Simvastatin prevents and reverses depigmentation in a mouse model of vitiligo

Abstract

Vitiligo is a common autoimmune disease of the skin that results in disfiguring white spots. There are no Food and Drug Administration (FDA)-approved treatments, and current treatments are time-consuming, expensive, and of low efficacy. We sought to identify new treatments for vitiligo, and first considered repurposed medications because of the availability of safety data and expedited regulatory approval. We previously reported that the IFN-γ-induced chemokine CXCL10 is expressed in lesional skin from vitiligo patients, and that it is critical for the progression and maintenance of depigmentation in our mouse model of vitiligo. We hypothesized that targeting IFN-γ signaling might be an effective new treatment strategy. Activation of signal transducer and activator of transcription 1 (STAT1) is required for IFN-γ signaling and recent studies revealed that simvastatin, an FDA-approved cholesterol-lowering medication, inhibited STAT1 activation in vitro. Therefore, we hypothesized that simvastatin may be an effective treatment for vitiligo. We found that simvastatin both prevented and reversed depigmentation in our mouse model of vitiligo, and reduced the number of infiltrating autoreactive CD8(+) T cells in the skin. Treatment of melanocyte-specific, CD8(+) T cells in vitro decreased proliferation and IFN-γ production, suggesting additional effects of simvastatin directly on T cells. Based on these data, simvastatin may be a safe, targeted treatment option for patients with vitiligo.

Figure 1. Simvastatin dose correlates with clinical response and reduction of melanocyte-specific CD8⁺ T cell in the ear skin

Vitiligo was induced and mice were treated with 0.2mg, 0.4mg, or 0.8 mg of simvastatin or vehicle control (No Tx) three times weekly for five weeks. (a) There is a strong correlation between the simvastatin dose and clinical response, with 0.8mg having the most significant effect. P-value calculated by ANOVA is shown. Post test for linear trend was also significant (p=0.0014). (b) A similar correlation was found between simvastatin dose and reduction of PMELs in the ear skin, but not skin draining lymph nodes. P-value calculated by ANOVA is shown. Post test for linear trend was also significant (p=0.0003). ns, not significant; *p<0.05; **p<0.01; ***p < 0.001.

Figure 2. Simvastatin prevents depigmentation and melanocyte-specific CD8⁺ T cell accumulation in the skin despite no global effect on T cell frequency

Vitiligo was induced and mice were treated with simvastatin (0.8mg) or vehicle control three times weekly for five weeks. (a) Representative mouse ears, noses, footpads and tails from each group are shown. (b) The effects of simvastatin on vitiligo score and (c) the total numbers of PMELs in the ear skin, tail skin, lymph nodes (LN), spleen and blood. (d) Representative flow cytometry plots are shown.

Figure 3. Acute treatment with simvastatin reduces T cell numbers in the epidermis

Vitiligo was induced and mice were treated with 1 dose or 3 daily doses of simvastatin (0.8mg) or vehicle control five weeks after vitiligo induction. (a) Representative flow plots show a reduced number of PMELs in the ear epidermis of the mice that received 1 or 3 daily doses of simvastatin when compared to vehicle. (b) The effects of acute treatment with simvastatin on the total number of PMELs on ear skin, tail skin, lymph nodes, and spleen. (c) Neutralization of IFN-γ has a similar effect on ear skin. (d) The relative expression of CXCL10 was unaffected in the ear skin of mice treated with 1 or 3 daily doses of simvastatin.

Figure 4. Simvastatin inhibits both the proliferation and IFN-γ production of melanocyte-specific CD8⁺ T cells in vitro through inhibition of the HMG-CoA reductase pathway

(a,b) PMEL CD8⁺ T cells were isolated, labeled with CFSE, and activated for 3 days in vitro using anti-CD3 and anti-CD8 antibodies in the presence of either 1, 5, 10 or 100 μM simvastatin or vehicle control. PMEL CD8⁺ T cells were then stained for intracellular IFN-γ and analyzed for (a) proliferation or (b) IFN-γ production by flow cytometry. (c,d) Simvastatin-treated PMEL cultures were supplemented with 1mM mevalonate or vehicle control and CD8⁺ T cell (c) proliferation and (d) IFN-γ production were analyzed by flow cytometry. Representative (e) histograms for CFSE dilution and (f) dot plots for IFN-γ. Blue represents control unstimulated samples.

Figure 5. Simvastatin reverses established vitiligo

Mice with extensive depigmentation on the tail (>50%) were treated with simvastatin (0.8mg) or vehicle control three times weekly beginning twelve weeks post vitiligo induction for a total of four to six weeks. Photographs of each tail before and after treatment were analyzed using ImageJ software to calculate the amount of repigmentation. (a) Simvastatin treatment in established vitiligo did not significantly affect total number of PMELs in skin. (b) The mean percent change in pigmentation from baseline was −3.1% and 8.4% for the mice treated with PBS or simvastatin, respectively. (c) Paired t-test showed a significant increase in percent tail pigmentation only in mice treated with simvastatin. (d) A representative tail from each group before and after treatment is shown.