Preservation of neuronal function as measured by clinical and MRI endpoints in relapsing-remitting multiple sclerosis: how effective are current treatment strategies?

<p><b>Introduction</b>: Approved medications for relapsing-remitting multiple sclerosis have shown to be effective in terms of their anti-inflammatory potential. However, it is also crucial to evaluate what long-term effects a patient can expect from current MS drugs in terms of preventing neurodegeneration. Here we aim to provide an overview of the current treatment strategies in MS with a specific focus on potential neuroprotective effects.</p> <p><b>Areas covered</b>: Randomized, double-blind and placebo or referral-drug controlled phase 2a/b and phase 3 trials were examined; non-blinded phase 4 studies (extension studies) were included to provide long-term data, if not otherwise available. Endpoints considered were expanded disability status scale, various neuropsychological tests, percent brain volume change and T1-hypointense lesions as well as multiple sclerosis functional composite, confirmed disease progression, and no evidence of disease activity.</p> <p><b>Expert commentary</b>: Overall, neuroprotective functions of classical MS therapeutics are not sufficiently investigated, but available data show limited effects. Thus, further research and development in neuroprotection are warranted. When counselling patients, potential long-term beneficial effects should be presented more conservatively.</p

Combination therapy with EGCG and GA alleviates disability in established EAE.

<p>For treatment of acute disease PLP_139–151-induced EAE mice were randomized into two groups after reaching a disease score of 2 and subsequently received 300 µg of oral EGCG or vehicle (0.9% NaCl) twice daily in addition to daily s.c. injections of 150 µg GA or vehicle (4% Mannitol). Mean disease score of control and combination therapy group is shown from day 0 to day 44 after immunization (A), *p<0.05 day 19, 20, 23, 24, 34, n = 6. Cumulative disease activity of control and combination therapy group was calculated (B), *p<0.05.</p

EGCG and GA together ameliorate CNS inflammatory pathology.

<p>Representative Hematoxylin and Eosin staining of transverse spinal cord sections of vehicle, EGCG, GA and EGCG and GA treated EAE mice is shown (A). 100× magnification, scale bar 500 µm. Inflammation in standardized areas of the spinal cord of all treatment groups was assessed and is presented semi-quantitatively as percentage of spinal cord quadrants with inflammatory foci relative to all assessed quadrants (B), *p<0.05, n = 8 per group.</p

EGCG and GA promote axonal growth and density <i>in vitro</i>.

<p>Representative photomicrographs of brain slices with axonal outgrowth after addition of EGCG (5 µg/ml), GA (6.25 µg/ml) and the combination of EGCG (5 µg/ml) and GA (6.25 µg/ml) are shown. 100× magnification, scale bar: 300 µm (A). The number of axons was measured by quantifying all intersections of axons with a standardized line parallel to the brain slice border, **p<0.001 (B). The density of outgrowing axons was determined by measuring the mean intensity in a standardized area parallel to the brain slice edge, **p<0.001 (C). Levels of GDNF (D) and BDNF (E) were measured in the supernatant of outgrowth assays by ELISA, n.d. = not detectable. **p<0.001 ***p<0.0001.</p

EGCG and GA in combination inhibit death of CNS cells <i>in vitro</i>.

<p>Survival of murine HT22 neuronal cells (A) and human glioblastomal LN18 cells (B) was determined in a crystal violet assay after induction of apoptosis by incubation with Glutamate (5 mM) (A) or TRAIL (20 ng/ml) (B). EGCG (10 µg/ml) and/or GA (12.5 µg/ml) were added to the culture 2 h before apoptosis induction, *p<0.05, **p<0.001, representative of 2 experiments.</p

Combination therapy with EGCG and GA delays disease onset and synergistically reduces EAE severity.

<p>For preventive treatment EGCG (300 µg) or vehicle (NaCl 0.9%) was administered orally twice daily from day −9 before immunization with PLP_139–151 until day 45. GA (50 µg) or vehicle (Mannitol 4%) in incomplete Freund's adjuvant was given s.c. once on day −7 before immunization. Mean disease score of control, EGCG, GA and the combination therapy group is shown from day 0 to day 45 after immunization (A), * p<0.05 day 13,15–18,20–22,24,26–27, **p<0.01 day 14,19,23,25,28,35–39,42, n = 8 per group. Onset of disease in control, GA, EGCG and combination therapy group is presented in a Kaplan-Meier survival curve as percentage of sick mice at a certain day after immunization (B), *p<0.05. Cumulative disease activity of control, GA, EGCG and combination therapy group was calculated as the area under the curve of the clinical score plots for each individual animal (C), *p<0.05.</p

Kinetics of IL-6 Production Defines T Effector Cell Responsiveness to Regulatory T Cells in Multiple Sclerosis

<div><p>In multiple sclerosis (MS) autoaggressive T effector cells (Teff) are not efficiently controlled by regulatory T cells (Treg) but the underlying mechanisms are incompletely understood. Proinflammatory cytokines are key factors facilitating Teff activity in chronic inflammation. Here we investigated the influence of IL-6 on Treg sensitivity of Teff from therapy-naïve MS patients with or without active disease. Compared to healthy volunteers and independent of disease course CD4⁺ and especially CD8⁺ MS-Teff were insensitive against functional active Treg from healthy controls. This unresponsiveness was caused by accelerated production of IL-6, elevated IL-6 receptor expression and phosphorylation of protein kinase B (PKB)/c-Akt in MS-Teff. In a positive feedback loop, IL-6 itself induced its accelerated synthesis and enhanced phosphorylation of PKB/c-Akt that finally mediated Treg resistance. Furthermore, accelerated IL-6 release especially by CD8⁺ Teff prevented control of surrounding Teff, described here as “bystander resistance”. Blockade of IL-6 receptor signaling or direct inhibition of PKB/c-Akt phosphorylation restored Treg responsiveness of Teff and prevented bystander resistance. In Teff of healthy controls (HC) exogenous IL-6 also changed the kinetics of IL-6 production and induced Treg unresponsiveness. This modulation was only transient in Teff from healthy volunteers, whereas accelerated IL-6 production in MS-Teff maintained also in absence of IL-6. Hence, we showed that the kinetics of IL-6 production instead of elevated IL-6 levels defines the Teff responsiveness in early Treg-T cell communication in MS independent of their disease course and propose IL-6 and associated PKB/c-Akt activation as effective therapeutic targets for modulation of Teff activity in MS.</p> </div

In a positive feedback loop IL-6 induces IL-6R upregulation and accelerated IL-6 production.

<p>(A) IL-6R expression after 24 h of culture with (grey) or without IL-6 (white) was analyzed by flow cytometry. Each point represents percentage of IL-6R⁺ cells within CD19⁺, CD14⁺, CD4⁺ or CD8⁺ cells from independent HC (n=4), differences were n.s.. (B) PBMC^depl. from HC were cultured for 24 h with (grey) or without (white) IL-6, washed extensively, stimulated with plate-bound anti-CD3 mAb and used for qRT-PCR. EF1-α served as housekeeping gene. Statistical analysis was determined by Mann-Whitney-test, <i>P</i>-values relative to Teff without IL-6 preincubation ** p<0.01. Bars represent mean of four independent experiments. (C) Left, IL-6 precultured HC PBMC^depl (red) were washed, cocultured with HC-Treg (grey) and stimulated with anti-CD3 mAb. Right, Teff (white) were cocultured in the lower chamber with Treg both from HC. IL-6 pre-cultured PBMC^depl. (red) were washed and placed in upper chamber. Culture was stimulated with anti-CD3 mAb in presence or absence of anti-IL-6R mAb. Proliferation in lower chamber was determined by ³H-Tdr incorporation and displayed as percentage of proliferation normalized to Teff alone as mean ± SEM (n=4), <i>P</i>-values to cocultures ** p<0.01. (D) Teff from HC or MS were cultured for 24 h in absence of IL-6, cocultured afterwards with Treg and stimulated with anti-CD3 mAb. Proliferation was determined on day three and displayed as mean ± SEM of (n=3) triplicate measurements.</p

Reduced Treg sensitivity of T effector cells in MS patients is independent of disease course.

<p>(A) We defined Teff as Treg-depleted PBMC stimulated with anti-CD3 mAb. Teff from HC were cocultured with syngeneic (black) or allogeneic Treg (white and grey) in different ratios and stimulated with anti-CD3 mAb. Teff proliferation was determined by ³H-Tdr incorporation on day three and displayed as mean ± SEM of triplicate measurements. One representative experiment of n=3 is shown. (B) CFSE-labeled Teff from RRMS patients with active disease (black) or HC (white) were cocultured with Treg and stimulated with anti-CD3 mAb. Teff proliferation of CD3⁺ T cells was determined on day three by flow cytometry. One representative result of six independent experiments is shown. (C) Teff from RRMS patients in remission (black) or HC (white) were cocultured with Treg and stimulated with anti-CD3 mAb. Teff proliferation was determined as described. Left: bars represent mean ± SEM of triplicates of one representative experiment. Right: curves show percentage of proliferation in presence of different Treg numbers normalized to proliferation of Teff alone as mean ± SEM of n=28, <i>P</i>-values relative to Teff of HC** p<0.01***, p<0.001 are shown.</p

Accelerated IL-6 synthesis is responsible for direct and bystander Treg resistance.

<p>(A) Isolated CFSE-labeled Teff of either HC (white) or MS patients (black) were cocultured with HC-Treg of an independent donor (ratio of 1:1) and stimulated with anti-CD3 mAb and T cell-depleted, irradiated PBMC. Proliferation of CD4⁺ (upper panel) or CD8⁺ Teff (lower panel) was determined within CD3⁺ T cells on day three. Percentage of proliferating cells is indicated; one experiment (n=3) is shown. (B) Treg-depleted PBMC, CD4⁺ or CD8⁺ Teff from HC or MS were left unstimulated or were activated with plate-bound anti-CD3 and anti-CD28 mAb. Expression levels of IL-6 mRNA were detected by RT-PCR. EF1-α used as housekeeping gene. One experiment (n=9) is shown. (C) Treg-depleted PBMC from HC or MS were stimulated with plate-bound anti-CD3 mAb and IL-6 or IL-2 mRNA levels were detected by qRT-PCR. EF1-α served as housekeeping gene. Bars represent mean of eight independent experiments; statistical analysis was determined by Mann-Whitney-test, measurements in duplets ***p<0.001. (D) We defined Teff as Treg-depleted PBMC stimulated with anti-CD3 mAb. Teff were cocultured with Treg and stimulated with anti-CD3 mAb in absence (white) or time-delayed addition (grey) of IL-6. Proliferation was determined by ³H-Tdr incorporation on day three and displayed as mean suppression ± SEM of triplicate measurements. One experiments of n=4 is shown.</p