Edited by: Sharon Glynn Lynch, University of Kansas Medical Center, United States
Reviewed by: Eva Havrdova, Charles University in Prague, Czechia; Amy Lovett-Racke, The Ohio State University, United States
This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology
†These authors have contributed equally to this work
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Dimethyl fumarate (DMF) is an approved first-line disease-modifying treatment for relapsing remitting multiple sclerosis (RRMS). DMF has multiple mechanisms of action (
Here we present an observational study aimed at investigating the long-term effects of DMF on different lymphocyte subsets in a cohort of relapsing-remitting multiple sclerosis patients.
This 2-years observational study included 38 patients aged from 22 to 58 years (mean age 38.0 ± 8.7 years, female sex 60.5%), with diagnosis of RRMS according to revised McDonald criteria. Patients were recruited at the Veneto Regional Multiple Sclerosis Center at University Hospital of Verona and were assigned to receive DMF at standard dose. Patients were examined every 3 months during the first year of treatment and every 6 months during the second year, with additional visits in case of relapses. A relapse was defined as a worsening of neurological impairment or appearance of a new symptom or abnormality attributable to MS, lasting at least 24 h and preceded by stability of at least 1 month (
Patients with no evidence of disease activity (NEDA) or with evidence of disease activity (EDA) were also identified. NEDA was defined as a composite score obtained from three related measures of disease activity: (i) no evidence of relapses; (ii) no confirmed disability progression as assessed by an increase of the EDSS score by at least 1 point sustained over 6 months; and (iii) no new or newly enlarging white matter T2 lesions at the MRI follow-up (
The Local Ethic Committee approved the present study. Informed consent was obtained from all patients.
Complete blood count including absolute lymphocyte counts (ALCs) and count of total CD3+ T cells, CD19+ B cells and CD3−CD56+ NK cells was performed by flow cytometry by mean of ADVIA 2120i Hematology analyzer (
Each patient underwent brain 3T MRI (at baseline, after 1 year and 2 years of clinical observation). MRI sequences were acquired by Philips Achieva 3T MR Scanner (Philips Medical Systems, Best, Netherlands). No software updating was carried out during the study period and the scanner underwent specific functioning test every 2 weeks to assure parameter stability and, thus, image reproducibility and comparability. For each subject 3D T2 weighted, 3D T1 weighted Fast Field Echo (FFE) and 3D Fluid Attenuated Inversion Recovery (FLAIR) image sets were acquired. Subjects were positioned for serial measurements according to published guidelines for serial MRI studies in MS (
Statistical analysis was performed using Prism 7.0 (GraphPad Software, La Jolla, CA) and R studio (Version 1.0.153); Wilcoxon matched-pairs signed-rank test for longitudinal analysis were applied;
As expected, mean lymphocyte absolute count decreased during the 2 years of treatment (see
Peripheral absolute values [× 109/L] of total lymphocyte count and T-cells, B-cells, and NK cells at baseline and after 3 (T3), 6 (T6), 12 (T12), and 24 (T24) months of DMF treatment. After 2 years of treatment total lymphocyte count persisted significantly decreased (
The mean absolute CD3+ T cells count progressively decreased during DMF exposure. Indeed, a 35% decrease was observed after 6 months of therapy, reaching 47.7% at the end of the second year of treatment (
Absolute values [× 109/L] and SD of T-cell subsets after 6, 12, and 24 months of DMF treatment compared to baseline values in a longitudinal analysis of a smaller cohort of patients.
CD4+ T | 623.2 |
506 (245.4) |
522 (282.0) |
503.2 (282.0) |
CD8+ T | 368.6 |
237.5 (150.4) |
150.7 (88.0) |
196 (98.0) |
CD4+/CD8+ | 2. |
2.4 (0.8) |
3.8 (1.5) |
2.7 (0.8) |
Th-1 | 69.8 |
102.6 (62.4) |
112.9 (61.5) |
53.9 (58.9) |
Th-2 | 64.8 |
78.5 (58.1) |
89.1 (36.2) |
38.5 (25.7) |
Th-17 | 97.5 |
76.6 (27.3) |
102.7 (37.6) |
101.5 (52.5) |
T-reg | 14.8 |
11.9 (11.5) |
7.4 (4.8) |
14.7 (7.4) |
Baseline | 41.2 (84.8) |
18.9 (80.5) |
75.3 (108.0) |
85.9 (98.2) |
3 months | −1.9 (75.0) |
58.8 (98.2) |
50.4 (106.0) |
|
6 months | 122 (213.0) |
101 (200.0) |
||
12 months | 31.5 (110.0) |
Within CD4+ T cells, Th-1 cells decrement was observed only after 2 years of treatment (
Unlike T and B cells, NK absolute count showed a strong variability over the 2-years longitudinal observation. Indeed, mean absolute counts increased by 85.9% after 2 years of treatment (
Among the 25 patients who completed the follow up, 8 showed evidence of disease activity (EDA); all of them had a new or increasing white matter T2 lesion while 5 of them also suffered a relapse.
After the two year follow up no significant differences were detected in total lymphocyte count (
In the present study, RRMS patients assigned to receive DMF treatment showed a significant decrease in total lymphocyte count since the first months of treatment. However, in line with previous studies (
In our longitudinal study a trend of preferential decrease in CD8+ T cells was observed in the first year of therapy, but was not confirmed in subsequent follow up; DMF mechanism of action on T cells appears more complex than a change in main lymphocyte subsets ratio and it probably implies a shift of immune response throughout a more “tolerogenic” profile of immune system (
Unlike T and B cell, NK cells showed a significant positive trend. The change over time appeared quite heterogeneous, with a consistent increase in percentage and a wide variance in trends of absolute cell count. With the limit of simple size, increasing percentage, and absolute count of NK cells over time did not correlate with the degree of lymphopenia (
Intriguingly, activation of NRF2 signaling pathway after DMF therapy has been observed to increase, among the others, the number of CD56bright natural killer cells and to be related with the absence of disease activity after 1 year of therapy (
Concluding, the effect of DMF on lymphocytes subsets still needs further investigations, especially with regard to NK cells count which appeared not in line with that of other lymphocytes.
The Ethic Committee for clinical research of Azienda Sanitaria di Padova approved the present study and the informed consent was obtained from all patients.
DM, AB, RM, and MC: conception and design, acquisition, analysis and interpretation of data, drafting the paper. FC, CZ, and SM: analysis and interpretation of data, drafting the paper. AP and CR: statistical analysis and interpretation of data, drafting the paper. SF, VS, BM: conception and design and acquisition of data, drafting the paper.
AB, SF, VS, and BM were employed by Data Medica Group, Synlab Limited, Padova, Italy. MC received payment for development of educational presentations including service on speakers bureaus by Biogen-Elan, Genzyme, TEVA, Bayer-Schering, he received travel/accomodation expenses by Novartis Pharma, Genzyme, Biogen idec, Merck Serono, Bayer-Schering, TEVA, and Advisory Board membership by Bayer-Shering, Genzyme, Biogen Idec and Novartis Pharma. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
Dimethyl fumarate
Relapsing Remitting Multiple Sclerosis
natural killer
nuclear factor erythroid 2-related factor 2
Monomethyl fumarate
Glyceraldehyde 3-phosphate dehydrogenase
T-helper 1
T-helper 17
T-helper 2
T-regulatory
Expanded Disability Status Scale
No Evidence of Disease Activity
Evidence of Disease Activity.