Abstract
In this study, we developed a three-dimensional (3D) in vitro infection model to investigate the crosstalk between phagocytes and microbes in inflammation using a nanofibrous membrane (NM). Poly(ε-caprolactone) (PCL)-NMs (PCL-NMs) were generated via electrospinning of PCL in chloroform. Staphylococcus aureus and phagocytes were able to adhere to the nanofibers and phagocytes engulfed S. aureus in the PCL-NM. The migration of phagocytes to S. aureus was evaluated in a two-layer co-culture system using PCL-NM. Neutrophils, macrophages and dendritic cells (DCs) cultured in the upper PCL-NM layer migrated to the lower PCL-NM layer containing bacteria. DCs migrated to neutrophils that cultured with bacteria and then engulfed neutrophils in two-layer system. In addition, phagocytes in the upper PCL-NM layer migrated to bacteria-infected MLE-12 lung epithelial cells in the lower PCL-NM layer. S. aureus-infected MLE-12 cells stimulated the secretion of tumor necrosis factor-α and IL-1α in 3D culture conditions, but not in 2D culture conditions. Therefore, the PCL-NM-based 3D culture system with phagocytes and bacteria mimics the inflammatory response to microbes in vivo and is applicable to the biomimetic study of various microbe infections.
Highlights
The recognition of pathogenic microorganisms is the first step of host defense
Phagocytosis of S. aureus by human neutrophils in suspension has been shown to depend on opsonization, while adherent neutrophils do readily internalize bacteria, independent of opsonization [24]
S. aureus and phagocytes adhered onto the surface of electrospun PCL nanofibers and infiltrated the PCL-nanofibrous membrane (NM), allowing 3D evaluation of phagocytosis in PCL-NM-based in vitro culture system
Summary
The recognition of pathogenic microorganisms is the first step of host defense. Components derived from bacteria, such as N-formylmethionine-leucyl-phenylalanine (fMLP), trigger chemotaxis and activation of phagocytes such as neutrophils and macrophages in infectious tissues [1]. A variety of infected host cells, such as fibroblasts, endothelial cells and epithelial cells, secrete phagocyte chemoattractants [2,3]. The assay is not appropriate for assessing migration of cells to a variety of bacteria because non-adherent bacteria grow as a suspension culture in the bottom chamber and can float, adhere, or migrate freely to the upper chamber through the permeable filter of transwell, inhibiting chemoattractant gradient formation. Huh et al developed a microfluidic lung-on-a-chip system consisting of a human alveolar cell layer and a human pulmonary microvascular endothelial cell layer separated by a porous membrane made of poly(dimethylsiloxane) (PDMS) and demonstrated that this system mimics the innate cellular response of bacteria to pulmonary infection [9]
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