Figure 2.

A. fumigatus Conidia Induce IFN-γ Production In Vitro and In Vivo

(A–C) IFN-γ gene expression (A) and production by immunofluorescence (B) and immunoblotting (C) in RAW264.7 cells pulsed with live A. fumigatus conidia.

(D and E) IFN-γ production by immunoblotting (D) and ELISA (E) in C57BL/6 lung macrophages exposed to live A. fumigatus conidia.

(F) IP10 gene expression in RAW264.7 cells pulsed with live conidia in the presence of rIFN-γ.

(G and H) IFN-γ production by immunoblotting (G) and ELISA (H) in lung macrophages from C57BL/6, Dectin1^−/−, or Myd88^−/− mice exposed to conidia.

(I) Immunoblotting of IFN-γ in RAW264.7 cells exposed to conidia and treated with the NF-kB inhibitor, SN50.

(J and K) Dapk1 gene (J) and protein (K) expression in lung macrophages stimulated as above. Gene expression was assessed by real-time qPCR (normalization was performed on Gapdh).

For immunoblotting, normalization was performed on mouse β-actin or Gapdh (the corresponding pixel density is indicated). For immunofluorescence, nuclei were counterstained with DAPI. Photographs were taken with a high-resolution microscope (Olympus BX51). Scale bars, 100 μm. IFN-γ fluorescence intensity was measured with the ImageJ software. Data (mean values ± SD) represent pooled results or representative images (immunofluorescence and immunoblotting) from three experiments. ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, conidia-pulsed versus unpulsed (none) or rIFN-γ treated versus untreated cells and knockouts versus wild-type mice.