title: Commercial cow milk contains physically stable extracellular vesicles expressing immunoregulatory TGF-β.
abstract: METHODS
  Extracellular vesicles, including exosomes, have been identified in all biological fluids and rediscovered as an important part of the intercellular communication. Breast milk also contains extracellular vesicles and the proposed biological function is to enhance the antimicrobial defense in newborns. It is, however, unknown whether extracellular vesicles are still present in commercial milk and, more importantly, whether they retained their bioactivity. Here, we characterize the extracellular vesicles present in semi-skimmed cow milk available for consumers and study their effect on T cells.
  
  
  RESULTS
  Extracellular vesicles from commercial milk were isolated and characterized. Milk-derived extracellular vesicles contained several immunomodulating miRNAs and membrane protein CD63, characteristics of exosomes. In contrast to RAW 267.4 derived extracellular vesicles the milk-derived extracellular vesicles were extremely stable under degrading conditions, including low pH, boiling and freezing. Milk-derived extracellular vesicles were easily taken up by murine macrophages in vitro. Furthermore, we found that they can facilitate T cell differentiation towards the pathogenic Th17 lineage. Using a (CAGA)12-luc reporter assay we showed that these extracellular vesicles carried bioactive TGF-β, and that anti-TGF-β antibodies blocked Th17 differentiation.
  
  
  CONCLUSIONS
  Our findings show that commercial milk contains stable extracellular vesicles, including exosomes, and carry immunoregulatory cargo. These data suggest that the extracellular vesicles present in commercial cow milk remains intact in the gastrointestinal tract and exert an immunoregulatory effect.
authors.affiliation: Experimental Rheumatology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Bartijn C H
authors.initials: BC
authors.lastname: Pieters
authors.affiliation: Experimental Rheumatology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Onno J
authors.initials: OJ
authors.lastname: Arntz
authors.affiliation: Experimental Rheumatology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Miranda B
authors.initials: MB
authors.lastname: Bennink
authors.affiliation: Experimental Rheumatology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Mathijs G A
authors.initials: MG
authors.lastname: Broeren
authors.affiliation: Experimental Rheumatology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Arjan P M
authors.initials: AP
authors.lastname: van Caam
authors.affiliation: Experimental Rheumatology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Marije I
authors.initials: MI
authors.lastname: Koenders
authors.affiliation: Experimental Rheumatology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Peter L E M
authors.initials: PL
authors.lastname: van Lent
authors.affiliation: Experimental Rheumatology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Wim B
authors.initials: WB
authors.lastname: van den Berg
authors.affiliation: Experimental Rheumatology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Marieke
authors.initials: M
authors.lastname: de Vries
authors.affiliation: Experimental Rheumatology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Peter M
authors.initials: PM
authors.lastname: van der Kraan
authors.affiliation: Experimental Rheumatology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Fons A J
authors.initials: FA
authors.lastname: van de Loo
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D002417:Cattle; D002454:Cell Differentiation / Q000276:immunology; D003612:Dairying / Q000592:standards*; D000067128:Extracellular Vesicles / Q000378:metabolism*; D005260:Female; D008156:Luciferases; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008892:Milk / Q000737:chemistry* / Q000276:immunology*; D053758:Nanoparticles; D060888:Real-Time Polymerase Chain Reaction; D018709:Statistics, Nonparametric; D060149:Tetraspanin 30 / Q000378:metabolism; D058504:Th17 Cells / Q000276:immunology*; D016212:Transforming Growth Factor beta / Q000276:immunology / Q000378:metabolism*
chemical_list: D000906:Antibodies; D060149:Tetraspanin 30; D016212:Transforming Growth Factor beta; D008156:Luciferases
references.citation: Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14751-6
references.pmid: 20679229
references.citation: Int Immunol. 2005 Jul;17(7):879-87
references.pmid: 15908444
references.citation: J Immunol. 2002 Apr 1;168(7):3235-41
references.pmid: 11907077
references.citation: PLoS One. 2013 Jul 02;8(7):e67554
references.pmid: 23844026
references.citation: Early Hum Dev. 2004 Apr;77(1-2):67-75
references.pmid: 15113633
references.citation: Annu Rev Cell Dev Biol. 2007;23:175-205
references.pmid: 17506695
references.citation: J Transl Med. 2011 Jan 14;9:9
references.pmid: 21235781
references.citation: PLoS One. 2008;3(11):e3694
references.pmid: 19002258
references.citation: Nutr J. 2013 Jul 25;12:103
references.pmid: 23883112
references.citation: Arthritis Res Ther. 2005;7(1):29-37
references.pmid: 15642151
references.citation: Nat Cell Biol. 2008 Dec;10(12):1470-6
references.pmid: 19011622
references.citation: J Nutr. 2005 Jan;135(1):1-4
references.pmid: 15623823
references.citation: Nature. 2006 May 11;441(7090):235-8
references.pmid: 16648838
references.citation: Immunity. 2006 Feb;24(2):179-89
references.pmid: 16473830
references.citation: Arthritis Rheum. 2009 Jun;60(6):1647-56
references.pmid: 19479869
references.citation: RNA Biol. 2013 Jul;10(7):1080-6
references.pmid: 23770773
references.citation: PLoS One. 2012;7(3):e30679
references.pmid: 22427800
references.citation: Immunol Cell Biol. 2000 Feb;78(1):74-9
references.pmid: 10651932
references.citation: Nat Rev Immunol. 2002 Aug;2(8):569-79
references.pmid: 12154376
references.citation: F1000 Biol Rep. 2011;3:15
references.pmid: 21876726
references.citation: Nat Med. 2008 Feb;14(2):170-5
references.pmid: 18223654
references.citation: J Endocrinol. 1996 Oct;151(1):77-86
references.pmid: 8943771
references.citation: Nat Med. 2001 Mar;7(3):297-303
references.pmid: 11231627
references.citation: J Pediatr. 2010 Feb;156(2 Suppl):S21-5
references.pmid: 20105660
references.citation: Am J Pathol. 2008 Jan;172(1):146-55
references.pmid: 18156204
references.citation: Cell Res. 2012 Mar;22(3):607-10
references.pmid: 22157651
references.citation: Gastroenterology. 2001 Aug;121(2):337-49
references.pmid: 11487543
references.citation: Cell Res. 2012 Jan;22(1):107-26
references.pmid: 21931358
references.citation: Silence. 2010 Mar 01;1(1):7
references.pmid: 20226005
references.citation: Nat Med. 1998 May;4(5):594-600
references.pmid: 9585234
references.citation: Biochem Biophys Res Commun. 2010 May 28;396(2):528-33
references.pmid: 20434431
references.citation: Cancer Res. 2007 Aug 1;67(15):7458-66
references.pmid: 17671216
references.citation: Curr Protoc Cell Biol. 2006 Apr;Chapter 3:Unit 3.22
references.pmid: 18228490
references.citation: Cell Calcium. 2005 Jul;38(1):53-7
references.pmid: 15936814
references.citation: EMBO J. 1998 Jun 1;17(11):3091-100
references.pmid: 9606191
references.citation: J Immunol. 2009 Jun 15;182(12):7846-54
references.pmid: 19494309
references.citation: Nature. 2006 May 11;441(7090):231-4
references.pmid: 16648837
references.citation: J Immunol. 2007 Aug 1;179(3):1969-78
references.pmid: 17641064
references.citation: Nat Cell Biol. 2007 Jun;9(6):654-9
references.pmid: 17486113
references.citation: Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6328-33
references.pmid: 20304794
references.citation: Nanomedicine. 2011 Dec;7(6):780-8
references.pmid: 21601655
references.citation: Clin Exp Immunol. 1993 Oct;94(1):220-4
references.pmid: 8403511
references.citation: Am J Clin Nutr. 2003 Jun;77(6):1537S-1543S
references.pmid: 12812151
references.citation: J Dairy Sci. 2012 Sep;95(9):4831-4841
references.pmid: 22916887
references.citation: J Nutr. 2009 Aug;139(8):1463-8
references.pmid: 19515744
references.citation: J Immunol. 2003 May 1;170(9):4665-72
references.pmid: 12707345
references.citation: Int J Biol Sci. 2012;8(1):118-23
references.pmid: 22211110
references.citation: Nat Rev Immunol. 2009 Aug;9(8):581-93
references.pmid: 19498381
references.citation: Nat Biotechnol. 2013 Nov;31(11):965-7
references.pmid: 24213763
references.citation: J Exp Med. 2005 Apr 4;201(7):1061-7
references.pmid: 15809351
references.citation: J Exp Med. 2003 Dec 15;198(12):1875-86
references.pmid: 14676299
references.citation: Traffic. 2010 May;11(5):675-87
references.pmid: 20136776
references.citation: J Extracell Vesicles. 2013 Jun 18;2:
references.pmid: 24009888
references.citation: J Exp Med. 2001 Aug 20;194(4):519-27
references.pmid: 11514607
references.citation: J Exp Med. 1996 Mar 1;183(3):1161-72
references.pmid: 8642258
references.citation: Gynecol Oncol. 2007 Dec;107(3):563-71
references.pmid: 17900673
references.citation: J Nutr. 2009 Jan;139(1):69-75
references.pmid: 19056655
references.citation: Proc Natl Acad Sci U S A. 2004 Sep 7;101(36):13368-73
references.pmid: 15326289