T cell cytokine signatures: Biomarkers in pediatric multiple sclerosis
Cather M. Cala b
, Carson E. Moseley c
, Chad Steele c
, Sarah M. Dowdy d
, Gary R. Cutter f
,
Jayne M. Ness d
, Tara M. DeSilva a,b,e,
⁎
a
Center for Glial Biology in Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United States
b
Department of Physical Medicine Rehabilitation, University of Alabama at Birmingham, Birmingham, AL 35294, United States
c
Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, United States
d
Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35294, United States
e
Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294, United States
f
Department of Biostatistics, University of Alabama at Birmingham, Birmingham, AL 35294, United States
a b s t r a c ta r t i c l e i n f o
Article history:
Received 11 December 2015
Received in revised form 13 April 2016
Accepted 26 April 2016
Although multiple sclerosis is predominantly regarded as a disease of young adulthood, up to 5% of MS patients
are diagnosed prior to age eighteen. The predominant form of MS is relapsing–remitting characterized by exacerbations
of symptoms followed by periods of remission. The majority of disease modifying drugs target T cell
proliferation or block migration into the central nervous system. Although these treatments reduce relapses, disease
progression still occurs, warranting therapeutic strategies that protect the CNS. Biomarkers to indicate relapses
would facilitate a personalized approach for add-on therapies that protect the CNS. A multiplex cytokine
bead array was performed to detect T cell associated cytokines in sera from patients 6–20 years of age with pediatric
onset MS clinically diagnosed in relapse or remission compared to healthy control patients. Of the 25 cytokines
evaluated, 17 were increased in patients clinically diagnosed in relapse compared to sera from control
patients in contrast to only 9 cytokines in the clinically diagnosed remission group. Furthermore, a linear regression
analysis of cytokine levels in the remission population showed 12 cytokines to be statistically elevated as a
function of disease duration, with no effect observed in the relapse population. To further explore this concept,
we used a multivariable stepwise discriminate analysis and found that the following four cytokines (IL-10, IL-
21, IL-23, and IL-27) are not only a signiﬁcant predictor for MS, but have important predictive value in determining
a relapse. Since IL-10 and IL-27 are considered anti-inﬂammatory and IL-21 and IL-23 are pro-inﬂammatory,
ratios of these cytokines were evaluated using a Duncan's multiple range test. Of the six possible combinations,
increased ratios of IL-10:IL-21, IL-10:IL-23, and IL-10:IL-27 were signiﬁcant suggesting levels of IL-10 to be a driving
force in predicting a relapse.
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords:
Pediatric multiple sclerosis
Cytokine
T cell
Multiplex array
1. Introduction
There are 2.5 million people diagnosed with multiple sclerosis (MS)
worldwide, 5% of whom are diagnosed prior to age eighteen(Renoux
et al., 2008). In MS, T cells are primed against unidentiﬁed white matter
or foreign antigens bound to major histocompatibility complexes
(MHCs) (Hemmer et al., 2002). This leads to destruction of myelin, the
lipid membrane of oligodendrocytes (OLs) that forms an insulating
sheath around axons allowing for increased action potential speed
(Funfschilling et al., 2012). Demyelination not only impairs conduction
of nerve impulses, but makes axons more vulnerable to injury contributing
to motor and cognitive decline. The most common form of MS is
relapsing–remitting (RRMS), whereby exacerbations of symptoms are
followed by a period of remission. A relapse is deﬁned as a clinical
event involving the onset of neurological symptoms caused by CNS inﬂammation.
The number of inﬂammatory events in the CNS of MS patients
is far greater than the number of relapses. Current treatments in
MS decrease the number of relapses, yet few eliminate relapses
(Smith et al., 2010) warranting treatment strategies that protect the
CNS. Predicting the time point before a relapse occurs is an important
intervention interval for CNS protective strategies, thus, identifying biomarkers
that predict relapses and/or disease progression highly clinically
relevant.
Current studies have utilized the number (Morgan et al., 2013;
Neuteboom et al., 2010) or location of lesions observed by MRI to predict
probability of a relapse (Filippi and Agosta, 2010). Assessing the
number of lesions in conjunction with relapses as a function of disease
duration has also been predictive of disease progression (Sormani
et al., 2011). However, imaging biomarkers may indicate a time point
Journal of Neuroimmunology 297 (2016) 1–8
⁎ Corresponding author at: 1825 University Blvd, Shelby 503, Center for Glial Biology
and Medicine, Department of Physical Medicine and Rehabilitation, University of
Alabama at Birmingham, Birmingham, AL 35294, United States.
E-mail address: desilvat@uab.edu (T.M. DeSilva).
http://dx.doi.org/10.1016/j.jneuroim.2016.04.015
0165-5728/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Contents lists available at ScienceDirect
Journal of Neuroimmunology
journal homepage: www.elsevier.com/locate/jneuroim
in the disease process where an intervention is no longer efﬁcacious.
Economical, easily accessible biomarkers that monitor a speciﬁc pathogenic
disease process, as an early predictor of relapse, would be
beneﬁcial.
Cytokines regulate immune cell inﬁltration into different areas of the
central nervous system resulting in different clinical manifestations in
animal models of MS (Pierson et al., 2012). Pro-inﬂammatory cytokine
levels in sera from patients with MS are elevated compared to healthy
controls (Martins et al., 2011; Farhadi et al., 2014; Kallaur et al., 2013).
These data provide evidence that serum cytokine levels may represent
an active disease process in patients with RRMS.
Naïve CD4+
T cells circulate between the blood and lymphatic system,
and differentiate upon encountering cognate peptides presented
by antigen presenting cells (APCs) in conjunction with co-stimulatory
molecules and cytokine signals. Differentiated effector T cells are considered
the principal orchestrators in MS pathogenesis (Komiyama
et al., 2006; Kebir et al., 2007). CNS inﬁltrating effector T cells and
their relevance to human disease are further conﬁrmed by the effectiveness
of disease modifying therapies that inhibit T cell proliferation/activation
(e.g. IFN-β, glatiramer acetate) or bind to adhesion molecules
(natalizumab) and prevent extravasation through blood vessels into
the CNS. Speciﬁc cytokines initiate the activation of discrete transcriptional
regulatory programs that segregate differentiating T helper cells
into one of four major subsets; Th1, Th17, Th2, and regulatory T cells
(Tregs) (Palmer and Weaver, 2010).
Despite their critical function in disease initiation and resolution, to
date no studies have extensively evaluated T cell associated cytokines
throughout the course of disease in the pediatric RRMS population.
Therefore, we evaluated a cytokine proﬁle speciﬁcally associated with
T helper signaling in sera from pediatric MS patients clinically diagnosed
in remission or relapse compared to healthy controls to provide important
clues about the disease process in remission distinct from relapse as
well as provide biomarkers to predict relapse.
2. Methods
2.1. Clinical samples and chart review
Serum samples were obtained with informed consent from parents
or guardians of patients with pediatric onset multiple sclerosis
(POMS) or healthy controls with no pathology under the approval of
the Institutional Review Board No. X130307001. Patients were diagnosed
with POMS based on the International Pediatric MS Study
Group criteria (Krupp et al., 2007, 2013) and categorized clinically as
being in MS relapse or remission when the sample was obtained. Patients
ranged in age from 6 to 17 years of age at disease onset and
from 8 to 20 years of age at time of sample collection. Control patients
ranged in age from 5 to 19 years. Patient data are reported in Table 1 including
corticosteroid treatment or disease modifying agents.
2.2. Multiplex cytokine array
Serum was isolated from collected blood samples and stored at
−80 °C until use for multiplex cytokine analysis. Serum cytokine levels
were measured using the MILLIPLEX MAP Human Th17 Magnetic Bead
Panel (catalog # HT17MG-14K-PX25) and the Luminex Multi-Analyte
Instrument (Luminex, Austin, TX) as per manufacturer's instructions
previously described (Singh et al., 2006), values were reported in
pg/mL. Luminex multiplex suspension array technology for analysis of
human samples was performed as previously described (Myers et al.,
2013; Morris et al., 2012; Sun et al., 2008).
2.3. Statistical analysis
Statistical analyses were performed using GraphPad Prism software
(San Diego, CA) and SAS (Version 9.4). All samples categorized by
disease states were compared to control samples using the two-tailed
nonparametric Mann–Whitney test. A p-value of ≤0.05 was used to determine
statistical signiﬁcance with no adjustments for multiple comparisons.
All of these values are reported in Table 2. DMTs were used
in 60% of the patients: 50% (7/14) of patients who were experiencing
a relapse were on a DMT compared to 65% (17/26) in the remission
group. To assess the inﬂuence of disease modifying therapy (DMT) on
cytokine levels, sera from patients on DMTs were compared to those
not on DMTs using a two-tailed Mann–Whitney test. No signiﬁcant difference
was observed in sera from patients on DMT compared to sera
from patients not on DMTs. The results were thus combined and reported
as their respective sub-group remission or relapse as indicated in
Table 2 without adjustment for DMT. To evaluate changes in cytokine
levels as a factor of disease duration within each disease group, a linear
regression was used as shown in Table 3. To assess strong predictors of
multiple sclerosis, a multivariable stepwise discriminate analysis was
performed as shown in Tables 4, 5, and 6.
3. Results
A major underlying pathogenesis in MS is T cell inﬁltration into the
CNS initiating an inﬂammatory cascade resulting in axonal demyelination
and eventual neurodegeneration. In an effort to elucidate potential
biomarkers to predict when relapses occur, a microarray panel of 25 cytokines
associated with T cell signaling was analyzed in sera from MS
patients clinically diagnosed in relapse or remission and compared to
levels in healthy control sera. Demographics for all patients enrolled in
the study are included in Table 1. Statistical signiﬁcance for cytokine
levels in serum samples from relapse compared to control as well as remission
compared to control is summarized in Table 2. These results are
described in the context of cytokines that regulate T cell differentiation
from CD4+ naïve T cells as well as cytokines released by these effector
sub-populations.
Chronic reactivity of CD4+ T cells to autoantigens plays a central
role in autoimmunity mainly by segregating into three major phenotypes,
Th1, Th2, and Th17. Th1 cells differentiate from naïve CD4+ T
cells in the presence of IL-12p70 and IFNγ to produce IL-2, IFNγ, TNFβ,
and GMCSF. Patients clinically diagnosed in MS relapse had signiﬁcantly
increased levels of IL-12p70 and TNFβ compared to controls whereas
neither of these cytokines were increased in patients diagnosed in remission
(Table 2). Th2 cells differentiate from naïve CD4+ T cells in
the presence of IL-4 and IL-33 to produce GMCSF, IL-4, IL-5, IL-10, ILTable
1
Demographics of pediatric relapsing–remitting MS and healthy control subjects.
Clinical information MS Controls
Relapse
(n = 14)
Remission
(n = 26)
(n = 11)
Age range⁎ 8.88–20.76 11.5–19.54 5.96–19.28
Mean age* ± SD 15.28 ± 3.30 16.38 ± 1.94 13.76 ± 3.37
Gender 10 female 21 female 5 female
4 male 5 male 6 male
Race 7 white 18 white 5 white
7 black 8 black 5 black
1 not reported
Steroids 3 yes 1 yes 11 no
11 no 25 no
Disease-modifying agents
Copaxone 2 6 None
Rebif 3 6 None
Avonex 2 3 None
Betaseron 0 1 None
Extavia 0 1 None
None 7 9 None
Age at symptom onset range⁎ 6.56–17.62 7.76–17.35 N/A
Mean age at symptom onset⁎ ± SD 13.54 ± 3.41 14.21 ± 2.38 N/A
Disease duration⁎ 1.74 ± 1.82 2.17 ± 2.11 N/A
⁎ Age is represented in years.
2 C.M. Cala et al. / Journal of Neuroimmunology 297 (2016) 1–8
Table 2
Statistical signiﬁcance (p-values) of T helper-associated cytokines in MS versus control as well as remission versus relapse (R vs R).
Mean Median Std dev Minimum Maximum p-Value⁎
Cytokine Ctl Remission Relapse Ctl Remission Relapse Ctl Remission Relapse Ctl Remission Relapse Ctl Remission Relapse Remission Relapse R vs
R
Th1-associated cytokines IFNg 0.899 16.830 5.539 0.00 0.00 0.00 2.052 62.064 10.428 0.00 0.00 0.00 5.97 313.31 31.21 0.22 0.18 0.81
IL-12p70 3.846 22.559 9.159 3.18 6.02 8.03 2.466 75.233 7.466 0.00 0.00 0.00 9.63 389.43 29.21 0.12 0.02 0.58
TNFb 0.000 0.045 0.038 0.00 0.00 0.00 0.000 0.104 0.076 0.00 0.00 0.00 0.00 0.36 0.25 0.08 0.05 0.82
IL-2 0.000 13.125 8.692 0.00 0.00 0.00 0.000 35.776 15.907 0.00 0.00 0.00 0.00 165.80 55.45 0.09 0.1 0.88
Th2-associated cytokines IL-4 0.002 0.033 0.056 0.00 0.00 0.00 0.006 0.102 0.096 0.00 0.00 0.00 0.02 0.50 0.29 0.39 0.12 0.22
IL-33 0.000 35.352 27.701 0.00 3.78 9.12 0.000 88.523 52.371 0.00 0.00 0.00 0.00 388.38 165.60 0.06 0.01 0.49
IL-5 0.132 8.523 1.989 0.00 0.37 0.61 0.381 20.406 4.369 0.00 0.00 0.00 1.27 80.53 16.45 0.04 0.04 0.73
IL-10 0.224 3.846 9.379 0.00 1.36 4.07 0.443 7.900 10.956 0.00 0.00 0.00 1.36 39.35 31.86 0.01 0.004 0.06
IL-13 13.933 35.379 36.059 13.87 29.01 32.39 14.649 39.575 30.234 0.00 0.00 0.00 43.85 154.51 93.57 0.1 0.04 0.59
IL-25/IL-17E 0.017 0.388 0.324 0.00 0.04 0.10 0.025 0.990 0.621 0.00 0.00 0.00 0.06 4.48 2.10 0.1 0.01 0.45
Th17-associated cytokines IL-6 1.711 10.817 8.930 0.00 0.00 0.00 5.674 32.219 17.254 0.00 0.00 0.00 18.82 158.42 47.73 0.11 0.22 0.78
IL-1β 1.127 3.237 4.410 0.00 0.00 0.00 3.739 8.042 7.818 0.00 0.00 0.00 12.40 35.69 25.35 0.15 0.15 0.76
IL-23 0.073 1.600 0.871 0.00 0.18 0.24 0.107 4.309 1.593 0.00 0.00 0.00 0.30 18.31 4.92 0.05 0.005 0.44
IL-17A 0.374 14.068 6.851 0.00 0.00 3.23 0.738 56.682 11.461 0.00 0.00 0.00 2.26 289.55 42.02 0.35 0.02 0.09
IL-17F 0.012 0.036 0.034 0.01 0.02 0.03 0.006 0.056 0.028 0.00 0.00 0.01 0.02 0.28 0.10 0.07 0.006 0.46
IL-21 5.885 18.304 18.219 4.70 13.59 14.43 3.983 22.093 12.998 0.00 0.00 4.23 14.45 111.44 51.30 0.02 0.001 0.55
IL-22 0.000 0.090 0.073 0.00 0.00 0.00 0.000 0.237 0.179 0.00 0.00 0.00 0.00 0.89 0.56 0.27 0.1 0.58
Cytokines associated with multiple T
helper cells
TNFa 12.145 19.554 20.434 10.98 15.28 17.16 6.895 18.602 13.241 0.00 0.00 2.32 23.16 96.58 58.94 0.1 0.03 0.38
GM-CSF 0.000 0.097 0.000 0.00 0.00 0.00 0.000 0.366 0.000 0.00 0.00 0.00 0.00 1.80 0.00 0.78 0.91 0.20
CCL20/MIP3a 11.883 27.122 26.546 11.19 20.46 24.86 6.075 21.577 9.926 2.99 0.00 10.52 20.85 104.90 44.78 0.004 0.0003 0.33
IL-9 0.000 7.895 6.152 0.00 0.00 0.00 0.000 22.648 9.131 0.00 0.00 0.00 0.00 112.26 22.67 0.04 0.1 0.92
IL-15 0.228 7.713 9.675 0.00 0.00 1.52 0.757 20.265 14.839 0.00 0.00 0.00 2.51 91.59 46.98 0.11 0.01 0.20
IL-27 0.189 1.109 1.066 0.13 0.59 0.66 0.185 1.530 0.948 0.00 0.21 0.36 0.52 6.98 3.46 0.0002 b0.0001 0.32
IL-28A 0.018 0.185 0.153 0.00 0.11 0.08 0.034 0.281 0.228 0.00 0.00 0.00 0.10 1.05 0.86 0.006 0.01 0.82
IL-31 0.005 0.184 0.126 0.00 0.02 0.04 0.008 0.537 0.240 0.00 0.00 0.00 0.02 2.67 0.75 0.01 0.002 0.60
3C.M.Calaetal./JournalofNeuroimmunology297(2016)1–8
13, and IL-25/IL-17E. All of these cytokines with the exception of IL-4
were signiﬁcantly elevated in MS relapse patients compared to control.
In contrast, only IL-5 and IL-10 were statistically elevated in serum from
patients in MS remission compared to controls. Th17 cells differentiate
from naïve CD4+ T cells in the presence of IL-6, TGFβ (not in panel),
IL-1β, and IL-23 to produce IL-17A, IL-17F, IL-21, IL-22, and GM-CSF.
IL-23, IL-17A, IL-17F, and IL-21 were signiﬁcantly elevated in sera from
patients in MS relapse compared to control, whereas only IL-23 and
IL-21 were elevated in sera from patients clinically diagnosed in
remission.
Other cytokines associated with multiple T cell subsets were also
assessed. TNF-α is associated with both Th1 and Th17 signaling and is
elevated during MS relapse, but not MS remission compared to control
sera. CCL20/MIP3a, which can recruit both Th17 cells and Tregs via the
chemokine receptor CCR6 (Reboldi et al., 2009; Arima et al., 2012;
Yamazaki et al., 2008; Villares et al., 2009), was increased during relapse
and remission. IL-9, which is released by a relatively unexplored subset
of T cells, Th9, is signiﬁcantly upregulated in MS remission serum samples,
but not in the MS relapse subset compared to control. Both IL-15
and IL-27 are associated with a negative regulatory role on Th17 responses
(Pandiyan et al., 2012; Batten et al., 2006) and were signiﬁcantly
increased in MS relapse, but only IL-27 was elevated in MS remission
compared to control samples. IL-28A and IL-31 are key mediators in Tcell
mediated inﬂammatory diseases (Siebler et al., 2007; Castellani
et al., 2010) and were signiﬁcantly elevated in sera from both relapse
and remission compared to sera from control samples.
While ﬁnding predictors of relapse may have important clinical ramiﬁcations
for add-on therapies, another approach in this regard is monitoring
changes in T cell associated cytokines during remission as a
function of disease duration. In RRMS, patients have exacerbations of
symptoms followed by a remission phase where symptoms subside
and clinical outcomes return to baseline. However, as disease duration
increases, the remission phase of the disease may not return to baseline.
Patients then advance into a secondary progressive phase precipitating
a downward decline in function. To assess changes in cytokine levels
during remission in relationship to disease duration, a heat map was
generated by plotting cytokine levels with increasing disease duration
(Fig. 1A). A linear regression analysis of cytokine levels in remission
sera as a function of disease duration was performed, and coefﬁcients
of determination (R2
) and p-values are reported in Table 3. Of the 25 cytokines
assessed, 12 were signiﬁcantly positively associated with disease
duration in the remission sera. These 12 cytokines were then
compared to their outcomes from Table 2. Five of the cytokines (TNFβ,
IL-33, IL-25, IL-17F, and IL-15) found signiﬁcant in the linear regression
analysis from remission sera (Table 3) were not elevated in the remission
compared to control sera, but were elevated in the relapse
compared to control sera (Table 2). Another ﬁve of the cytokines (IL-
10, IL-23, IL-27, IL-28A, and IL-31) found signiﬁcant in the linear regression
analysis from remission sera (Table 3) were elevated in both the remission
and relapse sera compared to control (Table 2). The last two
cytokines (IL-4 and Il-22) were signiﬁcant in the linear regression analysis
from the remission sera (Table 3), but were not elevated in the remission
or relapse sera compared to control. A linear regression analysis
of cytokine levels in relapse sera as a function of disease duration
(Table 3) demonstrated no statistically signiﬁcant differences, heat
map included in Fig. 1B. Collectively, these data suggest that monitoring
an individual's cytokine levels during subsequent remission phases may
be an important predictor of disease progression, and may potentially
provide a strong rationale for a more aggressive treatment strategy.
These data were further explored to assess how well cytokine data
could be used to predict if a patient has MS by comparing all three subsets
i.e. control, relapse, and remission. Using a multivariable stepwise
discriminate analysis, four cytokines (IL-23, IL-21, IL-10, and IL-27)
were shown to have a predictive value for multiple sclerosis. To further
evaluate the relevance of this four cytokine cluster in monitoring the remission
phase as a predictor of a relapse, we assessed the number of patients
with cytokine levels ﬁve times higher than the mean of the
control population for each cytokine (Table 4). This table shows that
9% of the control group, 79% in the relapse group, and 54% in the
Table 3
Linear regression analyses: duration of disease (years) and cytokine levels (pg/mL).
Cytokine MS remission MS relapse
Th1-associated cytokines
R2
p-Value R2
p-Value
IFNγ 0.002 0.82 0.001 0.89
IL-12p70 0.000 0.98 0.082 0.32
TNFβ 0.140 0.05 0.000 0.95
IL-2 0.057 0.24 0.015 0.67
Th2-associated cytokines
IL-4 0.274 0.006 0.007 0.76
IL-33 0.211 0.02 0.002 0.87
IL-5 0.015 0.55 0.007 0.78
IL-10 0.239 0.01 0.044 0.47
IL-13 0.102 0.11 0.014 0.69
IL-25/IL-17E 0.238 0.01 0.003 0.84
Th17-associated cytokines
IL-6 0.034 0.37 0.001 0.90
IL-1β 0.244 0.01 0.013 0.70
IL-23 0.202 0.02 0.006 0.79
IL-17A 0.000 0.94 0.009 0.74
IL-17F 0.302 0.003 0.012 0.70
IL-21 0.003 0.79 0.078 0.33
IL-22 0.177 0.03 0.000 0.99
Cytokines associated with multiple T helper cells
TNFα 0.025 0.44 0.012 0.71
GM-CSF 0.000 0.90 0.002 0.43
CCL20/MIP3α 0.018 0.50 0.003 0.84
IL-9 0.015 0.55 0.023 0.60
IL-15 0.287 0.004 0.004 0.82
IL-27 0.188 0.03 0.001 0.91
IL-28 A 0.083 0.15 0.000 0.99
IL-31 0.275 0.006 0.005 0.81
Table 4
Number of patients with one or more of the four cytokine cluster (IL-10, IL-21, IL-23, IL-27)
elevated ﬁve times above the mean of control levels.
# of four cytokines Control Relapse Remission
0 10 3 12
≥1 1 11 14
Percent ≥ 1 9% 79% 54%
Table 5
Predictive value of having one or more cytokines from the cluster elevated ﬁve times
above the normative baseline as a positive predicator (PPV) of a relapse compared to none
as a negative predictor of a relapse.
Sensitivity 11/14 79%
Speciﬁcity 12/26 46%
PPV 11/25 44%
NPV 12/15 80%
Table 6
Ratios derived from the four cytokine cluster, brackets demonstrate signiﬁcance of p 0.05
using Duncan's multiple range test.
Classiﬁcation Ratio
10:21
Ratio
10:23
Ratio
10:27
Ratio
21:23
Ratio
21:27
Ratio
23:27
Control
{0.027
{1.80
{
1.322 48.770 33.450 0.214
Relapse 0.827 36.89
{
12.547 57.970 21.749 1.432
Remission 0.333 9.81 3.264 52.980 24.247 0.684
Bracketed values indicate statistical signiﬁcance.
4 C.M. Cala et al. / Journal of Neuroimmunology 297 (2016) 1–8
remission group had a least one of the four cytokines in the cluster elevated
ﬁve times higher than the mean for the control population. These
values show an estimated sensitivity of 79% and a speciﬁcity of 46%
(Table 5). The positive predictive value (PPV) of having one or more cytokines
from the cluster elevated ﬁve times above the normative baseline
as a positive predicator (PPV) of a relapse is 44%. In comparison,
the probability of having none of the four cytokine clusters elevated
ﬁve times above the mean of the control population as a negative predictor
(negative predictive value, NPV) of a relapse is 80%. The identiﬁcation
of the four cytokine clusters supports the biology of the Th17 cell
as an important predictor of disease since IL-21 and IL-23 are central in
the pathogenesis for Th17 differentiation and both IL-10 and IL-27 are
important in the resolution of this effector state. Therefore, we next
assessed the ratios derived from these cytokines using a Duncan's multiple
range test to examine the consistency of the pattern of the cytokines
across the three classiﬁcation groups. If a zero (below the limit
of detection) was measured for a cytokine for a particular individual,
the ratio used was the smallest observed value for that cytokine in the
group (arguing that zero was merely below the limit of detection).
This avoids excluding the observation since one cannot divide by zero
and should be conservative as the ratio would exceed what is used in
the analysis. Of the six possible combinations, increased ratios for IL-
10:IL-21, IL-10:IL-23, and IL-10:IL-27 were signiﬁcant suggesting levels
of IL-10 to be a driving force in predicting a relapse (Table 6). Of note,
although ratios for IL-21:IL-23, IL-21:IL-27, and IL-23:IL-27 were not
statistically signiﬁcant, the ratios were appropriately rank ordered between
the control, relapse, and remission groups.
4. Discussion
Our data show that 17 of the 25 T helper cell associated cytokines are
elevated during pediatric MS relapse compared to healthy controls. In
Fig. 1. Heat map depicting levels of T helper cell associated cytokines in patients with multiple sclerosis during remission (A) and relapse (B). Columns represent individual patients and are
organized by increasing disease duration in years. Each box represents the percent difference between the patient and the average of healthy controls for the cytokines indicated to the left
of each row, both measured in pg/mL. Red, black, and green colors represent values above, equal to, or below the average of healthy controls, respectively. Values depicted at the top of the
ﬁgure represent the disease duration of each patient in years. A linear regression analysis was performed to determine which cytokines signiﬁcantly increased with disease duration, and
results are included in Table 3.
5C.M. Cala et al. / Journal of Neuroimmunology 297 (2016) 1–8
contrast, only 9 cytokines from this same cytokine proﬁle are elevated
during the remission phase compared to control subjects. Not only do
these data support well-known theories that T cells are involved in
MS, but they also provide evidence that serum cytokine levels have
the potential to conﬁrm diagnosis of an MS relapse. A linear regression
analysis of cytokine levels in sera from patients in remission as a function
of disease duration demonstrated statistical signiﬁcance in 12 of
the 25 cytokines from the panel. A linear regression analysis of cytokine
levels in sera from patients in relapse as a function of disease duration
demonstrated no statistically signiﬁcant difference. Furthermore, a multivariate
stepwise discriminate analysis demonstrated that four cytokines
(IL-10, IL-21, IL-23, and IL-27) have a predictive value for the
pediatric MS population studied. This four cytokine clusters provided a
more focused group of cytokines to track within individual patients as
a positive predictor of a relapse making it a potential biomarker. Since
IL-10 and IL-27 are considered anti-inﬂammatory and IL-21 and IL-23
are pro-inﬂammatory, the six possible combination ratios of these cytokines
were evaluated using a Duncan's multiple range test. Increased ratios
of IL-10:IL-21, IL-10:IL-23, and IL-10:IL-27 were signiﬁcant
suggesting that levels of IL-10 may be a driving force in predicting a relapse.
This study, like all studies has limitations. The focus on pediatric
MS by necessity limits the sample size and a matched case/control
study was not performed suggesting the possibility that matching age,
gender etc. may have provided a clearer comparison. This analysis also
did not control for therapeutic interventions although roughly only
50% were on DMTS. Finally, these studies warrant testing the IL-10 hypothesis
in larger cohorts that include MRI data and repeat samples to
compare relapse and remission periods within the same pediatric patient.
Since the number of inﬂammatory events in the CNS of MS patients
is far greater than the number of relapses, it will be appropriate
to evaluate serum cytokine levels with disease activity in the CNS as
measured by MRI as opposed to a clinical relapse. These ideas as well
as the importance of each cytokine in relationship to T cell effector functions
will be explored in the following discussion.
In experimental autoimmune encephalomyelitis (EAE), an animal
model of MS, Th17 cells have been implicated in playing a role in the disease
(Komiyama et al., 2006). In patients with MS, there is evidence for IL-
17-producing T cell upregulation in lesions (Kebir et al., 2007; Lock et al.,
2002) and cerebrospinal ﬂuid (CSF) (Ishizu et al., 2005). Here, we showed
that IL-17A and IL-17F were only elevated in sera from the relapse group
compared to control. Although elevated IL-17F levels have been documented
in sera from adult MS patients (Kallaur et al., 2013), only one
other adult study demonstrated an increase in IL-17A preferentially in
RRMS patients during relapse as opposed to patients in remission
(Brucklacher-Waldert et al., 2009). Of note, even when a single statistical
outlier for IL-17A in the remission group is eliminated, it is ﬁve standard
deviations above the mean which exceeds the usual statistical cutoff for
outliers, IL-17A is two times higher during relapse than the mean remission
value. Again, this is consistent with previous ﬁndings (BrucklacherWaldert
et al., 2009). Taken together, these data suggest the potential
use of IL17A and IL-17F as biomarkers to predict relapse. The time horizon
for the prediction is unknown, which is relevant for understanding the
therapeutic window to intervene. This is best assessed in a longitudinal
study and is not readily available from a cross-sectional survey.
Th1 cells also have a signiﬁcant role in the pathogenesis of MS, which
produce IFN-γ as their signature cytokine. Other serum cytokine proﬁle
studies in adult RRMS patients showed elevated levels of the Th1 signature
cytokine IFN-γ (Martins et al., 2011), however our study did not
observe any signiﬁcant changes in either of the MS groups. Explanations
for this disparity may be that the aforementioned study did not assess
relapse separately from remission groups or differences between adult
verses pediatric RRMS. Our study did identify, however, two cytokines
involved in the development of Th1 cells IL-12p70 and TNF-β to be elevated
in relapse compared to control sera and none were elevated in remission.
Again, emphasizing a discerning function of Th1 cells in MS
pathology with implications as a predictor of an MS relapse.
Th17 and Th1 cells are implicated in the pathogenesis of EAE, while
Th2 cells have typically been shown to have anti-inﬂammatory functions
in this setting. Interestingly, several cytokines associated with T
helper type 2 development IL-33, IL-5, IL-10, IL-13, IL-25/IL-17E are elevated
in the relapse group compared to control sera, whereas only IL-5
and IL-10 are elevated in the remission sera compared to control. These
data provide important implications for Th2-associated cytokines in MS
with their augmentation in the relapse phase of the disease, and suggest
that a greater understanding of the function of these cytokines in different
phases of disease may lead to new insights into the role of Th2 cells
in pediatric RRMS.
IL-28A (a Type III Interferon), similar to the Type I Interferons used
to treat MS, has been shown to have both potent anti-viral/pro-inﬂammatory
properties as well immuno-modulatory functions (Li et al.,
2009). In this study, it was shown here to be upregulated in both phases
of the disease. The CCL20/MIP3a chemokine was found to be associated
with Th17 entry speciﬁcally in the choroid plexus of mice in order to initiate
experimental autoimmune encephalomyelitis (EAE), the animal
model of EAE (Reboldi et al., 2009). Furthermore, mice deﬁcient in
CCR6, a receptor speciﬁc for the CCL20 chemokine, showed reduced T
cell entry into the ﬁfth lumbar spinal cord during the early stages of
EAE induction (Arima et al., 2012). Finally, CCL20/CCR6 signaling was
also shown to be required for T regulatory cell (Tregs) recruitment
into the inﬂamed CNS during EAE (Yamazaki et al., 2008; Villares
et al., 2009). Th1 and Th17 effector T cells have been shown to be drivers
of CNS inﬂammation in MS, while Tregs dampen inﬂammation and are
important for disease resolution. In our cohort, CCL20 was signiﬁcantly
increased both in MS relapse as well as remission. This suggests that this
chemokine may have a biphasic effect during disease, in which it may
enhance Th17 cell entry during relapses and promote disease resolution
during remission via Treg recruitment. IL-9, which is released by a relatively
unexplored subset of T cells, Th9, has been shown to promote expression
of CCL20 in tumor cells as well as activate T cells in
autoimmune inﬂammation (Lu et al., 2012; Li et al., 2011; Nowak
et al., 2009). IL-9 is signiﬁcantly upregulated in MS remission serum
samples, but not in the MS relapse subset compared to control.
Although this study does not represent a complete view of the
CD4+ T cell effector groups and their contribution to disease pathogenesis,
collectively, the cytokine proﬁle during relapse is distinct from the
remission group. Our analysis of cytokine levels in remission sera as a
function of disease duration demonstrated statistical signiﬁcance in 12
of the 25 cytokines from the panel. These data suggest that monitoring
cytokine concentrations in individuals during subsequent remission
phases compared to baseline levels may be a useful tool in monitoring
disease.
From our multivariable analyses we identiﬁed a four cytokine cluster
(IL-10, IL-21, IL-23, and IL-27) as a predictor of MS and then evaluated
the probability of having one or more cytokines within the cluster, elevated
above baseline during remission, as an indicator of a relapse. The
positive predictive value (PPV) of having one or more cytokines from
the cluster elevated ﬁve times above the normative baseline during remission
was 44%. While not a longitudinal assessment it is indicative of
some predictive value. Moreover, having none of the cytokine cluster elevated
above normative baseline during the remission phase estimated
that no relapse will occur 80% of the time. This cytokine cluster should
be evaluated in other pediatric RRMS populations as well as adults in
a prospective manner allowing the assessment of the predictive
power for elevations of these cytokines during remission and investigating
more carefully the time horizon for the predictor. Since IL-10 and IL-
27 are considered anti-inﬂammatory and IL-21 and IL-23 are proinﬂammatory,
ratios of these cytokines were evaluated. Speciﬁcally, increased
ratios of IL-10:IL-21, IL-10:IL-23, and IL-10:IL-27 were signiﬁcant,
suggesting that levels of IL-10 may be a driving force in
predicting a relapse. Even though it is generally assumed that effector
T cells mediate immunopathology in contrast to regulatory cells which
limit effector mechanisms, there are blurred boundaries in how these
6 C.M. Cala et al. / Journal of Neuroimmunology 297 (2016) 1–8
respective cytokines may play a role. For instance, IL-10 is secreted by
the effector T cell groups Th1, Th2, and Th17 as well as by T reg cells.
Therefore, increases in IL-10 during remission may be a consequence
of an increased T regulatory response, whereas elevated levels of IL-10
during relapse may indicate T cell effector populations. Interestingly, experimental
models and in vitro studies have shown that IL-27
upregulates IL-10 expression by T cells (Heinemann et al., 2014;
Apetoh et al., 2010), and the fact that both of these cytokines are coupregulated
with respect to disease course and duration suggests that
this relationship may extend to the pediatric RRMS population. Taken
together, these data support that ratios of the four cytokine clusters
may be a better biomarker to deﬁne the disease state taking into account
the interrelated expression of this four cytokine cluster in both
disease propagation and resolution.
The T cell is a known critical mediator in MS providing a strong rationale
to evaluate T cell associated cytokine signaling as a biomarker for relapse
as well as an indicator of disease progression for personalized
medicine approaches to clinical care. While it is not clear if signs of inﬂammation
in the serum can be an early predictor of an ensuing disease process
in the brain providing an important therapeutic time interval, based
on the critical role of T cells and the evidence provided here that their cytokine
proﬁle in remission is distinct from relapse, it seems a likely endeavor.
To advance such a concept, it would be beneﬁcial to evaluate T
cell cytokine proﬁles described here with lesion development assessed
by MRI imaging in a larger population. It should also be noted that our
study investigated cytokines from whole sera and not from distinct cellular
sources. Although CD4+
T cells are essential for MS pathogenesis, can
express numerous cytokine receptors, and are capable of secreting copious
amounts of cytokines upon stimulation, other immune cell populations
play a role in dictating the cytokine milieu in MS. Future studies to
ascertain the relevant cellular responders to and sources of the cytokines
measured here may further enhance the prognostic value of serum cytokine
analysis in MS and other neuro-inﬂammatory disorders such as
transverse myelitis and neuromyelitis optica.
Acknowledgments
This work was funded by NINDS P30-NS069324, The National Multiple
Sclerosis Society RG 4587-A-1, The Civitan International Research
Foundation, The Mike L. Jezdimir Transverse Myelitis Foundation, The
University of Alabama Health Services Foundation — General Endowment
Fund, Child Neurology Foundation Swaiman Scholarship
CY2013, The National Science Foundation 1355183.
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