Proteasome inhibitor bortezomib suppresses nuclear factor-kappa B activation and ameliorates eye inflammation in experimental autoimmune uveitis

Abstract

Bortezomib is a proteasome inhibitor used for hematologic cancer treatment. Since it can suppress NF-κB activation, which is critical for the inflammatory process, bortezomib has been found to possess anti-inflammatory activity. In this study, we evaluated the effect of bortezomib on experimental autoimmune uveitis (EAU) in mice and investigated the potential mechanisms related to NF-κB inactivation. High-dose bortezomib (0.75 mg/kg), low-dose bortezomib (0.15 mg/kg), or phosphate buffered saline was given after EAU induction. We found that the EAU is ameliorated by high-dose bortezomib treatment when compared with low-dose bortezomib or PBS treatment. The DNA-binding activity of NF-κB was suppressed and expression of several key inflammatory mediators including TNF-α, IL-1α, IL-1β, IL-12, IL-17, and MCP-1 was lowered in the high-dose bortezomib-treated group. These results suggest that proteasome inhibition is a promising treatment strategy for autoimmune uveitis.

Figure 1

Effect of bortezomib on clinical course of EAU induced with IRBP. (a) Comparison of clinical scores of EAU mice treated with high-dose (0.75 mg/kg) bortezomib (red line, n = 19), low-dose (0.15 mg/kg) bortezomib (black line, n = 17), or PBS (blue line, n = 19) in 0.1 mL. Data shown are the mean clinical score (ordinate) of each experiment group over time (abscissa) and the sum of three independent experiments. Comparison of (the course of the clinical symptoms) high-dose bortezomib-treated EAU mice (blue line) with saline-treated mice (red line) shows a significant difference and is indicated as (^*). Comparison of high-dose bortezomib-treated (blue line) with low-dose bortezomib-treated (black line) EAU mice also shows a significant difference and is indicated as (^*). ^* P < 0.05, via the Wilcoxon signed-rank test. (b), (c), (d), and (e): photomicrographs of H&E stained retinal tissue. Representative photomicrographs of paraffin-fixed H&E stained slides of the retina of (b): naïve C57BL/6 mice without EAU induction, (c): EAU mice that received 0.1 mL PBS treatment (^* indicates leukocytes in vitreous cavity; ^** indicates retinal folds), (d): EAU mice that received low-dose (0.15 mg/kg) bortezomib treatment, and (e): EAU mice that received high-dose (0.75 mg/kg) bortezomib treatment. The experiment was repeated three times with similar results. GCL: ganglion cell layer. INL: inner nuclear layer. ONL: outer nuclear layer. (f) Average clinical score over time of EAU in mice with high-dose (0.75 mg/kg) bortezomib (blue line, n = 9), etanercept (5 mg/kg) (black line, n = 6), or saline (0.1 mL/mouse) treatment (red line, n = 7). Data shown are the mean clinical score (ordinate) of each experiment group over time (abscissa) and the sum of two independent experiments. ^* P < 0.05, via the Wilcoxon signed-rank test.

Figure 2

The evaluation of protein expression of inflammatory mediators in retinas of EAU mice in Luminex analysis. Decreased expression of TNF-α (a), IL-1α (b), IL-1β (c), IL-17 (f), IL-12 (h), and MCP-1 (i) relative to the expression in the saline-treated group was noted in the high-dose bortezomib (Vel [H]) group but not in the low-dose bortezomib (Vel [L]) group except for TNF-α. In addition, there was no significant difference on the expression of IFN-γ (d), IL-4 (e), and IL-6 (g) in retinas between bortezomib and saline-treated mice. Data are expressed as the mean SD of three independent experiments (bar graph). ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001, via Student's t-test.

Figure 3

Evaluation of chymotrypsin-like and trypsin-like activity of the proteasome. Compared to the saline-treated EAU group, there was significantly decreased activity of chymotrypsin-like (a) and trypsin-like activity (b) in the low-dose [Vel (L)] and high-dose bortezomib [Vel (H)] groups. The activity was also markedly lowered in the high-dose bortezomib-treated group compared with the low-dose bortezomib-treated group. The data are expressed as the mean ± SD of the mean in 5 mice for each group (bar graph). ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001, via Student's t-test. The experiment was repeated three times with similar results.

Figure 4

EMSA for the evaluation of the NF-κB DNA-binding activity in naïve mice and different groups of EAU mice. Lane 1: p50 subunit of NF-κB. Lane 2: free probe (FP). Lane 3: naïve C57BL/6 mice. Lane 4: EAU mice with saline treatment. Lane 5: EAU mice treated with low-dose bortezomib (Vel [L]). Lane 6: EAU mice treated with high-dose bortezomib (Vel [H]). Lane 7: 100-fold molar excess of unlabeled NF-κB probe. Lane 8: anti-p65 subunit of NF-κB. The sample was pooled from both eyes of five mice in each group. Data are representative of results in three independent experiments.