Bacterial Infection-Mimicking Three-Dimensional Phagocytosis and Chemotaxis in Electrospun Poly(ε-caprolactone) Nanofibrous Membrane.

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.

Inotodiol From Inonotus obliquus Chaga Mushroom Induces Atypical Maturation in Dendritic Cells.

Dendritic cells (DCs) have the ability to stimulate naïve T cells that coordinate subsequent adaptive response toward an inflammatory response or tolerance depending on the DC differentiation level. Inotodiol, a lanostane triterpenoid found in Inonotus obliquus (wild Chaga mushroom), is a natural compound with a wide range of biological activities. In this study, we investigated whether inotodiol promotes the maturation of bone marrow-derived DCs (BMDCs) and inotodiol-treated BMDCs induce T cell activation. Inotodiol increased the expression of surface maturation markers, including MHC-I, MHC-II, CD86, and CD40, on BMDCs without affecting the production of various cytokines, including TNF-α and IL-12p40 in these cells. T cells primed with inotodiol-treated BMDCs proliferated and produced IL-2, without producing other cytokines, including IL-12p40 and IFN-γ. Injection of inotodiol into mice induced maturation of splenic DCs and IL-2 production, and the administration of inotodiol and inotodiol-treated BMDCs induced the proliferation of adoptively transferred CD8+ T cells in vivo. The phosphatidylinositol-3-kinase inhibitor wortmannin abrogated the upregulation of Akt phosphorylation and CD86 and MHC-II expression induced by inotodiol. However, inotodiol failed to induce phosphorylation of the IκB kinase and degradation of IκB-α, and increased expression of CD86 induced by inotodiol was not blocked by an IκB kinase inhibitor. These results suggest that inotodiol induces a characteristic type of maturation in DCs through phosphatidylinositol-3-kinase activation independent of NF-κB, and inotodiol-treated DCs enhance T cell proliferation and IL-2 secretion.

Co-Culturing of Endothelial and Cancer Cells in a Nanofibrous Scaffold-Based Two-Layer System.

Angiogenesis is critical for local tumor growth. This study aimed to develop a three-dimensional two-layer co-culture system to investigate effects of cancer cells on the growth of endothelial cells (ECs). Poly(ε-caprolactone) (PCL) nanofibrous membranes were generated via electrospinning of PCL in chloroform (C-PCL-M) and chloroform and dimethylformamide (C/DMF-PCL-M). We assembled a two-layer co-culture system using C-PCL-M and C/DMF-PCL-M for EC growth in the upper layer with co-cultured cancer cells in the lower layer. In the absence of vascular endothelial growth factor (VEGF), growth of bEND.3 ECs decreased on C/DMF-PCL-M but not on C-PCL-M with time. Growth of bEND.3 cells on C/DMF-PCL-M was enhanced through co-culturing of CT26 cancer cells and enhanced growth of bEND.3 cells was abrogated with anti-VEGF antibodies and sorafenib. However, EA.hy926 ECs displayed steady growth and proliferation on C/DMF-PCL-M, and their growth was not further increased through co-culturing of cancer cells. Moreover, chemical hypoxia in CT26 cancer cells upon treatment with CoCl2 enhanced the growth of co-cultured bEND.3 cells in the two-layer system. Thus, EC growth on the nanofibrous scaffold is dependent on the types of ECs and composition of nanofibers and this co-culture system can be used to analyze EC growth induced by cancer cells.