Internalization of mRNA lipoplexes by dendritic cells.
Lipoplexes, composed of Lipofectamine and mRNA encoding HIV Gag protein, were shown to be internalized by dendritic cells (DCs) and promote antigen presentation to stimulate HIV-specific T cell responses. Using confocal microscopy, we showed that one-third of fluorescently labeled mRNA containing lipoplexes are colocalized with late endosomes. We further investigated the effect of inhibitors, blocking phagocytosis, macropinocytosis, and clathrin- and caveolae-mediated endocytosis, on both the internalization of the lipoplexes by DCs and the transfection efficiency. We observed that chloropromazine had no effect on the cellular uptake or transfection efficiency, excluding the involvement of clathrin-mediated endocytosis. Cytochalasin D, inhibiting macropinocytosis and phagocystosis, strongly reduced internalization (50%) of the lipoplexes as well as protein expression (70%). Amiloride, which should specifically block macropinocytosis, induced only a modest reduction of uptake and transfection. Genistein and dynasore induced a strong reduction of on the level of protein expression (>70%), but not the overall uptake. Our results indicate that transfection-effective mRNA lipoplex internalization by DCs, i.e., uptake that results in protein expression, preferentially proceeds by macropinocytosis and/or phagocytosis.
Virology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine of Antwerp, Nationalestraat 155, Antwerp, Belgium. wdehaes@itg.be
Winni
W
De Haes
Greet
G
Van Mol
Céline
C
Merlin
Stefaan C
SC
De Smedt
Guido
G
Vanham
Joanna
J
Rejman
D021941:Caveolae / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D003713:Dendritic Cells / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008055:Lipids / Q000008:administration & dosage* / Q000493:pharmacokinetics*; D010587:Phagocytosis / Q000502:physiology; D010873:Pinocytosis / Q000502:physiology; D012333:RNA, Messenger / Q000008:administration & dosage* / Q000493:pharmacokinetics*; D014162:Transfection / Q000379:methods*
D002966:Clathrin; D008055:Lipids; C086724:Lipofectamine; D012333:RNA, Messenger