Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Abstract

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform. In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AMɸ) predominate in the lung parenchyma. AMɸ are highly efficient in clearing the lungs of microbial pathogens and cell debris. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response. AMɸ also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime naïve T cells. A relatively pure population of AMɸ (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AMɸ harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AMɸ from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Differential cellular responses in healthy mice and in mice with established airway inflammation when exposed to hematite nanoparticles

“…The results showed that the number of eosinophil in the lung tissue did not increase in sensitized mice exposed to hematite NPs, arguing against a significant cellular translocation to the interstitial compartments. Regarding the second alternative, cellular migration from the airways to the LDLN, it has been well described that both macrophages and lymphocytes may migrate to this compartment (Kirby et al, 2009;Chavez-Santoscoy et al, 2012;Caucheteux et al, 2013;Pabst and Binns, 1995). However, we consider this to be unlikely in this study since the total cell number in the lymph nodes was reduced, similarly to the cellular reduction observed in the BALF.…”

Effect of anti-podoplanin antibody administration during lipopolysaccharide-induced lung injury in mice

“… 8 Therefore, murine AMs were isolated following BAL washes and cultured in vitro for 24 hours to generate an inflammatory phenotype, as previously described. 11 Culture-induced iAMs were identified as F4/80 + CD11c + CD11b + cells using flow cytometry (online supplementary figure 1A ). Although variable, the percentage of iAMs present after 24 hours of culture did not alter significantly between conditions (P=0.113; online supplementary figure 1B ).…”

“…Alveolar macrophages were isolated post mortem via the trachea in 10× 1 mL PBS BAL washes from male or female Pdpn fl/fl VAV1cre + and floxed-only control mice, aged 8–17 weeks, as previously described. 11 Cells were cultured for 24 hours either untreated or treated with 0.1 µg/mL LPS in the presence or absence of an α-PDPN (clone 8.1.1) (10 µg/mL). Secreted cytokines and chemokines were measured in the supernatant by Fluorokine MAP Multiplex (R&D Systems, Abingdon, UK).…”

A critical role for ABC transporters in persistent lung inflammation in the development of emphysema after smoke exposure

“…Human peripheral blood monocytes (THP-1) cells (TIB-202; American Type Culture Collection, Manassas, VA, USA) were differentiated into macrophages by treatment with Phorbol 12-myristate 13-acetate (MilliporeSigma, Burlington, MA, USA) for 72 h. Mouse peritoneal macrophages were isolated from peritoneal cavities of 3% thioglycolate-injected mice 5 d after injection, during which time mice were exposed to room air or smoke (Reference Cigarette 3R4F; University of Kentucky) exposure conditions. Mouse alveolar macrophages were isolated from bronchoalveolar lavage fluid of mice exposed to 5 d of room air or smoke (31). Mouse BMDMs were obtained from LysMCreAbca1 flox/flox (ABCA1 KO) and Abca1 flox/flox (ABCA1 WT control) mice that were bred in a C57BL/6J background.…”