Figure 1.
HCNTs resuspended in 0.01% Tween-80 are adequately dispersed prior to use.
(A) A representative SEM image of HCNTs shows moderate variability in the diameter of individual tubes as well as sharp kinks and folding. The circled area highlights the diameter measurement using Gwyddion software (see supporting information). (B) Absorbance as a measurement of dispersion following 5 minutes of sonication. The decrease in absorbance over time is from dispersed HCNTs settling by gravity. A brief vortexing resuspends the HCNTs.
Figure 2.
Acute and repeated exposure to HCNTs result in phagocytosis of HCNTs by alveolar macrophages.
(A) Cytospin preparation of BALF from an HCNT treated mouse 24 hours after one administration. Many of the macrophages have phagocytized HCNTs (arrows). (B) Total protein and LDH levels measured from the BALF of mouse lungs acutely (1 day) and repeat (21 days) exposed to HCNTs. Black bars = HCNT, white bars = control. n = 3-4 for each group. * p < 0.05. Error bars indicate standard error of the mean.
Figure 3.
Acute and repeat exposure to HCNTs result in changes in the murine pulmonary leukocyte population.
(A) Quantitative analyses of the leukocyte populations in the BALF 1 day and 21 days after exposure to HCNTs. (B) KC and MCP-1 measured from the BALF of mouse lungs acutely (1 day) and repeatedly (21 days) exposed to HCNTs. KC levels were significantly elevated in the BAL following acute and repeated exposure to HCNTs.. Black bars = HCNT, white bars = control. n = 3-4 for each group. * p < 0.05. Error bars indicate standard error of the mean.
Figure 4.
Repeated exposure to HCNTs results in multiple pulmonary lesions.
(A) Mice exposed to the dispersal media (PBS + 0.01% Tween-80) showed no significant changes in the bronchi or alveolar spaces. (B) Mice exposed to HCNTs developed multiple granulomas (arrows). (C) A higher magnification of the rectangular inset from B. (D) HCNT laden macrophages can be found both within the terminal bronchiole (arrows) and alveolar spaces. (E) HCNTs can be found within the alveolar walls and epithelial cells (arrows) and can be distinguished from epithelial cell nuclei (arrowheads). (F) Alcian blue staining of the lungs repeatedly exposed to HCNTs show evidence of goblet cell hyperplasia in the larger conducting airways. Inset is a higher magnification of the bronchiolar epithelium.
Figure 5.
Repeated HCNT exposure enhances pulmonary inflammation without impeding clearance of P. aeruginosa.
Mice received HCNTs or PBS/Tween 80 twice/week for 3 weeks. Three days after the last treatment, P. aeruginosa strain PAO1 (107/mouse) was given to all mice. (A-B) Representative images of paraffin embedded lungs from mice 24 hours after PAO1 was administered show that HCNT treated mice had a more robust inflammatory response (A) compared to control mice (B). (C) Cytospin preparations from the BALF of mice 24 hours after PAO1 administration shows a significant increase in the numbers of neutrophils and macrophages from HCNT treated mice compared to controls. (D) KC and MCP-1 measured from the BALF of mouse lungs after PAO1 infection following repeated exposure to HCNTs or dispersal media. All parameters were elevated in HCNT exposed mice compared to controls, with MCP-1 reaching statistical significance. (E) Clearance of PAO1 in mice was determined by homogenization of whole lungs followed by serial dilutions and plating. Mice were infected 6-72 hours after the last HCNT treatment. Repeated administration of HCNTs did not affect the clearance of PAO1 from the lungs 24 hours after PAO1 infection. Black bars = HCNT, white bars = control. n = 5 to 6 mice for each group. * p < 0.05. Error bars indicate standard error of the mean.
Figure 6.
HCNTs inhibit phagocytosis of P. aeruginosa.
Macrophages from mouse lungs pre-exposed to HCNTs are less efficient at phagocytizing PAO1-GFP. n = 3 mice for each group, a minimum of 200 macrophages were counted per mouse. Black bars = HCNT, white bars = control. * p < 0.05. Error bars indicate standard error of the mean.
Figure 7.
HCNT pre-exposure affects clearance of P. aeruginosa in mice depleted of specific lineages of leukocytes.
(A) Preexposure to HCNTs in neutrophil depleted mice significantly increased the number of macrophages in the BAL in response to PAO1 infection. n = 5 mice for each group. * p < 0.05. Error bars indicate standard error of the mean. (B) HCNT exposed mice had increased clearance of PAO1 from the lungs than control mice when depleted of neutrophils prior to infection. (C) HCNT exposed mice had reduced clearance of PAO1 when systemically depleted of macrophages. Black bars = HCNT, white bars = control. n = 8 to 11 mice for each group. * p < 0.05. Error bars indicate standard error of the mean.