A JOURNAL OF NEUROLOGY
Micro-RNA dysregulation in multiple
sclerosis favours pro-inflammatory
Mireia Guerau-de-Arellano,1Kristen M. Smith,2Jakub Godlewski,3Yue Liu,2Ryan Winger,1
Sean E. Lawler,3Caroline C. Whitacre,2Michael K. Racke1and Amy E. Lovett-Racke2
1 Department of Neurology, The Ohio State University, 395 West 12th Avenue Columbus, OH 43210, USA
2 Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, 460 West 12th Avenue, Columbus,
OH 43210, USA
3 Department of Neurological Surgery, The Ohio State University, 400 West 12th Avenue, Columbus, OH 43210, USA
Correspondence to: Amy E. Lovett-Racke,
460 West 12th Avenue, Room 0784,
Columbus, OH 43210, USA
Pro-inflammatory T cells mediate autoimmune demyelination in multiple sclerosis. However, the factors driving their develop-
ment and multiple sclerosis susceptibility are incompletely understood. We investigated how micro-RNAs, newly described as
post-transcriptional regulators of gene expression, contribute to pathogenic T-cell differentiation in multiple sclerosis. miR-128
and miR-27b were increased in naı ¨ve and miR-340 in memory CD4+T cells from patients with multiple sclerosis, inhibiting Th2
cell development and favouring pro-inflammatory Th1 responses. These effects were mediated by direct suppression of B
lymphoma Mo-MLV insertion region 1 homolog (BMI1) and interleukin-4 (IL4) expression, resulting in decreased GATA3
levels, and a Th2 to Th1 cytokine shift. Gain-of-function experiments with these micro-RNAs enhanced the encephalitogenic
potential of myelin-specific T cells in experimental autoimmune encephalomyelitis. In addition, treatment of multiple sclerosis
patient T cells with oligonucleotide micro-RNA inhibitors led to the restoration of Th2 responses. These data illustrate the
biological significance and therapeutic potential of these micro-RNAs in regulating T-cell phenotypes in multiple sclerosis.
Keywords: multiple sclerosis; miRNA; autoimmune T cells; Th1; Th2
Abbreviations: IL = interleukin; miRNA = micro-RNA; UTR = untranslated region
Multiple sclerosis afflicts over two million people worldwide
(Rosati, 2001) and is the primary cause of non-traumatic neuro-
logical disability in young adults (Frohman et al., 2006). In mul-
tiple sclerosis, inflammatory attack of the CNS causes myelin and
axon damage, resulting in the nerve conduction defects that
underlie multiple sclerosis symptoms. About 80% of patients
present with relapsing–remitting multiple sclerosis, characterized
by disability attacks interspersed with periods of recovery, which
is followed by secondary progressive multiple sclerosis in which
progressive disability continues without remissions. Other patients
present with primary progressive multiple sclerosis, characterized
by steadily progressing neurological disability from the onset
(Compston and Coles, 2008). Although significant progress has
been made in the management of this disease, the understanding
doi:10.1093/brain/awr262Brain 2011: 134; 3575–3586 |
Received April 14, 2011. Revised July 1, 2011. Accepted July 23, 2011. Advance Access publication November 15, 2011
? The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: email@example.com
of multiple sclerosis pathogenesis, discovery of useful disease
biomarkers and development of new therapies remain important
A conundrum in multiple sclerosis pathogenesis is that myelin-
specific T cells exist in all individuals (Giegerich et al., 1992;
Lovett-Racke et al., 1998). While these cells remain tolerant in
(Allegretta et al., 1990; Lovett-Racke et al., 1998) with pro-
inflammatory Th1 (Olsson et al., 1990; Balashov et al., 1997;
Pelfrey et al., 2000; Crawford et al., 2004; Couturier et al.,
2011) and/or Th17 (Lock et al., 2002; Kebir et al., 2007;
Tzartos et al., 2008) features in multiple sclerosis, in contrast to
more benign Th2 responses, ultimately leading to CNS inflamma-
tion and demyelination. The factor(s) that predispose multiple
sclerosis naı ¨ve T cells to become activated and cause inflammation
in multiple sclerosis remain largely unknown, with a single report
demonstrating how a multiple sclerosis-associated polymorphism
in the tyrosine kinase 2 gene can drive Th1 responses (Couturier
et al., 2011).
Both genetic and environmental factors contribute to the patho-
genesis of multiple sclerosis (Ebers, 2008). While genome-wide
association studies have demonstrated genetic associations with
multiple sclerosis (Oksenberg et al., 2008; Baranzini et al.,
2009a, b; De Jager et al., 2009), a large portion of hereditary
susceptibility remains unknown (Oksenberg and Baranzini, 2010).
Micro-RNAs (miRNAs) have recently emerged as important regu-
lators of gene expression (Ambros, 2004; Bartel, 2004) and their
expression can be influenced by both genetic and environmental
factors, making them attractive candidates in multiple sclerosis
miRNAs are small RNAs, 19–24nucleotides in length, that bind
the 30-untranslated region (UTR) of target messenger RNAs, there-
by inhibiting translation or inducing messenger RNA degradation
(Ambros, 2004; Bartel, 2004). miRNAs contribute to disease sus-
ceptibility, are useful susceptibility biomarkers and have provided
novel therapeutic targets, particularly in cancer (Croce, 2009). The
role of miRNAs in autoimmunity is just beginning to be elucidated,
but several miRNAs have been associated with rheumatic diseases
(Alevizos and Illei, 2010). Studies in peripheral blood mononuclear
cells have revealed miRNA dysregulation in multiple sclerosis
(Keller et al., 2009; Otaegui et al., 2009), and miR-326 has
been associated with pro-inflammatory responses (Du et al.,
2009). However, it is unclear whether these miRNA differences
reflect differences in peripheral blood mononuclear cell compos-
ition or activation state during active multiple sclerosis, or whether
they underlie the aetiopathogenesis of the disease.
Here, we investigated miRNA expression in purified naı ¨ve CD4+
T cells of patients with multiple sclerosis. The naı ¨ve CD4+T-cell
population represents cells that have not been activated, allowing
us to take a snapshot of how T cells in patients with multiple scler-
osis are poised to differentiate into pro-inflammatory phenotypes.
We report that miR-128 and miR-27b levels were increased in
naı ¨ve CD4+T cells of patients with multiple sclerosis. In addition,
miR-340 was upregulated in memory CD4+T cells of patients
with multiple sclerosis. These miRNAs acted in concert to suppress
Th2 differentiation and set the stage for pro-inflammatory Th1
autoimmune responses, illustrating how these miRNAs may con-
tribute to multiple sclerosis pathogenesis.
Materials and methods
Blood was obtained by leukapheresis from healthy donors or patients
with multiple sclerosis after informed consent. Patients were in clinical
remission and were treatment-naı ¨ve for immunomodulatory drugs.
Peripheral blood mononuclear cells isolated over a Ficoll gradient were
stored in liquid nitrogen until further use. This study was performed
under OSU Internal Review Board protocol # 2006H0235 and
Mice were bred in specific pathogen-free conditions at the OSU
University Laboratory Animal Resources, under protocol # 2009A0142.
Human naı ¨ve and memory CD4+
T cell isolation
Naı ¨ve CD4+CD45RA+cells were isolated on an AUTOMACSPro with
the Dead Cell Removal Kit (Miltenyi) followed by the negative selec-
tion naı ¨ve T cell isolation Kit II (Miltenyi) by depleting cells expressing
CD8, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD45RO,
CD56, CD123, TCR?/?, HLA-DR and CD235a (Glycophorin A).
More than 95% CD4+CD45RA+pure samples were used in this ana-
CD25?CD45RO?, without any differences observed between healthy
donors and multiple sclerosis (Supplementary Fig. 1). The mean ?
standard deviation (SD) per cent purity of CD4+CD45RA+cells in
the various populations was as follows: healthy donors (96.8 ? 1.3),
primary progressive multiple sclerosis (98 ? 0.8), relapsing–remitting
multiple sclerosis (97 ? 1.47) and secondary progressive multiple scler-
osis (96.4 ? 1.9). There were no significant differences in purity be-
tween multiple sclerosis groups and healthy donors. Re-analysis of the
Taqman miRNA data limited to samples with a CD4+CD45RA+purity
of 99% confirmed the upregulation of miR-128 and miR-27b: fold
change ? SD: miR-128 [healthy donors (2.756 ? 2.750, n = 2), mul-
tiple sclerosis (9.072 ? 0.5854, n = 4)]; miR-27b [healthy donors
(1.055 ? 1.045, n = 2), multiple sclerosis (8.915 ? 2.748, n = 4)].
Memory CD4+CD45RO+T cells were isolated similarly, albeit using
the Human Memory CD4+T cell isolation kit (Miltenyi) by depleting
cells expressing CD45RA, CD8, CD14, CD16, CD19, CD56, CD36,
CD123, anti-TCR?/? and CD235a (glycophorin A). More than 95%
pure samples were used in this analysis. The
mean ? SD percent purity of CD4+CD45RO+cells in the various
populations was as follows: healthy donors (98.08 ? 0.9), primary pro-
gressive multiple sclerosis (98 ? 1.1), relapsing–remitting multiple
sclerosis (97.7 ? 0.7) and secondary progressive multiple sclerosis
(98.5. ? 0.5). There were no significant differences in purity between
multiple sclerosis groups and healthy donors.
RNA was isolated with the mirVana kit (Ambion) total RNA isolation
protocol, according to the manufacturer’s instructions, and stored
at ?80?C until analysis.
Brain 2011: 134; 3575–3586M. Guerau-de-Arellano et al.
Naı ¨ve CD4+T-cell miRNA Taqman
Naı ¨ve CD4+T-cell miRNA expression profiling was performed at the
OSU Nucleic Acid Shared Resource with the TaqMan Array Human
miRNA Panel (v.2, Applied Biosystems) containing 667 miRNA targets
and endogenous controls. RNA (50ng) was complementary DNA-
transcribed and underwent 14 pre-amplification cycles. A 1:20 dilution
of this reaction was loaded onto the Taqman array microfluidic cards.
Initial analysis of raw data, stable normalizer selection and statistical
analysis were performed using the high-throughput real-time polymer-
ase chain reaction analysis software Statminer (Integromics) at
OSUCCC Biomedical Informatics Shared Resource. The small nucleolar
RNAs RNU44 and RNU48 were confirmed to be stably expressed in
our sample set and their mean used as the normalizer value. A relative
fold change expression was calculated relative to the average Ctvalue
for the healthy donors group.
Memory CD4+T-cell miRNA analysis
Memory CD4+T-cell miRNA analysis was performed with the novel
Nanostring nCounter technology at the OSU Nucleic Acid Shared
Resource. This highly sensitive and specific methodology is based on
the direct digital detection and enumeration of RNA transcripts after
binding and labelling by specific capture and colour-coded reporter
probes (Geiss et al., 2008). The nCounter miRNA assays can accurately
distinguish between highly similar miRNAs with great specificity, with-
out the need of enzymatic amplification.
Real-time polymerase chain reaction
RNA (300ng) from profiled samples was complementary DNA tran-
scribed using random primers. Human (h)BMI1 Sybr Green real-time
polymerase chain reaction was performed using hBMI1 and 18S con-
trol primers, as described (Godlewski et al., 2008). Taqman hGATA3
real-time polymerase chain reaction was performed using Applied
Biosystems hGATA3 and hHPRT primer sets. Results were analysed
using the comparative Ctmethod after confirmation of similar ampli-
fication efficiency for test and control genes. Relative fold change
expression was calculated relative to the average healthy donors
A fragment of the human BMI1 gene (NM_005180.6: 2044- 2661),
GATA3 gene (NM_001002295.1: 1943-2522) or the entire interleu-
kin-4 (IL-4) gene 30-UTR were cloned into the XbaI site on the
PGL3 control vector (Promega). The miR-27 site or the two
miR-340 binding sites in the 30-UTR of the human BMI-1 gene
were mutated (miR27 site:GTCACTGTGAA!GTCCGCCCGAA;
(Agilent). The miR-340 binding site in the human IL-4 gene
(ATAAATATTGAG!ATGAG) was deleted using the QuickChange XL
kit. Vector (150ng) and 200pmol of precursor nonsense (NS1) or test
miRNA (Applied Biosystems) were transfected with Lipofectamine
(Invitrogen) overnight into cos-7 cells. After a 44-h incubation, cells
were lysed in Cell Culture Luciferase Lysis Buffer (Promega). Luciferase
activity in the lysate was measured using the Luciferase Assay System
(Promega) in a FLUOstar Optima plate reader (BMG Labtech).
Luciferase activity was normalized to protein content and percent rela-
tive luciferase units calculated relative to the NS1 control.
miRNA transfection and T-cell
Human peripheral blood mononuclear cells or mouse T cells/spleno-
cytes were transfected overnight with 1350pmol of double-stranded
miRNA mimics or inhibitors (Dharmacon) using the Transit-TKO
Reagent (Mirus). Mimic inhibitor sequences were nonsense control
(NS) (CTATGTCATCCGCTCCAC) or miR-27 (UUCACAGUGGCUAA
miR-340 (UUAUAAAGCAAUGAGACUGAUU). For human T-cell acti-
vation, transfected cells were plated onto anti-hCD3/CD28-coated
plates for 48h in the absence of exogenously added cytokines (Th
neutral conditions: ThN), harvested, rested for 7 days and restimulated
with anti-hCD3/CD28 for 72h. For mouse T-cell activation, TcR-tg
splenocytes were stimulated with irradiated wild-type splenocytes
(1:5 T cells:feeders) and 2mg/ml MBPAc1-11 for 72h in the absence
of exogenously added cytokines, rested 7 days and restimulated in the
same conditions. To estimate the minimum transfection efficiency in
human T cells, a fluorescein isothyocyanate (FITC)-labelled small RNA
was transfected into CD4+T cells and analysed by flow cytometry.
The observed FITC-positive CD4+T cells was 39% (Supplementary
Fig. 2). The expected transfection efficiency of untagged miRNA
would be significantly higher since the large FITC molecule would
impede transfection and this was confirmed in biological assays in
which miRNA targets were analysed.
Human T-cell activation for assessment
of IL-4 secretion
Purified (595% CD4+CD45RA+) naı ¨ve human T cells were stimu-
lated for 48h on anti-CD3/CD28 (1mg/ml) coated plates in Th neutral
conditions. Cells were then rested for a total of 7 days in the presence
of rhIL-2 (5ng/ml), prior to restimulation with Phorbol myristate
acetate (PMA)/Ionomycin (5h) in the presence of GolgiStop (BD) for
the last 3h of the incubation.
After a 15-min Fc block reagent incubation, cells were stained with
surface antibodies for 20min at 4?C, fixed with E-bioscience (Bmi-1
and GATA3 staining) or BD (IL-4 staining) FixPerm Reagent (30min,
4?C) and blocked with Fc block for 15min prior to staining with intra-
cellular antibodies (30–45min, 4?C). Data were acquired on a
FACSCantoII flow cytometer. The antibodies used were hCD3, hCD4
and hIL-4 (BD), hCD45RA (Biolegend), mCD4 and mCD62L(BD),
mCD44 and m/hGATA3 (eBioscience) and hBMI-1 (R&D).
Adoptive transfer experimental
Myelin basic protein peptide Ac1-11-specific ?b TcR-tg B10.PL mouse
splenocytes (Goverman et al., 1993) were transfected overnight
with NS or miR-27, miR-128 and miR-340 mimics. Cells were har-
vested, washed and stimulated with 2mg/ml of MBPAc1-11 in
the presence of irradiated B10.PL splenocytes for 72h in ThN condi-
tions. A suboptimal number of cells (4 ? 106/mouse) was transferred
intraperitoneally into B10.PL recipients. As expected, experimental
Inflammatory miRNAs in multiple sclerosisBrain 2011: 134; 3575–3586 |
autoimmune encephalomyelitis severity in control miRNA-transfected
mice was mild due to the Th neutral conditions used during T-cell
activation as well as the suboptimal number of adoptively transferred
cells. Experimental autoimmune encephalomyelitis was scored on a
scale of 0–6: 0 (no clinical disease), 1 (limp/flaccid tail), 2 (moderate
hind limb weakness), 3 (severe hind limb weakness), 4 (complete hind
limb paralysis), 5 (quadriplegia or premoribund state) and 6 (death).
Unless otherwise indicated, cytokines were detected in 72-h super-
natants post-stimulation by a sandwich enzyme-linked immunosorbent
assay (ELISA). Human and mouse interferon (IFN)-? and IL-4 reagents
were from BD and human and mouse IL-5 reagents from R&D DuoSet
ELISA. ELISA was performed as previously described (BD) (Yang et al.,
2009) or following the manufacturer’s instructions (R&D).
Th2 cell line generation
The Th2 cell line was generated with MBPAc1-11-specific TcR-tg naı ¨ve
CD4+T cells isolated by magnetic bead sorting and stimulated with
2mg/ml of MBPAc1-11 peptide and irradiated syngeneic feeder cells in
the presence of mouse (m) IL-4 (1500U/ml), anti-IFN-? (10mg/ml)
and anti-mIL-12 (10mg/ml). Three days post-primary stimulation,
cells were split and received human (h) IL-2 (5ng/ml). Cells were
subsequently restimulated every 7–10 days with 1mg/ml MBPAc1-11
with irradiated feeders and rhIL-2. Transfection with miR-340 mimic
was performed 18h prior to the fourth or later rounds of restimulation.
Statistical significance was determined with Statminer software para-
metric moderated Limma test optimized to adjust significance for the
large datasets of Taqman array data (healthy donors to all multiple
sclerosis or healthy donors to specific multiple sclerosis subtype com-
parisons in Taqman miRNA profiling). The parametric Limma test com-
putes the statistical significance of the detector expression moderating
the standard errors across all the detectors using a simple Bayesian
model (Smyth, 2004). The effectiveness of this approach has been
demonstrated on differential expression data sets (Tadesse et al.,
2005). A false discovery rate (FDR) q-value of 0.05 was used as a
cut-off to identify the miRNAs differentially regulated in multiple scler-
osis versus healthy donors. In the remaining comparisons, Student’s
t-test (single treatment to control comparisons), Dunnett’s post hoc
test after a significant one-way ANOVA (multiple comparisons of
treatment to control groups) or Mann–Whitney t-test (experimental
autoimmune encephalomyelitis control to treatment group compari-
son) were performed using GraphPad Prism software.
Increased miR-128 and miR-27
expression in naı ¨ve CD4+T cells
of patients with multiple sclerosis
In order to determine if there is a baseline miRNA dysregulation in
patients with multiple sclerosis, we isolated naı ¨ve CD4+CD45RA+
T cells (595% pure, Supplementary Fig. 1A) from peripheral blood
mononuclear cells of healthy donors (n = 16) or treatment-naı ¨ve
patients with multiple sclerosis (n = 22), falling into the primary
progressive (n = 5), relapsing–remitting (n = 12) or secondary pro-
gressive (n = 5) categories (Supplementary Table 1). The naı ¨ve
status of the CD4+
T cells was verified by post-purification
analysis ofCD45RA, CD45RO,
(Supplementary Fig. 1B–C). A Taqman array of 667 miRNAs was
The profiling data were analysed by comparing healthy donors
to either the entire multiple sclerosis patient population or individ-
ual multiple sclerosis subtypes. There were 85 miRNAs differen-
tially expressed (P50.05, FDR q50.05) in the comparison of
healthy donors to all patients with multiple sclerosis, irrespective of
multiple sclerosis subtype (primary progressive, relapsing–remitting
and secondary progressive; Supplementary Table 2). Individual
multiple sclerosis subtype versus healthy donors analysis yielded
61, 106 or 55 significantly different miRNAs (P50.05) in primary
progressive, relapsing–remitting or secondary progressive, respect-
ively (Supplementary Table 3). miR-128 was the second most sig-
nificant upregulated miRNA in patients with multiple sclerosis (16–
31-fold increase in the various multiple sclerosis subtypes, calcu-
lated relative to healthy donors geometric mean). In addition, it
became of interest because it was one of the few miRNAs that
were significantly upregulated in primary progressive, relapsing–
remitting and secondary progressive in our multiple sclerosis sub-
type analysis (Supplementary Table 3 and Fig. 1A and B).
Although primary progressive multiple sclerosis is clinically very
different from relapsing–remitting/secondary progressive multiple
sclerosis, they all involve immune attack on the CNS and a miRNA
commonly dysregulated in these three multiple sclerosis sub-
types may regulate a critical pathway in encephalitogenicity.
Furthermore, miR-128 has recently been shown to repress expres-
sion of BMI1 (Godlewski et al., 2008), which stabilizes the Th2
transcription factor GATA3 in T cells (Hosokawa et al., 2006). We
reasoned that increased miR-128 in multiple sclerosis naı ¨ve CD4+
T cells could target BMI1 to inhibit Th2 and promote pro-
inflammatory Th1 differentiation
In order to achieve their biological effects, miRNAs often act in
complex networks, with multiple miRNAs targeting the same gene
(Wu et al., 2010) or a single miRNA targeting multiple genes in
the same pathway (Boettger et al., 2009). Therefore, we investi-
gated whether other overexpressed miRNAs in multiple sclerosis T
cells identified in the miRNA profiling target BMI1 or other Th2
pathway genes. RNA hybrid software predicted targeting of BMI1
by two highly conserved miRNAs upregulated in multiple sclerosis:
miR-128 and miR-27b (Table 1). miR-27b was increased 5-fold in
the pooled multiple sclerosis population (Supplementary Table 2,
Fig. 1A) and particular multiple sclerosis groups (Supplementary
Table 3, Fig. 1B). miR-27a and miR-27b are highly homologous,
they share the same predicted targets, but are encoded by two
different chromosomes. We therefore analysed miR-27a in these
samples and observed a modest increase in patients with multiple
sclerosis (fold change = 2.2) that was not significant (P = 0.07;
Fig. 1A). Chromosome 9 encodes miR-27b and thus, is the
source of the overexpressed miR-27 observed in CD4+T cells of
patients with multiple sclerosis. miR-27 shares the third best
Brain 2011: 134; 3575–3586M. Guerau-de-Arellano et al.
context score among conserved miRNAs for BMI1 targeting after
miR-128, which is the top candidate (Table 1). In addition, when
we investigated whether other genes of the Th2 pathway were
targeted by multiple sclerosis-associated miRNAs, we found that
miR-27 and miR-128 were predicted to target GATA3, suggesting
that these miRNAs may be targeting two critical elements in the
Th2 differentiation pathway. Since IL-4 is the signature cytokine of
Th2 cells, we analysed whether any miRNAs were predicted to
target IL-4. miR-340 was one of only a few miRNAs predicted
to target IL-4 and interestingly, miR-340 was also predicted to
target BMI1.Although miR-340
overexpressed in the naı ¨ve CD4+T cells in patients with multiple
sclerosis,it washighly (36-fold
(P510?5) overexpressed in resting memory CD4+CD45RO+
T cells in relapsing–remitting multiple sclerosis and secondary pro-
gressive multiple sclerosis (Fig. 1C), where it could regulate IL-4
production. Importantly, the expression of these miRNAs was in-
dependent of sex or donor age, as confirmed by both linear re-
gression analysis and Pearson’s correlation analysis (Supplementary
Fig. 3 and Supplementary Table 4 and data not shown), ruling out
age as a factor affecting miR-128, miR-27 and miR-340 expres-
sion in patients with multiple sclerosis. Overall, it appeared that
there could be collaborative targeting of the Th2 pathway by sev-
eral miRNAs upregulated in naı ¨ve and memory CD4+T cells of
patients with multiple sclerosis.
change) and significantly
Multiple sclerosis-associated miRNAs
target genes of the Th2 pathway
To validate target prediction analyses, we determined whether the
BMI1, GATA3 and IL-4 transcripts were bona fide direct targets of
miR-128, -27 or -340. A luciferase vector containing the 30-UTR
of the hBMI1 transcript was transfected into cos-7 cells along with
NS or test miRNAs. miR-128, -27 and -340 significantly down-
regulated luciferase expression. Mutating the 30-UTR miRNA-
binding sites for miR-27 or miR-340 restored luciferase activity
(Fig. 2A), confirming direct and specific targeting of BMI1 by
these miRNAs. The specificity of miR-128 binding to the BMI1
30-UTR by restoration of luciferase activity upon miR-128 site
mutation has already been proven (Godlewski et al., 2008).
Figure 1 miR-128, miR-27 and miR-340 are overexpressed in
multiple sclerosis CD4+T cells. (A) miR-128 (left) and miR-27a
or miR-27b (right) expression in purified naı ¨ve CD4+T cells from
healthy donors (HD; n = 16) and multiple sclerosis (MS; n = 22,
including five primary progressive, 12 relapsing–remitting and
five secondary progressive) by Taqman real-time polymerase
chain reaction array. (B) Distribution of miR-128 and miR-27b
expression by Taqman array in primary progressive (PP) multiple
sclerosis, relapsing–remitting (RR) multiple sclerosis and
secondary progressive (SP) multiple sclerosis subtypes.
(C) miR-340 expression counts in purified memory CD4+T cells
from healthy donors (n = 17), all multiple sclerosis (n = 19),
primary progressive multiple sclerosis (n = 4), relapsing–remit-
ting multiple sclerosis (n = 11) or secondary progressive multiple
sclerosis (n = 4) by Nanostring nCounter detection. miRNA
fold-changes were calculated relative to healthy donors Ctvalue
geometric mean and geometric mean values for healthy
donors and patients with multiple sclerosis are shown by the
lines (A and B). Statminer’s Limma test P-values for the healthy
donors to multiple sclerosis groups comparisons shown in A–C.
Table 1 Th2 pathway miRNA targeting
Lower (more negative) TargetScan context scores indicate higher predicted
binding. m.f.e. = mean free energy.
Inflammatory miRNAs in multiple sclerosisBrain 2011: 134; 3575–3586 |
Importantly, we observed an inverse correlation of miR-128 ex-
pression and BMI1 transcripts in the profiled naı ¨ve CD4+T cells
(Fig. 2B) and miR-128, miR-27 and miR-340 repressed BMI1 pro-
tein expressed in glioma cells (Supplementary Fig. 4). In contrast,
none of the predicted GATA3 suppressing miRNAs repressed luci-
ferase expression from the GATA3-30-UTR-containing vector (Fig.
2C), indicating GATA3 is not a direct target of these miRNAs.
The predicted targeting of IL-4 by miR-340 (Table 1) was con-
firmed by the efficient repression of luciferase activity from an
IL-4-30-UTR-tagged luciferase vector (Fig. 2D). This shows direct
and specific targeting of the IL-4 gene by miR-340 and indicates
that, besides targeting the Th2 pathway at the differentiation
stage, miR-340 may additionally target the effector stage via inhi-
bition of IL-4 production. To confirm this, miR-340 was trans-
fected into a fully differentiated IL-4-producing Th2 cell line,
resulting in a reduction in IL-4 secretion (Fig. 2E). This reduction
of IL-4 in the Th2 cell line was not secondary to loss of
Th2-commitment, as shown by maintenance of GATA3 expression
and IL-5 secretion (Fig. 2E and Supplementary Fig. 5), yet in naı ¨ve
T cells miR-340 overexpression would be expected to inhibit Th2
cell differentiation. Overall, these results suggest that the dysre-
gulated miRNAs in patients with multiple sclerosis can suppress the
Th2 pathway through repression of BMI1 and IL-4 (Fig. 3).
Dysregulated miRNAs repress BMI1 and
GATA3 in T cells, exacerbating
We hypothesized that the increase in miR-128 and miR-27 in
multiple sclerosis naı ¨ve CD4+T cells would inhibit Th2 differenti-
ation and miR-340 in multiple sclerosis memory T cells and would
alter effector T-cell function. To test this, myelin basic protein-
specific T-cell receptor transgenic (TcR-tg) naı ¨ve mouse spleno-
cytes were transfected with miRNAs prior to activation. Indeed,
the percentage of BMI1+, GATA3+and BMI1+GATA3+CD4 T
cells representative of Th2 differentiation was decreased upon
transfection with individual miRNAs (Fig. 4A–B). The largest
Figure 2 Multiple sclerosis-associated miRNAs target Th2
pathway genes. (A) Relative luciferase units (RLU) from a luci-
ferase vector carrying the predicted multiple sclerosis-associated
miRNA wild-type (WT) or mutant binding sites (mut) from the
hBMI1 gene 30-UTR (indicated above bars) in cos-7 cells trans-
fected with multiple sclerosis-associated miRNAs. Mean relative
luciferase units ? SEM are shown as percentage of the control
NS miRNA. Results from three independent experiments are
shown. The miR-128 wild-type data have been previously re-
ported (Godlewski et al., 2008) and are shown here for con-
firmation and comparison of the relative effects of each miRNA.
The miR-128 mutant data have also been published (Godlewski
et al., 2008). (B) Non-linear regression Pearson’s correlation
analysis of the relationship between BMI1 and miR-128
expression in naı ¨ve CD4+T cells from healthy donors and
patients with multiple sclerosis. Pearson P (indicating statistical
significance) and r (indicating negative correlation) values shown.
Figure 2 Continued
(C) Luciferase assay with a hGATA3 30-UTR luciferase vector
after transfection of cos-7 cells with multiple sclerosis-associated
miRNAs predicted to bind GATA3 messenger RNA. Mean rela-
tive luciferase units ? SEM results from multiple replicates of
three independent experiments shown. (D) Luciferase assay with
a hIL-4 30-UTR luciferase vector after transfection with miR-340.
Mean relative luciferase units ? SEM results from multiple
replicates of two independent experiments shown. (E) IL-4 (left)
and IL-5 (right) expression determined by ELISA in 24-h
supernatants from a myelin basic protein Ac1-11–specific Th2
cell line transfected overnight with NS miRNA or miR-340 prior
to stimulation with myelin basic protein Ac1-11 peptide.
Mean ? SEM results shown are intra-experimental replicates,
representative of two independent experiments. Dunnett’s post
hoc (A and C–E) or t (E) test P-value: **P40.005. RU = relative
Brain 2011: 134; 3575–3586M. Guerau-de-Arellano et al.
reduction was observed when we combined all miRNAs to better
mimic the phenotype of multiple sclerosis T cells. BMI1 expression
was reduced on a per-cell basis, while the effects on GATA3 were
primarily visible as a reduction of the GATA3+population rather
than its mean fluorescence intensity (Fig. 4C). These results are
consistent with miR-27, miR-128 and miR-340 primarily inhibiting
BMI1 expression while loss of the GATA3+population, indicative
of Th2 phenotype, would be a consequence of BMI1 inhibition
and reduced Th2 differentiation. To determine if the reduced
number of BMI1+GATA3+T cells correlated with an enhanced
pathogenic phenotype, MBPAc1-11 TcR-tg cells transfected with
the miRNA combination were transferred into mice and monitored
for signs of experimental autoimmune encephalomyelitis (Fig. 4D).
Both the incidence and severity of experimental autoimmune en-
cephalomyelitis were enhanced in the mice that received the
miRNA-transfected T cells, illustrating that these miRNAs regulate
genes that modulate T-cell pathogenicity.
Multiple sclerosis-associated miRNAs
suppress Th2 and favour Th1 cytokine
To determine if these miRNAs were influencing Th1 and Th2 dif-
ferentiation, CD4+T cells isolated from Th1-prone C57BL/6 (B6)
mice were activated after transfection with miRNAs and miRNA
inhibitors, and effector cytokine secretion was measured by ELISA.
As expected, miR-27 and miR-128 increased IFN-? production,
while miR-27 and miR-128 inhibitors decreased it (Fig. 5A; no
IL-17 was detected). In these Th neutral (no exogenously added
cytokines) culture conditions, no IL-4 and only low levels of IL-5
were detected, consistent with the Th1 bias of B6 mice. To better
visualize the effects of these miRNAs on Th2 cytokine produc-
Th2-prone Balb/c mice were transfected with individual miRNAs,
prior to two rounds of activation in Th neutral conditions, allow-
ing differentiation into the Th2 pathway. IFN-? production was
increased in miR-27 transfected T cells, while both miRNAs sup-
pressed Th2 (IL-4 and IL-5) cytokine production (Fig. 5B,
Supplementary Fig. 6), indicating that these miRNAs influence
Dysregulated miRNAs repress BMI1 and
GATA3 in human CD4+T cells and their
inactivation restores Th2 cytokine
To determine whether these findings could be recapitulated in
humans, miRNAs were transfected into human peripheral blood
Figure 3 Model of the effects of multiple sclerosis-associated miRNAs on Th2 pathway genes. Th2 cell commitment is mediated by IL-4
receptor engagement during T-cell activation, resulting in STAT6 phosphorylation and transactivation of the GATA3 gene. In turn, GATA3
initiates transcription of the IL-4, IL-5 and IL-13 cytokine genes. BMI1 stabilizes GATA3, and BMI1 repression by increased multiple
sclerosis-associated miR-27, and miR-128 expression in naı ¨ve CD4+T cells would result in enhanced GATA3 degradation and reduced Th2
differentiation. In addition, increased miR-340 in memory CD4+T cells of patients with multiple sclerosis would directly target the IL-4
gene at the effector cell level.
Inflammatory miRNAs in multiple sclerosis Brain 2011: 134; 3575–3586 |
mononuclear cells prior to polyclonal stimulation. A decrease in the
BMI1+, GATA3+and BMI1+GATA3+T-cell populations with in-
dividual or combined miRNAs was observed (Fig. 6A and B), as
seen in mice. The mean expression of both BMI1 and GATA3 was
decreased (Fig. 6C). We hypothesized that overexpression of these
miRNAs in peripheral blood mononuclear cells from healthy pa-
tients should recapitulate the phenotype of patients with multiple
sclerosis, while inhibition of these miRNAs in T cells of patients
with multiple sclerosis should recapitulate the healthy donors
phenotype. To test this, healthy donors and multiple sclerosis pa-
tient samples were transfected with the miRNAs combination or
their inhibitors, respectively, and Th2 cytokine production was
analysed post-secondary stimulation. A reduction in IL-5 secretion
was observed in healthy donors samples treated with multiple
sclerosis-associated miRNAs, while miRNA inhibitor-treated mul-
tiple sclerosis samples increased IL-5 production (Fig. 6D). No
IL-4 was detected at this early point of differentiation and the
percentage of GATA3+cells increased in correlation with IL-5.
In addition, we determined whether there was a relationship be-
tween the endogenous miR-128 and miR-27 levels in humans and
the propensity of naı ¨ve CD4+T cells to differentiate into Th2 cells.
Naı ¨ve CD4+T cells were polyclonally activated under neutral con-
ditions, rested, reactivated and analysed for IL-4 expression. There
was an inverse relationship between the percentage of Th2 cells
and the level of miR-128 and miR-27 (Fig. 6E). This illustrates that
overexpression of these miRNAs in patients with multiple sclerosis
may predispose to the development of a Th1 response and
0246 8 10 12 14
Figure 4 Multiple sclerosis-associated miRNAs repress BMI1 and GATA3 in CD4+T cells and exacerbate experimental autoimmune
encephalomyelitis (EAE). (A) BMI1 versus forward scatter (FSC) (top), GATA3+versus FSC (middle) and BMI1 versus GATA3 (lower)
intranuclear staining of CD4+T cells from myelin basic protein-specific TcR-tg cells transfected with NS, miR-27, miR-128, miR-340 or
a combination of miR-27, miR-128 and miR-340 (miR mix) prior to stimulation with MBPAc1-11 peptide. Results are representative
of three independent experiments, which are pooled in the bar graphs in B (mean ? SEM, *t-test P40.05). (C) Histogram of BMI1
and GATA3 expression in cells from A to B, showing the mean fluorescence intensity in CD4+T cells transfected with control miRNA
or miR mix. The plots show individual results representative of three independent experiments. (D) Myelin basic protein Ac1-11-specific
TcR-tg splenocytes were transfected with control miRNA or miR mix before stimulation with myelin basic protein Ac1-11 in Th neutral
conditions for 72h, transferred to B10.PL mice and monitored for signs of experimental autoimmune encephalomyelitis. Four
out of seven mice receiving control miRNA-transfected cells and six out of seven mice receiving multiple sclerosis miRNA-transfected cells
developed experimental autoimmune encephalomyelitis. Mean ? SEM experimental autoimmune encephalomyelitis score is shown.
The plots show one experiment and are representative of two independent experiments. Significance was calculated using
Brain 2011: 134; 3575–3586 M. Guerau-de-Arellano et al.
Myelin-specific T cells exist in healthy individuals (Giegerich et al.,
1992; Lovett-Racke et al., 1998) indicating that the mere presence
of these cells does not result in the development of multiple scler-
osis. However, in patients with multiple sclerosis these T cells have
an activated phenotype (Allegretta et al., 1990; Olsson et al.,
1990; Balashov et al., 1997; Lovett-Racke et al., 1998; Pelfrey
et al., 2000; Crawford et al., 2004) and a tendency to differenti-
ate into pro-inflammatory phenotypes (Windhagen et al., 1998;
Couturier et al., 2011). Here, by comparing the miRNA profiles of
CD4+T cells of healthy donors and patients with multiple scler-
osis, we found that three miRNAs overexpressed in patients with
multiple sclerosis, miR-128, miR-27 and miR-340, had the ability
to inhibit Th2 differentiation (Fig. 3) and favour pathogenic Th1
differentiation in mouse and human cells, ultimately enhancing the
encephalitogenic capacity of myelin-specific T cells in adoptively
transferred experimental autoimmune encephalomyelitis.
The importance of miRNAs in multiple sclerosis is highlighted by
the number of recent studies showing miRNA expression differ-
ences in multiple sclerosis. However, little or no overlap has been
observed in the miRNAs identified by the different studies. This
can best be explained by the different tissues and cell populations
studied: brain (Junker et al., 2009), whole blood (Keller et al.,
2009; Cox et al., 2010), peripheral blood mononuclear cells
(Otaegui et al., 2009), total CD4+T cells (Du et al., 2009;
Lindberg et al., 2010) or CD4+CD25highT cells (De Santis
et al., 2010). Since miRNA expression varies between cell types,
miRNA changes may reflect different cellular composition or acti-
vation states. Our study is the first to analyse the miRNA profile of
highly purified naı ¨ve CD4+CD45RA+T cells. By focusing on naı ¨ve
(i.e. never activated) CD4+T cells, our experiments avoid non-
specific effects due to activation. In addition, this experimental
design allowed us to identify dysregulated miRNAs that may
cause or enhance susceptibility to multiple sclerosis. Interestingly,
we observed some healthy individuals displaying high levels of
these miRNAs, indicating they may have an increased risk for de-
velopment of multiple sclerosis or perhaps other pro-inflammatory
diseases. Besides miR-128 and miR-27, a number of additional
miRNAs were differentially expressed in multiple sclerosis com-
pared with naı ¨ve T cells of healthy donors, which may regulate
additional pathways. In addition, the identification of another Th2
targeting miRNA, miR-340, as overexpressed in patients with mul-
tiple sclerosis memory T-cell population, provides a potential mech-
anism by which Th2 response inhibition is perpetuated in multiple
Interestingly, the incidence of other Th1-mediated autoimmune
diseases, such as diabetes, thyroiditis and psoriasis, is increased in
patients with multiple sclerosis relative to the general population
(Roquer et al., 1987; Karni and Abramsky, 1999; Sloka, 2002;
Annunziata et al., 2003; Nielsen et al., 2006). In fact, it is possible
that the miRNA dysregulation observed in multiple sclerosis is not
specific to multiple sclerosis, but common to several autoimmune
diseases. In addition, multiple sclerosis has been associated with
reduced Th2-associated diseases, such as allergy and asthma
(Bergamaschi et al., 2009; Pedotti et al., 2009). The increased
expression of miR-27, miR-128 and miR-340 may enhance sus-
ceptibility to inflammation in a target organ-independent manner
while HLA genes, stochastic TcR rearrangement processes or epi-
genetic regulation and specific environmental triggers facilitate
CNS targeting in multiple sclerosis. Thus far, no studies have been
reported on the role of miRNAs in allergies, but one might predict
that miR-27, miR-128 and miR-340 would be decreased in
Figure 5 Multiple sclerosis-associated miRNAs suppress Th2
and favour Th1 cytokine production. (A) Per cent change (over
NS) in IFN-? secretion detected by ELISA in 72h supernatants of
purified CD4+T cells from Th1-prone B6 mice after stimulation
with plate-bound anti-CD3/CD28 in Th neutral conditions fol-
lowing overnight transfection with miRNAs (top) or miRNA in-
hibitors (bottom). Mean ? SEM data from multiple replicates of
two independent experiments. (B) IFN-? (top), IL-4 (middle)
and IL-5 (bottom) % change (over NS) detected by ELISA in
supernatants 72h after restimulation of DO11.10 TcR-tg Balb/c
mouse T cells with OVA 323–339 peptides. Initial stimulation
was performed 7–10 days earlier in Th neutral conditions, fol-
lowing overnight transfection with miRNAs or miRNA inhibitors.
Mean ? SEM data from multiple replicates of two independent
experiments. After significant ANOVA analysis, Dunnett’s post
hoc test P-values were calculated for the multiple sclerosis
miRNA to control miRNA comparison and are shown above each
group (**P40.001, *P40.05).
Inflammatory miRNAs in multiple sclerosisBrain 2011: 134; 3575–3586 |
individuals with atopy. Although Th17 cells have also been impli-
cated in multiple sclerosis, no IL-17 was detected using Th neutral
conditions in any of our assays and thus, the effect of these
miRNAs on IL-17 could not be assessed. However, the literature
demonstrates that suppressing the Th2 pathway enhances Th17
differentiation, suggesting that overexpression of miR-27, miR-
128 and miR-340 would enhance both Th1 and Th17 cell
development in the appropriate inflammatory environment (Park
et al., 2005).
Multiple mechanisms may contribute to altered expression of
miRNAs in multiple sclerosis. miRNAs can be located in intergenic
regions or within host gene introns, and can therefore be ex-
pressed using their own promoters or as bystanders when their
host gene is expressed. The fact that none of the miRNA genes
Figure 6 Multiple sclerosis-associated miRNAs suppress BMI1 and GATA3 in human CD4+T cells and Th2 cytokine production. (A) BMI1
versus forward scatter (FSC) (top), GATA3+versus FSC (middle) and BMI1 versus GATA3 (lower) intranuclear staining of CD4+T cells
from peripheral blood mononuclear cells of healthy donors transfected with NS, miR-27, miR-128, miR-340 or a combination of miR-27,
miR-128 and miR-340 (miR mix), stimulated with anti-CD3/CD28 in Th neutral conditions, and analysed for intranuclear staining by flow
cytometry. The panels in B show the mean ? SEM pooled results from three independent experiments (*t-test P40.05). (C) Histogram of
BMI1 and GATA3 expression in cells from A to B, showing the mean fluorescence intensity in CD4+T cells transfected with control miRNA
or a combination of multiple sclerosis-associated miRNAs. The plots show individual results representative of three independent experi-
ments. (D) Peripheral blood mononuclear cells from healthy donors (HD) were transfected with multiple sclerosis-associated or control
miRNAs, and peripheral blood mononuclear cells from patients with multiple sclerosis were transfected with miRNA inhibitors prior to
primary stimulation with anti-CD3/CD28 in Th neutral conditions. Cells were rested and restimulated with anti-CD3/CD28 and IL-5 was
measured by ELISA. Shows mean ? SEM of individual patient replicates, representative of two independent experiments. Dunnett’s post
hoc test P-values for the multiple sclerosis miRNA to control miRNA comparison are shown above each group (**P40.01, *P40.05).
(E) Association between endogenous miRNA expression and Th2 pathway differentiation. Healthy donors (black) or multiple sclerosis
(grey) purified naı ¨ve CD4+CD45RA+T cells with low or high, respectively, miR-27+ miR-128 expression (shown on right axis) were
stimulated with anti-CD3/CD28 in Th neutral conditions and, after differentiation, the percentage of IL-4 + Th2 cells assessed by flow
cytometry (shown on the left axis).
Brain 2011: 134; 3575–3586M. Guerau-de-Arellano et al.
studied here were encoded in loci identified in multiple sclerosis
genome-wide association studies published to date, suggests their
differential expression in multiple sclerosis may not be due to a
genetic change in the miRNA locus. However, some miRNA loci
were adjacent or nearby to identified loci (Supplementary Table 5),
which may affect chromatin structure and gene expression on
nearby loci. If miR-128 dysregulation was extended to CNS tis-
sues, it may be of significance in multiple sclerosis since miR-128 is
enriched in brain, and several miR-128 targets important in neur-
onal physiology have been identified, including NTRK3, Reelin,
DCX, E2F3a and SNAP25 (Eletto et al., 2008; Evangelisti et al.,
2009; Zhang et al., 2009; Guidi et al., 2010). It has also been
shown that miRNAs are expressed in response to infections to
silence viral replication (Pedersen et al., 2007), raising the possi-
bility that miRNA expression modulation during infections contrib-
utes to increased multiple sclerosis risk. Thus, it will be interesting
to determine whether the genetic variations and infections asso-
ciated with multiple sclerosis are related to the observed miRNA
The finding that miR-27, miR-128 and miR-340 are over-
expressed in patients with multiple sclerosis opens the door to pre-
ventative and/or therapeutic strategies that inhibit these miRNAs.
We found that treatment of T cells from patients with multiple
sclerosis with miRNA inhibitors specific for miR-27, miR-128 and
miR-340 was able to restore Th2 cytokine production. Although
the efficiency of this therapeutic strategy may be highest early in
the disease, the fact that miR-340 inhibitors are expected to re-
lieve the inhibition of Th2 pathway end-products indicates that
targeting these miRNAs may be able to inhibit pro-inflammatory
effector T cells, not merely prevent their differentiation.
By analysing miRNA differences in the CD4+T-cell population,
we have identified three miRNAs that target the Th2 pathway,
contributing to the susceptibility of patients with multiple sclerosis
to develop the pro-inflammatory myelin-specific T cells that me-
diate CNS pathology. These miRNAs have significant value as
potential multiple sclerosis biomarkers and therapeutic targets
(Ferracin et al., 2010).
We are grateful to Dr Hansjuerg Alder (OSUCCC Nucleic Acid
Shared Resource) for Taqman array expertise, Dr Kun Huang
and Dr Gulcin Ozer (OSUCCC Biomedical Informatics) for bio-
informatics analysis expertise, Dr Hayes-Ozello (OSU Veterinary
School Flow Cytometry Core) for AUTOMACS expertise and C
Pannell for expert animal care. We also thank Dr Joanne Turner,
Dr Virginia Sanders, Dr Terry Elton, Haiyan Peng, Dave Huss, Alan
Smith and members of the Neuroimmunology Seminar Series for
helpful discussions and critical reading of the article.
National Institutes of Health grants (R21 NS067383 to A.E.L.-R.),
(R01 NS067441 to A.E.L.-R.), (R01 NS037513 to M.K.R.) and
(K24 NS44250 to M.K.R.); National Multiple Sclerosis Society
grant (RG 3812 to A.E.L.-R.).
Supplementary material is available at Brain online.
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