Supplemental Digital Content Supplemental Figure Legends
Figure S1. Decreased hepcidin expression levels in airway epithelial cells from mice with E.
coli-induced bacterial pneumonia. The lungs were collected at the indicated time points after infection and were subjected to immunohistochemistry analyses. Representative images of lung sections at each time point after bacterial infection are shown. Scale bar, 20 μm.
Figure S2. Expression of ferroportin in the lungs of mice with pneumonia. A and B, Immunohistochemistry analysis of FPN expression in airway epithelium in lung tissues prepared at 0 hour (steady control) and 6 hours after infection. (A) Representative microscopic images. Scale bar, 20 μm. (B) Quantitative data are presented as the mean ± SD and were analyzed by Student's t test. Dots represent individual mice. C, Immunoblot analysis of FPN expression in alveolar macrophages prepared at 0 hour (steady control) and 6 hours after infection, using β- actin as an internal control. D and E, Immunofluorescence analysis of cell-surface and total FPN expression in pulmonary macrophages prepared at 0 hour (steady control) and 6 hours after infection. (D) Representative microscopic images. Scale bar, 20 μm.
(E) Quantitative data are presented as the mean ± SD and were analyzed by Student's t test.
FPN = ferroportin.
Figure S3. Reconstitution of hepcidin expression in the airway epithelium. Mice were intratracheally treated with adenovirus with Hamp1 overexpression or an adenovirus containing enhanced green fluorescent protein (GFP control). Nine days later, the lung tissues were observed under a fluorescence microscope. Representative microscopic images from three independent experiments are shown. Scale bar, 20 μm.
1
Figure S4. Suppression of airway epithelial cell-derived hepcidin leads to actin cytoskeleton rearrangement in alveolar macrophages. A, Alveolar macrophages were prepared from Hamp1-/- and WT mice and were stained with rhodamine conjugated phalloidin and 4’,6-diamidino-2-phenylindole. The cells were visualized using a confocal microscope.
Representative microscopic images from 3 mice in each group are shown. Bar, 10 µm. B, Bacterial pneumonia in mice was induced by the intratracheal instillation of E. coli. Alveolar macrophages were prepared 0 hour and 6 hours after infection and were stained with rhodamine conjugated phalloidin and 4’,6-diamidino-2-phenylindole. The cells were visualized using a confocal microscope. Representative microscopic images from 3 mice in each group are shown. Bar, 10 µm.
Figure S5. Effect of the suppression of airway epithelial cell-derived hepcidin on the activities of GTP-binding Rho family proteins. A and B, Alveolar macrophages were prepared from Hepcidin-1 knockout (Hamp1-/-) and wild-type (WT) mice and were subjected to a glutathione-S-transferase pull-down assay. Representative immunoblotting images from two independent experiments are shown. (A) Cdc42. (B) Rac1. PC = positive control.
2
0 h 6 h 12 h 24 h 48 h
E. coli
Figure S1
Steady E. coli
infection P. aeruginosa
infection
K. pneumoniae
infection S. pneumoniae
infection S. aureus
infection
Figure S2 A
B
350000 400000
250000 300000
FPN expression level in airway epithelia (IOD)
Steady E. coli
P. aeruginosa K. pneumoniaeS. pneumoniae S. aureus
C
FPN β-Actin
Steady E. coli
infection P. aeruginosa
infection Steady K. pneumoniae
infection S. pneumoniae infection Steady S. aureus
infection
Cell-surface Total
SteadyE. coli infection
D
E
FPN expression level in pulmonary macrophage (MFI) 250
100 0 50 150 200
Cell-surface Total
*
*
Steady E. coli
infection Steady E. coli infection
Hamp1 overexpression GFP control
Figure S3
Hamp1-/-
Figure S4
A WT
0 h 6 h
B
Cdc42-GTP
Total Cdc42
WT Hamp1-/- PC
Rac1-GTP
Total Rac1
WT Hamp1-/- PC
Figure S5 A
B
