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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

Cellular uptake of CpG-siRNA-FITC constructs by BMDCs.BMDCs on day 7 of culturing incubated with CpG-siRNA constructs linked to FITC. Confocal images of Nuc Blue (ex 405) stained CpG-siRNA A20-FITC (A) and CpG-siRNA scrambled-FITC (B) treated BMDCs from 0, 6, 12 hours and mean fluorescent intensity of FITC in 10 cells/time point over 24 hours.
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pone.0135444.g001: Cellular uptake of CpG-siRNA-FITC constructs by BMDCs.BMDCs on day 7 of culturing incubated with CpG-siRNA constructs linked to FITC. Confocal images of Nuc Blue (ex 405) stained CpG-siRNA A20-FITC (A) and CpG-siRNA scrambled-FITC (B) treated BMDCs from 0, 6, 12 hours and mean fluorescent intensity of FITC in 10 cells/time point over 24 hours.

Mentions: First, using confocal microscopy and fluorescence labeled CpG-siA20 constructs, we confirmed cellular uptake in BMDCs in vitro. Already 1 hour after FITC–labeled constructs were added to the cell culture the strong fluorescent signals were detected inside DCs (Fig 1). The efficiency of the uptake reached its peak (90% of FITC-positive cells) after 2h of incubation and approximately 80% of cells retained the FITC signal for 24 h. The uptake efficiency was equally efficient for A20-specific (Fig 1A) and scrambled control (Fig 1B) CpG-siRNA conjugates. Compared to cells incubated with control oligonucleotides (CpG alone or CpG linked to a scrambled control siRNA), the CpG-siA20-treated BMDCs expressed A20 mRNA at initially higher levels followed by marked decrease at later time points, as determined by quantitative real-time PCR (Fig 2A). This was specific for the siRNA directed at A20, since a construct containing a scrambled siRNA sequence (CpG-scr) did not have this effect. Similar results were obtained in JAWSII dendritic cell line (Fig 2B). The elevated expression of a NF-κB target, the IκBα gene in A20-depleted cells indicated enhanced activation of the canonical NF-κB pathway and confirmed the functionality of the constructs (Fig 2A and 2B). When analyzed on protein level, A20 was initially upregulated in all samples treated with CpG or with CpG-siRNA constructs irrespective of their specificity compared to non-treated control DCs. At later time points the amount of A20 protein decreased in cells with A20-specific siRNA fused to CpG. Four hours later the A20 protein signal was again elevated above the levels observed in control cells and CpG-treated cells. Most likely this displays the consequence of the prolonged and augmented NF-κB activity due to the A20 knockdown. (Fig 3)


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)

Cellular uptake of CpG-siRNA-FITC constructs by BMDCs.BMDCs on day 7 of culturing incubated with CpG-siRNA constructs linked to FITC. Confocal images of Nuc Blue (ex 405) stained CpG-siRNA A20-FITC (A) and CpG-siRNA scrambled-FITC (B) treated BMDCs from 0, 6, 12 hours and mean fluorescent intensity of FITC in 10 cells/time point over 24 hours.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135444.g001: Cellular uptake of CpG-siRNA-FITC constructs by BMDCs.BMDCs on day 7 of culturing incubated with CpG-siRNA constructs linked to FITC. Confocal images of Nuc Blue (ex 405) stained CpG-siRNA A20-FITC (A) and CpG-siRNA scrambled-FITC (B) treated BMDCs from 0, 6, 12 hours and mean fluorescent intensity of FITC in 10 cells/time point over 24 hours.
Mentions: First, using confocal microscopy and fluorescence labeled CpG-siA20 constructs, we confirmed cellular uptake in BMDCs in vitro. Already 1 hour after FITC–labeled constructs were added to the cell culture the strong fluorescent signals were detected inside DCs (Fig 1). The efficiency of the uptake reached its peak (90% of FITC-positive cells) after 2h of incubation and approximately 80% of cells retained the FITC signal for 24 h. The uptake efficiency was equally efficient for A20-specific (Fig 1A) and scrambled control (Fig 1B) CpG-siRNA conjugates. Compared to cells incubated with control oligonucleotides (CpG alone or CpG linked to a scrambled control siRNA), the CpG-siA20-treated BMDCs expressed A20 mRNA at initially higher levels followed by marked decrease at later time points, as determined by quantitative real-time PCR (Fig 2A). This was specific for the siRNA directed at A20, since a construct containing a scrambled siRNA sequence (CpG-scr) did not have this effect. Similar results were obtained in JAWSII dendritic cell line (Fig 2B). The elevated expression of a NF-κB target, the IκBα gene in A20-depleted cells indicated enhanced activation of the canonical NF-κB pathway and confirmed the functionality of the constructs (Fig 2A and 2B). When analyzed on protein level, A20 was initially upregulated in all samples treated with CpG or with CpG-siRNA constructs irrespective of their specificity compared to non-treated control DCs. At later time points the amount of A20 protein decreased in cells with A20-specific siRNA fused to CpG. Four hours later the A20 protein signal was again elevated above the levels observed in control cells and CpG-treated cells. Most likely this displays the consequence of the prolonged and augmented NF-κB activity due to the A20 knockdown. (Fig 3)

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