Pseudomonas aeruginosa Elastase Provides an Escape from Phagocytosis by Degrading the Pulmonary Surfactant Protein-A

Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute pneumonitis in immunocompromised patients and chronic lung infections in individuals with cystic fibrosis and other bronchiectasis. Over 75% of clinical isolates of P. aeruginosa secrete elastase B (LasB), an elastolytic metalloproteinase that is encoded by the lasB gene. Previously, in vitro studies have demonstrated that LasB degrades a number of components in both the innate and adaptive immune systems. These include surfactant proteins, antibacterial peptides, cytokines, chemokines and immunoglobulins. However, the contribution of LasB to lung infection by P. aeruginosa and to inactivation of pulmonary innate immunity in vivo needs more clarification. In this study, we examined the mechanisms underlying enhanced clearance of the ΔlasB mutant in mouse lungs. The ΔlasB mutant was attenuated in virulence when compared to the wild-type strain PAO1 during lung infection in SP-A+/+ mice. However, the ΔlasB mutant was as virulent as PAO1 in the lungs of SP-A-/- mice. Detailed analysis showed that the ΔlasB mutant was more susceptible to SP-A-mediated opsonization but not membrane permeabilization. In vitroand in vivo phagocytosis experiments revealed that SP-A augmented the phagocytosis of ΔlasB mutant bacteria more efficiently than the isogenic wild-type PAO1. The ΔlasB mutant was found to have a severely reduced ability to degrade SP-A, consequently making it unable to evade opsonization by the collectin during phagocytosis. These results suggest that P. aeruginosa LasB protects against SP-A-mediated opsonization by degrading the collectin

Following Tetraploidy in Maize, a Short Deletion Mechanism Removed Genes Preferentially from One of the Two Homeologs

Following genome duplication and selfish DNA expansion, maize used a heretofore unknown mechanism to shed redundant genes and functionless DNA with bias toward one of the parental genomes

Helical Carbon Nanotubes Enhance the Early Immune Response and Inhibit Macrophage-Mediated Phagocytosis of <i>Pseudomonas aeruginosa</i>

<div><p>Aerosolized or aspirated manufactured carbon nanotubes have been shown to be cytotoxic, cause pulmonary lesions, and demonstrate immunomodulatory properties. CD-1 mice were used to assess pulmonary toxicity of helical carbon nanotubes (HCNTs) and alterations of the immune response to subsequent infection by <i>Pseudomonas aeruginosa</i> in mice. HCNTs provoked a mild inflammatory response following either a single exposure or 2X/week for three weeks (multiple exposures) but were not significantly toxic. Administering HCNTs 2X/week for three weeks resulted in pulmonary lesions including granulomas and goblet cell hyperplasia. Mice exposed to HCNTs and subsequently infected by <i>P. aeruginosa</i> demonstrated an enhanced inflammatory response to <i>P. aeruginosa</i> and phagocytosis by alveolar macrophages was inhibited. However, clearance of <i>P. aeruginosa</i> was not affected. HCNT exposed mice depleted of neutrophils were more effective in clearing <i>P. aeruginosa</i> compared to neutrophil-depleted control mice, accompanied by an influx of macrophages. Depletion of systemic macrophages resulted in slightly inhibited bacterial clearance by HCNT treated mice. Our data indicate that pulmonary exposure to HCNTs results in lesions similar to those caused by other nanotubes and pre-exposure to HCNTs inhibit alveolar macrophage phagocytosis of <i>P. aeruginosa</i>. However, clearance was not affected as exposure to HCNTs primed the immune system for an enhanced inflammatory response to pulmonary infection consisting of an influx of neutrophils and macrophages. </p> </div

HCNTs inhibit phagocytosis of <i>P. aeruginosa</i>.

<p>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.</p

Repeated exposure to HCNTs results in multiple pulmonary lesions.

<p>(<b>A</b>) Mice exposed to the dispersal media (PBS + 0.01% Tween-80) showed no significant changes in the bronchi or alveolar spaces. (<b>B</b>) Mice exposed to HCNTs developed multiple granulomas (arrows). (<b>C</b>) A higher magnification of the rectangular inset from B. (<b>D</b>) HCNT laden macrophages can be found both within the terminal bronchiole (arrows) and alveolar spaces. (<b>E</b>) HCNTs can be found within the alveolar walls and epithelial cells (arrows) and can be distinguished from epithelial cell nuclei (arrowheads). (<b>F</b>) 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.</p

Acute and repeat exposure to HCNTs result in changes in the murine pulmonary leukocyte population.

<p>(<b>A</b>) Quantitative analyses of the leukocyte populations in the BALF 1 day and 21 days after exposure to HCNTs. (<b>B</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.</p

HCNT pre-exposure affects clearance of <i>P. aeruginosa</i> in mice depleted of specific lineages of leukocytes.

<p>(<b>A</b>) 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.</p