Fig 2: Initiation of classical complement activation at the bases of SAP-mediated phagocytosis.a FITC-labeled AF conidia (1.6 × 106) phagocytosis by freshly isolated human neutrophils (2 × 105) in presence of sera depleted (-) of complement components and the effect of human SAP opsonisation after 1 and 30 min. AF conidia (2 × 108) opsonisation with 100 µg of human native SAP. Five percent (left) or 10% (right) of human sera were used. Mean ± SEM of two experiments performed in triplicate and merged. Each spot corresponds to a replicate. Left, NS vs. NS + SAP, **P = 0.009; ***P = 0.002. MBL- S vs. MBL- S + SAP, ***P = 0.002 (two tailed, Mann–Whitney U-test). Right, NS vs. NS + SAP, *P = 0.01; ***P = 0.002. MBL- S vs. MBL- S + SAP, *P = 0.04 (two tailed, Mann–Whitney U-test). b FACS analysis of phagocytosis of FITC-labeled AF conidia (5 × 106/200 µl of blood; 20 min) opsonized or not with murine SAP in whole blood of wt, C3-, C1q-, MBL1/2-, and Factor B-deficient mice. Each figure refers to independent experiments performed. Results of C3-/-, C1q-/-, and Mbl1/2-/- are mean ± SEM of one experiment, whereas for Fb-/- two experiments were merged. Correspondent wt littermates were used as control for C1q-, MBL1/2- (C57BL/6J) and Factor B-deficient mice (C57BL6/NJ). Each spot represents a single mouse. Left, n = 11 wt, n = 10 C3-/-, n = 8 C1q-/-. wt vs. C3-/-, *P = 0.02; wt vs. C1q-/-, *P = 0.02 (two-sided, unpaired t-test). Middle, n = 5 wt, n = 5 Mbl1/2-/-. wt vs. Mbl1/2-/-, *P = 0.01; wt vs. wt + Sap, ***P = 0.004; Mbl1/2-/- vs. Mbl1/2-/- + Sap, ***P = 0.002 (two-sided, unpaired t-test). Right, n = 12 wt, n = 14 Fb-/-. wt vs. wt + Sap, *P = 0.02 (two-sided, unpaired t-test); Fb-/- vs. Fb-/- + Sap, **P = 0.005 (two tailed, Mann–Whitney U-test). c FACS analysis of C1q deposition on AF conidia (1 × 107) in presence of plasma from wt (n = 9) or Apcs-/- (n = 7) mice. Mean ± SEM; *P = 0.05 (two-sided, unpaired t-test). d Effect of blocking of Fc? receptors in SAP-mediated phagocytosis. Human SAP-pre-opsonized FITC-labeled AF conidia (1.6 × 106) phagocytosis by freshly isolated human neutrophils (2 × 105; 5 min) in presence of NS and anti-CD16, anti-CD32, anti-CD64 or an irrelevant mouse IgG1 (all used at 10 µg/ml). Each spot corresponds to a single healthy donor (n = 4) performed in duplicate. Mean ± SEM. e–g Effect of IgG depletion in SAP-mediated phagocytosis (f) and complement deposition (g). e Western blot analysis for SAP and IgGs in human plasma and after their depletion. 1 µl/lane of plasma-heparin from five healthy donors (A–E) and 10 µg/lane of proteins eluted from the anti-SAP column were loaded. Ponceau red staining was shown. Human SAP (0.1 µg/lane) was used as control. f FACS analysis of phagocytosis by neutrophils (2 × 105) isolated from A–E donors after challenge with FITC-labeled AF conidia (1.6 × 106) opsonized or not with human SAP (100 µg/1 × 109 conidia/ml) in the presence of autologous plasma-heparin (10%) depleted of SAP (SAP-) and IgGs (IgG-). g C3 deposition on AF conidia (1 × 107) opsonized or not with human SAP. SAP- and IgG-depleted plasma-heparin (10%) from A–E donors were used. f, g Each spot refers to mean of a duplicate per donor. Mean ± SEM.

Fig 3: SAP-mediated complement activation on A. fumigatus.a FACS analysis (frequency, left; MFI, right) of C3 from wt (-, n = 10; + Sap, n = 10) and Apcs-/- (-, n = 10; + Sap, n = 10) mouse plasma on AF conidia (1 × 107) opsonized or not with murine SAP. a Left, wt vs. Apcs-/-, *P = 0.01 (1 min), *P = 0.02 (5 min), ***P = 0.003 (10 min), **P = 0.007 (20 min). wt vs. wt + Sap, *P = 0.02 (1 min). Apcs-/- vs. Apcs-/- + Sap, **P = 0.008 (1 min), ***P = 0.003 (5 min), *P = 0.05 (10 min), *P = 0.05 (20 min) (two-sided, unpaired t-test). a Right, wt vs. Apcs-/-, ***, P = 0.002 (10 min), **P = 0.006 (20 min). wt vs. wt + Sap, *P = 0.01 (1 min), **P = 0.005 (5 min). Apcs-/- vs. Apcs-/- + Sap, ***P = 0.0003 (1 min), ***P = 0.002 (5 min), *P = 0.04 (10 min and 20 min) (two-sided, unpaired t-test). b Murine plasma C5a levels after incubation with AF conidia shown in a. n = 11 mice/genotype. wt vs. Apcs-/-, **P = 0.008 (5 min), ***P = 0.003 (10 min), **P = 0.005 (20 min). wt vs. wt + Sap, **P = 0.009 (20 min). Apcs-/- vs. Apcs-/- + Sap, ***P = 0.003 (1 min), **P = 0.007 (5 min), ***P = 0.002 (10 min), *P = 0.04 (20 min) (two-sided, unpaired t-test). a, b A representative independent experiment shown out of 3 performed. Each spot represents plasma from a single mouse. a, b Mean ± SEM. c FACS analysis of SAP-mediated MAC deposition on AF conidia (1 × 107) in the presence of human sera depleted of complement components. Mean ± SEM of two experiments in quadruplicate and merged out of 4 performed. Each spot corresponds to a replicate. NS vs. NS + SAP, ***P = 0.001; **P = 0.008 (two-sided, unpaired t-test). MBL- S vs. MBL- S + SAP, **P = 0.006; ***P = 0.003 (two-sided, unpaired t-test). FB- S vs. FB- S + SAP, ***P = 0.002; **P = 0.009 (two-sided, unpaired t-test). d Quantitative analysis of AF viability performed using a resazurin-based assay. AF viability assay performed in sera depleted of complement components. Results are mean ± SEM of the relative fluorescence intensity. Effect of Posaconazole (POC; 1 µM) exposure is also shown. Figure summarizes one experiment out of 5 performed with n = 5 replicates each. *P = 0.01; **P = 0.009 (two-sided, unpaired t-test).

Fig 4: Effects of SAP on myelomonocytic cell function.a FACS analysis of phagocytosis (1 and 20 min) of FITC-labeled AF conidia opsonized with murine SAP by peripheral murine neutrophils (Ly6G+ CD11b+), monocytes (Ly6C+ CD11b+) and DCs (CD11c+) (1 × 107 conidia/200 µl of blood). N = 7 wt, n = 8 Apcs-/-. wt vs. wt + Sap, ****P < 0.0001 (1 min), ***P = 0.003 (20 min) (neutrophils); ****P < 0.0001 (1 min), ***P = 0.002 (20 min) (monocytes). Apcs-/- vs. Apcs-/- + Sap, ***P = 0.0004 (1 min), ****P < 0.0001 (20 min) (neutrophils); **P = 0.005 (1 min), ****P < 0.0001 (20 min) (monocytes) (two-sided, unpaired t-test). b Effect of human SAP opsonisation on in vitro phagocytosis by isolated human monocytes (n = 4 donors) (1 × 105 cells and 1 × 106 FITC-labeled AF conidia; 30 min; 10% of autologous serum). *P = 0.03 (two-sided, unpaired t-test). c Levels of CXCL1, CXCL2, TNF-a, and IL-6 in supernatants of bone marrow-derived macrophages from wt and Apcs-/- mice stimulated (24 h) with A. fumigatus conidia (ratio 1:5, cell:conidia) in the presence of correspondent mouse serum (10%). LPS, 10 ng/ml. n = 4 mice per genotype. d FACS analysis of FITC-labeled AF conidia phagocytosis by neutrophils (CD45+ Ly6G+), macrophages (CD45+ Ly6C+ F4/80+) and DCs (CD45+ CD11c+) collected from BALFs of wt (-, n = 7; Sap, n = 5) and Apcs-/- (-, n = 8; Sap, n = 5) mice after i.t. injection of 5 × 107 conidia (4 h) opsonized with murine SAP. d Neutrophils, wt vs. Apcs-/-, *, P = 0.05; wt vs. wt + Sap, *, P = 0.03; ***, P = 0.0003. Macrophages, *P = 0.030; ***P = 0.004 (two-sided, unpaired t-test). a–d Each spot corresponds to a single mouse (a, c, d) or single healthy donor (b). a–d Mean ± SEM of independent experiments.

Fig 5: Therapeutic efficacy of SAP against invasive aspergillosis in immunosuppressed hosts.a Percent survival of transiently immunosuppressed wt mice after i.t. injection with the indicated doses of AF conidia. The curve of untreated mice (n = 3) with cyclophosphamide is also shown. Human SAP (4 mg/kg) was i.p. injected at 2 h and 24 h after infection. AF 5 × 107, n = 17 mice; AF 5 × 107 + SAP, n = 15 mice; AF 1 × 107, n = 7 mice; AF 1 × 107 + SAP, n = 8 mice; AF 5 × 106, n = 8 mice; AF 5 × 106 + SAP, n = 10 mice. An independent experiment shown. SAP-treated vs. saline, P = 0.002, AF 5 × 107; P = 0.001, AF 1 × 107; P = 0.004, AF 5 × 106 (Log-rank, Mantel-Cox test). b Lung CFU in mice treated with cyclophosphamide and infected with 1 × 107 AF conidia. N = 5 AF, n = 5 AF + SAP. Mean ± SEM of an independent experiment. *P = 0.02 (two-sided, Mann–Whitney U-test). c, d Effect of local administration of SAP on survival (c) and lung CFU (d) after i.t. injection of 5 × 106 in wt mice treated with saline (50 µl/mouse; n = 13) or human SAP (50 µg/50 µl/mouse; n = 12) 2 h after infection. ***P = 0.003 (Log-rank, Mantel-Cox test). c One experiment shown out of 2 performed with similar results. d Each spot corresponds to a single mouse. Mean ± SEM of an independent experiment. **P = 0.008 (two-sided, Mann–Whitney U-test).