PTX3-mediated increase of A fumigatus conidia phagocytosis is serum and complement dependent but not Ig-dependent. (A) For the phagocytic assay, A fumigatus conidia were preincubated or not (CTR) with 1.1μM PTX3 and then incubated for 30 minutes with neutrophils in the presence of 1%, 3%, or 10% NHS or HHS. Results are mean ± SEM of 3-6 pooled experiments. (B) The phagocytosis assay was performed with 10% NHS or Ig-depleted human serum (NHS-Ig). Results are mean ± SEM of 3 pooled experiments. (C) The phagocytosis assay was performed with A fumigatus conidia pre-incubated with different doses of PTX3 (0.27-2.22μM) in the presence or not of 10% of NHS. Results are representative of 1 of 2 experiments. (D) FACS analysis of PTX3-opsonized FITC-conidia phagocytosis. MFI results are mean ± SEM (n = 7 donors). (E) Full-length PTX3 (PTX3_full), N-terminal domain (PTX3_N-term), and C-terminal domain of PTX3 (PTX3_C-term) were incubated with A fumigatus conidia at different concentrations (6.9-444nM). The binding was detected as described in supplemental Methods. MFI results are representative of 1 of 3 independent experiments. (F) FACS analysis of FITC-conidia phagocytosis after opsonization with full-length PTX3, N-terminal, or C-terminal domains. Results (neutrophil MFI) are representative of 1 of 3 independent experiments. (G) Conidia were pre-incubated (white and gray bars) or not (black bar) with 1.1 μM PTX3 for the indicated time (0, 60, 120 minutes) and eventually washed to eliminate unbound PTX3 before the phagocytosis assay (gray bar) in the presence of 10% NHS. Results are mean ± SEM of 2-6 pooled experiments. *P ≤ .05; **P < .01; ***P = .0005, Student paired t test.