Activation of endothelial cells by extracellular vesicles derived from <i>Mycobacterium tuberculosis</i> infected macrophages or mice

<div><p>Endothelial cells play an essential role in regulating an immune response through promoting leukocyte adhesion and cell migration and production of cytokines such as TNFα. Regulation of endothelial cell immune function is tightly regulated and recent studies suggest that extracellular vesicles (EVs) are prominently involved in this process. However, the importance of EVs in regulating endothelial activation in the context of a bacterial infection is poorly understood. To begin addressing this knowledge gap we characterized the endothelial cell response to EVs released from <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) infected macrophages. Our result showed increased macrophage migration through the monolayer when endothelial cells were pretreated with EVs isolated from <i>Mtb</i>-infected macrophages. Transcriptome analysis showed a significant upregulation of genes involved in cell adhesion and the inflammatory process in endothelial cells treated with EVs. These results were validated by quantitative PCR and flow cytometry. Pathway analysis of these differentially expressed genes indicated that several immune response-related pathways were up-regulated. Endothelial cells were also treated with EVs isolated from the serum of <i>Mtb</i>-infected mice. Interestingly, EVs isolated 14 days but not 7 or 21 days post-infection showed a similar ability to induce endothelial cell activation suggesting a change in EV function during the course of an <i>Mtb</i> infection. Immunofluorescence microscopy result indicated that NF-κB and the Type 1 interferon pathways were involved in endothelial activation by EVs. In summary, our data suggest that EVs can activate endothelial cells and thus may play an important role in modulating host immune responses during an <i>Mtb</i> infection.</p></div

CPE activates ERK and AKT signaling pathways in primary cultured rat hippocampal neurons.

<p>Top panels: Representative Western blot analyses of hippocampal neuron lysates showing p-ERK 1/2 (<b>A</b>) and p-AKT (<b>B</b>) after 0, 15, 30 and 60 min treatment with 0.4 µM CPE. Total ERK (t-ERK 1/2) and total AKT (t-AKT) were also analyzed and served as internal controls. Bottom panels: Bar graphs showing the quantification of the p-ERK 1/2 (<b>A</b>) and p-AKT (<b>B</b>) signals normalized to t-ERK 1/2 and t-AKT, respectively. Note the increase in p-ERK and p-AKT after 15, 30 and 60 min treatment with CPE. Statistical analysis for <b>A</b> and <b>B</b> by one way ANOVA with Tukey post-hoc test, n = 4, *p<0.05 when compared to untreated control cells.</p

Analysis of the endothelial cell gene expression profile following treatment with EVs isolated from <i>Mtb</i>-infected and uninfected macrophages.

<p>Total RNA was sequenced for two independent biology replicates. <b>(A)</b> Venn diagram of the genes that showed a minimum two fold up- or down-regulation in endothelial cells following treatment with EVs from <i>Mtb</i>-infected (RvEV) or uninfected (UnEV) macrophages compared to each other and to untreated cells. <b>(B)</b> Hierarchical cluster analysis of differentially expressed genes in endothelial cells treated with EVs isolated from <i>Mtb</i>-infected or uninfected macrophages. The analysis was conducted with a minimal 2-fold change compared with resting endothelial cells.</p

Purified recombinant CPE protein protects against H₂O₂-induced neurotoxicity in primary cultured rat hippocampal neurons.

<p><b>A)</b> Bar graphs showing WST activity, indicative of cell viability, of hippocampal neurons treated with and without H₂O₂. Note the reduced cell viability after H₂O₂ treatment that was significantly attenuated by the pretreatment of the neurons with 0.4 µM and 1 µM purified CPE. <b>B)</b> Bar graphs showing LDH activity, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071578#pone-0071578-g001" target="_blank">figure 1</a>, in the culture media of hippocampal neurons treated with and without H₂O₂. Note that the H₂O₂-induced cytotoxicity was significantly attenuated by the pretreatment of the neurons with 0.4 µM and 1 µM purified CPE. <b>C)</b> Bar graphs showing WST activity of hippocampal neurons treated with purified CPE. Note that cell viability increased significantly by treatment with 0.4 µM and 1 µM CPE. <b>D)</b> Photomicrographs of hippocampal neurons with and without H₂O₂ treatment stained by TUNEL (green) and DAPI (blue). Note that the number of dead cells (green) increased significantly after H₂O₂ treatment (middle, left panel) and that pretreatment with 0.4 µM CPE protected the neurons (bottom, left panel). The bar graph represents the quantification of the dead cells as a % of the total number of cells determined by the DAPI staining. 500 cells were counted in each of 6 different dishes generated from embryos from 2 rats. <b>A–C</b>, one way ANOVA with Tukey post-hoc test, n = 5, *p<0.05 when compared to control cells; ^#p<0.05 compared to H₂O₂ treated only cells. <b>D</b>, Students t test, n = 6, ***p<0.001 compared to control cells; ^##p<0.01 compared to H₂O₂ treated only cells. Bar = 100 µm.</p

Purified recombinant CPE rescues cell death in CPE knock-out mouse hippocampal neurons.

<p><b>A)</b> Photomicrographs of 2 week cultures of hippocampal neurons derived from WT and CPE-KO embryos stained by TUNEL (green) and DAPI (blue). Note the higher number of dead cells (green) in the CPE-KO culture (KO) compared to the WT culture (WT). Culturing the CPE-KO neurons in the presence of 0.4 µM CPE significantly reduced the number of dead cells (KO+CPE). <b>B)</b> The bar graph represents the quantification of the dead cells as a % of the total number of cells determined by the DAPI staining. Students t test, n = 4, *p<0.05 compared to WT neurons; ^#p<0.05 compared to KO neurons not treated with CPE. Bar = 100 µm.</p

Neuroprotection by CPE involves BCL-2 and Caspase-3.

<p><b>A)</b> Bar graph showing the quantification by qRT-PCR of <i>Bcl-2</i> mRNA in primary cultured rat hippocampal neurons after treatment with 0.4 µM CPE for 3 h. Data is normalized against 18S RNA and presented as a % compared to untreated control (Ctrl) cells, (t-test, n = 3, *p<0.05). <b>B)</b> Representative Western blot analysis of BCL-2 protein in primary cultured hippocampal neurons, pretreated with 0.4 µM CPE for 24 h and subsequently challenged or not with H₂O₂ for 24 h. Actin was also analyzed and served as an internal control for protein load. <b>C)</b> Bar graphs showing the quantification of BCL-2 protein normalized to Actin and expressed as a % compared to untreated control (Ctrl) cells. Note that CPE significantly inhibited the H₂O₂-induced decrease in BCL-2 protein in primary cultured hippocampal neurons, (t-test, n = 4, ** p<0.01 compared to Ctrl; ^# p<0.05 compared to H₂O₂ treated only cells) <b>D)</b> Western blot analysis of cleaved caspase-3 in primary cultured hippocampal neurons pretreated with 0.4 µM CPE for 24 h and subsequently challenged or not with H₂O₂ for 24 h. Note that the H₂O₂-induced activation of caspase-3 is blocked by CPE. Western blot is representative of 2 independent experiments.</p

Mechanism of neuroprotection by CPE in primary cultured hippocampal neurons.

<p>CPE signals the MEK/ERK and PI3-K/AKT signaling pathways which up-regulate the expression of the anti-apoptotic protein, BCL-2, and inhibit the caspase cascade leading to a decrease in cleaved caspase-3, the apoptotic executioner.</p

CPE containing conditioned medium protects against H₂O₂-induced cytotoxicity in primary cultured rat hippocampal neurons.

<p><b>A)</b> Western blot showing the presence of endogenous CPE in the cell lysate (CE) and conditioned medium (CM) of hippocampal neurons. No signal was obtained from the non-conditioned or fresh medium (FM). <b>B)</b> Western blot showing CPE protein in conditioned medium from primary hippocampal neurons transduced with adenovirus harboring LacZ and WT or E300Q CPE constructs. <b>C)</b> Bar graphs showing LDH activity, as an indicator of cellular cytotoxicity, in the culture media of hippocampal neurons treated with and without H₂O₂. Note the increased cytotoxicity after H₂O₂ treatment (LacZ/H₂O₂) compared to control cells (LacZ) that was significantly attenuated by the presence of either WT (CPE/H₂O₂) or E300Q (E300Q/H₂O₂) CPE. Students t test, n = 4, ***p<0.001 compared to LacZ control; ^###p<0.001, compared to LacZ/H₂O₂.</p

Treatment of SVEC4-10 cells with EVs from <i>Mtb</i>-infected macrophages induces NF-κB nuclear localization.

<p>SVEC4-10 cells were seeded in collagen-coated cover slips and incubated for three days to produce a cell monolayer. The cells were left untreated or treated for 4hrs with EVs released from non-infected or <i>Mtb</i>-infected macrophages (40μg/mL). Cells were fixed and stained with rabbit anti-mouse NF-κB antibody. FITC-conjugated goat anti rabbit was used as the secondary antibody. DAPI was used for nuclear staining. Cover slips were mounted on slides in mounting media and observed at 40x using a Nikon c2 confocal fluorescent microscope. <b>(A)</b> Representative images of the different treatment groups from two independent experiments. <b>(B)</b> Quantification of the number of cells with NF-κB nuclear localization. Approximately 100 cells in 4–5 randomly selected fields per coverslip were counted. (*) indicates a p value < 0.05 compared to RC or UnEV treatment. RC: untreated cells, RvEV: EVs from H37Rv-infected macrophages, UnEV: EVs from non-infected macrophages.</p

EVs derived from the serum of <i>Mtb</i>-infected mice can activate endothelial cells <i>ex vivo</i>.

<p><b>(A)</b> SVEC4-10 cell monolayers were left untreated or stimulated for 3 hrs with EVs derived from non-infected or <i>Mtb</i>-infected mice. CFSE-labeled mouse BMMs were added to SVEC4-10 cells. The fluorescently-labeled macrophages which migrated through the SVEC4-10 cell monolayer into the bottom of the Transwell filter were imaged. The number of BMMs in seven randomly selected fields were counted and the total number of cells for each condition defined. The data is the average of three independent mouse <i>Mtb</i> infections +SD with (*) indicating a p value < 0.05 compared to RC. <b>(B)</b> Quantitative RT-PCR was performed on endothelial cells that were left untreated or treated for 4 hours with EVs derived from uninfected or <i>Mtb</i>-infected macrophages. Total RNA was extracted followed by cDNA synthesis. Fold change of gene expression was calculated by comparative Ct method. Data is from two independent mouse <i>Mtb</i> infections. <b>(C)</b> Scatter plots of flow cytometry analysis of CCL2 expression. Endothelial cells were left untreated or treated for 16 hours with EVs derived from non-infected or <i>Mtb</i>-infected macrophages. Permeabilized cells were stained with PE-conjugated anti-mouse CCL2 antibody or PE-labeled IgG as an isotype control. Gate was set for isotype control and was maintained for all subsequent analysis. RC: untreated cells. Un-EV: serum-derived EVs from uninfected mice, D7-EV, D14-EV, D21-EV: serum derived EVs from mice infected for 7, 14 and 21 days respectively. Data is representative of the CCL2 expression from two independent experiments.</p