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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

Survival curves of B16 melanoma-bearing mice after CpG/ CpG-construct treatment.Injection of 1x10^6 B16 melanoma cells s.c., repetitive treatment (daily) with CpG/ CpG-scrambled RNA/ CpG-siRNA A20. (A) Treatment started on day 7 until end of experiment on day 14 (tumor diameter ≥10 mm). (B) Treatment started on day 4 until day 10. Shown is percentage of surviving mice till the end of the experiment (tumor diameter ≥10 mm).
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pone.0135444.g009: Survival curves of B16 melanoma-bearing mice after CpG/ CpG-construct treatment.Injection of 1x10^6 B16 melanoma cells s.c., repetitive treatment (daily) with CpG/ CpG-scrambled RNA/ CpG-siRNA A20. (A) Treatment started on day 7 until end of experiment on day 14 (tumor diameter ≥10 mm). (B) Treatment started on day 4 until day 10. Shown is percentage of surviving mice till the end of the experiment (tumor diameter ≥10 mm).

Mentions: B16 melanoma cells developed aggressive tumors, which invariably killed the animals in the absence of treatment. The stimulation and activation of the innate and adaptive immune response in response to application of CpG or CpG-siRNA constructs resulted in less aggressive growth of B16 melanoma tumors. In the first set of experiments, administration of ODN-constructs was started 7 days after B16 cells had been inoculated. At this time point the tumors had reached approximately 5 mm in diameter. With this treatment schedule a slight inhibition of tumor growth could be achieved in mice injected with CpG-siA20 constructs (CpG-siA20_5, CpG-siA20_6). Mice from these two groups survived 3 to 4 days longer than the control animals until the tumor reached the limiting size (Fig 8A). Next, we modified the ODNs administration schedule: the oligonucleotides were injected earlier, 4 days after inoculation of the tumors. This modification further improved the survival of the mice, which received siA20-specific conjugates. Now tumor growth was markedly inhibited (S3 Fig) and in one group, CpG-siA20_5, half of the animals survived until day 18 on which they were sacrifice due to the tumor diameter approaching 10 mm (Fig 8B). The analysis of tumor growth curves at different treatment conditions revealed that the average tumor diameter remained smaller in animals treated with CpG-siA20 compared to PBS or CpG/CpG-siSCR(Fig 9). The most prominent effects were observed after doubling the dose of injected conjugates (Fig 9C). This trend failed to reach statistical significance when experiments were analyzed separately. However, integrated results obtained from all three presented experiments at the final phase of the treatment, indicated significantly smaller tumor diameter in A20-knockdown groups compared to CpG alone. In contrast, the scrambled CpG-siSCR conjugate and CpG treated tumors progressed with similar speed (Fig 9D).


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)

Survival curves of B16 melanoma-bearing mice after CpG/ CpG-construct treatment.Injection of 1x10^6 B16 melanoma cells s.c., repetitive treatment (daily) with CpG/ CpG-scrambled RNA/ CpG-siRNA A20. (A) Treatment started on day 7 until end of experiment on day 14 (tumor diameter ≥10 mm). (B) Treatment started on day 4 until day 10. Shown is percentage of surviving mice till the end of the experiment (tumor diameter ≥10 mm).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556692&req=5

pone.0135444.g009: Survival curves of B16 melanoma-bearing mice after CpG/ CpG-construct treatment.Injection of 1x10^6 B16 melanoma cells s.c., repetitive treatment (daily) with CpG/ CpG-scrambled RNA/ CpG-siRNA A20. (A) Treatment started on day 7 until end of experiment on day 14 (tumor diameter ≥10 mm). (B) Treatment started on day 4 until day 10. Shown is percentage of surviving mice till the end of the experiment (tumor diameter ≥10 mm).
Mentions: B16 melanoma cells developed aggressive tumors, which invariably killed the animals in the absence of treatment. The stimulation and activation of the innate and adaptive immune response in response to application of CpG or CpG-siRNA constructs resulted in less aggressive growth of B16 melanoma tumors. In the first set of experiments, administration of ODN-constructs was started 7 days after B16 cells had been inoculated. At this time point the tumors had reached approximately 5 mm in diameter. With this treatment schedule a slight inhibition of tumor growth could be achieved in mice injected with CpG-siA20 constructs (CpG-siA20_5, CpG-siA20_6). Mice from these two groups survived 3 to 4 days longer than the control animals until the tumor reached the limiting size (Fig 8A). Next, we modified the ODNs administration schedule: the oligonucleotides were injected earlier, 4 days after inoculation of the tumors. This modification further improved the survival of the mice, which received siA20-specific conjugates. Now tumor growth was markedly inhibited (S3 Fig) and in one group, CpG-siA20_5, half of the animals survived until day 18 on which they were sacrifice due to the tumor diameter approaching 10 mm (Fig 8B). The analysis of tumor growth curves at different treatment conditions revealed that the average tumor diameter remained smaller in animals treated with CpG-siA20 compared to PBS or CpG/CpG-siSCR(Fig 9). The most prominent effects were observed after doubling the dose of injected conjugates (Fig 9C). This trend failed to reach statistical significance when experiments were analyzed separately. However, integrated results obtained from all three presented experiments at the final phase of the treatment, indicated significantly smaller tumor diameter in A20-knockdown groups compared to CpG alone. In contrast, the scrambled CpG-siSCR conjugate and CpG treated tumors progressed with similar speed (Fig 9D).

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