Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis

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.

Figure 1

C57BL/6 wild type, LMP2^−/−, MECL-1^−/−, and LMP7^−/− mice were immunized with MOG_35–55 peptide. Mice were monitored daily for clinical symptoms of EAE. Clinical score ( y-axis) is plotted versus time post immunization ( x-axis). Data points represent mean ± s.e.m. of six mice. The experiments were performed three times, yielding similar results.

Figure 2

ONX 0914 prevents MOG35–55-induced EAE.Mice were immunized with MOG_35–55 peptide and were monitored daily for clinical symptoms of EAE. Data are presented as mean clinical score s.e.m. of six mice. * P < 0.05; ** P < 0.01; *** P < 0.001.

1. C57BL/6 mice were treated with 10 mg/kg ONX 0914 or vehicle three times a week beginning on the day of immunization. The experiments were performed five times, yielding similar results.

2. LMP7^−/− mice were treated with 10 mg/kg ONX 0914 or vehicle three times a week beginning on the day of immunization. The experiments were performed twice, yielding similar results.

3. C57BL/6 mice were treated with 2 mg/kg PR-825 or vehicle three times a week beginning on the day of immunization. The experiments were performed twice, yielding similar results.

4. LMP7^−/− mice were treated with 1 mg/kg PR-825 or vehicle three times a week beginning on the day of immunization. The experiments were performed twice, yielding similar results.

5. C57BL/6 wild type mice were irradiated and reconstituted with bone marrow derived from wild type (wild type → wild type) or LMP7^−/− (LMP7^−/− → wild type) mice. Mice were treated with 10 mg/kg ONX 0914 or vehicle, three times a week beginning on the day of immunization. The experiments were performed twice, yielding similar results.

Figure 3

Reduced infiltration into the CNS in ONX 0914 treated mice.C57BL/6 mice were immunized with MOG_35–55 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.

1. Flow cytometric analysis of lymphocytes and myeloid cells invading the brain (upper panels) or spinal cord (lower panels). Graphs show the mean absolute numbers ± s.e.m. of CNS invading CD4⁺ lymphocytes, CD45^highCD11b⁻ lymphocytes, and CD45^highCD11b⁺ myeloid cells. Representative flow cytometry profiles of CNS infiltrating cells are depicted on the right side. n.d.: not detected.

2. Representative histological spinal cord sections (left side) of indicated mice (H&E, original magnification ×5 [upper panels] and ×40 [lower panels]). Semiquantitative histopathologic assessment (right side) of CNS infiltration. Data points represent mean ± s.e.m.

Figure 4

Reduced CNS inflammations in ONX 0914 treated mice.C57BL/6 mice were immunized with MOG_35–55 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.

1. 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.

2. B Brain infiltrating CD4⁺ lymphocytes were restimulated in vitro with MOG_35–55 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.

Figure 5

ONX 0914 blocks GM-CSF production of T cells.Cytokine concentrations measured by ELISA are presented as the mean s.e.m. from triplicate wells. Data represent one out of three experiments.

1. GM-CSF production of ONX 0914 (300 nM) treated splenocytes stimulated with plate bound anti-CD3/anti-CD28 Abs for 96 h as analyzed by ELISA.

2. GM-CSF production of ONX 0914 (300 nM) treated splenocytes stimulated with plate bound anti-CD3/anti-CD28 Abs in the presence of neutralizing Abs to IFN-c and IL-12 for 96 h as analyzed by ELISA.

3. Assessment of GM-CSF and IL-23 production of human PBMCs. PBMCs were incubated with indicated concentrations of ONX 0914 for 2 h and stimulated with plate bound anti-CD3/anti-CD28 Abs (GM-CSF) or LPS (IL-23) for 48 h.

Figure 6

ONX 0914 inhibits progression of MOG35–55-induced EAE.

1. C57BL/6 were immunized with MOG_35–55 peptide and were daily scored for clinical symptoms. On the day of disease onset (d 15) mice were treated three times a week with intravenous administration of 6 mg/kg ONX 0914, 10 mg/kg ONX 0914, or vehicle or once a week with 10 mg/kg ONX 0914. Data, presented as the mean clinical score ± s.e.m. ( n = 10 per group), are from one experiment of three performed with similar results. The arrow indicates the time point when treatment was initiated. * P < 0.05; ** PA < 0.01; *** P < 0.001.

2. Histopathological analysis of spinal cords from MOG_35–55-immunized mice at day 25 after immunization. Data are a representative of 2 separate experiments.

3. MOG_35–55-immunized mice received either vehicle or 10 mg/kg ONX 0914 three times per week starting on day 14 after immunization. Individual spinal cords were harvested on day 25 from a cohort of animals in each group and analyzed by quantitative RT–PCR (β-actin normalized) for expression of the indicated genes. Data presented are the mean normalized value ± s.e.m. ( n = 5 per group) and P values were derived from an unpaired t-test.

4. In vitro restimulated MOG_35–55 reactive T cells were adoptively transferred into C57BL/6 mice. Mice were treated three times a week with 6 mg/kg ONX 0914 or vehicle for 14 days and were daily monitored for clinical symptoms. Data are presented as mean clinical score ± s.e.m. ( n = 5 per group).

5. C57BL/6 were immunized with MOG_35–55 peptide and treated with 10 mg/kg ONX 0914 or vehicle, three times a week beginning on the day of immunization. On day 9 post immunization draining lymph node cells were restimulated in vitro with MOG_35–55 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 mean percentages ± s.e.m. ( n = 6 per group) 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.

Figure 7

ONX 0914 ameliorates PLP139–151-induced EAE.

1. SJL/J mice were immunized with PLP_139–151 and were monitored daily for clinical symptoms of EAE. From day 11 on (indicated by an arrow), mice were treated with 10 mg/kg ONX 0914 three times a week (indicated 3×), 10 mg/kg ONX 0914 once a week (indicated weekly), or vehicle. Data, presented as the mean clinical score s.e.m. ( n = 10 per group), are from one experiment of three performed with similar results. *** P < 0.001 by two-way ANOVA followed by Bonferroni post-hoc comparison at the end of study.

2. Representative histological spinal cord sections of indicated mice. From day 11 on, mice were treated with10 mg/kg ONX 0914 three times a week. Mice were analyzed on day 14 post immunization.

3. SJL/J mice were immunized with PLP_139–151 and were monitored daily for clinical symptoms of EAE. From day 19 on (indicated by an arrow), mice were treated with 10 mg/kg ONX 0914 three times a week (indicated 3×), 10 mg/kg ONX 0914 once a week (indicated weekly), or vehicle. Data, presented as the mean clinical score s.e.m. ( n = 10 per group), are from one experiment of three performed with similar results. *** P < 0.001 by two-way ANOVA followed by Bonferroni post-hoc comparison at the end of study.

4. In vitro restimulated PLP_139–151–reactive T cells were adoptively transferred into SJL/J mice. Mice were treated three times a week with 10 mg/kg ONX 0914 or vehicle beginning on day 9 and were monitored daily for clinical symptoms. Data are presented as mean clinical score s.e.m. ( n = 9–10 per group). * P < 0.05; ** P < 0.01; *** P < 0.001.