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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

Expression of IL-6 and TNF mRNA in CpG/ CpG-construct treated BMDCs and JAWSII cells.IL-6 and TNF mean expression measured at 0–24h by qRT-PCR (∆∆CT normalized to β-2-microglobulin) in BMDCs (A) and JAWSII dendritic cell line (B); Lower panels show integrated results of all time points calculated as areas under curves (AUC) defined by 9 time points; The results from three technical replicates of one representative experiment are shown; n.s. indicates no significant differences, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001 compared to CpG treated cells.
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pone.0135444.g004: Expression of IL-6 and TNF mRNA in CpG/ CpG-construct treated BMDCs and JAWSII cells.IL-6 and TNF mean expression measured at 0–24h by qRT-PCR (∆∆CT normalized to β-2-microglobulin) in BMDCs (A) and JAWSII dendritic cell line (B); Lower panels show integrated results of all time points calculated as areas under curves (AUC) defined by 9 time points; The results from three technical replicates of one representative experiment are shown; n.s. indicates no significant differences, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001 compared to CpG treated cells.

Mentions: In order to assess the consequences of CpG-siRNA-mediated A20 knockdown on DC function, we analyzed the mRNA levels of the pro-inflammatory cytokines IL-6 and TNF-α in BMDCs and JAWSII cells that were left untreated or incubated with CpG only or CpG-conjugates. Both in murine BMDCs (Fig 4A) and the immature murine DC line JAWSII (Fig 4B) the stronger induction of IL-6 and TNF-α expression was observed in CpG-siA20 treated cells. In BMDCs the induction of both cytokines reached its peak between 2–6h and was slightly higher than in JAWSII. Next, we verified the activity of the CpG-siA20 construct in vivo. Naïve healthy mice were treated with PBS or 1 nmol of CpG or CpG-siRNA constructs, respectively. After intraperitoneal injection, the serum levels of pro-inflammatory cytokines were measured at four time points up to 48 hours using the cytokine beads assay (CBA). As expected from former CpG studies, the assay revealed significantly higher concentrations of IL-6, TNF-α, but also IFN-γ and IL-10 in serum isolated from animals treated with CpG or with CpG-siRNA constructs compared to PBS-injected mice. Notably, in case of IL-6, TNF-α and IFN-γ the cytokine concentrations induced by the CpG-siA20 constructs were substantially higher than those triggered by treatment with CpG alone or with the CpG-scr construct indicating an A20-specific effect (Fig 5).


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)

Expression of IL-6 and TNF mRNA in CpG/ CpG-construct treated BMDCs and JAWSII cells.IL-6 and TNF mean expression measured at 0–24h by qRT-PCR (∆∆CT normalized to β-2-microglobulin) in BMDCs (A) and JAWSII dendritic cell line (B); Lower panels show integrated results of all time points calculated as areas under curves (AUC) defined by 9 time points; The results from three technical replicates of one representative experiment are shown; n.s. indicates no significant differences, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001 compared to CpG treated cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135444.g004: Expression of IL-6 and TNF mRNA in CpG/ CpG-construct treated BMDCs and JAWSII cells.IL-6 and TNF mean expression measured at 0–24h by qRT-PCR (∆∆CT normalized to β-2-microglobulin) in BMDCs (A) and JAWSII dendritic cell line (B); Lower panels show integrated results of all time points calculated as areas under curves (AUC) defined by 9 time points; The results from three technical replicates of one representative experiment are shown; n.s. indicates no significant differences, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001 compared to CpG treated cells.
Mentions: In order to assess the consequences of CpG-siRNA-mediated A20 knockdown on DC function, we analyzed the mRNA levels of the pro-inflammatory cytokines IL-6 and TNF-α in BMDCs and JAWSII cells that were left untreated or incubated with CpG only or CpG-conjugates. Both in murine BMDCs (Fig 4A) and the immature murine DC line JAWSII (Fig 4B) the stronger induction of IL-6 and TNF-α expression was observed in CpG-siA20 treated cells. In BMDCs the induction of both cytokines reached its peak between 2–6h and was slightly higher than in JAWSII. Next, we verified the activity of the CpG-siA20 construct in vivo. Naïve healthy mice were treated with PBS or 1 nmol of CpG or CpG-siRNA constructs, respectively. After intraperitoneal injection, the serum levels of pro-inflammatory cytokines were measured at four time points up to 48 hours using the cytokine beads assay (CBA). As expected from former CpG studies, the assay revealed significantly higher concentrations of IL-6, TNF-α, but also IFN-γ and IL-10 in serum isolated from animals treated with CpG or with CpG-siRNA constructs compared to PBS-injected mice. Notably, in case of IL-6, TNF-α and IFN-γ the cytokine concentrations induced by the CpG-siA20 constructs were substantially higher than those triggered by treatment with CpG alone or with the CpG-scr construct indicating an A20-specific effect (Fig 5).

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