131 Alveolar Macrophage Degranulation Initiates the Spatially Restricted Innate Immune Response During Ventilator-Assisted Nanoparticle Inhalation

Abstract

Abstract To visualize and measure in real-time the cellular pulmonary innate immune response elicited by inhaled NPs, we apply state-of-the-art intravital microscopy on the alveolar region of the murine lung. Fluorescent carboxyl-quantum-dot-NPs (deposited dose of 16 cm2/g) applied via ventilator-assisted inhalation accumulated within seconds as distinct fluorescent spots at the alveolar walls. Already at 30min, cQD-NPs elicited an increase in microvascular neutrophil numbers as compared to controls, which subsequently transmigrated into the alveolar space. This early immune response was not specific to cQD-NPs, since a comparable increase in neutrophil numbers was also observed upon inhalation of a bioequivalent dose of carbon black NPs, a typical component of urban air pollution. AMs actively accumulated cQD-NPs over time, starting few minutes after inhalation. Significantly more neutrophils were attracted in proximity to cQD-NP-laden AMs, as compared to NP-free AMs or control areas with epithelial associated cQD-NP. Prior airway application of a cellular degranulation inhibitor prevented cQD-NP and CNP induced neutrophil recruitment at 2h and 24 h after inhalation. Inhibition of Fcγ-receptor mediated AM cQD-NP uptake by prior application of anti-CD64 mAbs or neutralizing of TNFα by anti-TNFα mAbs in the airspace, abolished this early immune response. Importantly, initial ImageStream flow cytometry analysis of BAL samples indicated an increase of macrophage-derived microvesicles upon NP inhalation. Taken together, our data links NP-induced AM degranulation and cytokine release to the rapid and site-specific recruitment of neutrophils, suggesting this process to be a key event in mounting the immune response upon NP inhalation.