Search for specific biomarkers of IFNβ bioactivity in patients with multiple sclerosis.
ABSTRACT Myxovirus A (MxA), a protein encoded by the MX1 gene with antiviral activity, has proven to be a sensitive measure of IFNβ bioactivity in multiple sclerosis (MS). However, the use of MxA as a biomarker of IFNβ bioactivity has been criticized for the lack of evidence of its role on disease pathogenesis and the clinical response to IFNβ. Here, we aimed to identify specific biomarkers of IFNβ bioactivity in order to compare their gene expression induction by type I IFNs with the MxA, and to investigate their potential role in MS pathogenesis. Gene expression microarrays were performed in PBMC from MS patients who developed neutralizing antibodies (NAB) to IFNβ at 12 and/or 24 months of treatment and patients who remained NAB negative. Nine genes followed patterns in gene expression over time similar to the MX1, which was considered the gold standard gene, and were selected for further experiments: IFI6, IFI27, IFI44L, IFIT1, HERC5, LY6E, RSAD2, SIGLEC1, and USP18. In vitro experiments in PBMC from healthy controls revealed specific induction of selected biomarkers by IFNβ but not IFNγ, and several markers, in particular USP18 and HERC5, were shown to be significantly induced at lower IFNβ concentrations and more selective than the MX1 as biomarkers of IFNβ bioactivity. In addition, USP18 expression was deficient in MS patients compared with healthy controls (p = 0.0004). We propose specific biomarkers that may be considered in addition to the MxA to evaluate IFNβ bioactivity, and to further explore their implication in MS pathogenesis.
- [show abstract] [hide abstract]
ABSTRACT: Mx proteins are interferon-induced GTPases that belong to the dynamin superfamily of large GTPases. Similarities include a high molecular weight, a propensity to self-assemble, a relatively low affinity for GTP, and a high intrinsic rate of GTP hydrolysis. A unique property of Mx GTPases is their antiviral activity against a wide range of RNA viruses, including bunya- and orthomyxoviruses. The human MxA GTPase accumulates in the cytoplasm of interferon-treated cells, partly associating with the endoplasmic reticulum. In the case of bunyaviruses, MxA interferes with transport of the viral nucleocapsid protein (N) to the Golgi compartment, the site of virus assembly. In the case of Thogoto virus (an orthomyxovirus), MxA prevents the incoming viral nucleocapsids from being transported into the nucleus, the site of viral transcription and replication. In both cases, the GTP-binding and carboxy-terminal effector functions of MxA are required for target recognition. In general, Mx GTPases appear to detect viral infection by sensing nucleocapsid-like structures. As a consequence, these viral components are trapped and sorted to locations where they become unavailable for the generation of new virus particles.Traffic 11/2002; 3(10):710-7. · 4.65 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Interferon-beta (IFNbeta) has proven to be an important advance in the therapy of multiple sclerosis (MS), but optimal markers for bioactivity have not been identified. To accurately measure bioactivity in MS patients treated with IFNbeta, we developed and tested a real-time reverse transcriptase (RT)-PCR assay for gene expression of MxA, an IFNbeta-induced gene in the peripheral blood of patients treated with IFNbeta. We compared IFNbeta-treated patients with MS to controls in expression of MxA relative to the house-keeping gene, GAPDH. 2'-5'oligoadenylate synthetase (OAS) gene expression was also tested by real-time RT-PCR on RNA from the same patient specimens. Anti-IFNbeta antibody was measured by ELISA and a cytopathic effect assay. Seven of 54 patients were found to have complete loss of bioactivity. MxA expression correlated well with OAS expression. All patients with lost bioactivity had high levels of binding antibodies or neutralizing antibodies. This is the first demonstration that a real-time RT-PCR assay can be used to monitor therapy with interferons. These data identify MxA mRNA as an excellent biomarker for INFbeta action on the IFN receptor, and clarify the relationship between anti-IFNbeta antibodies and bioactivity in patients with MS treated with IFNbeta.Molecular Diagnosis 02/2003; 7(1):17-25.
- [show abstract] [hide abstract]
ABSTRACT: Biological activity of interferon-beta (IFNbeta) can be assessed by measuring IFN-stimulated genes (ISGs). Among them, myxovirus resistance protein A (MxA) appears to have the highest specificity, but it has no role in the pathogenesis of multiple sclerosis (MS). To investigate the reliability of MxA as a biomarker, we compared its expression to that of two other ISGs: TNF-related apoptosis-inducing ligand (TRAIL) and X-linked inhibitor of apoptosis factor-1 (XAF-1). Both were shown to be involved in immunoregulatory mechanisms and might play a role in MS. Quantitative-PCR measurements were performed in peripheral blood mononuclear cells from 73 MS patients after short-term and long-term treatment with IFNbeta. A time-dependent response for multiple ISGs was observed in all patients after short-term treatment. In contrast, long-term treatment induced concurrent inhibition of ISGs in 12.3% (9/73) of patients, in whom neutralizing antibodies (NAbs) were detectable. Besides, 22% (16/73) of chronically treated patients showed a non-NAbs-related abrogation of TRAIL expression. In summary, 1) MxA expression was significantly higher than both TRAIL and XAF-1, and 2) MxA was the most sensitive gene to detect decreased bioavailability due to NAbs. These findings identify MxA as an appropriate biomarker for IFNbeta, although there is no evidence for a functional role of it in MS.Multiple Sclerosis 03/2006; 12(1):47-57. · 4.47 Impact Factor
Search for Specific Biomarkers of IFNb Bioactivity in
Patients with Multiple Sclerosis
Sunny Malhotra1, Marta F. Bustamante1, Francisco Pe ´rez-Miralles1, Jordi Rio1, Mari Carmen Ruiz de
Villa2, Esteban Vegas2, Lara Nonell1, Florian Deisenhammer3, Nicola ´s Fissolo1, Ramil N. Nurtdinov1,
Xavier Montalban1, Manuel Comabella1*
1Centre d’Esclerosi Mu ´ltiple de Catalunya, CEM-Cat, Unitat de Neuroimmunologia Clı ´nica, Hospital Universitari Vall d9Hebron (HUVH), Barcelona, Spain, 2Departament
d9Estadı ´stica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain, 3Department of Neurology, Innsbruck Medical University, Innsbruck, Austria
Myxovirus A (MxA), a protein encoded by the MX1 gene with antiviral activity, has proven to be a sensitive measure of IFNb
bioactivity in multiple sclerosis (MS). However, the use of MxA as a biomarker of IFNb bioactivity has been criticized for the
lack of evidence of its role on disease pathogenesis and the clinical response to IFNb. Here, we aimed to identify specific
biomarkers of IFNb bioactivity in order to compare their gene expression induction by type I IFNs with the MxA, and to
investigate their potential role in MS pathogenesis. Gene expression microarrays were performed in PBMC from MS patients
who developed neutralizing antibodies (NAB) to IFNb at 12 and/or 24 months of treatment and patients who remained NAB
negative. Nine genes followed patterns in gene expression over time similar to the MX1, which was considered the gold
standard gene, and were selected for further experiments: IFI6, IFI27, IFI44L, IFIT1, HERC5, LY6E, RSAD2, SIGLEC1, and USP18. In
vitro experiments in PBMC from healthy controls revealed specific induction of selected biomarkers by IFNb but not IFNc,
and several markers, in particular USP18 and HERC5, were shown to be significantly induced at lower IFNb concentrations
and more selective than the MX1 as biomarkers of IFNb bioactivity. In addition, USP18 expression was deficient in MS
patients compared with healthy controls (p=0.0004). We propose specific biomarkers that may be considered in addition to
the MxA to evaluate IFNb bioactivity, and to further explore their implication in MS pathogenesis.
Citation: Malhotra S, Bustamante MF, Pe ´rez-Miralles F, Rio J, Ruiz de Villa MC, et al. (2011) Search for Specific Biomarkers of IFNb Bioactivity in Patients with
Multiple Sclerosis. PLoS ONE 6(8): e23634. doi:10.1371/journal.pone.0023634
Editor: Josef Priller, Charite ´-Universita ¨tsmedizin Berlin, Germany
Received December 24, 2010; Accepted July 21, 2011; Published August 23, 2011
Copyright: ? 2011 Malhotra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors thank the ‘‘Red Espan ˜ola de Esclerosis Mu ´ltiple (REEM)’’ sponsored by the ‘‘Fondo de Investigacio ´n Sanitaria’’ (FIS), Ministry of Science and
Innovation, Spain, and the ‘‘Ajuts per donar Suport als Grups de Recerca de Catalunya (SGR 2005-1081)’’, sponsored by the ‘‘Age `ncia de Gestio ´ d’Ajuts Universitaris
i de Recerca’’ (AGAUR), Generalitat de Catalunya, Spain. This work was supported by a grant from Merck-Serono (Ayudas a la Investigacio ´n - Fundacio ´n Salud
2000). SM is early stage researcher funded by the European Community’s Seventh Framework Programme ([FP7/2007–2013] under grant agreement nu 212877
(UEPHA*MS). RN is an experienced researcher also funded by the European Community’s Seventh Framework Programme ([FP7/2007–2013] under grant
agreement nu 212877 (UEPHA*MS), and has a contract with INSERM U563, University of Toulouse, France. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
In 1993, IFNb became the first FDA-approved drug for the
treatment of relapsing-remitting MS (RRMS), and since then it
has widely been used in clinical practice. IFNb has demonstrated
beneficial effects on decreasing the number of clinical relapses and
disease activity measured by magnetic resonance imaging [1–3].
The mechanisms of action by which IFNb produces its therapeutic
effects in MS are not yet fully understood, however, IFNb
beneficial effects are most likely associated with its immunomod-
IFNb is a type I IFN that binds a heterodimeric cell surface
receptor composed of the IFN receptor 1 (IFNAR1) and 2
(IFNAR2) subunits and activates the JAK-STAT signaling
pathway. As a result, IFN-stimulated gene factor 3 (ISGF3)
complexes are formed and translocated to the nucleus where they
bind to IFN-stimulated response elements (ISREs) and initiate the
transcription of type I IFN-responsive genes . Among the
different type I IFN-responsive genes, myxovirus resistance protein
A (MxA), a GTPase protein encoded by the MX1 gene with potent
antiviral activity , has proven to be one of the most sensitive and
specific biomarkers of IFNb bioactivity [6,7]. MxA expression is
significantly reduced during the development of neutralizing
antibodies (NABs) [8–10], and its measurement has provided the
basis for in vitro and in vivo assays to determine the presence of
NABs [11,12]. However, there is a lack of clear roles of MxA as a
biomarker on disease pathogenesis or in the therapeutic response
In the present study, we aimed to identify new biomarkers of
IFNb bioactivity in order to compare their specificities as genes
induced by type I IFNs with the MxA, and evaluate their potential
implication in MS pathogenesis.
Microarray studies identify biomarkers of IFNb bioactivity
with similar gene expression patterns to the MX1
We first performed gene expression microarrays in PBMC
collected at different time points from IFNb-treated patients.
Supplementary Tables S1 and S2 show the top canonical
PLoS ONE | www.plosone.org1August 2011 | Volume 6 | Issue 8 | e23634
pathways that were identified in up- and down-regulated genes
respectively during IFNb treatment compared to the baseline
condition. As expected, the type I IFN signaling pathway was one
of the most significant pathways identified among up-regulated
In order to identify new markers of IFNb bioactivity, we
stratified patients based on the presence and absence of NABs at
12 and/or 24 months of IFNb treatment. Nine genes fulfilled the
conditions described in the Methods section and followed patterns
of gene expression over time similar to the MX1, the gold standard
gene, and were chosen for further experiments (Table 1). As shown
in Figure 1A, selected genes were significantly induced by IFNb
treatment after 3 months of treatment and their expression levels
were reduced by the presence of NABs and reversed in NAB
Further analysis of potential transcription factors binding to the
promoter region of selected genes revealed that seven of them
(MX1, IFI27, IFIT1, RSAD2, USP18, IFI44L, and HERC5) had
ISRE responding elements (STAT1 transcription factor binding
sites) in upstream regions very close to the annotated transcription
initiation sites, findings that support the specificity of selected
biomarkers as type I IFN induced genes.
We next performed real time RT-PCR of selected genes in
order to validate microarray findings. As depicted in Figure 1B,
mRNA expression levels measured by PCR over time in NAB
positive and negative patients mirrored those obtained with gene
Selected IFNb bioactivity markers are specifically induced
by type I IFNs
As a next step, we performed in vitro experiments to
characterize the specific induction of selected biomarkers by type
I (IFNb) but not type II (IFNc) IFNs. First, we cultured PBMC
from healthy controls for 8 hours in the presence or absence of
different concentrations of Avonex, Rebif, Betaferon, and IFNc.
As shown in Figure 2, all genes were selectively induced by IFNb,
as indicated by the differences in gene expression observed for
IFNb and IFNc. The different types of IFNb resulted in similar
levels of gene expression and were considered together for
calculations. Four genes had a lower limit of quantification
(LLOQ) of 0.1 IU/ml: HERC5 (p=0.007), USP18 (p=0.01),
IFI27 (p=0.02), and IFI6 (p=0.03)(Figure 2, arrows). The
remaining genes, included MX1, reached statistical significance
in their gene expression inductions at higher IFNb concentrations
(LLOQ: 1 IU/ml). Except for RSAD2, all the selected biomarkers
were shown to be more selective than the MX1 gene, as indicated
by the p-values associated with the area under the curve (AUC) of
the difference between IFNb and IFNc. USP18 had the lowest p-
value (p=2.3610217) and was considered to be the most selective
IFNb biomarker. Four genes (IFI27, IFIT1, RSAD2, and USP18)
had stronger inductions in gene expression by IFNb compared
with the MX1, whereas IFI6, IFI44L, HERC5 and SIGLEC1
showed gene expression levels comparable to the MX1. Finally,
Ly6E was up-regulated at lower levels (Figure 2).
From these dose-dependent experiments, a concentration of
100 IU/ml was considered optimal for gene expression induction
and selected for further experiments.
Next, we cultured PBMC from healthy controls at different time
points with 100 IU/ml of IFNb and IFNc. As depicted in Figure 3,
comparisons of the AUC obtained for gene expression at the
different time points revealed HERC5 (p=2.4610219) and USP18
(p=2.6610216) as the genes showing the highest differences in
their expression levels between IFNb and IFNc. The remaining
genes showed lower selectivity values compared with the MX1
(p=2.2610215). Similar to the dose-dependent induction, IFI27,
IFIT1, RSAD2, and USP18 were more up-regulated at the different
time points by IFNb than the MX1. On the other hand, IFI6,
IFI44L, HERC5, and SIGLEC1 showed comparable levels of gene
expression induction to the MX1, whereas Ly6E was the least
induced gene at all time points (Figure 3).
For most of the biomarkers, peak levels of gene expression
occurred after 8 hours of cell culture and this time point was
chosen for further experiments.
These data indicate that, although all the selected genes are
specifically induced by type I but not type II IFNs, several
biomarkers appear to be induced at lower IFNb concentrations
and more selective than the MX1.
Gene expression of selected biomarkers is gradually
inhibited by increasing NAB titres
We next evaluated the capacity of high and low NAB titres to
inhibit the expression of selected IFNb bioactivity biomarkers. As
depicted in Supplementary Figure S1, all biomarkers showed
similar profiles of gene expression inhibition by different NAB
dilutions, and gene expression was greatly reduced by high NAB
titres (undiluted serum and serum dilutions ranging from 1:3 to
1:27). At lower NAB titres (1:81 serum dilutions), except for
SIGLEC1 gene expression of selected biomarkers was reduced by
more than 50% of the expression levels obtained for the positive
control. At 1:243 serum dilutions, except for SIGLEC1, IFI44L,
and Ly6E gene expression of the remaining biomarkers was
reduced by greater than 25% of the positive control expression
levels. Interestingly, RSAD2 showed the highest degree of
inhibition in gene expression by low NAB titres, and was the only
IFNb bioactivity biomarker whose expression was reduced by
more than 25% of the positive control condition at the highest
serum dilutions (1:729), and greater than 50% after 1:243 dilutions
(Supplementary Figure S1).
Despite similar levels of NAB-induced gene expression inhibi-
tion observed for selected biomarkers, these results point to RSAD2
as the most sensitive biomarker to capture the blocking effect of
low NAB titres.
Abrogation of gene expression of selected biomarkers
following cell activation
To evaluate whether selected biomarkers could be indirectly
induced via the production of cytokines other than IFNb, PBMC
from healthy controls were non-specifically activated with LPS
plus PHA in the presence or absence of a high-titre NAB positive
serum. As shown in Figure 4, IFNb accounted for the majority of
gene expression induced by non-specific cell activation, as IFNb
blocking was associated with a more than 80% reduction in the
expression levels for MX1, IFI44L, HERC5, and Ly6E, and greater
than 90% reduction for IFI6, IFI27, IFIT1, RSDA2, SIGLEC1, and
USP18. As expected from dose- and time-dependent experiments,
IFNc contributed little to cell activation-induced gene expression,
and IFNc blocking only resulted in a small additional decrease in
gene expression that ranged from 1.5% for SIGLEC1 to 7.1% for
IFI44L (Figure 4).
These findings indicate that cell activation-induced up-regula-
tion of selected biomarkers is mostly mediated by the effects of
IFNb, and other cytokines included IFNc appear to contribute
little to their expression.
USP18 expression is deficient in MS patients
We finally aimed to evaluate the potential implication of selected
biomarkers in MS pathogenesis. To achieve this, expression levels
IFNß Bioactivity Markers in Multiple Sclerosis
PLoS ONE | www.plosone.org2August 2011 | Volume 6 | Issue 8 | e23634
for these biomarkers were compared between untreated RRMS
patients and healthy controls. Interestingly, only USP18 survived
correction for multiple testing, and expression levels for this gene
were significantly lower in MS patients compared with controls
(p=0.0004)(Figure 5). Trends towards lower expression in MS
patients were also observed for HERC5 (p=0.018) and Ly6E
(p=0.012), although differences did not reach the threshold for
statistical significance after Bonferroni correction (alpha=0.005).
Expression levels for the remaining genes were similar between MS
patients and healthy controls (Figure 5).
No significant correlations were observed between USP18
expression levels and variables such as gender, age at onset, EDSS
scores at the time of blood collection, number of relapses in the 2
previous years, and disease duration (p.0.05).
MxA is specifically induced by type I IFNs and has
demonstrated to be a reliable and sensitive measure of the
biological response to IFNb [6,7]. However, it has no confirmed
roles in MS pathogenesis or in the clinical response to IFNb. By
applying gene expression microarrays to PBMC from patients who
developed NABs to IFNb and patients who remained NAB
negative, we identified 9 biomarkers that followed changes in gene
expression over time similar to the MX1, the gold standard gene.
While some of these biomarkers have been used in previous studies
to evaluate the biological response to IFNb [13,14] (Supplemen-
tary Table S3), others have not been tested yet. In the present
study, we compared the potential for selected biomarkers to
evaluate IFNb bioactivity. Interestingly, although MX1 induction
was highly selective for type I IFNs, dose- and time-dependent
induction experiments revealed several biomarkers of IFNb
bioactivity that were more selective, and significantly induced by
lower IFNb concentrations and at higher levels than the MX1.
The finding of similar profiles of gene expression inhibition by
different NAB dilutions for all selected biomarkers supports their
use to measure the in vivo effects of NABs on IFNb bioactivity.
Finally, the gene expression abrogation experiments following
non-specific stimulation indicate that cytokines other than IFNb
contribute little to the expression of selected biomarkers and
reinforce their specificity by type I IFNs. Although not proven in
the present study, the low gene expression levels that remained
after inhibiting the effects of both IFNb and IFNc were most likely
due to the action of IFNa, another type I IFN.
USP18 was one of the most selective biomarkers of IFNb
bioactivity, and was significantly induced at the lowest IFNb
concentration and up-regulated to a greater degree by type I IFNs
compared to the MX1 gene. Furthermore, it was the only
biomarker found to be differentially expressed between MS
patients and controls, which suggests that USP18 may play a role
in the pathogenesis of MS. USP18 codes for a type I IFN-inducible
cysteine protease that deconjugates ISG15, a ubiquitin-like
protein, from target proteins . Interestingly, USP18 has been
shown to negatively regulate the type I IFN signalling pathway,
and its deficiency results in enhanced and prolonged STAT1
phosphorylation [15–17]. This action appears to be independent
of its protease activity and mediated by the specific binding of
USP18 to IFNAR2, which then blocks the interaction between
JAK1 and the IFN receptor and results in inhibition of the
downstream phosphorylation cascade . Although further
studies are needed, it is tempting to speculate that a deficient
expression of USP18 in MS patients may lead to overactivation of
the type I IFN pathway and have implications in the therapeutic
response to IFNb. In fact, overexpression of type I IFN-responsive
genes has been associated with a decrease biological and clinical
response to IFNb in MS patients [19,20]. Whether or not
responders and non-responders to IFNb differ in their allelic
frequencies for USP18 is an open question.
Together with USP18, HERC5 was highly selective as IFNb
bioactivity biomarker, significantly induced at the lowest IFNb
concentration, and showed induction levels comparable to the
MX1. HERC5 codes for a protein ligase that is involved in the
ISG15 conjugation process (ISGylation) upon stimulation with
type I IFNs .
Similar to the MX1, RSAD2 was significantly induced at a
concentration of IFNb of 1 IU/ml, but showed stronger induction in
gene expression by type I IFNs although with lower selectivity. RSAD2
(also known as viperin) encodes an antiviral protein that is involved in
innate immunityagainstthe infectionofmanyDNAand RNAviruses.
Of note, RSAD2 showed the highest degree of inhibition in gene
inhibiting effect of high and low NAB titres on RSDA2 expression was
also evaluated in a recent study . These findings suggest that
Table 1. Selected markers of IFNb bioactivity from gene expression microarrays.
probe setSymbol Description
Other aliases and
202086_atMX1*myxovirus (influenza virus) resistance 1, interferon-inducible protein p78
204415_atIFI6interferon, alpha-inducible protein 6 IFI-6-16, G1P31p35
202411_at IFI27interferon, alpha-inducible protein 27 ISG1214q32
204439_atIFI44L interferon-induced protein 44-like C1orf291p31.1
203153_at IFIT1 interferon-induced protein with tetratricopeptide repeats 1IFI56, ISG5610q25-q26
219863_at HERC5hect domain and RLD 5 CEB1, CEBP14q22.1
202145_atLY6E lymphocyte antigen 6 complex, locus ERIGE 8q24.3
213797_at/242625_atRSAD2 radical S-adenosyl methionine domain containing 2viperin2p25.2
219519_s_atSIGLEC1 sialic acid binding Ig-like lectin 1, sialoadhesinCD169 20p13
219211_atUSP18 ubiquitin specific peptidase 18ISG43 22q11.21
*MX1 was used as the gold standard gene.
IFNß Bioactivity Markers in Multiple Sclerosis
PLoS ONE | www.plosone.org3August 2011 | Volume 6 | Issue 8 | e23634
RSAD2 measurement may be considered in the design of new and
more sensitive assays to determine NABs.
SimilartotheMX1,SIGLEC1 andLy6EhadaLLOQof1 IU/ml.
In dose-dependent induction experiments, they were shown to be
more selective than the MX1 as IFNb bioactivity biomarkers.
However, Ly6E induction levels were the lowest following stimula-
tion with IFNb, and SIGLEC1 was the least sensitive biomarker to
capture the blocking effect of low NAB titres. SIGLEC1 (also known
as CD169) codes for a macrophage-restricted sialic acid receptor,
which mediates adhesive interactions with lymphoid and myeloid
cells . Although little is known on the function of Ly6E, Ly6
proteins may be playing roles in cell signalling and cell adhesion
processes[23,24].Interestingly,SIGLEC1and Ly6E werefound to be
up-regulated in peripheral blood cells, mainly monocytes, from
patients with other autoimmune disorders such as systemic sclerosis
and systemic lupus erythematosus compared with healthy controls
[25–28],and mRNAand protein levelswere shown to correlate with
disease activity in lupus patients [25,27,28]. Studies correlating
SIGLEC1 and Ly6E levels with disease activity or the response to
IFNb have not been performed in MS.
In dose-dependent induction experiments, IFI6 and IFI27 were
significantly induced at lower IFNb concentrations and more
Figure 1. (A) Changes in gene expression observed with microarrays for selected IFNb bioactivity markers at baseline (T=0), and
after 3, 12, and 24 months of treatment. Four patients developed NABs at 12 and/or 24 months (Patients 1–4) and 4 patients remained NAB
negative at these time points (Patients 5–8). (B) Validation of microarray findings by real time RT-PCR in representative patients belonging to the
different categories (Patients 1, 2, 3, and 5). Given the much stronger induction in gene expression observed for IFI27, graphs corresponding to its
expression were depicted separately for the sake of clarity. Open squares: Ly6E; open circles: IFIT1; open triangles: IFI6; open inverted triangles: USP18;
open diamonds: HERC5; asterisks: IFI44L; solid squares: MX1; solid circles: SIGLEC1; solid inverted triangles: IFI27; solid diamonds: RSAD2. Q: refers to
induction in gene expression after 3 months of treatment. +: NAB positive determination. -: NAB negative determination.
IFNß Bioactivity Markers in Multiple Sclerosis
PLoS ONE | www.plosone.org4August 2011 | Volume 6 | Issue 8 | e23634
selective than the MX1. While IFI6 showed comparable induction
levels to the MX1, IFI27 was by far the most up-regulated gene
following stimulation with type I IFNs. Of note, IFI27 was
proposed as a sensitive marker of IFNb bioactivity in a recent
study , and in a one-year time course transcriptomic study
IFI6 was found among the genes consistently up-regulated by
IFNb . IFI6 and IFI27 belong to the FAM14 family of proteins
and have roles in the regulation of apoptosis. IFI6 encodes an anti-
apoptotic protein that inhibits depolarization of mitochondrial
membrane potential, cytochrome c release, and caspase-3 activity
. Interestingly, IFI6 has also been shown to antagonize the
effects of TRAIL (TNF-related apoptosis-inducing ligand) by
inhibiting the intrinsic apoptotic pathway through mitochondrial
stabilization . The protein encoded by IFI27 associates with or
inserts into the mitochondrial membrane, and its up-regulation
has been reported to lead to decreased viable cell numbers and
enhanced sensitivity to DNA-damage induced apoptosis .
Given the important role that apoptosis plays in the pathogenesis
of MS, further studies to explore the implication of IFI6 and IFI27
in disease pathogenesis are warranted.
Finally, LLOQ of IFIT1 and IFI44L was the same as the MX1
(1 IU/ml). Whereas in the dose-dependent experiments IFI44L
showed similar selectivity and induction levels to the MX1, IFIT1
appeared to be more selective and induced to a higher degree
compared to the MX1. IFIT1 encodes a protein that is rapidly
induced in response not only to viral infections but also non-viral
stimuli such as LPS, IL-1 and TNFa [33,34], and may be involved
in cell apoptosis via interaction with the eukaryotic elongation
factor-1A (eEF1A). Little evidence exists in the literature
regarding the function of the protein encoded by IFI44L.
However, it is important to mention that a related gene, IFI44,
and IFIT1 were found to be among the genes that best predicted
the response to IFNb treatment in MS patients .
In summary, we propose specific biomarkers that may be
considered in addition to the MxA to measure the biological
response to IFNb and the in vivo effects of NABs. Although more
Figure 3. Time-dependent induction in gene expression of selected IFNb bioactivity markers. PBMC from 7 healthy controls were
cultured at different time points in the presence or absence of 100 IU/ml of Avonex (asterisks), Rebif (open squares), Betaferon (solid squares), and
recombinant IFNc (solid circles). At each time point, mRNA expression levels for each gene were determined by real time RT-PCR, as described under
Methods. Results are expressed as fold change in gene expression relative to the uncultured condition (0 h) after subtraction of the expression levels
obtained for untreated cells. Bars represent SEM. AUC: area under the curve (SEM) of the difference between IFNb and IFNc inductions, together with
the associated p-value (selectivity).
Figure 2. Dose-dependent induction in gene expression of selected IFNb bioactivity biomarkers. PBMC from 6 healthy controls were
cultured for 24 hours with Avonex (asterisks), Rebif (open squares), Betaferon (solid squares), and recombinant IFNc (solid circles) at different
concentrations (Conc; x-axis). After cell culture, mRNA expression levels were determined by real time RT-PCR, as described in Methods. Results are
expressed as fold change in gene expression relative to the untreated condition (0 IU/ml). Bars represent SEM. AUC: area under the curve (SEM) of the
difference between IFNb and IFNc inductions, together with the associated p-value (selectivity). Arrows indicate the p-values resulting from the
comparisons in gene expression between the different IFNb concentrations and the untreated conditions (lower limit of quantification).
IFNß Bioactivity Markers in Multiple Sclerosis
PLoS ONE | www.plosone.org5 August 2011 | Volume 6 | Issue 8 | e23634
studies are needed, findings from the present study suggest that
some of these selected biomarkers may also be playing roles in the
pathogenesis of MS and/or the therapeutic response to IFNb.
Materials and Methods
The study was approved by the Hospital Universitari Vall
dHebron Ethics Committee [PR(AG)33/2008] and all patients
gave written informed consent to be included in the study.
Gene expression microarrays
PBMC from RRMS patients were collected before and during
IFNb treatment and stored in liquid nitrogen until used. Gene
expression microarrays (Affymetrix Human Genome U133 Plus
2.0) were performed in PBMC from 8 RRMS patients at baseline
and after 3, 12 and 24 months of IFNb treatment. All patients
were females and the mean age (SD) was 43.1 years (8.8). Four
patients were treated with subcutaneous IFNb -1b (Betaferon), and
the remaining received subcutaneous IFNb-1a (Rebif). Four
patients were negative for NABs at 12 and 24 months and 4
patients developed NABs at 12 and/or 24 months (one patient was
NAB positive at 12 and 24 months, another patient was negative at
12 and positive at 24 months, and 2 patients were positive at 12
and negative at 24 months).
Quality control, preprocessing and analysis of microarray data
were performed as previously described . We aimed to identify
genes that followed temporal expression patterns similar to the
Figure 4. Abrogation of gene expression of selected biomarkers following non-specific cell activation (A–J). PBMC from 3 healthy
controls were cultured for 8 hours in preincubated medium with PHA plus LPS in the presence or absence of undiluted high-titre NAB positive serum
with and without anti-IFNc antibodies, as described in Methods. Results are expressed as fold change in gene expression relative to a condition of
unstimulated cells and with a value of 1 after normalization (not shown in the graphs for the sake of clarity). PBMC cultured with 100 IU/ml of
Betaferon in the presence or absence of high-titre NAB positive serum were used as positive controls (graphs on the left). Bars represent SEM. Arrows
indicate the difference in gene expression observed after the addition of anti-IFNc antibodies. NAB: neutralizing antibodies to IFNb.
IFNß Bioactivity Markers in Multiple Sclerosis
PLoS ONE | www.plosone.org6 August 2011 | Volume 6 | Issue 8 | e23634
MX1, which was chosen as our ‘gold standard’ gene. To achieve
this purpose, graphics of MX1 gene expression (202086_at affy ID)
over time were generated for the 8 patients included in the study,
and searched for genes that followed the same pattern in gene
expression. First, for each patient, behaviour of MX1 was analyzed
at each time point and determined whether MX1 gene expression
decreased or increased in each time interval: 0–3 months, 3–12
months, and 12–24 months. Next, genes that followed the same
increase-decrease pattern in gene expression to the MX1 were
selected. The final list of genes was generated with all common
genes in the 8 study patients. The absolute value of change in gene
expression was set at 0.8, because 0.83 was the minimum increase
in gene expression observed for MX1 in one of the patients from
baseline to the 3 months time point. Pathway analysis was
performed with Ingenuity Pathway Analysis (Ingenuity Systems,
version 9.0 www.ingenuity.com) using two separate lists of genes,
on one side the 816 unique transcripts of up-regulated genes and,
on the other side, the 329 unique transcripts list of down-regulated
genes. Search of potential binding sites for transcription factors in
promoter regions of selected genes was performed using the
TRANSFAC database  (see Supplementary Methods S1 for a
more detailed description of this type of analysis). Microarray data
are stored in the Gene Expression Omnibus (GEO) repository and
following entry number: GSE26104.
NABs were determined in serum samples at baseline and after
12 and 24 months of treatment by means of the myxovirus A
induction bioassay, as described elsewhere , and titers equal or
higher than 20 neutralizing units were deemed positive results.
Validation of selected IFNb bioactivity markers by real
In 4 patients, expression levels of selected genes were also
determined by real time RT-PCR in order to validate microarray
findings. Total RNA was taken from the same samples that had
been used for the microarrays. cDNA was synthesized from total
RNA using the High Capacity cDNA Archive Kit (Applied
Biosystems, Foster City, CA, U.S.A). Amplifications were
performed in duplicate using Taqman probes specific for the
genes selected from microarray studies (Applied Biosystems). The
housekeeping gene GAPDH was used as an endogenous control.
The threshold cycle (CT) value for each reaction, and the relative
level of gene expression for each sample were calculated using the
22DDCTmethod . In brief, GAPDH was employed for the
normalization of the quantity of RNA used. Its CTvalue was
subtracted from that of the specific genes to obtain a DCT value.
The differences (DDCT) between the DCT values obtained for the
untreated baseline samples (calibrators) and the DCT values for
the IFNb-treated samples (at 3, 12 and 24 months) were
determined. The relative quantitative value for the treated samples
was then expressed as 22DDCT, representing the fold change in
gene expression normalized to the endogenous control and relative
to the calibrators.
Dose- and time-dependent induction of selected IFNb
For dose-dependent experiments, fresh PBMC from 6 healthy
controls [3 females/3 males; mean age: 27.5 years (7.1)] were
isolated by Ficoll-Isopaque density gradient centrifugation (Gibco
BRL, Life Technologies LTD, UK), washed twice and resus-
pended in culture medium (RPMI medium 1640 supplemented
with 10% fetal bovine serum, 4 mM L-glutamine, 25 mM Hepes
buffer, 50 units/ml penicillin, and 50 mg/ml streptomycin (Gibco
BRL). PBMC (26106cells/ml) were cultured for 24 hours with
intramuscular IFNb-1a (Avonex), Rebif, Betaferon, and human
recombinant IFNc at different concentrations: 0.1, 10, 100, and
1000 IU/ml. After cell culture, mRNA expression levels of
selected IFNb bioactivity markers were determined by real time
RT-PCR, as previously described. Changes in gene expression
were always compared with cells cultured in the absence of IFNb
(referred to as 0 IU/ml; calibrators).
For time-dependent experiments, freshly isolated PBMC from 7
healthy controls [3 females/4 males; mean age: 27.5 years (5.7)]
were cultured as previously described in the presence or absence of
100 IU/ml of Avonex, Rebif, Betaferon, and human recombinant
IFNc for 2, 4, 6, 8, and 24 hours. After cell culture, gene
expression levels for selected markers were determined by real
time RT-PCR, as described above. Changes in gene expression
were always referred to a baseline uncultured condition (0 h;
calibrators). Previously, gene expression levels obtained for the
Figure 5. Comparison of gene expression levels of selected biomarkers in MS patients and controls. PBMC were collected from
untreated RRMS patients (N=14) and healthy controls (N=15) and the mRNA expression levels for each gene were determined by real time RT-PCR.
The y-axis represents the threshold cycle (CT) values obtained for each individual. CTis inversely related to quantity, and higher CTvalues are
indicative of lower mRNA expression levels. MS: untreated RRMS patients. HC: healthy controls.
IFNß Bioactivity Markers in Multiple Sclerosis
PLoS ONE | www.plosone.org7 August 2011 | Volume 6 | Issue 8 | e23634
different biomarkers in untreated cells cultured for the same time
points were subtracted from the values obtained after treatment
with IFNb and IFNc.
NAB-induced gene expression inhibition
Undiluted and serially diluted serum (1:3, 1:9, 1:27, 1:81, 1:243,
1:729) collected from a patient treated with Betaferon who developed
NABs at high titres (.1280) was preincubated for 1 hour in the
presence or absence of 100 IU/ml of Betaferon. Subsequently, freshly
isolated PBMC from 3 healthy controls [2 females/1 male; mean age:
markers were determined by real time RT-PCR, as described above.
IFNb-induced expression levels were compared with those obtained
from cells cultured without IFNb in the presence of serum from a
NAB negative patient (calibrators). PBMC cultured with 100 IU/ml
of Betaferon was used as positive control.
Abrogation of gene expression induced by non-specific
Freshly isolated PBMC from 3 healthy controls [2 females/1
male; mean age: 29.3 years (4.9)] were cultured for 8 hours in
preincubated medium with PHA (5 mg/ml) plus LPS (1 mg/ml) in
the presence or absence of undiluted high-titre NAB positive
serum (.1280) with and without anti-IFNc antibodies (100 ng/
ml) at 37uC for 1 hour. After cell culture, gene expression of
selected biomarkers was determined by real time RT-PCR, as
previously described. Cell activation-induced expression levels
were compared with those obtained from unstimulated cells
cultured in the presence of serum from a NAB negative patient
(calibrators). PBMC cultured with 100 IU/ml of Betaferon in the
presence or absence of high-titre NAB positive serum were used as
positive controls of NAB-induced inhibition.
Gene expression levels for selected bioactivity markers in
MS patients and controls
Fresh PBMC were isolated from 14 untreated RRMS patients
[64.3% females; mean age (standard deviation): 42.1 years (9.6);
mean number of relapses in the previous 2 years: 0.9 (0.9); mean
disease duration: 12.4 years (7.1); median EDSS at the time of
blood collection (interquartile range): 2.0 (1.5–3.0)]. A group of 15
healthy controls [53.3% females; mean age: 30.5 years (6.2)] was
also included in the study.
After RNA extraction, mRNA expression levels for selected
biomarkers were determined by real time RT-PCR, as described
above. Gene expression values obtained for MS patients were
referred to the expression levels observed in controls (calibrators).
For dose-dependent experiments, the following parameters were
considered:(i) Sensitivity wasevaluatedbytheLLOQanddefined as
the minimum IFNb concentration that induced a statistically
significant increase in gene expression when compared with the
untreated condition, and it was calculated by paired t-tests adjusting
for multipletesting using theBonferroni approach. (ii)Selectivity was
defined, for each gene, as the difference observed in gene expression
between different concentrations of type I and type II IFNs, and it
was calculatedbycomparing theAUC obtained for IFNb and IFNc.
The p-value associated with the AUC of the difference was
calculated by means of a t-type statistic that uses the critical value
from a t-distribution with Satterthwaite’s approximation  to the
degrees of freedom for calculation of confidence intervals.
Similarly, for time-dependent selectivity was defined as the
difference observed in gene expression between type I and type II
IFNs at the different time points of in vitro cell culture, and it was
NAB-induced gene expression inhibition was evaluated by
comparing the NAB-positive serum dilutions that were associated
with reductions in gene expression of selected biomarkers greater
than 25% and 50% of the expression levels obtained for the
positive control condition.
A Mann-Whitneys test was used to test for significant differences
in gene expression levels between MS patients and controls.
Insomuch as 10 different genes were evaluated, Bonferroni
correction was used to correct the alpha level for multiple
Statistical calculations were performed with R language and the
SPSS 11.5 package (SPSS Inc, Chicago, IL) for MS-Windows.
biomarkers. Undiluted and increasingly diluted serum from an
IFNb-treated patient who developed high NAB titres were
preincubated for 1 hour in the presence or absence of 100 IU/
ml of Betaferon, and then added to PBMC from 3 healthy controls
for 8 hours, as described in Methods. Results are expressed as fold
change in gene expression relative to a condition of cells cultured
without IFNb and with a value of 1 after normalization (not shown
in the graphs for the sake of clarity). Bars represent SEM. Dotted
lines indicate the expression levels that correspond to 25% and
50% reductions in gene expression of the positive control
condition. US: undiluted serum. PC: positive control. NAB:
neutralizing antibodies to IFNb.
NAB-induced gene expression inhibition of selected
ment with IFNb.
Top canonical pathways up-regulated during treat-
treatment with IFNb.
Top canonical pathways down-regulated during
Summary of studies related with selected IFNb
The authors would like to thank the Fundacio ´ Esclerosi Mu ´ltiple (FEM).
Conceived and designed the experiments: SM MFB MC. Performed the
experiments: SM MFB FD NF. Analyzed the data: MCRdV EV LN RNN.
Contributed reagents/materials/analysis tools: FM JR XM. Wrote the
paper: SM MC.
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