Supporting Information - Extracellular Vesicle - Cytokine Crosstalk in the Gene Expression

Supporting information

Supplementary Figure 1. Flow cytometry characterization of CCRF cell-derived extracellular vesicles (EVs)

In order to characterize surface molecules of CCR cell-derived EVs, we stained purified CCRF MVs with fluorochrome-conjugated antibodies. Anti-CD63-PE and anti-CD9 FITC antibodies were purchased from Sigma-Aldrich (B and C), annexin V-FITC (A) , anti-CD5 FITC (D), anti-CD71 FITC (E) and anti-CD33 PE (F) were purchased from BD Biosciences). MVs were found positive for Annexin V, CD63, CD71, CD33 and weakly positive for CD5, while they were negative for CD9 (black lines). Positive staining diminished after adding 0.01% Triton-X (gray lines), demonstrating that the positive events were indeed associated with membrane surrounded vesicles. Background staining is indicated by thin grey lines.

Supplementary Figure 2. Interaction network of genes modulated by EV treatment or TNF treatment compared with control

GeneMania analysis was used to prepare an interaction network of genes significantly differentially expressed after an EV treatment compared to the control in terms of co-expression, co-localization, genetic or physical interactions as well as shared functions or domains. Upregulation is indicated by red, downregulation by blue colour, grey diamond shapes represent functional domains, shared by proteins translated from upregulated genes. We found that genes involved in vesicular trafficking, oxidative metabolism and those connected to IL-8-like domain were differentially regulated by EVs. In addition, a combined TNF and EV treatment also changed the expression of genes connected with 7-transmembrane G-protein-coupled receptor domain.

Supplementary Figure 3. Validation of the microarray results using a CCL2 ELISA

To further validate the microarray experiments, we carried out a CCL2 and IL8 ELISA experiments. As shown on the figure, we found slightly, but not significantly elevated CCL2 levels after all three treatments of U937 cells.

Supplementary Figure 4. Testing for endogenous TNF in our EV samples

A) Measuring TNF concentration in EVs

In order to elucidate if EVs carried TNF, we performed TNF ELISA testing the concentration of the cytokine present in the conditioned medium of CCRF cells or in isolated CCRF derived MVs. We found a TNF content of isolated MVs lower than the values measured in EV-free and cell-free supernatants. Importantly, the TNF levels in all three types of samples were in the picogram/mL range which was an order of magnitude lower than the concentration of recombinant TNF used in our U937 stimulating experiments (10ng/ml).

B) Testing if TNF was adsorbed to the surface of purified MVs.

The presence of TNF receptors has been demonstrated on U937-derived MVs2. Since TNF receptors are universally present on immune cells, it is possible that they are also present on T-cell-derived MVs. We wanted to test for the possibility that MVs modify the effects of TNF due to the TNF being captured on the surface of MVs. Therefore, we prepared pure MVs derived from CCRF cells, and treated U937 cells in serum-free RPMI with pure MVs, a mixture of MVs and TNF, and ‘washed MV+TNF’. In this latter treatment, we mixed purified MPs with an appropriate amount of TNF, and after an incubation period of 30 min at 37 oC, the MVs were washed once in PBS, in order to remove unbound TNF. U937 cells were incubated for 24 hours at 37 oC in 5% CO2/air, harvested by spinning at 300 g for 5 min and washed once in PBS before RNA extraction. IL-8 Taqman assays were carried out. Results are expressed in relative gene expression (Gex) referred to HPRT (mean ± SEM, n=3). Data were analyzed by one-way ANOVA assuming non-normal distribution (Kruskal-Wallis test) with Dunn’s post test. Differences with p0.05 were considered statistically significant.

Supplementary Figure 5. GSEA Enrichment Map

Biological function was also derived from GO terms linked to genes with an altered expression. The network of individual words in enriched GO terms was plotted using the Wordcloud plugin 1. GO terms related to gene expression changes were analysed by GSEA. Based on the number of overlapping genes between individual terms, enrichment map networks were created, using Cytoscape. Node size indicates the number of genes included in the ontology. Closely linked nodes of the network were grouped and a word cloud of node names is shown as a label for each group. A: TNF treatment, B: combined treatment, C: EV treatment. As visualized by this enrichment map, there was an enrichment of many signal transduction-related terms after treatment with TNF or EVs+TNF. After a treatment where EVs were present (either combined with TNF or not), cellular respiration and mitochondrion-related terms were downregulated.

Supplementary Figure 6. List of genes associated with significantly altered probes in the microarray

The figure shows the list of genes associated with significantly altered probes in the microarray. The simplified heatmap (corresponding to the one shown in Figure 3.) is presented here with a legible list of gene names. Blue color indicates downregulation, while red color indicates upregulation. Borders between clusters, as shown on Figure 3, are also shown here.

References

1. Oesper L, Merico D, Isserlin R, Bader GD. WordCloud: a Cytoscape plugin to create a visual semantic summary of networks. Source Code Biol Med. 2011;6:Published online 2011 April 2017. doi: 2010.1186/1751-0473-2016-2017.

2. Kolowos W, Gaipl US, Sheriff A, et al. Microparticles Shed from Different Antigen-Presenting Cells Display an Individual Pattern of Surface Molecules and a Distinct Potential of Allogeneic T-Cell Activation. Scandinavian Journal of Immunology. 2005;61:226-233.

1