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

Upregulation of TLR2, VCAM-1 and CCL2 on endothelial cells following exposure to EVs from <i>Mtb</i>-infected macrophages.

<p>Endothelial cells were left untreated or treated for 16 hrs with LPS (1μg/mL) or EVs derived from non-infected or <i>Mtb</i>-infected macrophages. <b>(A)</b> Cells were stained with FITC conjugated anti-mouse TLR2 antibody or FITC conjugated anti-mouse IgG1 antibody as an isotype control. <b>(B)</b> Cells were surface-stained with FITC-labeled rat anti-mouse VCAM1 or FITC labeled anti-rat IgG2a antibody as an isotype control. <b>(C)</b> Cells were permeabilized and stained for intracellular CCL2 using PE-conjugated anti-mouse CCL2 antibody or PE-labeled IgG as an isotype control. Gates were set to approximately 1% for isotype control and were maintained for all subsequent analysis. RC: untreated cells, RvEV: Treatment with EVs from H37Rv-infected macrophages, UnEV: Treated with EVs from non-infected macrophages. Data is representative of the protein expression from three independent experiments.</p

Read and alignment information for each sample sequenced.

<p>Roughly 76% of all high quality reads was used for differential expression analysis.</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

EV-induced gene expression in endothelial cells.

<p><b>(A)</b> Endothelial cells were treated 4hrs with 40μg/mL EVs derived from either non-infected or <i>Mtb</i>-infected macrophages or left untreated. Total RNA was sequenced for two independent biology replicates. Venn diagram indicating the number of genes whose expression was >2-fold upregulated with a false discover rate (q<0.05) for each indicated comparison. <b>(B)</b> Quantitative RT-PCR was performed for a subset of genes defined as upregulated in the sequence analysis. Endothelial cells were left untreated or treated 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. <i>Dnaja2</i> whose expression by sequence analysis did not change before and after EV treatment was selected as a negative control. Expression data was normalized to <i>gapdh</i>. RC: Untreated cells, RvEV: Treatment with EVs from H37Rv-infected macrophages, UnEV: Treated with EVs from non-infected macrophages. Graph indicates fold change of gene expression from two independent experiments +/- SD.</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

Shared pathways significantly upregulated in endothelial cells treated with EVs.

<p>Shared pathways significantly upregulated in endothelial cells treated with EVs.</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

VCF of all SNPs

SNPs from 38 members of An. gambiae and An. coluzzi

VCF of Bamako SNPs

SNPs retained and filtered for the Bamako population onl