Regulation of Treg-associated CD39 in multiple sclerosis and effects of corticotherapy during relapse.

567215

research-article2015

MSJ0010.1177/1352458514567215Multiple Sclerosis JournalN Muls, HA Dang

MULTIPLE SCLEROSIS MSJ JOURNAL

Original Research Paper

Regulation of Treg-associated CD39 in multiple sclerosis and effects of corticotherapy during relapse

Multiple Sclerosis Journal 1–13 DOI: 10.1177/ 1352458514567215 © The Author(s), 2015. Reprints and permissions: http://www.sagepub.co.uk/ journalsPermissions.nav

Nathalie GV Muls, Hong Anh Dang, Christian JM Sindic and Vincent van Pesch

Abstract Background: Accumulating data highlight proinflammatory processes leading to MS relapses. Whether anti-inflammatory mechanisms are concomitantly activated is unclear. The ectonucleotidase CD39 has been described as a novel T regulatory cell (Treg) marker. The purpose of this study was to explore whether regulatory mechanisms are activated during MS relapses and reinforced by intravenous methylprednisolone (ivMP). Methods: Blood samples were collected from stable and relapsing MS patients and healthy controls. We used FOXP3 methylation-specific qPCR and CD4+CD25highFOXP3+ analysis to quantify Tregs. Cytokine mRNA expression levels were measured in peripheral blood mononuclear cells (PBMCs) and in CD4+ T cells. CD39 expression was determined by flow cytometry in monocytes, NK, T and B cells. CD39 enzymatic activity was assessed by ATP luminometry. Results: The proportion of Tregs was similar in relapsing MS patients and healthy controls. CD39 mRNA level was higher in PBMCs of relapsing MS patients than in controls. The proportion of CD39-expressing Tregs was higher in MS patients. IvMP decreased the overall proportion of Tregs while it increased CD39 mRNA levels, the proportions of CD39-expressing Tregs and monocytes as well as CD39 ectonucleotidase activity. Conclusions: Our data suggest that immunoregulatory mechanisms are ongoing in MS patients, particularly during relapses, and strengthened by ivMP.

Keywords: Multiple sclerosis, CD39, regulatory T cells, corticosteroids, relapse, immunoregulation Date received: 3 June 2014; revised: 11 November 2014; accepted: 11 December 2014 Introduction Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). In recent years, T helper (Th) cells producing interleukin-17 (IL-17) have emerged as one of the crucial players in the pathogenesis of MS and its mouse model, experimental autoimmune encephalomyelitis (EAE). MS patients have a higher proportion of circulating IL-17+ T cells and higher levels of IL-17 messenger RNA (mRNA) in cerebrospinal fluid (CSF) cells during relapse.1,2 Furthermore, IL-17 is detected in CD4+ and CD8+ T cells from active MS brain lesions.3 Some data suggest that altered regulatory T cell (Treg) function could be part of the immune dysregulation leading to MS.4 In humans, the analysis of this cell

subpopulation is made difficult by the lack of specific markers. Although the surface marker CD25 is widely used to identify these cells, its specificity is limited because its expression is also induced upon activation of conventional T cells. However, two recently identified properties could help in the characterization of regulatory T cells. Firstly, the demethylation of the first intron of FOXP3 (FOXP3i1) is specific to natural Tregs (nTregs).5 FOXP3i1 methylation status has never been investigated in Treg cells from MS patients. Secondly, CD39 has been described as a novel functional marker of regulatory effector/memory-like T (TREM) and suggested to be regulated by the transcription factor aryl hydrocarbon receptor (AHR).6,7 CD39 degrades extracellular adenosine triphosphate (ATP) to adenosine monophosphate (AMP). This conversion

Correspondence to: V. van Pesch Service de Neurologie, Cliniques Universitaires Saint-Luc, Avenue Hippocrate 10, 1200 Bruxelles, Belgium. vincent.vanpesch@ uclouvain.be Nathalie GV Muls Hong Anh Dang Christian JM Sindic Vincent van Pesch Unité de Neurochimie, Institute of Neuroscience, Université catholique de Louvain, Belgium

http://msj.sagepub.com 1

Downloaded from msj.sagepub.com at University of Sussex Library on September 18, 2015

Multiple Sclerosis Journal is considered as one of the mechanisms by which Treg cells reduce inflammation.7 Glucocorticoids (GCs) have been used for the treatment of MS relapses and other immune-mediated diseases since the early 1950s.8 High-dose methylprednisolone (MP), a synthetic GC, exerts its therapeutic effects notably by downregulating cytokine expression.1 However, the precise effects of GCs on immunoregulatory mechanisms have not been fully investigated. The aim of our study was to investigate, firstly, immune regulatory processes during MS relapses and, secondly, the mechanisms of action of MP. We studied the ex vivo anti-inflammatory cytokine expression profile in peripheral blood mononuclear cells (PBMCs) of stable and relapsing MS patients. CD39 expression was investigated both at the mRNA and protein level in several immune cells. In addition, we studied other markers and cytokines related to Tregs (CD39, AHR, FOXP3 and EBI3). The effect of intravenous methylprednisolone (ivMP) treatment on these markers and on CD39 enzymatic activity was also assessed. Materials and methods Study population and sample collection Blood samples were collected from a total of 44 relapsing MS patients, two patients with a clinically isolated syndrome (optic neuritis), 16 clinically stable MS patients and 26 healthy controls (HCs). Among the 44 relapsing patients, three were treated with interferon beta (IFN-β), one of whom had anti-IFN-β neutralizing antibodies. Among the stable patient cohort, four were treated with glatiramer acetate. Demographic and clinical features are summarized in Supplementary Table 1. The study was approved by our local ethics committee and informed consents were obtained from all patients. MS relapse was defined by a mono- or multifocal neurological deficit, compatible with MS, lasting for more than 24 hours and not associated with fever or infection. Relapsing MS patients were given 1 g ivMP/day for five days. Samples were collected before treatment on Day 1 (relapsing MS) and before the last administration of ivMP on Day 5 (ivMP MS). The mean interval between relapse onset and first blood sampling was 14 ± 13 days (mean ± standard deviation). PBMC isolation and culture Human PBMCs were prepared by Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation.

Cells were stored in liquid nitrogen until further use. For in vitro studies, PBMCs were cultured in X-VIVOTM10 medium (Lonza) with IL-2 (50 U/ml). Methylation-specific-quantitative polymerase chain reaction (methylS-qPCR) for FOXP3i1 Genomic DNA (gDNA) was prepared from 106 PBMCs using the PureLink DNA Mini Kit (Invitrogen). gDNA was treated with sodium bisulfite using EpiTect Plus DNA Bisulfite Kit (Qiagen). Realtime PCR of methylated and demethylated FOXP3i1 sequences was performed with the Rotor-Gene Probe PCR Kit (Qiagen). Sequences of primers and probes are indicated in Supplementary Table 2. The percentage of demethylated sequences is calculated as follows: 2^(Ct methylated – Ct demethylated)/[2^(Ct methylated – Ct demethylated) +1]*100. Fluorescence-activated cell sorting (FACS) For Treg cells, PBMCs were stained for surface antigens then fixed and permeabilized prior to FOXP3 staining (clone 236A/E7, eBioscience). For CD39 analysis, PBMCs were stained with a combination of antibodies conjugated with the appropriate fluorochrome. The following antibodies were used: CD3, CD4, CD14, CD33, natural killer (NK)p46 and FOXP3 (eBioscience), CD8, CD19, CD39 and CD25 (BioLegend). Acquisition was performed on an LSR Fortessa cytometer (BD Biosciences). Data were analyzed using FlowJo software (TreeStar Inc). CD4+ T cells isolation T cells were polyclonally stimulated overnight with the antibody-based polyclonal stimulation reagent CytoStim (Miltenyi Biotec).9 CD4+ T cells were selected by automated magnetic-activated cell sorting (AutoMACS, Miltenyi Biotec) using anti-human CD4 MicroBeads (Miltenyi Biotec). cDNA synthesis and qPCR RNA from PBMCs or immunopurified CD4+ T cells were isolated using the miRNeasy mini kit (Qiagen) according to the manufacturer’s protocol and reverse transcribed to cDNA using Superscript Reverse Transcriptase (Invitrogen). qPCR assays were performed as previously described.1 Relative amount of transcripts was determined by normalizing for the Abelson gene (ABL) using the comparative Ct method (2–∆∆Ct). ABL mRNA levels were not affected by ivMP treatment. Sequences of primers are detailed in Supplementary Table 3.

2 http://msj.sagepub.com


N Muls, HA Dang et al. ATP hydrolysis assay A total of 5.104 PBMCs were incubated in medium containing 25 µM ATP with or without 100 µM of the ATPase inhibitor ARL67156 (Sigma-Aldrich) for 30 minutes. ATP concentration in the supernatant was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). ARL67156 inhibits ATPase activity and was therefore used to determine the maximum ATP concentration in the experimental setting. Following incubation, ATP was measured and compared to the control condition. Spontaneous ATP release in wells containing only cells in X-VIVOTM10 medium (Lonza) accounted for only 0.13% of the ATP concentrations measured in the other conditions. Statistical analysis Statistical analysis was performed using GraphPad Prism 5 software. To test for differences before and after ivMP treatment, nonparametric Wilcoxon’s signed rank tests were performed. MS patients and HCs were compared using nonparametric MannWhitney tests. P values ≤ 0.05 were considered as statistically significant. Supplementary materials Underlying research materials related to the manuscript can be accessed by contacting the corresponding author. Results Ex vivo CD39 and AHR mRNA levels are increased in PBMCs from relapsing MS patients We measured the ex vivo mRNA expression level of several immune regulatory markers (AHR, CD39, FOXP3, EBI3 and IL23R) in total PBMCs of stable and relapsing MS patients as well as HCs. No significant differences were observed between stable and relapsing MS patients (Figure 1, Table 1). Stable patients showed a two-fold decreased level of IL23R mRNA in comparison to HCs. In relapsing MS patients, CD39 and AHR mRNA were significantly increased in comparison to HCs. AHR and CD39 mRNA expression levels were highly correlated in MS patients (p < 0.0001). Furthermore, AHR and CD39 were both highly correlated to the percentage of circulating monocytes in relapsing MS patients (Supplementary Figure 1). The mRNA levels of FOXP3 and EBI3 did not show significant differences between subgroups.

CD39 cellular distribution is modified in MS patients As we observed an increase in CD39 mRNA expression level in relapsing MS patients, we aimed to characterize CD39-expressing cells. In HCs, CD39 was expressed on 99% of monocytes, 94% of CD19+ B cells, 6.8% of CD4+ T cells, 2.7% of NK cells and 2.3% of CD8+ T cells (Table 2(a)). The median proportion of CD39-expressing CD4+CD25highFOXP3+ Tregs was 10% higher in relapsing MS patients (77.3%) than in stable patients (69.85%), although this result did not reach statistical significance (p = 0.77) (Figure 2). On the contrary, in comparison to HCs, the proportion of CD39+ Tregs was significantly increased by 26.3% and 39.8% respectively in stable (p = 0.016) and relapsing MS patients (p = 0.033). A bimodal distribution of CD39+ Treg within the three subgroups was observed. Relapsing MS patients also showed an increased proportion of CD39-expressing CD8+ T cells in comparison to HCs (5.0% versus 2.3%; p = 0.018) (Table 2(b)). Median fluorescence intensity (MFI) for CD39 was comparable between MS patients and HCs in all cell subpopulations. As the proportion of CD39+ Tregs was increased in MS patients, we wanted to characterize the effects of ivMP on Treg-related parameters. Firstly, we quantified Tregs before and after ivMP treatment. nTregs were measured by analyzing FOXP3i1 demethylation status and overall CD4+CD25highFOXP3+ Tregs were assessed by flow cytometry. The proportion of nTregs was comparable between relapsing MS patients and HCs. ivMP treatment reduced this proportion by 60% (p = 0.0493) (Figure 3(a)). These results were confirmed by flow cytometry. The overall proportion of Tregs defined by the CD4+CD25hiFOXP3+ subset within the lymphoid cell population was similar in relapsing MS and HCs but decreased by 25% in MS patients following ivMP treatment (p < 0.0001, Figure 3(b)). ivMP also decreased the Treg proportion within CD4+ T cells (p = 0.04, Figure 3(c)). Of note, there was an overall 40% decrease in the proportion of CD4+ T cells following ivMP treatment (data not shown). ivMP impact on ex vivo mRNA levels in PBMCs and on in vitro-stimulated CD4+ T cells After four days of ivMP treatment, there was a significant increase in the ex vivo median mRNA levels of CD39 (69%, p = 0.0005), AHR (75%, p = 0.0005) and EBI3 (300%, p = 0.0015) (Figure 4, Table 3(a)).



Multiple Sclerosis Journal

Figure 1. Ex vivo immune regulatory markers in total PBMCs of MS patients and healthy controls. Scatter dot plots show relative mRNA expression levels in PBMCs from stable (stable MS, n = 16), relapsing MS patients (relapsing MS, n = 15) and healthy controls (HCs, n = 10). For each target, individual mRNA levels were expressed as relative values to the mean level of the control group. Horizontal lines represent the median value in all subgroups. * indicates a p value of ≤0.05. PBMCs: peripheral blood mononuclear cells; mRNA: messenger RNA; MS: multiple sclerosis; AHR: aryl hydrocarbon receptor.

On the contrary, FOXP3 (p = 0.0013) and IL23R (p = 0.0005) mRNA levels were decreased by 40% and 80% respectively. The mRNA levels of IL-27p28 and IL-12p35 did not show significant change (data not shown).

Additionally, we studied mRNA levels in in vitrostimulated CD4 + T cells and observed that, contrarily to FOXP3, AHR and IL23R, CD39 expression was increased (Figure 5, Table 3(b)). In opposition to what was observed ex vivo, the mRNA



N Muls, HA Dang et al. Table 1. Ex vivo mRNA levels in PBMCs of stable and relapsing MS patients as well as HCs. Median and range of mRNA levels are expressed as relative values to the mean level of the control group. Ex vivo PBMC relative mRNA levels

Stable MS

Relapsing MS

Median

Range

Median

AHR CD39 FOXP3 EBI3 IL23R

1.17 1.06 0.78 1.047 0.58

0.52–1.75 0.72–1.971 0.04–2.19 0.0–8.923 0.06–1.33

1.37 1.236 1.03 0.3725 0.68

HCs

Range

p value (stable MS vs relapsing MS)

Median

0.93–2.42 0.58–2.03 0.21–2.41 0.0–8.042 0.28–2.42

0.06 0.31 0.21 0.1 0.066

1.02 0.99 0.91 0.67 1.35

Range

p value (stable MS vs HCs)

p value (relapsing MS vs HCs)

0.69–1.4 0.7–1.3 0.46–1.94 0.0–2.282 0.19–2.44

0.51 0.48 0.16 0.23 0.03

0.014 0.048 0.8 0.65 0.56

mRNA: messenger RNA; PBMCs: peripheral blood mononuclear cells; MS: multiple sclerosis; HCs: healthy controls; AHR: aryl hydrocarbon receptor.

Table 2. CD39+-expressing-cell analysis by flow cytometry of (a) HCs, (b) stable and relapsing MS patients. Median percentage and range of CD39+ cells in different immune cell subpopulations are indicated. (a)

% of CD39+ cells in

HCs

CD8+ T cells CD4+ T cells NKp46+ cells CD14+ monocytes CD19+ B cells CD4+ CD25hiFOXP3+ T cells

2.3 (1.26–5.25) 6.8 (3.87–11.15) 2.7 (1.2–6.2) 99.4 (98.1–99.65) 94.25 (82.05–96.7) 55.3 (24.4–69.6)

(b)

% of CD39+ cells in

Stable MS

Relapsing MS

p value (stable MS vs relapsing MS)

HCs

p value (stable MS vs HCs)

p value (relapsing MS vs HCs)

CD8+ T cells CD4+CD25highFOXP3+ T cells

3.17 (0.84–7.49) 69.85 (22.15–92.4)

5.0 (1.38–14.8) 77.3 (33.2–93.6)

0.14 0.84

2.3 (1.26–5.25) 55.3 (24.4–64.1)

0.76 0.016

0.018 0.033

HCs: healthy controls; MS: multiple sclerosis; NK: natural killer.

expression level of EBI3 was reduced in polyclonally restimulated CD4+ T cells that had been exposed to ivMP. CD39 cellular distribution is modified by ivMP As ivMP increased the ex vivo mRNA level of CD39, we analyzed whether the treatment affected the surface expression of CD39 on circulating cell subsets. The percentage of CD39-expressing monocytes increased above 99% in nine of 10 patients after ivMP (Figure 6). In contrast, CD39+CD8+ T cells were

decreased in eight of 11 patients. The proportion of CD39-expressing NK and B cells was not modified by ivMP treatment (Table 4(a)). Interestingly, although an overall decrease in the proportion of CD39+CD4+ T cells was observed following ivMP treatment, the proportion of CD39+ cells specifically increased in the CD4+CD25highFOXP3+ cellular subset (Figure 6(c) and (d)). Indeed, CD39+ Tregs were enriched by 15.8% after ivMP (p = 0.024). This increase was linked to an increasing shift in MFI values, contrary to other cell types (monocytes, B, CD8+ and NK cells) (Table 4(b)).




Figure 2. Analysis of CD39-expressing Tregs of MS patients and healthy controls by flow cytometry. PBMCs of stable (stable MS, n = 16), relapsing MS patients (relapsing MS, n = 15) and healthy controls (HCs, n = 10) were analyzed ex vivo by flow cytometry. (a) PBMCs gating strategy: CD25highFOXP3+ cells are selected within CD4+ cells and then analyzed for CD39 staining. (b) Comparison of CD39 expression in CD4+CD25highFOXP3+ Tregs between HCs and MS patients. The horizontal lines represent the median value in all subgroups. * indicates a p value of ≤0.05. MS: multiple sclerosis; PBMCs: peripheral blood mononuclear cells; Tregs: T regulatory cells.

Figure 3. Treg proportion in relapsing MS patients before and after ivMP treatment and healthy controls. (a) Total PBMCs of MS patients during relapse before (relapsing MS, n = 20) or after (ivMP MS, n = 16) ivMP treatment and healthy controls (HCs, n = 13) were analyzed by methylS-qPCR to quantify cells with demethylated FOXP3i1. (a) Results represent the percentage of circulating natural Tregs with demethylated FOXP3i1 sequences. (b) Scatter plot of CD4+CD25highFOXP3+ cells analyzed by flow cytometry and represented as percentage of lymphoid cells or (c) of CD4+ cells. The horizontal lines represent the median value in all subgroups. * and *** indicate p values of ≤0.05 and ≤0.001, respectively. MS: multiple sclerosis; ivMP: intravenous methylprednisolone; PBMCs: peripheral blood mononuclear cells; Tregs: T regulatory cells.



N Muls, HA Dang et al.

Figure 4. Ex vivo immune regulatory markers in total PBMCs of ivMP-treated MS patients. Scatter dot plots show relative mRNA expression levels in paired samples from MS patients before (relapsing MS) and after ivMP treatment (ivMP MS). For each target, individual mRNA levels were expressed as relative values to mean level of the relapsing group. Horizontal lines represent the median value in all subgroups. ** and *** indicate p values of ≤0.01 and ≤0.001, respectively. PBMCs: peripheral blood mononuclear cells; ivMP: intravenous methylprednisolone; MS: multiple sclerosis; mRNA: messenger RNA; Tregs: T regulatory cells; AHR: aryl hydrocarbon receptor.

Ex vivo CD39 enzymatic activity is increased by ivMP To test for the functional activity of CD39, the consumption of exogenous ATP by PBMCs was assessed. Ectonucleotidase activity increased after ivMP in 11/14 patients. ivMP treatment increased ATP consumption by a mean of 26% (p = 0.01) (Figure 7). Discussion Accumulating data highlight the proinflammatory processes activated in relapsing MS patients.1,10,11

Notably, peripherally activated Th1 and Th17 cells are thought to cross the blood-brain barrier and to induce inflammation within the CNS.12 Whether antiinflammatory mechanisms are also activated is still unclear. As other immune-mediated diseases, MS might also arise from impaired regulatory functions.4,13 In the literature, conflicting data are found regarding circulating Treg proportion in RRMS patients. Some studies show a decreased proportion of these cells in MS patients while most reports do not find a difference.14–17 To date, the most specific marker for human nTregs is the DNA demethylation of the first intron of FOXP3 (FOXP3i1), also known



Multiple Sclerosis Journal Table 3. Median mRNA levels before and after ivMP in (a) ex vivo PBMCs and in (b) in vitro polyclonally restimulated CD4+ T cells. mRNA levels are expressed as a relative value to the mean level of the relapsing group. The ranges and p values are indicated. (a)

Relative mRNA levels in PBMCs AHR (n = 20) CD39 (n = 17) EBI-3 (n = 14) FOXP3 (n = 18) IL23R (n = 20)

Relapsing MS

ivMP MS

Median

Range

Median

Range

1.20 0.92 0.77 0.95 0.9

(0.68–2.12) (0.45–2) (0–1.86) (0.5–3.07) (0.48–4.1)

2.10 1.53 2.48 0.49 0.28

(1.04–3.38) (0.51–3.15) (0–21.8) (0.15–1.22) (0.07–1.12)

(b) CD4+ T cell relative mRNA levels AHR (n = 13) CD39 (n = 19) FOXP3 (n = 19) EBI-3 (n = 19) IL23R (n = 10)

p value (relapsing MS vs ivMP MS) 0.0005 0.0005 0.0015 0.0013 0.0005

Relapsing MS

ivMP MS

Median

Range

Median

Range

1.04 0.86 0.86 0.83 0.98

(0.66–1.81) (0.33–4.73) (0.53–2.3) (0.2–6.7) (0.26–2.8)

0.98 1.520 0.84 0.53 0.81

(0.31–1.54) (0.2–3.54) (0.22–2.68) (0.04–3.42) (0.39–2.51)

p value (relapsing MS vs ivMP MS) 0.62 0.006 0.126 0.013 0.88

mRNA: messenger RNA; PBMCs: peripheral blood mononuclear cells; MS: multiple sclerosis; ivMP: intravenous methylprednisolone; AHR: aryl hydrocarbon receptor.

as TSDR5 or CNS2.18 To our knowledge, this property has never been used to investigate the frequency of Tregs in MS patients. Here, we used both Treg CD4+CD25highFOXP3+ phenotype and FOXP3i1 demethylation to assess the proportion of Tregs. These two techniques led to the same conclusions. In agreement with previous studies, we found that the proportion of Tregs is similar between relapsing MS patients and HCs.16,19 The in vivo effect of GCs on the number of circulating Tregs is still a matter of debate. Several studies on autoimmune diseases highlight an increased proportion of Tregs after GC therapy while other studies conducted in animals and humans conclude the opposite.20,21 Since Tregs play a crucial role in controlling immune responses, we wondered whether ivMP might increase their number in MS patients. On the contrary, our results demonstrate that ivMP rather decreases the proportion of Tregs. ivMP treatment largely decreases the CD4+ population and consequently the proportion of Tregs in PBMCs. Reduction in the CD4+ subset is consistent with the proapoptotic effect of GCs.22 Consequently, changes in the proportion of circulating Tregs do not account for the therapeutic effects of ivMP. These data are in line with the results obtained by Wüst et al. in EAE, but do not exclude that Treg function could be affected by GCs.23

We found indeed a significant increase of the surface marker CD39 on Tregs. This could also influence the inflammatory response in MS. CD39 degrades extracellular ATP to AMP. ATP stimulation of immune cells induces largely pro-inflammatory responses such as activation of the inflammasome and subsequent IL-1β maturation.24 CD39+ Tregs have been reported to selectively suppress IL-17 production by cell contact.25 An increase in the proportion of CD39+ Tregs has been shown in relapsing MS patients compared to patients in the remitting phase, with some conflicting results.6,25–27 Discrepancies could be explained by divergences in experimental settings, including patient selection and cellular markers used to analyze Tregs. In this study, we performed analyses at both the mRNA and protein levels on various immune cell types of relapsing and stable MS patients. Furthermore, we investigated the impact of GCs on those parameters. Our results show an increase in CD39 mRNA levels in the PBMCs of relapsing MS patients. This expression is further increased after ivMP treatment. Using flow cytometry experiments, we demonstrated that the proportion of CD39expressing CD8+ T cells and Tregs is increased in relapsing MS patients in comparison to HCs. CD39expressing Tregs are also increased in stable MS patients. Although the proportion of CD39-expressing



N Muls, HA Dang et al.

Figure 5. In vitro immune regulatory markers in CD4+ T cells of ivMP-treated MS patients. Scatter dot plots show relative mRNA expression levels in paired samples from MS patients before (relapsing MS) and after (ivMP MS) ivMP treatment. For each target, individual mRNA levels were expressed as relative values to the mean level of the relapsing group. Horizontal lines represent the median value in all subgroups. * and ** indicate p values of ≤0.05 and ≤0.01, respectively. ivMP: intravenous methylprednisolone; MS: multiple sclerosis; mRNA: messenger RNA; PBMCs: peripheral blood mononuclear cells; AHR: aryl hydrocarbon receptor.

CD4+ T cells is slightly reduced following ivMP, we observed a specific increase in the MFI of CD39 in this subpopulation, also observed in CD4 + CD25highFOXP3+ cells. This effect is highly specific as it is not observed in other cell types. This is in line with our results in vitro, showing an increase in CD39 mRNA following exposure to ivMP. In addition, in CD4+CD25highFOXP3+, we observed a specific increase in the proportion of CD39-expressing cells upon ivMP treatment, indicating that this cell

subpopulation is regulated differently from the overall CD4+ population. We have shown that an increase in CD39+ cells after ivMP is correlated with upregulated ectonucleotidase activity. As CD39 mediates anti-inflammatory effects, this suggests that regulatory mechanisms are ongoing in MS patients and that ivMP further reinforces them. This is an additional mechanism by which ivMP downregulates inflammation, in addition to stimulating IL-10 and transforming growth factor beta (TGF-β) production.1 It is




Figure 6. Effect of ivMP on CD39-expressing immune cells analyzed by flow cytometry. PBMCs of MS patients before (relapsing MS) or after (ivMP MS) ivMP treatment were analyzed ex vivo by flow cytometry. (a) CD39 expression in CD8+ T cells (n = 11), (b) CD14+ monocytes (n = 10) and (C) CD4+CD25highFOXP3+ Tregs (n = 11). (d) Treg proportion within the CD39+CD4+ cell population before and after ivMP treatment. Horizontal lines represent the median value in all subgroups. * and ** indicate p values of ≤0.05 and ≤0.01, respectively. ivMP: intravenous methylprednisolone; PBMCs: peripheral blood mononuclear cells; MS: multiple sclerosis; mRNA: messenger RNA; Tregs: T regulatory cells.

Table 4. CD39+-expressing-cell analysis by flow cytometry. (a) Median percentage of CD39+ cells in different immune cell subpopulations and (b) MFI with ranges and p values. (a) CD39+

% of

CD8+

T cells CD4+ T cells NKp46+ cells CD14+ monocytes CD19+ B cells CD4+CD25highFOXP3+ T cells

Relapsing MS

ivMP-treated MS

p value

3.2 (1.2–7.8) 10.3 (4.38–23.2) 2.4 (1.1–8.5) 99.65 (98.7–99.9) 91.7 (83.9–97.7) 58.35 (19.2–85.8)

2.9 (0.7–5.9) 7.7 (2.4–16.8) 2 (1.1–7.2) 99.9 (98.7–99.9) 94.2 (82.3–97.1) 67.6 (21.5–83.9)

0.02 0.019 0.13 0.034 0.45 0.024

(b)

CD39 MFI of

Relapsing MS

ivMP-treated MS

p-value

CD8+

393 (282.5–749.5) 433 (291–885.5) 762 (394–1100) 3530 (2669–4412) 1241 (819–2004) 1181 (418–2797)

367 (252.5–617.5) 521.5 (272–1034) 685 (365–1270) 2907 (2043–3613) 1182 (775–1773) 1349 (475–2178)

0.131 0.042 0.03 0.004 0.001 0.024

T cells CD4+ T cells NKp46+ cells CD14+ monocytes CD19+ B cells CD4CD25highFOXP3+ T cells

MS: multiple sclerosis; MFI: median fluorescence intensity; ivMP: intravenous methylprednisolone; NK: natural killer.



N Muls, HA Dang et al. IL-27 acts on DCs, in an autocrine manner, to suppress T cell response by inducing expression of CD39.30 Moreover, IL-27 directly inhibits development of Th17 cells.31

Figure 7. Effect of ivMP on CD39 ATPase activity. Nucleotidase activity was assessed with 25 µM ATP for 30 minutes and determined by luminometric assay in paired PBMC samples (n = 14) from MS patients before (relapsing MS) or after ivMP treatment (ivMP MS). Horizontal lines represent the median value in all subgroups. * indicates a p value of ≤0.05. ivMP: intravenous methylprednisolone; PBMCs: peripheral blood mononuclear cells; MS: multiple sclerosis; ATP: adenosine triphosphate.

interesting to point out a bimodal distribution in the CD39+ Treg percentages. This observation was not correlated to any patient characteristics including age, disease duration or disability.27 In our experiments, this distribution is present in patients and age- and gender-matched controls. Therefore, this is likely unrelated to the disease. Under certain conditions, the activation of the transcription factor AHR promotes the expression of CD39 but does not affect FOXP3 expression.6 Interestingly, we have demonstrated that AHR mRNA level is induced following ivMP treatment, in agreement with Bielefeld et al., who showed an increase in AHR mRNA in a mouse hepatoma cell line following treatment by dexamethasone.28 These data suggest that AHR might be implicated in the cellular response to ivMP and responsible for the clinical benefits by mediating the transcription of immune regulatory genes such as CD39. Our results reveal an increase in EBI3 mRNA level in total PBMCs after ivMP treatment but a decrease (p = 0.013) in stimulated CD4+ T cells. Together with p35, EBI3 constitutes IL-35, an anti-inflammatory cytokine produced by Tregs and mediating their function.29 In association with p28, EBI3 also forms IL-27. IL-27 is produced by activated monocytes, macrophages and dendritic cells (DCs). Therefore, these results could be explained by an increased expression of IL-27 by antigen-presenting cells, present in the PBMCs, in response to ivMP. On the contrary, in CD4+ T cells, IL-35 might be decreased owing to the reduced frequency of Tregs after ivMP. Interestingly,

We have shown that ivMP decreases IL-23R expression. It has been firmly demonstrated that IL-23 is essential in the development of several autoimmune diseases notably by driving Th17 expansion.32 Therefore, preventing cellular response to IL-23 could be an additional mechanism by which ivMP downregulates inflammation, in addition to shutting down all pro-inflammatory Th1, Th17 and Th2 responses.1 In conclusion, we have shown that the overall population of Tregs and of nTregs is unchanged in relapsing MS, but that CD39 mRNA expression in PBMCs is increased. MS patients also have an increased proportion of CD39+ Tregs. CD39 expression is further reinforced by ivMP treatment in Tregs and in CD14+ monocytes. This result has functional relevance as CD39-mediated nucleotidase activity is increased following ivMP. This work underlines that immune regulatory mechanisms are activated in MS patients and could be further strengthened by ivMP treatment. In addition to shutting down the inflammatory response of T helper cells, we suggest that ivMP could also act on antigen-presenting cells through induction of IL-27 and CD39. Further research is needed to determine the mechanisms by which ivMP exerts its effects, notably on EBI3 expression and CD39 regulation and function. Acknowledgements We thank Professors S. Lucas, J. Van Snick and N. Dauguet for invaluable discussions (de Duve Institute, Brussels). Conflict of interest None declared. Funding This work was supported by the FNRS, Bayer educational grants, the Belgian Charcot Foundation and the Walloon Region “Désordres Inflammatoires dans les Affections Neurologiques” project.

References 1. Muls N, Jnaoui K, Dang HA, et al. Upregulation of IL-17, but not of IL-9, in circulating cells of CIS and relapsing MS patients. Impact of corticosteroid therapy on the cytokine network. J Neuroimmunol 2012; 243: 73–80.



Multiple Sclerosis Journal 2. Matusevicius D, Kivisakk P, He B, et al. Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis. Mult Scler 1999; 5: 101–104.

15. Serpero LD, Filaci G, Parodi A, et al. Fingolimod modulates peripheral effector and regulatory T cells in MS patients. J Neuroimmune Pharmacol 2013; 8: 1106–1113.

3. Tzartos JS, Friese MA, Craner MJ, et al. Interleukin-17 production in central nervous systeminfiltrating T cells and glial cells is associated with active disease in multiple sclerosis. Am J Pathol 2008; 172: 146–155.

16. Haas J, Hug A, Viehover A, et al. Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis. Eur J Immunol 2005; 35: 3343– 3352.

4. Viglietta V, Baecher-Allan C, Weiner HL, et al. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med 2004; 199: 971–979. 5. Baron U, Floess S, Wieczorek G, et al. DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells. Eur J Immunol 2007; 37: 2378–2389.

17. Huan J, Culbertson N, Spencer L, et al. Decreased FOXP3 levels in multiple sclerosis patients. J Neurosci Res 2005; 81: 45–52. 18. Zheng Y, Josefowicz S, Chaudhry A, et al. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 2010; 463: 808–812.

6. Gandhi R, Kumar D, Burns EJ, et al. Activation of the aryl hydrocarbon receptor induces human type 1 regulatory T cell-like and Foxp3(+) regulatory T cells. Nat Immunol 2010; 11: 846–853.

19. Feger U, Luther C, Poeschel S, et al. Increased frequency of CD4+ CD25+ regulatory T cells in the cerebrospinal fluid but not in the blood of multiple sclerosis patients. Clin Exp Immunol 2007; 147: 412–418.

7. Borsellino G, Kleinewietfeld M, Di Mitri D, et al. Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: Hydrolysis of extracellular ATP and immune suppression. Blood 2007; 110: 1225–1232.

20. Braitch M, Harikrishnan S, Robins RA, et al. Glucocorticoids increase CD4CD25 cell percentage and Foxp3 expression in patients with multiple sclerosis. Acta Neurol Scand 2009; 119: 239–245.

8. Rudick. Multiple sclerosis therapeutics. London: Martin Dunitz, 1999, p.574.

21. Sbiera S, Dexneit T, Reichardt SD, et al. Influence of short-term glucocorticoid therapy on regulatory T cells in vivo. PLoS One 2011; 6: e24345.

9. Dodero A, Carniti C, Raganato A, et al. Haploidentical stem cell transplantation after a reduced-intensity conditioning regimen for the treatment of advanced hematologic malignancies: Posttransplantation CD8-depleted donor lymphocyte infusions contribute to improve T-cell recovery. Blood 2009; 113: 4771–4779. 10. Li Y, Wang H, Long Y, et al. Increased memory Th17 cells in patients with neuromyelitis optica and multiple sclerosis. J Neuroimmunol 2011; 234: 155–160. 11. Steinman L. Immunology of relapse and remission in multiple sclerosis. Annu Rev Immunol 2014; 32: 257–281. 12. Brucklacher-Waldert V, Stuerner K, Kolster M, et al. Phenotypical and functional characterization of T helper 17 cells in multiple sclerosis. Brain 2009; 132: 3329–3341.

22. Sionov RV, Cohen O, Kfir S, et al. Role of mitochondrial glucocorticoid receptor in glucocorticoid-induced apoptosis. J Exp Med 2006; 203: 189–201. 23. Wüst S, van den Brandt J, Tischner D, et al. Peripheral T cells are the therapeutic targets of glucocorticoids in experimental autoimmune encephalomyelitis. J Immunol 2008; 180: 8434–8443. 24. Martinon F, Mayor A and Tschopp J. The inflammasomes: Guardians of the body. Annu Rev Immunol 2009; 27: 229–265. 25. Fletcher JM, Lonergan R, Costelloe L, et al. CD39+Foxp3+ regulatory T Cells suppress pathogenic Th17 cells and are impaired in multiple sclerosis. J Immunol 2009; 183: 7602–7610.

13. Gonsette RE. Self-tolerance in multiple sclerosis. Acta Neurol Belg 2012; 112: 133–140.

26. Dalla Libera D, Di Mitri D, Bergami A, et al. T regulatory cells are markers of disease activity in multiple sclerosis patients. PLoS One 2011; 6: e21386.

14. Praksova P, Stourac P, Bednarik J, et al. Immunoregulatory T cells in multiple sclerosis and the effect of interferon beta and glatiramer acetate treatment on T cell subpopulations. J Neurol Sci 2012; 319: 18–23.

27. Peelen E, Damoiseaux J, Smolders J, et al. Th17 expansion in MS patients is counterbalanced by an expanded CD39+ regulatory T cell population during remission but not during relapse. J Neuroimmunol 2011; 240–241: 97–103.



N Muls, HA Dang et al. 28. Bielefeld KA, Lee C and Riddick DS. Regulation of aryl hydrocarbon receptor expression and function by glucocorticoids in mouse hepatoma cells. Drug Metab Dispos 2008; 36: 543–551. 29. Collison LW, Workman CJ, Kuo TT, et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 2007; 450: 566–569. 30. Mascanfroni ID, Yeste A, Vieira SM, et al. IL27 acts on DCs to suppress the T cell response and autoimmunity by inducing expression of the

immunoregulatory molecule CD39. Nat Immunol 2013; 14: 1054–1063. 31. Batten M, Li J, Yi S, et al. Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing T cells. Nat Immunol 2006; 7: 929–936. 32. Langrish CL, Chen Y, Blumenschein WM, et al. IL23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 2005; 201: 233–240.

Visit SAGE journals online http://msj.sagepub.com

SAGE journals



Relapse in multiple sclerosis.

Characterizing cognitive function during relapse in multiple sclerosis.

Multiple sclerosis: Atacicept increases relapse rates in multiple sclerosis.

Seasonal variation in multiple sclerosis relapse.

Immunology of relapse and remission in multiple sclerosis.

Emerging Approaches for Validating and Managing Multiple Sclerosis Relapse.

The Lived Experience of Multiple Sclerosis Relapse: How Adults with Multiple Sclerosis Processed Their Relapse Experience and Evaluated Their Need for Postrelapse Care.

Urinary trypsin inhibitor levels are reduced in cerebrospinal fluid of multiple sclerosis and neuromyelitis optica patients during relapse.

How to run a multiple sclerosis relapse clinic.

Expression, regulation and function of microRNAs in multiple sclerosis.

Headache in relapse and remission phases of multiple sclerosis: A case-control study.

Role of prolactin in B cell regulation in multiple sclerosis.

Late relapse of non-seminomatous testicular cancer during treatment of multiple sclerosis with interferon β-1a: A case report.

Long-term effect of ACTH treatment of relapse in multiple sclerosis.

Sex as a determinant of relapse incidence and progressive course of multiple sclerosis.

Severe relapse in a multiple sclerosis patient associated with ipilimumab treatment of melanoma.

Seasonal variation of relapse rate in multiple sclerosis is latitude dependent.

Regulation of the T Cell Response by CD39.

Atorvastatin calcium in combination with methylprednisolone for the treatment of multiple sclerosis relapse.

Categorization of multiple sclerosis relapse subtypes by B cell profiling in the blood.

The UK patient experience of relapse in Multiple Sclerosis treated with first disease modifying therapies.

Poor early relapse recovery affects onset of progressive disease course in multiple sclerosis.

Increased anti-KIR4.1 antibodies in multiple sclerosis: could it be a marker of disease relapse?

Platform Therapy Compared with Natalizumab for Multiple Sclerosis: Relapse Rates and Time to Relapse Among Propensity Score-Matched US Patients.