title: Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages.
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.
  
  
  METHODS
  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.
authors.affiliation: Krefting Research Centre, Sahlgrenska Academy, University of Gothenburg, Box 424, 405 30 Gothenburg, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Lässer
authors.forename: Vesta Seyed
authors.initials: VS
authors.lastname: Alikhani
authors.forename: Karin
authors.initials: K
authors.lastname: Ekström
authors.forename: Maria
authors.initials: M
authors.lastname: Eldh
authors.forename: Patricia Torregrosa
authors.initials: PT
authors.lastname: Paredes
authors.forename: Apostolos
authors.initials: A
authors.lastname: Bossios
authors.forename: Margareta
authors.initials: M
authors.lastname: Sjöstrand
authors.forename: Susanne
authors.initials: S
authors.lastname: Gabrielsson
authors.forename: Jan
authors.initials: J
authors.lastname: Lötvall
authors.forename: Hadi
authors.initials: H
authors.lastname: Valadi
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D037281:Calnexin / Q000378:metabolism; D055354:Exosomes / Q000737:chemistry / Q000378:metabolism*; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000378:metabolism*; D008895:Milk, Human / Q000737:chemistry / Q000378:metabolism*; D010949:Plasma / Q000737:chemistry / Q000378:metabolism*; D012313:RNA / Q000032:analysis / Q000378:metabolism*; D012463:Saliva / Q000737:chemistry / Q000378:metabolism*; D060225:Tetraspanin 28
chemical_list: D015703:Antigens, CD; C554798:CD81 protein, human; D050883:HSC70 Heat-Shock Proteins; D060225:Tetraspanin 28; D037281:Calnexin; D012313:RNA