Samples were analyzed using a Zeiss epifluorescence photomicroscope (Zeiss, Jena, Germany) and a set of 200 cells was examined for the presence of S. pneumoniae. In addition, the percentage of cells with associated bacteria (adhered or internalized) was calculated as follows: number of infected cells/200 cells × 100. Confocal microscopy Cells were seeded at a density of 1.2 × 106 cells/ml in DMEM F-12 medium plus 10% FCS on poly-L-lysine plus laminin-coated glass coverslips for 30 min VX-689 chemical structure at 37°C and mounted in N-propylgallate (Sigma) in PBS-glycerol. The samples were placed under a Leica TCS SP5 confocal microscope (Leica Microsystems, Heidelberg, Germany) and all images were acquired
with a 63X glycerol selleck chemicals llc immersion objective lens. Image treatment was performed using the Image Processing Leica Confocal and ImageJ Software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). The three-dimensional sections perpendicular to the plane of the monolayer and parallel to the x or y axis were reconstructed using Leica Application Suite Advanced Fluorescence (LAS AF) software. Statistical analysis Statistical analyses of the data from assays of competition and of cell/bacteria association were performed with One-way ANOVA followed by the Tukey test for multiple comparisons. In case of single comparisons, the Student t test was applied. P values
equal to or less than 0.05 were considered statistically significant. Results and discussion The present study is focused on the interaction between S. pneumoniae, a major agent of bacterial meningitis, and glial cells, which are currently considered as part of the innate immune system, forming a first
line of defense against infections of the nervous system. We used a model of infection of glial cells by S. pneumoniae. This model was AZD1390 research buy improved Protein kinase N1 during previous studies by our group, which showed that the bacterial load and time course of infection are crucial in this in vitro model . Recent studies have shown that glial cells are highly reactive to pathogens, through regulating inflammation, and participating in innate and adaptive immunity [5,31–34]. In the specific case of SCs, it has been shown that, similarly to microglia in the brain, they may act as sentinel cells in the PNS and thus orchestrate the induction of a host defense response [35,8]. Recent data from our group indicate that SCs from the rat sciatic nerve and a human SC line (ST88-14) express MR in a functional state capable of internalizing mannosylated ligand [20,7]. We also have previously shown that cells egress from sciatic nerve explant cultures treated with IFN-γ, MHC class II staining colocalized with internalized neoglycoprotein in perinuclear areas of cells phenotypically identified as SC . These findings are consistent with a possible role of SC in the clearance of DAMPs and PAMPs, acting as facultative antigen-presenting cells during inflammation.