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.
Highlights
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
For the results presented in this work, the surface marker panel consists of antibodies against MHC II, CD86, CD40 and CIRE (CD209)
Measuring the activation of the resident phagocytic cell populations in the lungs induced by this vaccine delivery platform permits evaluation of its potential capability to promote adaptive immune responses
Summary
Resuspend the cell pellet by gently raking the centrifuge tube across the top of a test tube rack. Add 1 mL of cAMɸ medium to resuspend the cells. 4. Dilute cells to 2.5 x 105 per mL using cAMɸ medium. 6. To enrich for AMɸ, incubate culture dishes inside a 37 °C humidified incubator with a 5% CO2 atmosphere for 6 h to allow cells to adhere to the bottom of the well. 8. Incubate cells overnight inside a 37 °C humidified incubator with a 5% CO2 atmosphere prior to addition of stimulants. Incubate cells overnight inside a 37 °C humidified incubator with a 5% CO2 atmosphere prior to addition of stimulants After this enrichment step, approximately 87% of the cells are positive for the macrophage markers CD11b and F4/80 (Figure 2c)
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