In Vivo Silencing of A20 via TLR9-Mediated Targeted SiRNA Delivery Potentiates Antitumor Immune Response.
A20 is an ubiquitin-editing enzyme that ensures the transient nature of inflammatory signaling pathways induced by cytokines like TNF-α and IL-1 or pathogens via Toll-like receptor (TLR) pathways. It has been identified as a negative regulator of dendritic cell (DC) maturation and attenuator of their immunostimulatory properties. Ex vivo A20-depleted dendritic cells showed enhanced expression of pro-inflammatory cytokines and costimulatory molecules, which resulted in hyperactivation of tumor-infiltrating T lymphocytes and inhibition of regulatory T cells. In the present study, we demonstrate that a synthetic molecule consisting of a CpG oligonucleotide TLR9 agonist linked to A20-specific siRNAs silences its expression in TLR9+ mouse dendritic cells in vitro and in vivo. In the B16 mouse melanoma tumor model, silencing of A20 enhances the CpG-triggered induction of NFκB activity followed by elevated expression of IL-6, TNF-α and IL-12. This leads to potentiated antitumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. Our findings confirm the central role of A20 in controlling the immunostimulatory potency of DCs and provide a strategy for simultaneous A20 silencing and TLR activation in vivo.
Clinic of Internal Medicine C, Department of Molecular Hematology, University Medicine Greifswald, Greifswald, Germany.
Floriane C M
FC
Braun
Central Core & Research Facility of Laboratory Animals, University of Greifswald, Greifswald, Germany.
Jens
J
van den Brandt
Clinic of Internal Medicine C, Department of Molecular Hematology, University Medicine Greifswald, Greifswald, Germany.
Sören
S
Thomas
Clinic of Internal Medicine C, Department of Molecular Hematology, University Medicine Greifswald, Greifswald, Germany.
Sandra
S
Lange
Clinic of Internal Medicine C, Department of Molecular Hematology, University Medicine Greifswald, Greifswald, Germany.
Juliane
J
Schrank
Clinic of Internal Medicine C, Department of Molecular Hematology, University Medicine Greifswald, Greifswald, Germany.
Claudia
C
Gand
Clinic of Internal Medicine C, Department of Molecular Hematology, University Medicine Greifswald, Greifswald, Germany; Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
Grzegorz K
GK
Przybylski
Institute of Immunology and Transfusion Medicine, University of Greifswald, Greifswald, Germany.
Katrin
K
Schmoeckel
Institute of Immunology and Transfusion Medicine, University of Greifswald, Greifswald, Germany.
Barbara M
BM
Bröker
Clinic of Internal Medicine C, Department of Molecular Hematology, University Medicine Greifswald, Greifswald, Germany.
Christian A
CA
Schmidt
Clinic of Internal Medicine C, Department of Molecular Hematology, University Medicine Greifswald, Greifswald, Germany.
Piotr
P
Grabarczyk
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000187:drug effects*; D016207:Cytokines / Q000378:metabolism; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology; D005260:Female; D020868:Gene Silencing; D056724:Immunity, Humoral / Q000187:drug effects / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins / Q000187:drug effects*; D008546:Melanoma, Experimental / Q000188:drug therapy* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D058990:Molecular Targeted Therapy; D016328:NF-kappa B / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051217:Toll-Like Receptor 9 / Q000187:drug effects*; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3
D016207:Cytokines; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D034741:RNA, Small Interfering; C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D003546:Cysteine Endopeptidases; C513261:Tnfaip3 protein, mouse