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Pharmacological prion protein silencing accelerates central nervous system autoimmune disease via T cell receptor signalling.

Hu W, Nessler S, Hemmer B, Eagar TN, Kane LP, Leliveld SR, Müller-Schiffmann A, Gocke AR, Lovett-Racke A, Ben LH, Hussain RZ, Breil A, Elliott JL, Puttaparthi K, Cravens PD, Singh MP, Petsch B, Stitz L, Racke MK, Korth C, Stüve O - Brain (2010)

Bottom Line: Disruption of cellular prion protein signalling augmented antigen-specific activation and proliferation, and enhanced T cell receptor signalling, resulting in zeta-chain-associated protein-70 phosphorylation and nuclear factor of activated T cells/activator protein 1 transcriptional activity.Cellular prion protein silencing with small interfering ribonucleic acid also resulted in the worsening of actively induced and adoptively transferred experimental autoimmune encephalomyelitis.Thus, central nervous system autoimmune disease was modulated at all stages of disease: the generation of the T cell effector response, the elicitation of T effector function and the perpetuation of cellular immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Texas Southwestern Medical Center at Dallas, TX, USA.

ABSTRACT
The primary biological function of the endogenous cellular prion protein has remained unclear. We investigated its biological function in the generation of cellular immune responses using cellular prion protein gene-specific small interfering ribonucleic acid in vivo and in vitro. Our results were confirmed by blocking cellular prion protein with monovalent antibodies and by using cellular prion protein-deficient and -transgenic mice. In vivo prion protein gene-small interfering ribonucleic acid treatment effects were of limited duration, restricted to secondary lymphoid organs and resulted in a 70% reduction of cellular prion protein expression in leukocytes. Disruption of cellular prion protein signalling augmented antigen-specific activation and proliferation, and enhanced T cell receptor signalling, resulting in zeta-chain-associated protein-70 phosphorylation and nuclear factor of activated T cells/activator protein 1 transcriptional activity. In vivo prion protein gene-small interfering ribonucleic acid treatment promoted T cell differentiation towards pro-inflammatory phenotypes and increased survival of antigen-specific T cells. Cellular prion protein silencing with small interfering ribonucleic acid also resulted in the worsening of actively induced and adoptively transferred experimental autoimmune encephalomyelitis. Finally, treatment of myelin basic protein(1-11) T cell receptor transgenic mice with prion protein gene-small interfering ribonucleic acid resulted in spontaneous experimental autoimmune encephalomyelitis. Thus, central nervous system autoimmune disease was modulated at all stages of disease: the generation of the T cell effector response, the elicitation of T effector function and the perpetuation of cellular immune responses. Our findings indicate that cellular prion protein regulates T cell receptor-mediated T cell activation, differentiation and survival. Defects in autoimmunity are restricted to the immune system and not the central nervous system. Our data identify cellular prion protein as a regulator of cellular immunological homoeostasis and suggest cellular prion protein as a novel potential target for therapeutic immunomodulation.

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Decreased PrPc expression levels on peripheral leucocytes but not in the CNS in mice treated with Prnp-siRNA. (A) Transfection with Prnp-siRNA decreased PrPc expression in murine splenocytes, compared with nonsense (NS)-siRNA treatment. On Day 3 after in vivo treatment of (B) unimmunized SJL/J mice and (C) SJL/J mice immunized with PLPp139−151 with 50 μg of Prnp-siRNA, PrPc expression was decreased in splenocytes, but not in (D) brain homogenate of immunized mice. (A–D) Densitometry was performed, and relative PrPc expression was determined by normalizing the PrPc to β-actin levels. (E) Using flow cytometry three days after immunization and siRNA treatment, lymph node cells (LNC) and splenocytes were gated on CD3 and PrPc. The top two panels show the percentage of PrPc+ cells within the CD3+ cell population from nonsense-siRNA-treated murine cells. The lower two panels show the percentage of PrPc+ cells within the CD3+ cell population from Prnp-siRNA-treated murine cells. PrPc expression was decreased by ∼70% following Prnp-siRNA treatment. (F) After injecting Cy3-labelled siRNA or DY547-labelled siRNA intravenously, fluorescence was detectable 72 h later in liver and kidney tissue but not in the brain of naïve mice or mice in which EAE had been induced by active immunization (data not shown).
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Figure 1: Decreased PrPc expression levels on peripheral leucocytes but not in the CNS in mice treated with Prnp-siRNA. (A) Transfection with Prnp-siRNA decreased PrPc expression in murine splenocytes, compared with nonsense (NS)-siRNA treatment. On Day 3 after in vivo treatment of (B) unimmunized SJL/J mice and (C) SJL/J mice immunized with PLPp139−151 with 50 μg of Prnp-siRNA, PrPc expression was decreased in splenocytes, but not in (D) brain homogenate of immunized mice. (A–D) Densitometry was performed, and relative PrPc expression was determined by normalizing the PrPc to β-actin levels. (E) Using flow cytometry three days after immunization and siRNA treatment, lymph node cells (LNC) and splenocytes were gated on CD3 and PrPc. The top two panels show the percentage of PrPc+ cells within the CD3+ cell population from nonsense-siRNA-treated murine cells. The lower two panels show the percentage of PrPc+ cells within the CD3+ cell population from Prnp-siRNA-treated murine cells. PrPc expression was decreased by ∼70% following Prnp-siRNA treatment. (F) After injecting Cy3-labelled siRNA or DY547-labelled siRNA intravenously, fluorescence was detectable 72 h later in liver and kidney tissue but not in the brain of naïve mice or mice in which EAE had been induced by active immunization (data not shown).

Mentions: Prnp-specific siRNA and control NS-siRNA were generated to investigate the tissue specific role of PrPc in activating and perpetuating cellular immune responses. By western blot analyses, we demonstrated that in vitro transfection of murine splenocytes with Prnp-siRNA substantially decreased PrPc expression (Fig. 1A). On Day 3 after in vivo intravenous treatment of naïve SJL/J mice (Fig. 1B), and SJL/J mice immunized for EAE with 50 μg of Prnp-siRNA (Fig. 1C), PrPc expression was decreased in splenocytes, but not in the brain (Fig. 1D). When evaluated by flow cytometry, PrPc expression was found to be decreased by ∼70% in CD3+ T cells from Prnp-siRNA-treated mouse lymph node cells and splenocytes compared with nonsense-siRNA-treated samples (Fig. 1E). To investigate the bioavailability of Prnp-siRNA further, Cy3-labelled siRNA or DY547-labelled siRNA was injected intravenously into naïve experimental animals, or into mice in which EAE had been induced by active immunization. Fluorescence was detectable in the tissue sections of liver and kidney 72 h after injection (Fig. 1F), but not in the brain and spinal cord of naïve mice (Fig. 1F) or mice with active EAE (data not shown). Thus, Prnp-siRNA treatment created chimeric mice that expressed normal levels of PrPc in the CNS, but substantially decreased levels of PrPc in peripheral organs.Figure 1


Pharmacological prion protein silencing accelerates central nervous system autoimmune disease via T cell receptor signalling.

Hu W, Nessler S, Hemmer B, Eagar TN, Kane LP, Leliveld SR, Müller-Schiffmann A, Gocke AR, Lovett-Racke A, Ben LH, Hussain RZ, Breil A, Elliott JL, Puttaparthi K, Cravens PD, Singh MP, Petsch B, Stitz L, Racke MK, Korth C, Stüve O - Brain (2010)

Decreased PrPc expression levels on peripheral leucocytes but not in the CNS in mice treated with Prnp-siRNA. (A) Transfection with Prnp-siRNA decreased PrPc expression in murine splenocytes, compared with nonsense (NS)-siRNA treatment. On Day 3 after in vivo treatment of (B) unimmunized SJL/J mice and (C) SJL/J mice immunized with PLPp139−151 with 50 μg of Prnp-siRNA, PrPc expression was decreased in splenocytes, but not in (D) brain homogenate of immunized mice. (A–D) Densitometry was performed, and relative PrPc expression was determined by normalizing the PrPc to β-actin levels. (E) Using flow cytometry three days after immunization and siRNA treatment, lymph node cells (LNC) and splenocytes were gated on CD3 and PrPc. The top two panels show the percentage of PrPc+ cells within the CD3+ cell population from nonsense-siRNA-treated murine cells. The lower two panels show the percentage of PrPc+ cells within the CD3+ cell population from Prnp-siRNA-treated murine cells. PrPc expression was decreased by ∼70% following Prnp-siRNA treatment. (F) After injecting Cy3-labelled siRNA or DY547-labelled siRNA intravenously, fluorescence was detectable 72 h later in liver and kidney tissue but not in the brain of naïve mice or mice in which EAE had been induced by active immunization (data not shown).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Decreased PrPc expression levels on peripheral leucocytes but not in the CNS in mice treated with Prnp-siRNA. (A) Transfection with Prnp-siRNA decreased PrPc expression in murine splenocytes, compared with nonsense (NS)-siRNA treatment. On Day 3 after in vivo treatment of (B) unimmunized SJL/J mice and (C) SJL/J mice immunized with PLPp139−151 with 50 μg of Prnp-siRNA, PrPc expression was decreased in splenocytes, but not in (D) brain homogenate of immunized mice. (A–D) Densitometry was performed, and relative PrPc expression was determined by normalizing the PrPc to β-actin levels. (E) Using flow cytometry three days after immunization and siRNA treatment, lymph node cells (LNC) and splenocytes were gated on CD3 and PrPc. The top two panels show the percentage of PrPc+ cells within the CD3+ cell population from nonsense-siRNA-treated murine cells. The lower two panels show the percentage of PrPc+ cells within the CD3+ cell population from Prnp-siRNA-treated murine cells. PrPc expression was decreased by ∼70% following Prnp-siRNA treatment. (F) After injecting Cy3-labelled siRNA or DY547-labelled siRNA intravenously, fluorescence was detectable 72 h later in liver and kidney tissue but not in the brain of naïve mice or mice in which EAE had been induced by active immunization (data not shown).
Mentions: Prnp-specific siRNA and control NS-siRNA were generated to investigate the tissue specific role of PrPc in activating and perpetuating cellular immune responses. By western blot analyses, we demonstrated that in vitro transfection of murine splenocytes with Prnp-siRNA substantially decreased PrPc expression (Fig. 1A). On Day 3 after in vivo intravenous treatment of naïve SJL/J mice (Fig. 1B), and SJL/J mice immunized for EAE with 50 μg of Prnp-siRNA (Fig. 1C), PrPc expression was decreased in splenocytes, but not in the brain (Fig. 1D). When evaluated by flow cytometry, PrPc expression was found to be decreased by ∼70% in CD3+ T cells from Prnp-siRNA-treated mouse lymph node cells and splenocytes compared with nonsense-siRNA-treated samples (Fig. 1E). To investigate the bioavailability of Prnp-siRNA further, Cy3-labelled siRNA or DY547-labelled siRNA was injected intravenously into naïve experimental animals, or into mice in which EAE had been induced by active immunization. Fluorescence was detectable in the tissue sections of liver and kidney 72 h after injection (Fig. 1F), but not in the brain and spinal cord of naïve mice (Fig. 1F) or mice with active EAE (data not shown). Thus, Prnp-siRNA treatment created chimeric mice that expressed normal levels of PrPc in the CNS, but substantially decreased levels of PrPc in peripheral organs.Figure 1

Bottom Line: Disruption of cellular prion protein signalling augmented antigen-specific activation and proliferation, and enhanced T cell receptor signalling, resulting in zeta-chain-associated protein-70 phosphorylation and nuclear factor of activated T cells/activator protein 1 transcriptional activity.Cellular prion protein silencing with small interfering ribonucleic acid also resulted in the worsening of actively induced and adoptively transferred experimental autoimmune encephalomyelitis.Thus, central nervous system autoimmune disease was modulated at all stages of disease: the generation of the T cell effector response, the elicitation of T effector function and the perpetuation of cellular immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Texas Southwestern Medical Center at Dallas, TX, USA.

ABSTRACT
The primary biological function of the endogenous cellular prion protein has remained unclear. We investigated its biological function in the generation of cellular immune responses using cellular prion protein gene-specific small interfering ribonucleic acid in vivo and in vitro. Our results were confirmed by blocking cellular prion protein with monovalent antibodies and by using cellular prion protein-deficient and -transgenic mice. In vivo prion protein gene-small interfering ribonucleic acid treatment effects were of limited duration, restricted to secondary lymphoid organs and resulted in a 70% reduction of cellular prion protein expression in leukocytes. Disruption of cellular prion protein signalling augmented antigen-specific activation and proliferation, and enhanced T cell receptor signalling, resulting in zeta-chain-associated protein-70 phosphorylation and nuclear factor of activated T cells/activator protein 1 transcriptional activity. In vivo prion protein gene-small interfering ribonucleic acid treatment promoted T cell differentiation towards pro-inflammatory phenotypes and increased survival of antigen-specific T cells. Cellular prion protein silencing with small interfering ribonucleic acid also resulted in the worsening of actively induced and adoptively transferred experimental autoimmune encephalomyelitis. Finally, treatment of myelin basic protein(1-11) T cell receptor transgenic mice with prion protein gene-small interfering ribonucleic acid resulted in spontaneous experimental autoimmune encephalomyelitis. Thus, central nervous system autoimmune disease was modulated at all stages of disease: the generation of the T cell effector response, the elicitation of T effector function and the perpetuation of cellular immune responses. Our findings indicate that cellular prion protein regulates T cell receptor-mediated T cell activation, differentiation and survival. Defects in autoimmunity are restricted to the immune system and not the central nervous system. Our data identify cellular prion protein as a regulator of cellular immunological homoeostasis and suggest cellular prion protein as a novel potential target for therapeutic immunomodulation.

Show MeSH
Related in: MedlinePlus