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. 2011 Jan 14:9:9.
doi: 10.1186/1479-5876-9-9.

Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages

Cecilia Lässer  1 , Vesta Seyed Alikhani, Karin Ekström, Maria Eldh, Patricia Torregrosa Paredes, Apostolos Bossios, Margareta Sjöstrand, Susanne Gabrielsson, Jan Lötvall, Hadi Valadi
Affiliations

Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages

Cecilia Lässer et al. J Transl Med. .

Abstract

Background: Exosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages.

Method: Exosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy.

Results: RNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages.

Conclusions: Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.

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Figures

Figure 1
Figure 1
Exosomes from saliva, plasma and breast milk detected with electron microscopy. Exosomes from human saliva (A, B), plasma (C) and breast milk (D) were examined in the electron microscope. No isotype control antibody (A), but anti-CD63 antibody (B-D), was detected by 10 nm gold labelled secondary antibody. The scale bars represent 100 nm.
Figure 2
Figure 2
Flow cytometry detection of surface molecules on exosomes from saliva, plasma and breast milk. Exosomes from saliva, plasma and breast milk captured on anti-MHC class II beads were immunostained by using monoclonal antibodies against the tetraspanins CD9, CD63 and CD81 and analysed by flow cytometry. The antibodies (open peaks) were compared with their appropriate isotype controls (filled peaks).
Figure 3
Figure 3
Characterisation of breast milk exosomes by Western blot. The exosomal proteins from breast milk exosomes were loaded onto a 10% acrylamide gel and transferred to a nitrocellulose membrane. The breast milk exosomes are positive for Hsc70 and CD81, but negative for the endoplasmic reticulum protein, calnexin. Macrophage protein ("Cells") was used as positive control.
Figure 4
Figure 4
Exosomal RNA analysed using a Bioanalyzer. Total RNA was isolated from saliva, plasma and breast milk exosomes using Trizol® and analysed with a Bioanalyzer. The results show that exosomes from human saliva, plasma and breast milk contain a dissimilar RNA content compared to cellular RNA from HMC-1 cells, as exosomes contain little or no ribosomal RNA.
Figure 5
Figure 5
Detection of mRNA in plasma exosomes using a Bioanalyzer. The exosomal RNA was transcribed to cDNA using an oligo (dT) primer. The results show that a portion of the RNA in plasma exosomes is mRNA. Arrows show the peaks for the lower and upper markers. The peaks in between these markers indicate the presence of cDNA synthesised from plasma exosomal RNA.
Figure 6
Figure 6
Uptake of saliva and breast milk exosomes by human macrophages. 10 μg of the PKH67-labelled saliva exosomes, PKH67-labelled breast milk exosomes or a PKH67-PBS control were added per 200 000 macrophages and incubated at 37ºC or 4ºC for 2 h. The uptake of the fluorescently labelled saliva and breast milk exosomes by macrophages was detected with both flow cytometry (A and B respectively) and fluorescence microscopy (C and D respectively). The uptake was reduced at 4ºC, indicating a biologically active uptake. In the fluorescence microscopy pictures (C and D), 7-AAD was used to detect the nucleus of the macrophages (red) and PKH67 was used to label the exosomes (green). MFI data are shown as mean ± SEM for saliva exosomes n = 3 and for breast milk exosomes n = 4.
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