Lung epithelial cell TRAF3 expression is down-regulated under A. fumigatus infection. Expression of TRAF3 was detected in A549 cells stimulated or unstimulated with A. fumigatus by qRT-PCR and western blot (A, B, C). The expression and localization of TRAF3 in A549 cells were determined by immunofluorescence (D, E). Results are expressed as the mean ± SD from three independent experiments. **P < 0.01, ***P < 0.001. Scale bars: 50 µm.

TRAF3 overexpression promotes lung epithelial cell adhesion and internalization of A. fumigatus spores. A549 cells were transfected with pIRES-TRAF3-EGFP plasmid (TRAF3) and control pIRES-EGFP plasmid (Vector). mRNA (A) and protein (B, C) expression of TRAF3 were determined by qRT-PCR and western blot. A. fumigatus spores were co-incubated with A549 cells with or without TRAF3 overexpression for 6 h. A. fumigatus spores were labeled with fluorescent white (CFW), then fixed with 4% paraformaldehyde. Cells were dead after fixation and then Annexin-V-stained cell membrane; the number of A. fumigatus spores adhered to lung epithelial cells was determined by fluorescence (D). (E) is the number of A. fumigatus spores adhered to each cell. The number of internalized A. fumigatus spores with or without TRAF3 overexpression in A549 cells was determined by nystatin protection assay (F). Results are expressed as the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001. Scale bars: 50 µm.

TRAF3 inhibits the inflammatory response of A. fumigatus-stimulated activated lung epithelial cells. IL-1β (A), TNF-α (C), IL-6 (E), IL-8 (G) mRNA levels were detected by quantitative real-time PCR in A549 cells stably overexpressing TRAF3 or vector control with or without A. fumigatus stimulation for 6 h; the secretion of IL-1β (B), TNF-α (D), IL-6 (F), and IL-8 (H) was detected by ELISA in A549 cell culture supernatants. Results are expressed as the mean ± SD of three independent experiments. *, # P < 0.05; **, ## P < 0.01; ***P < 0.001.

TRAF3 inhibits macrophage migration and macrophage cytokine production by lung epithelial cells infected with A. fumigatus. (A) Schematic of co-culture of A. fumigatus, lung epithelial cells, and macrophages. Macrophages that migrated to the lower chamber were fixed with 4% paraformaldehyde, stained with Giemsa staining solution, and counted (B, C). The expression of cytokines in the macrophages in the upper chamber of Transwell was analyzed by quantitative real-time PCR (D). Fluorescent staining and nystatin protection assay were performed on lung epithelial cells at the bottom of 24-well plates to detect A549 cells adhesion (E, F) and internalization of A. fumigatus spores (G). Results are expressed as the mean ± SD of three independent experiments. *, # P < 0.05; **, ## P < 0.01; ***, ### P < 0.001. Scale bars: 50 µm.

TRAF3 overexpression inhibits the activation of NF-κB and MAPK signaling pathways in A. fumigatus-infected lung epithelial cells. Protein expression of p-p65, p65, p-IκBα, IκBα, JNK, p-ERK, ERK, p-AKT, and AKT was detected by western blot for 6 h in A549 cells stimulated with or without TRAF3 overexpression by A. fumigatus (A, B, F, G). The protein expression of p65 in the nucleus and cytoplasm of A549 cells was detected by western blotting (C, D). The expression and localization of p65 in A549 cells were determined by immunofluorescence (E). Results are expressed as the mean ± SD of three independent experiments. ns stands for no statistically significant difference; *, # P < 0.05; **, ## P < 0.01; ***P < 0.001. Scale bars: 50 µm.

TRAF3 overexpression reduces survival and promotes fungal load in vivo in A. fumigatus-infected zebrafish. (A) Images of zebrafish larvae transgenic for TRAF3 and expressing EGFP at 3 dpf. qRT-PCR was used to detect the transcriptional expression levels of TRAF3 at various time points after zebrafish infection with A. fumigatus (B). Zebrafish larvae were immersed in E2 medium containing 1 × 10⁷A. fumigatus spores and replaced with sterile E2 medium after 2 h. The effect of TRAF3 on the survival rate of zebrafish larvae infected with A. fumigatus (C) and the fungal load of zebrafish larvae at various time points (D) were examined.The expression levels of pro-inflammatory cytokines (E–H) in zebrafish infected with A. fumigatus at various time points were measured by qRT-PCR. ns stands for no statistically significant difference; *, # P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 50 µm.

Schematic representation of epithelial cells infected with A. fumigatus affecting the migration of macrophages. (Left) When lung epithelial cells are infected with A. fumigatus spores, a large number of macrophages migrate to the infection site. (Right) TRAF3 overexpression causes lung epithelial cells to adhere to and internalize a large number of A. fumigatus spores; fewer A. fumigatus spores are recognized by macrophages, and only a small number of macrophages migrate to the infection site.