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Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis.

Basler M, Mundt S, Muchamuel T, Moll C, Jiang J, Groettrup M, Kirk CJ - EMBO Mol Med (2014)

Bottom Line: The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes.Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing-remitting model.These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, Switzerland.

ABSTRACT
Multiple sclerosis (MS) is a chronic demyelinating immune mediated disease of the central nervous system. The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes. Recently, we demonstrated a novel function of immunoproteasomes in cytokine production and T cell differentiation. In this study, we investigated the therapeutic efficacy of an inhibitor of the immunoproteasome (ONX 0914) in two different mouse models of MS. ONX 0914 attenuated disease progression after active and passive induction of experimental autoimmune encephalomyelitis (EAE), both in MOG₃₅-₅₅ and PLP₁₃₉₋₁₅₁-induced EAE. Isolation of lymphocytes from the brain or spinal cord revealed a strong reduction of cytokine-producing CD4(+) cells in ONX 0914 treated mice. Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing-remitting model. An analysis of draining lymph nodes after induction of EAE revealed that the differentiation to Th17 or Th1 cells was strongly impaired in ONX 0914 treated mice. These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.

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Reduced CNS inflammations in ONX 0914 treated mice.C57BL/6 mice were immunized with MOG35–55 peptide, treated three times a week with 10 mg/kg ONX 0914 or vehicle beginning on the day of immunization and analyzed on day 19 post immunization. The experiments were performed twice ( n = 6 per group and n = 2 naïve mice), yielding similar results. * P < 0.05; ** P < 0.01; *** P < 0.001.The TNF-α, IL-23, IL-17, IL-1β, and IL-6 mRNA content in spinal cords was analyzed by real-time RT–PCR. The values were normalized to the expression of hypoxanthineguanine phosphoribosyl transferase in the same organs. Shown are the mean fold expression ± s.e.m.B Brain infiltrating CD4+ lymphocytes were restimulated in vitro with MOG35–55 peptide for 6 h and analyzed by flow cytometry after staining for CD4 and intracellular IFN-γ, IL-17, TNF-α, or GM-CSF. Shown are the percentages of IFN-γ-, IL-17-, TNF-α-, or GM-CSF-positive cells of CD4+ T cells ( y-axis) as determined by flow cytometry. Unstimulated cells (no peptide) were used as a negative control.
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fig04: Reduced CNS inflammations in ONX 0914 treated mice.C57BL/6 mice were immunized with MOG35–55 peptide, treated three times a week with 10 mg/kg ONX 0914 or vehicle beginning on the day of immunization and analyzed on day 19 post immunization. The experiments were performed twice ( n = 6 per group and n = 2 naïve mice), yielding similar results. * P < 0.05; ** P < 0.01; *** P < 0.001.The TNF-α, IL-23, IL-17, IL-1β, and IL-6 mRNA content in spinal cords was analyzed by real-time RT–PCR. The values were normalized to the expression of hypoxanthineguanine phosphoribosyl transferase in the same organs. Shown are the mean fold expression ± s.e.m.B Brain infiltrating CD4+ lymphocytes were restimulated in vitro with MOG35–55 peptide for 6 h and analyzed by flow cytometry after staining for CD4 and intracellular IFN-γ, IL-17, TNF-α, or GM-CSF. Shown are the percentages of IFN-γ-, IL-17-, TNF-α-, or GM-CSF-positive cells of CD4+ T cells ( y-axis) as determined by flow cytometry. Unstimulated cells (no peptide) were used as a negative control.

Mentions: Experimental autoimmune encephalomyelitis is characterized by the invasion of autoreactive T helper cells into the CNS, leading to inflammation and demyelination. Infiltration of immune cells into the brain and spinal cord on day 19 of MOG35–55-induced EAE was measured by flow cytometry. Inhibition of LMP7 reduced infiltration of CD4+ T helper cells, activated lymphocytes (CD45highCD11b−), and activated myeloid cells (CD45highCD11b+) (Fig 3A). The degree of inflammation in H&E stained cross-sections of the spinal cord was microscopically assessed and semiquantitatively graded from 0 to 4 blinded to sample identity (Fig 3B). In contrast to vehicle treated mice, ONX 0914 treated mice had only mild signs of inflammation. Several soluble mediators of inflammation, like proinflammatory cytokines, are elevated in EAE and play a crucial role in the course of the disease (Petermann & Korn, 2011). To investigate whether ONX 0914 treatment altered cytokine expression in mice after EAE induction, the mRNA levels for TNF-α, IL-1β, IL-6, IL-17, and IL-23 were determined by real-time RT–PCR in spinal cords on day 19 after immunization with MOG35–55 peptide (Fig 4A). Cytokines were upregulated in vehicle treated mice, whereas ONX 0914 treated mice showed significantly reduced TNF-α, IL-1β, and IL-6 mRNA production. To investigate whether autoreactive T helper cells of ONX 0914 treated mice were able to promote and sustain inflammation, we analyzed CD4+ T cells from the brains and spinal cords for their cytokine expression profile. While CD4+ cells derived from the brain of vehicle treated mice produced IFN-γ, TNF-α, IL-17, and GM-CSF upon in vitro restimulation with MOG35–55 (Fig 4B), the few CD4+ cells recovered from the brains of ONX 0914 treated mice were barely expressing these cytokines. Although some CD4+ T cells were able to invade the brain of ONX 0914 treated mice, these data suggest that LMP7 inhibition dampens the ability of these cells to produce cytokines.


Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis.

Basler M, Mundt S, Muchamuel T, Moll C, Jiang J, Groettrup M, Kirk CJ - EMBO Mol Med (2014)

Reduced CNS inflammations in ONX 0914 treated mice.C57BL/6 mice were immunized with MOG35–55 peptide, treated three times a week with 10 mg/kg ONX 0914 or vehicle beginning on the day of immunization and analyzed on day 19 post immunization. The experiments were performed twice ( n = 6 per group and n = 2 naïve mice), yielding similar results. * P < 0.05; ** P < 0.01; *** P < 0.001.The TNF-α, IL-23, IL-17, IL-1β, and IL-6 mRNA content in spinal cords was analyzed by real-time RT–PCR. The values were normalized to the expression of hypoxanthineguanine phosphoribosyl transferase in the same organs. Shown are the mean fold expression ± s.e.m.B Brain infiltrating CD4+ lymphocytes were restimulated in vitro with MOG35–55 peptide for 6 h and analyzed by flow cytometry after staining for CD4 and intracellular IFN-γ, IL-17, TNF-α, or GM-CSF. Shown are the percentages of IFN-γ-, IL-17-, TNF-α-, or GM-CSF-positive cells of CD4+ T cells ( y-axis) as determined by flow cytometry. Unstimulated cells (no peptide) were used as a negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: Reduced CNS inflammations in ONX 0914 treated mice.C57BL/6 mice were immunized with MOG35–55 peptide, treated three times a week with 10 mg/kg ONX 0914 or vehicle beginning on the day of immunization and analyzed on day 19 post immunization. The experiments were performed twice ( n = 6 per group and n = 2 naïve mice), yielding similar results. * P < 0.05; ** P < 0.01; *** P < 0.001.The TNF-α, IL-23, IL-17, IL-1β, and IL-6 mRNA content in spinal cords was analyzed by real-time RT–PCR. The values were normalized to the expression of hypoxanthineguanine phosphoribosyl transferase in the same organs. Shown are the mean fold expression ± s.e.m.B Brain infiltrating CD4+ lymphocytes were restimulated in vitro with MOG35–55 peptide for 6 h and analyzed by flow cytometry after staining for CD4 and intracellular IFN-γ, IL-17, TNF-α, or GM-CSF. Shown are the percentages of IFN-γ-, IL-17-, TNF-α-, or GM-CSF-positive cells of CD4+ T cells ( y-axis) as determined by flow cytometry. Unstimulated cells (no peptide) were used as a negative control.
Mentions: Experimental autoimmune encephalomyelitis is characterized by the invasion of autoreactive T helper cells into the CNS, leading to inflammation and demyelination. Infiltration of immune cells into the brain and spinal cord on day 19 of MOG35–55-induced EAE was measured by flow cytometry. Inhibition of LMP7 reduced infiltration of CD4+ T helper cells, activated lymphocytes (CD45highCD11b−), and activated myeloid cells (CD45highCD11b+) (Fig 3A). The degree of inflammation in H&E stained cross-sections of the spinal cord was microscopically assessed and semiquantitatively graded from 0 to 4 blinded to sample identity (Fig 3B). In contrast to vehicle treated mice, ONX 0914 treated mice had only mild signs of inflammation. Several soluble mediators of inflammation, like proinflammatory cytokines, are elevated in EAE and play a crucial role in the course of the disease (Petermann & Korn, 2011). To investigate whether ONX 0914 treatment altered cytokine expression in mice after EAE induction, the mRNA levels for TNF-α, IL-1β, IL-6, IL-17, and IL-23 were determined by real-time RT–PCR in spinal cords on day 19 after immunization with MOG35–55 peptide (Fig 4A). Cytokines were upregulated in vehicle treated mice, whereas ONX 0914 treated mice showed significantly reduced TNF-α, IL-1β, and IL-6 mRNA production. To investigate whether autoreactive T helper cells of ONX 0914 treated mice were able to promote and sustain inflammation, we analyzed CD4+ T cells from the brains and spinal cords for their cytokine expression profile. While CD4+ cells derived from the brain of vehicle treated mice produced IFN-γ, TNF-α, IL-17, and GM-CSF upon in vitro restimulation with MOG35–55 (Fig 4B), the few CD4+ cells recovered from the brains of ONX 0914 treated mice were barely expressing these cytokines. Although some CD4+ T cells were able to invade the brain of ONX 0914 treated mice, these data suggest that LMP7 inhibition dampens the ability of these cells to produce cytokines.

Bottom Line: The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes.Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing-remitting model.These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, Switzerland.

ABSTRACT
Multiple sclerosis (MS) is a chronic demyelinating immune mediated disease of the central nervous system. The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes. Recently, we demonstrated a novel function of immunoproteasomes in cytokine production and T cell differentiation. In this study, we investigated the therapeutic efficacy of an inhibitor of the immunoproteasome (ONX 0914) in two different mouse models of MS. ONX 0914 attenuated disease progression after active and passive induction of experimental autoimmune encephalomyelitis (EAE), both in MOG₃₅-₅₅ and PLP₁₃₉₋₁₅₁-induced EAE. Isolation of lymphocytes from the brain or spinal cord revealed a strong reduction of cytokine-producing CD4(+) cells in ONX 0914 treated mice. Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing-remitting model. An analysis of draining lymph nodes after induction of EAE revealed that the differentiation to Th17 or Th1 cells was strongly impaired in ONX 0914 treated mice. These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.

Show MeSH
Related in: MedlinePlus