Mitoxantrone Induces Natural Killer Cell Maturation in Patients with Secondary Progressive Multiple Sclerosis

Mitoxantrone is one of the few drugs approved for the treatment of progressive multiple sclerosis (MS). However, the prolonged use of this potent immunosuppressive agent is limited by the appearance of severe side effects. Apart from its general cytotoxic effect, the mode of action of mitoxantrone on the immune system is poorly understood. Thus, to develop safe therapeutic approaches for patients with progressive MS, it is essential to elucidate how mitoxantrone exerts it benefits. Accordingly, we initiated a prospective single-arm open-label study with 19 secondary progressive MS patients. We investigated long-term effects of mitoxantrone on patient peripheral immune subsets using flow cytometry. While we corroborate that mitoxantrone persistently suppresses B cells in vivo, we show for the first time that treatment led to an enrichment of neutrophils and immunomodulatory CD8low T cells. Moreover, sustained mitoxantrone applications promoted not only persistent NK cell enrichment but also NK cell maturation. Importantly, this mitoxantrone-induced NK cell maturation was seen only in patients that showed a clinical response to treatment. Our data emphasize the complex immunomodulatory role of mitoxantrone, which may account for its benefit in MS. In particular, these results highlight the contribution of NK cells to mitoxantrone efficacy in progressive MS

Treatment of Chronic Experimental Autoimmune Encephalomyelitis with Epigallocatechin-3-Gallate and Glatiramer Acetate Alters Expression of Heme-Oxygenase-1.

We previously demonstrated that epigallocatechin-3-gallate (EGCG) synergizes with the immunomodulatory agent glatiramer acetate (GA) in eliciting anti-inflammatory and neuroprotective effects in the relapsing-remitting EAE model. Thus, we hypothesized that mice with chronic EAE may also benefit from this combination therapy. We first assessed how a treatment with a single dose of GA together with daily application of EGCG may modulate EAE. Although single therapies with a suboptimal dose of GA or EGCG led to disease amelioration and reduced CNS inflammation, the combination therapy had no effects. While EGCG appeared to preserve axons and myelin, the single GA dose did not improve axonal damage or demyelination. Interestingly, the neuroprotective effect of EGCG was abolished when GA was applied in combination. To elucidate how a single dose of GA may interfere with EGCG, we focused on the anti-inflammatory, iron chelating and anti-oxidant properties of EGCG. Surprisingly, we observed that while EGCG induced a downregulation of the gene expression of heme oxygenase-1 (HO-1) in affected CNS areas, the combined therapy of GA+EGCG seems to promote an increased HO-1 expression. These data suggest that upregulation of HO-1 may contribute to diminish the neuroprotective benefits of EGCG alone in this EAE model. Altogether, our data indicate that neuroprotection by EGCG in chronic EAE may involve regulation of oxidative processes, including downmodulation of HO-1. Further investigation of the re-dox balance in chronic neuroinflammation and in particular functional studies on HO-1 are warranted to understand its role in disease progression

MX-induced NK cell maturation is associated with the clinical response.

<p>Thawed PBMCs from SPMS patients stratified into responders and non-responders to MX, were stained for expression of CD62L, CD27, CD57, NKp30, and NKp46 on NK cells. (A) Following 12 months of treatment the responder cohort showed decreased CD62L, NKp46 and NKp30 expression on NK cells. (B) The non-responder cohort did not show any difference in CD62L, NKp46 and NKp30 expression. *p<0.05; mth, months.</p

MX treatment leads to a persistent reduction of B cells and enrichment of neutrophils and CD8^low T cells.

<p>Peripheral blood samples from SPMS patients were surface stained for CD16, CD19, CD14, CD3, CD4, and CD8. (A) A representative flow cytometry dot plot shows the gating strategy for leucocytic populations: R1, comprising all cells without the debris and R2, representing the lymphocytes. Representative flow cytometry plots showing MX-induced changes over time in neutrophils, B cells and CD8^low T cells. (B) The T helper cell (CD4+), the cytotoxic T cell (CD8^high), and the monocyte (CD14+) populations did not change over time. (C) The B cell (CD19+) population decreased significantly after six months and 12 months of treatment. The cytotoxic T cell (CD8^low) population increased significantly after six months and 12 months of treatment, and the neutrophil (CD16+) population was enriched after 12 months of treatment. (D) Comparison of frequencies of B cells, CD8^low cells and neutrophils before and after treatment and in patients with stable MS and healthy individuals. B cell levels presented no difference. CD8^low T cell levels after 12 months of treatment were normalized to healthy controls levels. Neutrophil levels after 12 months of treatment were increased compared to the controls. Repeated-measures ANOVA *p<0.05; **p<0.01; ***p<0.001; One-way ANOVA #p<0.05; ###p<0.001. FSC, forward scatter; SSC, side scatter; mth, months.</p

MX treatment induces NK cell maturation.

<p>Thawed PBMCs from MX-treated SPMS patients were stained for expression of CD62L, CD27, CD57, NKp30, and NKp46 on NK cells. (A) Following 12 months of treatment, the population of cells expressing the maturation marker CD62L decreased, but no change was detected for CD27 and CD57 expressing NK cells. (B) The population of cells expressing the activatory receptors NKp46 and NKp30 showed a significant decrease after 12 months of treatment. *p<0.05; **p<0.01; mth, months.</p

Clinical data of the 15 SPMS patients included in the study.

<p>nd, not determined; R, responder; NR, non-responder; na, not available.</p

MX treatment promotes NK cell enrichment.

<p>Thawed PBMCs from SPMS patients were stained for NK cells and major subsets using CD56 and CD16. (A) Representative flow cytometry plots show the NK cell gating strategy and MX-induced changes over time in NK cells. (B) Shows the population percentages. After six months of treatment, the NK cell population was significantly enriched and then decreased from six to 12 months of treatment. The CD56^dim and CD56^bright NK cells subsets were significantly increased after six months of treatment, but no difference was detected from six to 12 months. (C) Frequencies of NK cells and NK cell subsets in SPMS patients before and after MX treatment compared to the frequency observed in matched healthy individuals. (D) Shows absolute counts of NK cells and CD56 subsets: NK cells, CD56^dim and CD56^bright NK cell subsets remained unchanged over time. *p<0.05; **p<0.01; FSC, forward scatter; SSC, side scatter; mth, months; ns. non significant.</p

The combined application of GA and EGCG treatment does not alter the iron chelator activity but enhanced HO-1 expression compared to EGCG.

<p>A: Soluble iron content in blood serum was quantified day 26 after immunization by using a modification of the ferrozine-based assay. B: Relative mRNA expression of HO-1 in cerebellar and cerebral regions of the CNS of mice included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130251#pone.0130251.g003" target="_blank">Fig 3</a> treated with EGCG alone, or in combination with GA. (t-test) *p<0.05.</p

Effects of EGCG alone and in combination with a suboptimal dose of GA in established EAE.

<p>A: Disease severity of control, GA, EGCG and combination therapy group. Analysis includes data from three independent experiments (Mann-Whitney test). B: Mean clinical scores of animals are shown. Data are given as mean ± SEM. Cumulative disease activity is represented as the area under the curve of control, GA, EGCG and combination therapy group (Kruskal-Wallis). C: Proliferation of MOG-specific CD4⁺ T cells at day 62 after immunization. CFSE-labeled lymph node cells were incubated for 72h with MOG (50μg/ml). As a positive control, cells were cultured with 3 μg/ml anti-CD3 antibody and 2.5 μg/ml anti-CD28 antibody. For the negative control, cells were incubated alone, in the absence of antigen. To assess cell division cells were stained with anti-CD4 Alexa Fluor 647 and analyzed by flow cytometry (ANOVA). *p<0.05, **p<0.01, ***p<0.001.</p

Addition EGCG therapy leads to decreased expression of heme oxygenase-1 (HO-1).

<p>A: Relative mRNA expression of HO-1 in cerebral and cerebellar regions as well as spinal cords of mice treated with vehicle control, EGCG alone, or in combination with GA. (ANOVA). B: Relative expression of HO-1 in mice treated with EGCG compared to mice treated with EGCG+GA C: Relative expression of HO-1 in mice treated with EGCG compared to control vehicle treated animals (t-test).</p