated with dectin-1 blocking antibody or left untreated and stimulated with heat-inactivated hgclΔ yeast-locked C. albicans (MOI = 40). Arrowheads indicate incomplete decondensation. Arrows indicate NET release. Confocal images were taken every 30 seconds. Panels represent frames of the indicated times. Scale bars = 20 μm. (d) Human peripheral neutrophils stimulated with hgc1Δ yeast-locked C. albicans. NE (red), CD63 (green) and DNA (DAPI, blue) localization after 1 h. NE in the phagosome (arrows). NE in the nucleus (asterisks). Scale bar = 5 μm. (e) NET release in the lungs of dectin-1 KO mice and WT (C57BL/6) controls infected intratracheally with 1×10⁶ c.f.u. A. fumigatus assessed 48 h post infection by immunofluorescence staining against citrullinated histone H3 (H3-cit), MPO and DNA (DAPI). Scale bars = 20 μm. Data are representative of two independent experiments. UT, untreated. US, unstimulated.

(a) Survival of WT (C57BL/6) and dectin-1 deficient mice, untreated or treated with NEi or AREG, infected intratracheally with 1×10⁷ c.f.u. hgc1Δ yeast-locked C. albicans (n=4). Statistics by Log-rank (Mantel-Cox) test, NS p>0.5, * p<0.05, ** p<0.01. (b) NET release in lungs of WT (C57BL/6) and dectin-1-deficient mice treated with NEi prior to infection or left untreated, infected with 3×10⁶ c.f.u. hgc1Δ yeast-locked C. albicans. NET release 24 h post infection. Lungs sections stained for DNA (DAPI, blue), citrullinated histone H3 (cit-H3, red) and MPO (green) and analyzed by immunofluorescence confocal microscopy. Scale bars = 50 μm. (c) Quantification of fibrin deposition (i, ii) and bleeding (iii, iv) in lungs of WT (C57BL/6) and dectin-1 deficient mice infected with of 3×10⁶ c.f.u. hgc1Δ yeast-locked C. albicans, assessed 36 h post infection with scores from 0 to 3. Average score for each mouse (i, iii) and percent of analyzed images from all mice that fall within each score (ii, iv). Fibrin score: 0 = none, 1 = >1, 2 = <50%, 3 = <50%. Bleeding score: 0 = none, 1 = mild, 2 = moderate, 3 = severe. Statistics by two-way ANOVA, followed by Tukey’s multiple comparison post test: ** p<0.01, *** p<0.001, **** p<0.0001. Data are representative of two independent experiments.