2013TellabatiAKPhD.pdf (5.49 MB)
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Effects of Nano Particles on Alveolar Macrophages

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posted on 25.09.2013, 15:19 by Anantha Kalyan Tellabati
Background: Epidemiological studies suggest that inhalation of carbonaceous particulate matter from increases susceptibility to bacterial pneumonia. In vitro studies report that phagocytosis of carbon black by alveolar macrophages (AM) impairs killing of Streptococcus pneumoniae. We therefore aimed to use a mouse model to test the hypothesis that high levels of carbon loading of AM in vivo increases susceptibility to pneumococcal pneumonia. We also want to develop an air-tissue interface model to assess DNA damage to airway macrophages due to inhalation of manufactured nanoparticals. Methods: Female outbred mice were treated with either intranasal phosphate buffered saline (PBS) or ultrafine carbon black (UF-CB in PBS; 500 μg on day 1 and day 4), and then infected with S. pneumoniae strain D39 on day 5. Survival was assessed over 72 h. The effect of UF-CB on AM carbon loading, airway inflammation, and a urinary marker of pulmonary oxidative stress was assessed in uninfected animals. The human monocyte cell line Mono Mac 6 and primary alveolar macrophages were used to assess DNA damage. To measure DNA damage in macrophages, we used the alkaline Comet assay. After nanoparticle exposure, a total of 100 macrophages were analysed per sample, as n=50 duplicate slides. Tail length, percentage of DNA in the tail of the comet (% tail DNA), tail extent moment and olive tail moment, were calculated for each cell using the Komet Analysis software. Results: Instillation of UF-CB in mice resulted high levels of carbon loading in alveolar macrophages. In uninfected animals, UF-CB treated animals had increased urinary 8-oxodG (P = 0.055), and an increased airway neutrophil differential count (P < 0.01). All PBS-treated mice died within 72 h after infection with S. pneumoniae, whereas morbidity and mortality after infection was reduced in UF-CB treated animals (median survival 48 h vs. 30 h, P < 0.001). At 24 hr post-infection, UF-CB treated mice had lower lung and the blood S. pneumoniae colony forming unit counts, and lower airway levels of keratinocyte-derived chemokine/growth-related oncogene (KC/GRO), interferon gamma( IFN-γ) and other inflammatory cytokines. No increase in DNA damage was observed when cells were placed in the nitrogen gas flow for 10 mins compared with cells placed in air (insert control vs air control).Exposure to nanoparticles from all three metals for 10 min caused a significant increase in DNA damage in human Mono Mac 6 cells compared to insert control. There was no significant difference between DNA damage caused by gold (Au), silver (Ag) and Iron (Fe) nanoparticles. Conclusion: Acute high level loading of AM with ultrafine carbon black particles per se does not increase the susceptibility of mice to pneumococcal infection in vivo. We found significant DNA damage in macrophages cultured for 24 h with doses of up to 10 mg/cm² aerosolized iron, gold or silver nanoparticles.



Grigg, Jonathan; Andrew, Peter; Howes, Paul

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University of Leicester

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