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. 2017 Aug 30;7(1):9954.
doi: 10.1038/s41598-017-10449-0.

Immune activated monocyte exosomes alter microRNAs in brain endothelial cells and initiate an inflammatory response through the TLR4/MyD88 pathway

Pranjali Dalvi  1 , Bing Sun  1 , Norina Tang  1 , Lynn Pulliam  2   3
Affiliations

Immune activated monocyte exosomes alter microRNAs in brain endothelial cells and initiate an inflammatory response through the TLR4/MyD88 pathway

Pranjali Dalvi et al. Sci Rep. .

Abstract

The host immune response is critical for homeostasis; however, when chronic low level activation of the immune response with or without the driver continues, a cascade of events can trigger immunological dysfunction. Monocytes are key peripheral sensors of the immune response and their activation is instrumental in the development of cognitive impairment. Here, we show that monocytes activated by interferon alpha, lipopolysaccharide or a combination of both generate exosomes carrying significantly altered microRNA profiles compared to non-activated monocytes. These exosomes alone can activate human brain microvascular endothelial cells to stimulate adhesion molecules, CCL2, ICAM1, VCAM1 and cytokines, IL1β and IL6. This activation is through the toll like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88) pathway that activates nuclear factor-κB and increases monocyte chemotaxis. Inhibition of monocyte exosome release reverses endothelial cell activation and monocyte chemotaxis. Our study suggests that activated monocytes have an impact on brain vascular function through intercellular exosome signaling.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Exosomes from LPS and I/L activated monocytes increase migration towards brain endothelial cells. (A) HBMECs receiving exosomes from calcein AM dye (green) stained, nonstimulated human monocytes in the upper-well (left panel). Monocytes were treated or not with the exosome inhibitor GW4869 (EXOi) (right panel). Scale bar: 50 µm. Representative picture of triplicates. (B) Migration of activated monocytes toward HBMECs was quantified and compared to that of monocytes treated with EXOi (n = 6). Shaded boxes indicate range of the data, horizontal bars indicate mean. Two-sided paired Student’s t tests with multiple comparison correction were used.
Figure 2
Figure 2
Increase in brain endothelial cell activation is due to monocyte derived exosomes. (A) HBMECs were cocultured with exosomes derived from IFNα, LPS or I/L activated monocytes. Selected genes were analyzed by real time qPCR (n = 3). (B) I/L activated monocytes were incubated with or without exosome inhibitor (EXOi) and cocultured with HBMECs in a cell culture insert. qPCR was performed on HBMECs after 3 h (n = 3), nonstimulated (NS) and I/L are the same samples as in Fig. 2A. (C) ELISA from conditioned media of HBMECs (n = 5 or 6) cocultured with exosomes derived from NS, IFNα, LPS or I/L activated monocytes. (D) Western blot of HBMECs exposed to exosomes from NS, IFNα, LPS or I/L activated monocytes. The blots are a representative of four experiments. The bar graph shows the average densitometry analysis using ImageJ software (n = 4). The shaded boxes in (A) and (C) represent the range and the horizontal bars in each box is the mean. Quantitation data in (D) are presented as mean ± s.d. Two-sided paired Student’s t-tests with multiple comparison correction were used.
Figure 3
Figure 3
miRNAs are significantly modulated by IFNα and LPS stimulation of monocytes. (A) miRNA arrays were performed on normal human monocytes isolated from blood and stimulated with IFNα, LPS or both (I/L) (n = 3). Differentially expressed genes between groups are shown. Full heatmap is shown in supplemental Figure S1. (B) Heatmap of selected miRNA expressions from monocyte derived exosomes (n = 3) using real time qPCR. (C) Venn diagram representing differentially up or down regulated monocyte miRNAs overlapping between the groups as shown in (A). Red circles show selected miRNAs of interest.
Figure 4
Figure 4
Monocyte derived exosomes transfer functional miRNAs to HBMECs. (A) HBMECs were co-cultured with exosomes derived from IFNα, LPS or I/L activated monocytes. HBMECs were analyzed by real time qPCR for selected miRNAs. The first treatment group in every graph represents HBMECs without exosomes. The second treatment group represents HBMECs treated with nonstimulated exosomes. Each dot represents the mean of technical triplicates. The shaded boxes represent the range and the line in each box is the mean of the group. (B) I/L stimulated monocytes were incubated with HBMECs in the presence or not of exosome inhibitor (EXOi) in the upper-well of a dual chamber cell culture system. HBMECs were analyzed by real time qPCR for selected miRNAs. NS, nonstimulated. Experiments were performed in triplicate for each of 3 different blood donors. Data in (B) are presented as mean ± s.d. Two-sided paired Student’s t-tests with multiple comparison correction were used.
Figure 5
Figure 5
Inhibition of TLR4 reduces exosome mediated adhesion molecules/cytokines and miRNAs in a dose dependent manner and normalizes differential miRNA transcription. IFNα and/or LPS treated monocyte exosomes were added to HBMECs followed by representative western blots for (A) TLR4 and (B) MyD88. Graphs represent densitometry analysis (n = 3). (C and D) HBMECs were pretreated with various doses of TLR4 inhibitor, TAK-242. Nonstimulated (NS), LPS or IFNα and LPS (I/L) treated monocyte exosomes were added to HBMECs for 24 h. qPCR was performed in triplicate for each of 4 different blood donors. Each dot represents the mean of the triplicate. The shaded box represents the range and the horizontal line is the mean of the group. Two-sided paired Student’s t tests with multiple comparison correction were used. For repeated measures, Page’s trend tests showed all gene expressions had a decreasing trend with increasing dose of TAK-242 in both LPS and I/L exosome treated HBMECs (P < 0.01 for all genes, except IL1B and IL6 with I/L-exosome treatment were P < 0.05).
Figure 6
Figure 6
Inhibition of NF-κB reduces exosome mediated adhesion molecules/cytokines and miRNAs in a dose dependent manner. (A) HBMECs were incubated with or without exosomes derived from nonstimulated (NS) or stimulated (IFNα, LPS, I/L) monocytes, and analyzed by western blot (n = 2). The graph represents densitometry analysis for protein expression. (B and C) HBMECs were pretreated with NF-κB inhibitor PTN. Exosomes derived from nonstimulated (NS) or stimulated monocytes (LPS, I/L) were added for 24 h. qPCR was performed in triplicates for each of 4 different blood donors. Each dot represents the mean of the triplicate. The shaded box represents the range and the horizontal line is the mean of the group. Two-sided paired Student’s t tests with multiple comparison correction were used. For repeated measures, Page’s trend tests showed all gene expressions had a significant decrease with increasing PTN dose in both LPS and I/L exosome treated HBMECs (P < 0.01 for all genes, except CCL2 and VCAM1 with LPS-exosome treatment were P < 0.05).
Figure 7
Figure 7
Schematic representing the key role of IFNα and LPS-activated monocyte-derived exosomes in brain endothelial stimulation. We propose that IFNα and LPS together cause significant changes in the monocyte exosome cargo, specifically miRNAs. These exosomes are taken up by the brain endothelial cells leading to damage in the form of abnormal upregulation of adhesion molecules, chemoattractants and pro-inflammatory cytokines. This mechanism is regulated by the TLR4/NF-κB pathway and involves dysregulated epigenetic operation by miRNAs known to be involved in these pathways. These miRNAs are constitutively expressed in endothelial cells (left panel). Stimulation of monocytes by IFNα and/or LPS changes the miRNA content within the exosomes and thereby the recipient endothelial cells (middle). Inhibiting the release of exosomes or TLR4 or NF-κB reverse this effect (right).
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