Dimethyl Fumarate Treatment Mediates an Anti-Inflammatory Shift in B Cell Subsets of Patients with Multiple Sclerosis This information is current as Rui Li, Ayman Rezk, Mathab Ghadiri, Felix Luessi, Frauke of September 29, 2021. Zipp, Hulun Li, Paul S. Giacomini, Jack Antel and Amit Bar-Or J Immunol 2017; 198:691-698; Prepublished online 14 December 2016; doi: 10.4049/jimmunol.1601649 Downloaded from http://www.jimmunol.org/content/198/2/691 Supplementary http://www.jimmunol.org/content/suppl/2016/12/14/jimmunol.160164 Material 9.DCSupplemental http://www.jimmunol.org/ References This article cites 32 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/198/2/691.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • No Triage! 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The Journal of Immunology Dimethyl Fumarate Treatment Mediates an Anti-Inflammatory Shift in B Cell Subsets of Patients with Multiple Sclerosis Rui Li,* Ayman Rezk,* Mathab Ghadiri,* Felix Luessi,*,† Frauke Zipp,† Hulun Li,‡ Paul S. Giacomini,* Jack Antel,* and Amit Bar-Or* The therapeutic mode of action of dimethyl fumarate (DMF), approved for treating patients with relapsing-remitting multiple sclerosis, is not fully understood. Recently, we and others demonstrated that Ab-independent functions of distinct B cell subsets are important in mediating multiple sclerosis (MS) relapsing disease activity. Our objective was to test whether and how DMF influences both the phenotype and functional responses of disease-implicated B cell subsets in patients with MS. High-quality PBMC were obtained from relapsing-remitting MS patients prior to and serially after initiation of DMF treatment. Multiparametric flow Downloaded from cytometry was used to monitor the phenotype and functional response-profiles of distinct B cell subsets. Total B cell counts decreased following DMF treatment, largely reflecting losses of circulating mature/differentiated (but not of immature transitional) B cells. Within the mature B cell pool, DMF had a greater impact on memory than naive B cells. In keeping with these in vivo effects, DMF treatment in vitro remarkably diminished mature (but not transitional B cell) survival, mediated by inducing apoptotic cell death. Although DMF treatment (both in vivo and in vitro) minimally impacted B cell IL-10 expression, it strongly reduced B cell ex- pression of GM-CSF, IL-6, and TNF-a, resulting in a significant anti-inflammatory shift of B cell response profiles. The DMF- http://www.jimmunol.org/ mediated decrease in B cell proinflammatory cytokine responses was further associated with reduced phosphorylation of STAT5/6 and NF-kB in surviving B cells. Together, these data implicate novel mechanisms by which DMF may modulate MS disease activity through shifting the balance between pro- and anti-inflammatory B cell responses. The Journal of Immunology, 2017, 198: 691–698. ultiple sclerosis (MS) is a chronic inflammatory demy- tively little is known about the impact of DMF on B cell subset elinating disease of the CNS (1). An imbalance between responses, which are now strongly implicated in relapsing MS M proinflammatory immune effectors and anti-inflammatory disease activity based on the clinical success of B cell depleting by guest on September 29, 2021 immune regulators has been implicated in MS disease pathogenesis, therapy. with a traditional focus on the role of particular T cell subsets (1). Interestingly, B cell depletion using anti-CD20 monoclonal Abs Oral dimethyl fumarate (DMF) has been recently approved for treat- effectively decreased MS disease activities without apparently ing patients with relapsing-remitting MS (RRMS) (2–4), yet the affecting the abnormal Ab levels in the CSF of MS patients (12– mode of action for DMF is still not fully understood. Although 15), suggesting that secreting pathogenic autoantibodies may emerging evidence has suggested that DMF can downregulate not be the primary mechanism by which B cells contribute T cell and myeloid cell proinflammatory responses (5–11), rela- to MS relapses. Indeed, we and others have shown that Ab- independent functions of B cells, such as Ag presentation and production of proinflammatory cytokines by functionally dis- *Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Mon- tinct B cell subsets, are important contributors to MS disease † treal, Quebec H3A 2B4, Canada; Department of Neurology, Focus Program Trans- activity (16–23). lational Neuroscience and Immunotherapy, Rhine-Main Neuroscience Network, University Medical Center of the Johannes Gutenberg University Mainz, Mainz In this study we examined whether and how DMF may influence 55131, Germany; and ‡Department of Neurobiology, Harbin Medical University, both the phenotypes and functional response profiles of distinct NanGang District, Harbin 150086, Heilongjiang, China B cell subsets. We show that total B cell counts diminish substan- ORCIDs: 0000-0003-3594-4880 (M.G.); 0000-0002-1231-1928 (F.Z.); 0000-0002- tially following the initiation of DMF treatment, a decrease that 1346-3042 (P.S.G.). largely reflects the loss of circulating differentiated but not of Received for publication September 22, 2016. Accepted for publication November 14, 2016. immature transitional B cells in treated patients. In vitro treatment This work was supported by the research foundation of the Multiple Sclerosis Society with DMF mirrored the in vivo effects, directly inducing mature of Canada (A.B.-O.), Banque National Fellowship (R.L.), and National Natural Sci- B cell but not transitional B cell apoptosis. The functional analysis ence Foundation (Grant 81430035, R.L. and H.L.). further revealed that treatment with DMF (both in vivo and Address correspondence and reprint requests to Prof. Amit Bar-Or, Neuroimmunol- in vitro) decreased B cell expression of proinflammatory (GM-CSF, ogy Unit and Experimental Therapeutics Program, Montreal Neurological Institute a and Hospital, McGill University, 3801 University Street, Room 123, Montreal, QC IL-6, and TNF- ) but minimally impacted anti-inflammatory H3A 2B4, Canada. E-mail address: [email protected] (IL-10) B cell response profiles, associated not only with prefer- The online version of this article contains supplemental material. ential apoptosis, but also with reduced phosphorylation of STAT5/6 Abbreviations used in this article: DMF, dimethyl fumarate; HC, healthy control; and NF-kB in surviving B cells. Our study suggests that the ca- ICS, intracellular cytokine staining; MMF, monomethyl fumarate; MS, multiple scle- pacity of DMF to limit new MS inflammatory disease activity may, rosis; PI, propidium iodide; RRMS, relapsing-remitting MS. in part, relate to its ability to mediate an anti-inflammatory shift in Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 the balance of phenotypically and functionally distinct B cell subsets. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601649 692 DMF REGULATES THE BALANCE OF B CELL SUBSETS IN MS PATIENTS interassay variability. Where indicated, magnetic bead sorting (Miltenyi Materials and Methods + Subjects and study design Biotec) was used to positively select CD19 B cells from fresh PBMC with purities routinely .98% as confirmed by flow cytometry. Isolated B cells A total of 13 patients (11 females, 2 males) with McDonald criteria– were plated in U-bottom 96-well plates at 2 3 105/well in a total volume of confirmed RRMS, mean age 41 (range 20–60), were prospectively fol- 200 ml of serum-free x-vivo medium (Lonza), and stimulated with soluble lowed at a single center in Montreal, Canada, prior to and following CD40L (1 mg/ml; Enzo Life Sciences), goat anti-human BCR cross- treatment initiation with DMF. Patients were assessed every 3 mo with linking Ab (Xab) (10 mg/ml; Jackson ImmunoResearch) with or without clinical review, physical examination, and expanded disability status score. IL-4 (20 ng/ml; R&D Systems) for 48 h, at which time supernatants were At study entry, patients had an average expanded disability status score of collected and frozen (270) for subsequent quantification of cytokine secretion 2.5 (range 1.0–4.0), preceding annualized relapse rate of 0.8 (0–2), and by ELISA and the cells were analyzed by flow cytometry (as described be- disease duration of 9.6 y (range 1–27 y). Of the 13 patients, 11 had pre- low). B cells were cultured in parallel wells in either medium alone, vehicle viously been treated with either IFN or glatiramer acetate, one had re- (DMSO), monomethyl fumarate (MMF), or DMF (Sigma Aldrich, Oakville, ceived a single dose of ofatumumab 18 mo prior to recruitment,