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In Vivo Silencing of A20 via TLR9-Mediated Targeted SiRNA Delivery Potentiates Antitumor Immune Response.

Braun FC, van den Brandt J, Thomas S, Lange S, Schrank J, Gand C, Przybylski GK, Schmoeckel K, Bröker BM, Schmidt CA, Grabarczyk P - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Clinic of Internal Medicine C, Department of Molecular Hematology, University Medicine Greifswald, Greifswald, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

B16 tumor cell apoptosis after CpG/ CpG-construct treatment.Injection of 1x10^6 B16 melanoma cells s.c. followed by repetitive treatment (day 4–10) with CpG/ CpG-scrambled RNA/ CpG-siRNA A20. Apoptosis measured in tumors isolated at the end of experiment. (A) Shown are mean percentage of early (Annexin V positive) and late (7-AAD and Annexin V positive) apoptotic cells (6 mice/ group); n.s. indicates not significant, *indicates p<0.05 compared to CpG. (B) Dot plots showing percentage of apoptotic cells for one representative animal per group.
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pone.0135444.g012: B16 tumor cell apoptosis after CpG/ CpG-construct treatment.Injection of 1x10^6 B16 melanoma cells s.c. followed by repetitive treatment (day 4–10) with CpG/ CpG-scrambled RNA/ CpG-siRNA A20. Apoptosis measured in tumors isolated at the end of experiment. (A) Shown are mean percentage of early (Annexin V positive) and late (7-AAD and Annexin V positive) apoptotic cells (6 mice/ group); n.s. indicates not significant, *indicates p<0.05 compared to CpG. (B) Dot plots showing percentage of apoptotic cells for one representative animal per group.

Mentions: Since activation of the cytotoxic arm of the immune system should result in killing of infected or transformed cells, we checked the tumors for the presence of dead or dying cells using Annexin V/7-AAD staining assay. While CpG alone and CpG-scr construct did not cause significant apoptosis within the tumor, the percentage of dead cells in A20-depleted groups was substantially elevated, reaching up to 25% of all B16 cells (Fig 12).


In Vivo Silencing of A20 via TLR9-Mediated Targeted SiRNA Delivery Potentiates Antitumor Immune Response.

Braun FC, van den Brandt J, Thomas S, Lange S, Schrank J, Gand C, Przybylski GK, Schmoeckel K, Bröker BM, Schmidt CA, Grabarczyk P - PLoS ONE (2015)

B16 tumor cell apoptosis after CpG/ CpG-construct treatment.Injection of 1x10^6 B16 melanoma cells s.c. followed by repetitive treatment (day 4–10) with CpG/ CpG-scrambled RNA/ CpG-siRNA A20. Apoptosis measured in tumors isolated at the end of experiment. (A) Shown are mean percentage of early (Annexin V positive) and late (7-AAD and Annexin V positive) apoptotic cells (6 mice/ group); n.s. indicates not significant, *indicates p<0.05 compared to CpG. (B) Dot plots showing percentage of apoptotic cells for one representative animal per group.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556692&req=5

pone.0135444.g012: B16 tumor cell apoptosis after CpG/ CpG-construct treatment.Injection of 1x10^6 B16 melanoma cells s.c. followed by repetitive treatment (day 4–10) with CpG/ CpG-scrambled RNA/ CpG-siRNA A20. Apoptosis measured in tumors isolated at the end of experiment. (A) Shown are mean percentage of early (Annexin V positive) and late (7-AAD and Annexin V positive) apoptotic cells (6 mice/ group); n.s. indicates not significant, *indicates p<0.05 compared to CpG. (B) Dot plots showing percentage of apoptotic cells for one representative animal per group.
Mentions: Since activation of the cytotoxic arm of the immune system should result in killing of infected or transformed cells, we checked the tumors for the presence of dead or dying cells using Annexin V/7-AAD staining assay. While CpG alone and CpG-scr construct did not cause significant apoptosis within the tumor, the percentage of dead cells in A20-depleted groups was substantially elevated, reaching up to 25% of all B16 cells (Fig 12).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Clinic of Internal Medicine C, Department of Molecular Hematology, University Medicine Greifswald, Greifswald, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus