Real time in vivo investigation of nanoparticle dynamics and immune response during ventilator-assisted nanoparticle inhalation

Liu, Qiongliang (2023): Real time in vivo investigation of nanoparticle dynamics and immune response during ventilator-assisted nanoparticle inhalation. Dissertation, LMU München: Medizinische Fakultät

Abstract

Many lung diseases have been associated with exposure to various factors of air pollution including (nano)particles. In the past, research investigating cell-particle interactions was mainly based on cultured cells or ex-vivo tissues. Such experimental methods cannot reproduce in full, the complex reactions of the immune system, which might be triggered by nanoparticles (NPs) in vivo. To visualize and measure in real-time the cellular pulmonary innate immune response elicited by different inhaled NPs, we apply state of the art intravital microscopy on the peripheral alveolar region of the murine lung, in combination with ventilator-assisted inhalation of nebulized Quantum-Dot and carbon black NP aerosols. Fluorescent Quantum-Dot NPs (with carboxyl-, amine-PEG- or PEG-surface modifications) became visible within seconds after the onset of inhalation and accumulated as distinct fluorescent spots at the alveolar walls. As early as 60 min after inhalation, a deposited dose of 16 cm2/g (NP surface area /mass lung) elicited an increase in neutrophil numbers only for cQDs, but neither for aPEG-QDs nor PEG-QDs. Neutrophils preferentially arrested in microvessels in close proximity to the site of cQD deposition, where they exhibited probing and crawling behavior, followed by rapid translocation into the alveolar space, where neutrophils ingested cQDs. This early immune response was not specific to cQDs as a comparable increase in neutrophil numbers was also observed upon inhalation of an equivalent dose of carbon black NPs (CNP), a typical component of urban air pollution. The neutrophil response was accompanied by resident alveolar macrophages (AMs) increasingly internalizing cQDs up to the maximum observation time of 90 min, and again only little uptake was detected for aPEG-QDs. Similar results were observed in in vitro experiments using the AM-like M-HS cell line. Analysis of neutrophil numbers in a 30 µm radius (which roughly corresponds to one alveolar diameter) around cQD-positive AMs clearly showed increased neutrophil amounts, whereas no local increase in neutrophil numbers was detected neither around cQD-negative AMs, nor close to cell-free cQD, thus indicating a central role of AMs in the initiation of a spatially restricted innate-immune response. Intriguingly, cQDs as well as CNPs increased the migration velocity of AMs in the alveoli of exposed mice, whereas aPEG-QDs exposure decreased AM crawling velocity, as compared to sham controls. In order to decipher the chain of effects leading to NP-induced neutrophil recruitment, we applied specific blocking antibodies and inhibitors in order (1) to weaken AM-epithelial bonds to inhibit AM migration and affect NP uptake (anti-ICAM1 and anti-LFA1 mAbs), (2) to inhibit NP uptake and AM stimulation via Complement 5a receptor 1 (anti-CD88 mAbs) and Fcγ receptor (anti-CD64 mAbs), (4) to block NP-induced ROS formation via N-acetyl cysteine (NAC) application, (5) to prevent NP induced cellular degranulation (cromolyn), (6) and to assess if fast acting inflammatory mediator release (TNF-α) contributes to the NP induced immune cell recruitment. These mechanistic studies suggest that the induction of NP-elicited neutrophil recruitment depends on several key events: 1. NP-induced neutrophil recruitment is initiated by AMs and related to NP internalization/uptake by AMs. 2. The particle uptake efficacy depends on three factors: (1) NP surface modification; (2) the speed of macrophage patrolling movement in the alveoli; (3) AMs phagocytic/internalizing capability. (1) PEGylation of QDs which are deposited in alveoli renders the NPs invisible to AMs via avoiding protein corona formation and impairs subsequent NP-induced neutrophil recruitment. (2) Alveolar AM patrolling mainly involves ICAM-1 and LFA-1 interactions and can be blocked by respective antibodies applied to the airway side but not to the vascular side. Blocking ICAM-1 and LFA-1 also effectively impaired particle-triggered neutrophil recruitment. (3) The macrophage receptors C5aR1 and FcγRI mediate particle internalization by AMs. Blocking C5aR1 or FcγRI also completely blocks Neutrophile recruitment. 3. NP-triggered neutrophil recruitment requires cellular degranulation and can be inhibited by cromolyn treatment. 4. Recruitment of neutrophils further requires TNFα as an anti-TNFα application into the airways to reduce the neutrophilic inflammatory response. 5. Scavenging ROS via NAC also decreased cQDs-induced neutrophil recruitment to some extent but was less effective than ICAM-1/LFA-1 blocking. Overall, our data indicate a close relation between AM activity (phagocytosis, migration) and the rapid and site-specific recruitment of neutrophils during the early phase (1h and 24h) of particle inhalation, demonstrating a specific role of AMs in triggering the immune response by different NPs.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: nanoparticle inhalation, innate immune response, lung intravital microscopy, alveolar macrophage activity, neutrophil recruitment
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 5. April 2023
• 1. Bericht­erstatter:in: Rehberg, Markus