Fig. 2: SAP acts as opsonin for phagocytosis of A. fumigatus.

a Induction of SAP levels in mice after i.t. injection of 5 × 10⁷ AF conidia; −, sham mice; LPS, 0.8 mg/kg. Mean ± SD of an independent experiment; n = 3, −; n = 4, AF 4 h; n = 3, AF 16 h; n = 3, LPS. b Confocal images depicting SAP localization in infected lung (4 h; 5 × 10⁷ fluorescein (FITC)-labeled AF conidia; n = 3 mice). Left, merged image of a lung stained for SAP (red), C3 (blue) and neutrophils (Ly6G⁺ cells; cyan). Conidia are evident from the detection of FITC (green) signal. Dashed white arrowheads indicate regions of SAP localization on AF conidia. A representative staining of lung of a non-infected mouse is also shown (basal; left box). Middle, close-up image of the white dashed region (left) showing SAP localization on AF. The image refers to extracted AF and SAP signals also separately shown in the boxes within the image. Right, a section of infected lung of Apcs^−/− mouse is also shown. Scale bar, 50 µm. c Number of CFU per lung at 16 h after infection with 5 × 10⁷ conidia. Each spot corresponds to a single mouse (wt, n = 9; Apcs^−/− = 11). One experiment out of 2 performed with similar results. Mean ± SEM. P < 0.0001 (two-sided, Mann–Whitney U-test). d FACS analysis of in vivo phagocytosis in BALF neutrophils 4 h after injection of 5 × 10⁷ FITC-labeled AF conidia. The figure shows results of two pooled experiments with n = 10 wt and n = 9 Apcs^−/− mice. Mean ± SEM. *P = 0.05 (two-sided, unpaired t-test). e, f Interaction of SAP with A. fumigatus. e FACS analysis of binding of biotin-conjugated (b-) murine SAP (Sap; 10 µg/ml) to viable dormant or germinating conidia of AF (1 × 10⁸). Human SAP (50 µg/ml) and CRP (50 µg/ml) were also used. Mean ± SD of one quadruplicate experiment of two performed. f b-Sap (10 µg/ml) interaction with AF. Images refer to maximum intensity projection of z-stacks. Scale bar, 5 µm. g, h Effect of SAP on A. fumigatus phagocytosis by neutrophils. g, h FACS analysis (one experiment shown out of 3 performed) of phagocytosis (g) and CD11b internalization (h) in neutrophils after challenge with FITC-labeled AF conidia (1 × 10⁷/200 µl of blood) opsonized or not with murine SAP is shown. g neutrophil phagocytosis in whole blood of wt and Apcs^−/− mice. h CD11b expression in neutrophils of g. g, h n = 12 wt and n = 11 Apcs^−/− mice. Mean ± SEM. g 1 min, *P = 0.05; ***P = 0.002; ****P < 0.0001. 20 min, *P = 0.01, wt vs. Apcs^−/−; *P = 0.03 wt vs. wt + Sap or Apcs^−/− vs. Apcs^−/− + Sap. h 1 min, *P = 0.01; ***P = 0.0004; ****P < 0.0001; 20 min, *P = 0.04; ***P = 0.004. g, h (two-sided, unpaired t-test).