Journal of Affective Disorders 237 (2018) 65–72

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Journal of Affective Disorders

journal homepage: www.elsevier.com/locate/jad

Research paper

Different levels of pro- and anti-inflammatory cytokines in patients with T

unipolar and bipolar depression
Ruizhi Maoa, Chen Zhanga, Jun Chena, Guoqing Zhaoa,d, Rubai Zhoua, Fan Wanga, Jingjing Xua,
Tao Yanga, Yousong Sua, Jia Huanga, Zhiguo Wua, Lan Caoa, Yong Wanga, Yingyan Hua,
⁎
Chengmei Yuana, Zhenghui Yia, Wu Honga, Zuowei Wange, Daihui Penga, Yiru Fanga,b,c,
a
Division of Mood Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China
b
CAS Center for Excellence in Brain Science and Intelligence Technology, China
c
Shanghai Key Laboratory of Psychotic Disorders, China
d
Department of Psychology, Provincial Hospital Affiliated to Shandong University, Jinan, 250021, China
e
Division of Mood Disorders, Hongkou District Mental Health Center of Shanghai, Shanghai, 200083, China

A R T I C LE I N FO A B S T R A C T

Keywords: Background: Immune system dysregulation is critical in the physiopathology of major depressive disorder (MDD)
Major depressive disorder and bipolar disorder (BD). However, it is unclear whether both diseases present the same inflammatory patterns
Bipolar disorder during depressive episodes. We explored the differences in pro- and anti-inflammatory cytokines between uni-
Depressive episode polar and bipolar depression (BDD) and the trajectory of these cytokines after acute-phase treatment.
Cytokines
Methods: Sixty-four MDD patients, 61 BDD patients, and 62 healthy controls (HCs) were enrolled. We assessed
the clinical features and cytokines plasma levels at baseline and week 12. The pro-inflammatory cytokines (IL-6,
TNF-α) and anti-inflammatory cytokines (IL-4, IL-13) of all subjects were assessed by multiplexed sandwich
ELISA-based quantitative arrays.
Results: Before acute-phase treatment, the initial levels of TNF-α and IL-13 were significantly lower in the BDD
patients than in the MDD patients. The results demonstrated that there was no relationship between each cy-
tokine level and clinical features of unipolar and bipolar depressions. After 12 weeks, TNF-α, IL-4, and IL-13
levels became lower in MDD patients than in the other two groups regardless of the patients’ response to
treatment while the levels of TNF-α and IL-4 increased only in the BDD responders.
Limitations: The effects of different drugs on inflammatory cytokines in MDD or BDD could not be explored
further due to the relatively small sample size.
Conclusion: Even within the same depressive states, MDD and BDD patients present different inflammatory
features, particularly in regard to pro-inflammatory TNF-α and anti-inflammatory IL-13. In addition, the fluc-
tuations of cytokines induced by medication may provide a hint regarding the prediction of treatment response.

1. Introduction disorders, particularly as an aberrant peripheral cytokine network.

Major depressive disorder (MDD) and bipolar disorder (BD) are
chronic, severe mood disorders, leading to substantial health and eco-
nomic burdens to patients worldwide (Merikangas et al., 2011; Smith,
2014). In China, the lifetime prevalence rates of MDD and BD are re-
latively high at approximately 3.3% and 1.5%, respectively (Hou et al.,
2016; Huang et al., 2016; Merikangas et al., 2011). Although neuro-
chemical, neuroendocrine and other hypotheses have been proposed for
decades, their pathological mechanism remains unclear. To date, an
increasing amount of evidence has indicated that the immune system
plays an important role in the psychopathology and treatment of mood
Peripheral cytokines can alter the activation of the brain and then affect
emotion and behavior in several ways, including penetration of the
brain through weak regions of the blood-brain barrier (BBB), trans-
mission by specific transporters or activation of endothelial cells to
release secondary messengers within the brain (Eyre and Baune, 2012).
Abundant evidence has shown that MDD and BD are both related to
peripheral inflammatory dysregulation. A meta-analysis including 82
studies indicated that interleukin-6 (IL-6), tumor necrosis factor-α
(TNF-α), IL-10, IL-13, IL-18 and CeC chemokine ligand 2 are elevated
in MDD patients compared to healthy controls (HCs) (Kohler et al.,
2017). Concerning BD, a systematic review and meta-analysis

⁎
Corresponding author at: Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Wan Ping Nan Road, Xuhui District, Shanghai 200030, China.
E-mail addresses: zhangchen645@gmail.com (C. Zhang), yirufang@aliyun.com (Y. Fang).

https://doi.org/10.1016/j.jad.2018.04.115
Received 5 December 2017; Received in revised form 18 April 2018; Accepted 24 April 2018
Available online 25 April 2018
0165-0327/ © 2018 Elsevier B.V. All rights reserved.
R. Mao et al.
illustrated that levels of the soluble IL-2 receptor, TNF-α, sIL-6R, and IL-
4 were significantly higher in BD patients than in healthy subjects
(Munkholm et al., 2013). Although the anti-inflammatory properties of
antidepressants are uncertain, some studies have shown that anti-
depressants decreased systemic inflammation (Kohler et al., 2018). Our
previous work supported this view by verifying that venlafaxine could
inhibit the upregulation of plasma TNF-α in Chinese patients diagnosed
with MDD (Li et al., 2013). Cytokines can be regulated by the inter-
action between T helper-1/2 lymphocytes and neurotransmitters such
as noradrenaline and serotonin, which can be altered by anti-
depressants (Wiedlocha et al., 2018). Mood stabilizers, such as lithium,
can target parts of the arachidonic acid cascade, which includes cy-
clooxygenase-2 (COX-2) and cytosolic phospholipase A (2) (cPLA (2))
(Bosetti et al., 2002). Another study from our team found that the levels
of IL-17, IL-23, TNF-α and transforming growth factor (TGF)-β1 could
be reduced by typical first-line treatments (lithium and quetiapine) for
BD with an acute manic episode (Li et al., 2015).
As mentioned above, most previous studies have explored the in-
flammatory features of MDD or BD patients compared with HCs.
However, few of these studies have attempted to directly compare MDD
and BD patients to determine the inflammatory differences between
these disorders (Bai et al., 2015; Chang and Chen, 2017; Mota et al.,
2013). The heterogeneity of affective states should be taken into con-
sideration as an important confounder in these studies as well
(Munkholm et al., 2013). Patients with bipolar depression (BDD), as
well as MDD subjects, can exhibit depressive symptoms, resulting in a
high misdiagnosis rate. It remains unknown whether BDD and MDD
patients present similar inflammatory patterns during depressive epi-
sodes and whether different immune characteristics change or not after
treatment. Evidence exists for an association among IL-6, TNF-α and
depressive episodes both in bipolar and unipolar depression (Goldsmith
et al., 2016; Kohler et al., 2018; Rosenblat and McIntyre, 2017; Zhang
et al., 2016). Meanwhile, these two pro-inflammatory cytokines have
been reported to be related to depression-like behavior in mice (Hodes
et al., 2014; Yamada et al., 2000). Anti-inflammatory cytokines, in-
cluding IL-4, IL-13 and IL-10, could induce microglia to antagonize pro-
inflammatory responses. These anti-inflammatory cytokines may also
present abnormal levels in MDD or BD patients as mentioned above
(Kohler et al., 2017; Munkholm et al., 2013). Therefore, it is necessary
to explore both pro-inflammatory and anti-inflammatory mediators at
the same time, as the two classes of molecules co-exist and cause effects
jointly in actual pathological processes (Takahashi et al., 2016; Tang
and Le, 2016).
We hypothesized that MDD and BDD might present different plasma
levels of pro- and anti-inflammatory cytokines even in the same affec-
tive state and that the differences may change along with the ther-
apeutic process. Therefore, we performed a longitudinal study to
identify the different plasma levels of IL-6, TNF-α, IL-4, IL-13, and IL-10
between MDD and BDD before treatment. In addition, we observed the
trajectory of these cytokines in both types of patients after acute-phase
treatment.
2. Material and methods
2.1. Participants
Sixty-four MDD and 61 BDD outpatients were recruited from the
Shanghai Mental Health Center. All patients were screened by a spe-
cialized psychiatrist using the Structured Clinical Interview for DSM-IV-
TR Axis I Disorders-Patient Edition (SCID-I/P). The inclusion criteria
included the following: (1) age between 18 and 50 years; (2) diagnosis
of MDD or BD based on the DSM-IV criteria; (3) total score on the
Hamilton Rating Scale for Depression-17 (HAMD-17) ≥ 17 and item
1 ≥ 2 and total score on the Young Mania Rating Scale (YMRS) ≤ 12;
and (4) no psychotropic drugs were taken for at least 1 month. The
exclusion criteria included the following: (1) comorbidity with another
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Journal of Affective Disorders 237 (2018) 65–72
Axis I psychiatric disorders within the previous 6 months, including
schizophrenia or other substance abuse disorder; (2) severe physical
illness (e.g., cardiac disease, organic brain disease or other serious
medical condition); (3) pregnant or breastfeeding; (4) alcohol users or
cigarette smokers; (5) infectious or allergy diseases within the prior
month; and (6) autoimmune disorders or the use of anti-inflammatory
drugs, corticosteroids or antibiotics. A control group consisted of 62
healthy volunteers whose gender, age, education, and BMI were mat-
ched with the patients. The MDD patients were treated with selective
serotonin reuptake inhibitors (SSRIs) or with selective noradrenaline
reuptake inhibitors (SNRIs). The BDD patients were treated according
to the Chinese treatment guidelines for bipolar disorder (see supple-
mentary Table S1). Assessments were conducted independently at
baseline and week 12 by two highly trained psychiatrists. We mon-
itored the medication adherence every four weeks through a face-to-
face interview with the revisiting patients or through telephone follow-
ups if they did not have time to come to the clinics. We asked the pa-
tients whether they took the medicine on time, and the treatment
emergent symptom scale (TESS) was used to evaluate adverse drug
reactions. The patients who finished acute-phase treatment had good
medication adherence. Response was defined as a 50% reduction in the
HAMD-17 score from baseline to the endpoint. Remission was defined
as a total HAMD-17 score ≤ 7 and a YRMS score ≤ 12 at week 12.
Informed consent was obtained from each participant and all the
procedures for this study were reviewed and approved by the
Institutional Review Boards of the Shanghai Mental Health Center (No.
2012–05).
2.2. Sample collection and measurement of cytokines
Samples of whole blood (5 ml) were collected from each fasting
participant into EDTA tubes via venipuncture between 07:00 and 09:00
am. The plasma was obtained by centrifugation at 3000 r/min for 15
minutes at 4 °C, and samples were kept frozen at −80 °C until analysis.
Plasma levels of the cytokines were measured with the Quantibody
Human Th1/Th2 Array from Ray Biotech (Norcross, GA). Compared
with a traditional sandwich-based ELISA, this array kit uses an array
format that detects multiple cytokine-specific captured antibodies on a
glass support, enabling the simultaneous quantitative detection of
multiple cytokines with small sample quantities. A laser scanner
equipped with a Cy3 wavelength (green channel), such as Axon
GenePix, was used to visualize the signals at a 532-nm wavelength
excitation. The limitation of detection was 2 pg/mL for IL-4 and TNF-α
and 1 pg/mL for IL-6, IL-10, and IL-13. Since IL-10 failed to be detected
in more than half of all participants, this cytokine was excluded from
the consequent analysis.
2.3. Statistical analyses
The Statistical Package for the Social Sciences (SPSS) version 24 was
used for data analysis. Unless otherwise stated, the statistical tests were
two-tailed, and the significance level was set at P < 0.05. Demographic
data and clinical characteristics were compared between the groups
using chi-squared tests, Student's t-test or one-way ANOVA. Because the
data distribution was skewed, we used the Kruskal-Wallis test for
identifying differences in the plasma cytokines among the three groups
and Mann-Whitney U test for pairwise comparisons. The Bonferroni-
adjusted significance tests were used for multiple comparisons. A par-
tial correlational analysis was used to explore the relationship between
clinical features and cytokines, controlling for age, gender, and BMI as
covariates. The Wilcoxon test for two related samples was used to
compare the plasma levels of cytokines pre- and post-treatment.
R. Mao et al. Journal of Affective Disorders 237 (2018) 65–72

Table 1
Demographic and clinical features of participants.
HC MDD BDD F(t)/χ2 P
(n = 62) (n = 64) (n = 61)

Demographic features
Age(years)a 30.81 ± 4.67 31.08 ± 5.76 29.52 ± 5.93 1.418 0.245
Education(years)a 15.97 ± 2.58 15.80 ± 2.39 15.65 ± 2.38 0.235 0.791
BMIa 23.10 ± 2.96 22.61 ± 3.49 21.74 ± 3.52 2.629 0.075
Gender(Male/Female)b 29/33 31/33 30/31 0.075 0.963
Marriage(single/married)b 37/25 37/27 45/16 5.065 0.131

Clinical features
Age of first onset with depressionc NA 28.75 ± 16.98 24.23 ± 11.82 1.043 0.309
Duration of current episode (weeks)c NA 33.27 ± 45.13 28.54 ± 32.63 0.561 0.455

Comorbidity(positive/negative)
Generalized anxiety disorderb NA 8/56 19/42 6.413 0.011
Social phobiab NA 12/52 6/55 2.051 0.156
Obsessive-compulsive disorderb NA 3/61 9/52 3.647 0.056
HAMD-17c NA 20.63 ± 3.10 21.75 ± 4.04 −1.746 0.084
HAMAc NA 17.20 ± 5.84 16.36 ± 6.72 0.749 0.455
YMRSc NA 1.22 ± 1.61 1.39 ± 1.68 −0.595 0.553

Note: Data were presented as mean ± SD.

MDD major depressive disorder, BDD bipolar depression, HC healthy control, BMI body mass index, HAMD-17 Hamilton Rating Scale for Depression-17, HAMA
Hamilton Rating Scale for Anxiety, YMRS Young Mania Rating Scale.
a
one-way ANOVA.
b
chi-squared test.
c
Student's t test.

3. Results
3.1. Demographic and clinical features
We enrolled sixty-two HCs, 64 MDD and 61 BDD (17 bipolar I dis-
order and 44 bipolar II disorder) patients in this study. The three groups
were compared in terms of demographic characteristics and clinical
features. As seen in Table 1, there were no significant differences in age,
gender, education, marital status or BMI among the three groups; there
were also no differences in age at the first onset of depression or in the
duration of the current episode. The BDD group was more susceptible
than the MDD group to a comorbidity with generalized anxiety disorder
(F = 6.413, P = 0.011). At baseline, there were no differences in the
total HAMD-17, Hamilton Rating Scale for Anxiety (HAMA) or YMRS
scores between the BDD and MDD groups (P > 0.05).
3.2. Plasma levels of cytokines at baseline
The baseline concentrations of two pro-inflammatory (IL-6 and
TNF-α) and anti-inflammatory (IL-4 and IL-13) cytokines in the HC,
MDD, and BDD groups are presented in Table 2. Using nonparametric
tests, we found no difference among the three groups in the IL-4 or IL-6
levels at baseline. After adjusting with Bonferroni's correction, the le-
vels of IL-13 and TNF-α in the BDD group were much lower than those
in the MDD group (P = 0.033 and P = 0.022, respectively). Table 2
shows that there were no significant differences when the two patient
groups were compared with the HC group (P > 0.05). The results of the
partial correlational analysis showed that the initial cytokine levels
were not related to clinical features in either MDD or BDD depressive
patients after controlling for age, gender and BMI as covariates
(P > 0.05; Table 3).
3.3. Changes in cytokines after treatment in the MDD and BDD groups
At the end of week 12, there were 49 MDD patients and 29 BDD
patients remaining for follow-up assessments (the drop-out rates were
23% and 53% for MDD and BDD groups, respectively). The drop-out
rate was relatively high in the BDD group, but there was no difference
in the initial demographic and clinical data between these 29 BDD
patients and the 61 BDD patients enrolled at baseline (see supplemen-
tary Table S2). The results of a linear regression analysis indicated that
neither age, gender, nor BMI was a confound for each cytokine level at
baseline or week 12 (P > 0.05). After 12 weeks of treatment, the levels
of IL-4, IL-13 and TNF-α became much lower in the MDD group than in
the HC group (P < 0.05), and the level of IL-13 was lower in the BDD
group than in the HC group (P = 0.000). Moreover, the levels of IL-4
and TNF-α were much lower in the MDD patients than in the BDD
patients (P = 0.000 and P = 0.014, respectively) (Table 2).
There were 42 MDD and 22 BDD patients who showed responses
after the 12-week treatment. The BDD patients who showed a response
to treatment had lower levels of IL-4, IL-13 and TNF-α than both MDD
and HC participants at baseline (P < 0.05). No difference was seen in
the initial IL-6 levels among the MDD responders, BDD responders and
healthy subjects (P = 0.995, Fig. 1). After acute-phase treatment, no
difference in IL-6 was found among the three groups, a finding that was
similar to the results at baseline (P = 0.164). Nevertheless, the levels of
IL-4 and TNF-α in the MDD responders were lower than those in the
other two groups (P < 0.05). In regard to IL-13, the level in the HC
group level was higher than that in the MDD or BDD groups (P < 0.01),
but the difference between the two patient groups was not statistically
significantly. In addition, the differences in IL-4 and TNF-α between
BDD and HC groups at baseline disappeared at week 12 (P > 0.05;
Fig. 2). Moreover, the levels of the four cytokines in the 38 MDD and 17
BDD patients who were in remission at the end of 12 weeks were also
analyzed. As shown in Fig. 3, the differences in the levels of the four
cytokines between the two groups of remitted patients were similar to
those in the responders.
As for non-responders, there were no differences in the initial levels
of the four cytokines among the three groups (IL-4, P = 0.259; IL-6,
P = 0.600; IL-13, P = 0.264; and TNF-α, P = 0.646). At week 12, the
levels of IL-4, IL-13, and TNF-α were lower in MDD non-responders
than in HCs, and the differences in the IL-4 and TNF-α levels were still
significant after multiple corrections (IL-4, P = 0.013; IL-3, P = 0.057;
and TNF-α, P = 0.023). No differences were found between the BDD
and HC groups at the study endpoint (P > 0.05).

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R. Mao et al. Journal of Affective Disorders 237 (2018) 65–72

Table 2
Comparisons of IL-4, IL-6, TNF-α and IL-13 in patients with unipolar depression, patients with bipolar depression and healthy control persons.
HC MDD BDD Pd MDD vs HC BDD vs HC MDD vs BDD

Mean ± SD Mean ± SD Mean ± SD Z P Z P Z P

Baseline
IL-4(pg/ml) 1.51 ± 1.20 1.35 ± 0.90 2.03 ± 4.123 0.205 −0.622 1.000 −1.712 0.261 −1.205 0.684
IL-6(pg/ml) 3.63 ± 3.83 3.48 ± 3.53 4.01 ± 4.43 0.495 −0.576 1.000 −1.181 0.714 −0.627 1.000
IL-13(pg/ml) 16.93 ± 13.14 20.74 ± 13.89 15.87 ± 13.82 0.037 1.600 0.329 −0.937 1.000 2.537 0.033
TNF-α(pg/ml) 15.28 ± 16.02 15.62 ± 13.12 14.59 ± 21.69 0.022 0.153 0.458 −1.253 0.631 2.687 0.022
12-week
IL-4(pg/ml) 1.51 ± 1.20 0.54 ± 0.95 4.40 ± 6.56 0.000 −6.751 0.000 0.778 1.000 −6.255 0.000
IL-6(pg/ml) 3.63 ± 3.83 2.71 ± 2.13 6.34 ± 8.84 0.164 −0.796 1.000 −1.094 0.822 −2.052 0.120
IL-13(pg/ml) 16.93 ± 13.14 8.04 ± 7.97 11.76 ± 19.66 0.000 −4.836 0.000 −4.543 0.000 0.416 1.000
TNF-α(pg/ml) 15.28 ± 16.02 2.83 ± 2.77 12.20 ± 23.96 0.000 −5.482 0.000 −1.723 0.255 −2.819 0.014

Note: MDD major depressive disorder, BDD bipolar depression, HC healthy control, IL-4 interleukin-4, IL-6 interleukin-6, IL-13 interleukin-13, TNF-α tumor necrosis
factor-α.
P-values of pairwise comparisons in Table 2 were Bonferroni-corrected p-values (uncorrected p-values were multiplied by 3 because of 3 pairwise comparisons).
d
Kruskal-Wallis test.

4. Discussion
The present study showed that plasma levels of two cytokines,
namely, TNF-α and IL-13, were lower in BDD patients than in MDD
patients during depressive states. After an acute-phase treatment, IL-4,
IL-13, and TNF-α levels were significantly reduced in the MDD patients
regardless of whether the antidepressants were effective or not, while
the levels of IL-4 and TNF-α were elevated only in the BDD responders.
The results indicated that these two classes of mood disorders may
present different inflammatory features, even in depressive episodes
and that plasma levels are state-related indicators that are affected by
medication.
A few studies have compared the inflammatory features of MDD and
BDD directly, but the results were not consistent. One study
(Chang et al., 2017) reported that patients with BD II exhibited sig-
nificantly higher HAMD-17 scores and CRP levels than patients with
MDD during depressive episodes. These results indicated that CRP, at a
level of 621.6 ng/ml, can be used as a biomarker to differentiate MDD
from BD II in patients during both depressed and euthymic states. An-
other study (Mota et al., 2013) showed that IL-1β levels were higher
than in MDD but not in different episodes of BD (euthymic, depressed,
manic or mixed). In accordance with these findings, we found that there
were differences in cytokines between MDD and BDD patients with
depression as well, not only in pro-inflammatory TNF-α but also in anti-
inflammatory IL-13. While the four cytokine levels did not differ be-
tween the patient groups and controls at baseline, which was incon-
sistent with the findings of previous studies. The results may partially
be attributed to the relatively small sample size. In addition, although
we controlled for some confounders, peripheral inflammatory cytokines
could also be affected by other factors, such as stress (Liu et al., 2017)
and physical activities (Euteneuer et al., 2017). These factors could lead
to a large variation in plasma concentrations but did not significantly
change the levels of cytokines between the patient groups and HCs. One
study obtained similar results after analyzing the plasma concentrations
of IL-1β, IL-6, IL-8, IL-10, TNF-α, interferon-α, and interferon-γ in 53
MDD patients and 60 HCs in China. They also found no significant
differences in these inflammatory factors between MDD patients and
healthy individuals. (Wei et al., 2018). Further studies with larger
sample sizes should be conducted in China in the futures.
Our study also found that not only TNF-α levels but also those of the
other two anti-inflammatory cytokines IL-4 and IL-13 were decreased
after antidepressant treatment. One meta-analysis yielded 32 studies
that showed a significant decrease in IL-4, IL-6, and IL-10 levels in MDD
patients after antidepressant treatment, which agreed with our results

Table 3
Partial correlation analysis between cytokines and clinical characteristics.
HAMD-17 HAMA Reductive ratio of HAMD-17 Age of first onset of depression Duration of current episode

MDD BDD MDD BDD MDD BDD MDD BDD MDD BDD

e
IL-4
R 0.151 −0.012 0.015 0.016 −0.084 −0.051 0.139 −0.208 −0.160 −0.083
P 0.247 0.953 0.910 0.938 0.581 0.803 0.287 0.308 0.222 0.688

e
IL-6
R −0.115 −0.030 0.142 −0.157 0.076 −0.066 0.080 −0.193 −0.037 −0.063
P 0.377 0.884 0.276 0.445 0.615 0.749 0.537 0.344 0.777 0.760

e
IL-13
R 0.164 0.073 0.133 −0.093 −0.042 −0.034 0.003 −0.152 −0.240 −0.241
P 0.207 0.723 0.307 0.652 0.783 0.870 0.979 0.458 0.065 0.236

TNF-α e
R −0.032 −0.183 0.024 −0.343 −0.005 −0.283 −0.043 −0.145 −0.077 −0.266
P 0.808 0.371 0.854 0.086 0.975 0.161 0.744 0.480 0.557 0.189

Note: MDD major depressive disorder, BDD bipolar depression, HAMD-17 Hamilton Rating Scale for Depression-17, HAMA Hamilton Rating Scale for Anxiety, IL-4
interleukin-4, IL-6 interleukin-6, IL-13 interleukin-13, TNF-α tumor necrosis factor-α.
e
Partial correlation analysis controlling age, gender and BMI as covariate.

68
R. Mao et al.
Fig. 1. Comparisons of IL-4, IL-6, TNF-α and IL-13 in unipolar depression responders, bipolar depression responders and healthy control persons at
baseline. a: plasma levels of IL-4, b: plasma levels of IL-6, c: plasma levels of IL-13, d: plasma levels of TNF-α; Data was present as mean ± SE; *: P < 0.05, **:
P < 0.01; MDD major depressive disorder, BDD bipolar depression, HC healthy control; IL-4 interleukin-4, IL-6 interleukin-6, IL-13 interleukin-13, TNF-α tumor
necrosis factor-α.
to some extent (Wiedlocha et al., 2018). TNF-α is a pro-inflammatory
cytokine. Thus, its reduction may be the result of the anti-inflammatory
action of the antidepressants. In contrast, IL-4 and IL-13 are anti-in-
flammatory cytokines, and their decrease may be due to other more
complicated mechanisms that require further exploration. Additionally,
we found an increase in TNF-α and IL-4 in BDD responders at week12,
which could be explained by the effect of mood stabilizers, e.g., lithium.
Twenty of the 29 BDD patients were prescribed with lithium mono-
therapy or a combination with quetiapine in the present study. Pe-
tersein et al. found that IL-1β, IL-6 and TNF-α levels increased in the
simulated blood of 30 healthy subjects supplemented with lithium
alone, or in combination with antidepressants. They explained the re-
sults as the pro-inflammatory properties of lithium (Petersein et al.,
2015). Another study investigated the effect of mood stabilizers, in-
cluding lithium and antiepileptic drugs, on cytokines using the OKT3/
5C3-stimulated blood of 14 healthy participants. The results showed
that lithium induced a nominal but not statistically significant increase
in IL-1β, IL-2, and TNF-α (Himmerich et al., 2014). Our study indicated
that not only TNF-α levels but also those of the anti-inflammatory IL-4
increased in BDD responders, a finding that aligned with the results of
Rapaport and Manji (2001). The role of mood stabilizers, such as li-
thium or valproate, in the cytokine network has not been identified due
to the complex bidirectional interaction among gene expression, cel-
lular signal transduction pathways and immune indicators. Our data
69
Journal of Affective Disorders 237 (2018) 65–72
may provide a hint as to the effect of mood stabilizers on the balance of
pro- and anti-inflammatory cytokines in BD. Unlike in the MDD pa-
tients, IL-4 and TNF-α increased in BDD patients who showed a re-
sponse or remission, but there was no change in the four cytokines in
BDD non-responders. This finding indicated that these cytokines may
change with the state of BDD, which is consistent with the findings of
other studies (Bai et al., 2014; Jacoby et al., 2016).
A strength of the present work was that the patient groups were
restricted to individuals with depression to guarantee homogeneity.
And it took both pro- and anti-inflammatory cytokines into considera-
tion at the same time. Classical activation of neuro-inflammatory sys-
tems in the central nervous system (CNS) is associated with the secre-
tion of TNF- α, IL-6, superoxide, nitric oxide (NO) and others.
Meanwhile, anti-inflammatory cytokines, including IL-4, IL-13, IL-10,
and TGF-β, induce microglia to antagonize the pro-inflammatory re-
sponses (Tang and Le, 2016; Zhou et al., 2012). IL-4 and IL-13 partially
share the common IL-13 receptor alpha 1 chain; as a result, these two
cytokines are often investigated together (Mori et al., 2016). The het-
erodimeric subunit IL-13 receptor alpha 1 has been reported in dopa-
minergic neurons of the ventral tegmental area and the substantia nigra
pars compacta, indicating that these two cytokines affect neurons that
regulate mood, reward and motor coordination (Morrison et al., 2012).
These cytokines have been proposed to be anti-inflammatory cytokines
that have effects on tissue repair and the extracellular matrix by
R. Mao et al. Journal of Affective Disorders 237 (2018) 65–72

Fig. 2. Comparisons of IL-4, IL-6, TNF-α and IL-13 in unipolar depression responders, bipolar depression responders and healthy control persons at week
12. a: plasma levels of IL-4, b: plasma levels of IL-6, c: plasma levels of IL-13, d: plasma levels of TNF-α; Data was present as mean ± SE; *: P < 0.05, **: P < 0.01;
MDD major depressive disorder, BDD bipolar depression, HC healthy control; IL-4 interleukin-4, IL-6 interleukin-6, IL-13 interleukin-13, TNF-α tumor necrosis factor-
α.

promoting the M2 microglia phenotype (Won et al., 2013; Yang et al.,
2006). Our study used TNF-α, IL-6, IL-4, and IL-13 as representatives of
pro- and anti-inflammatory cytokines, which was in accordance with
actual neuro-inflammatory reactions. Gender, BMI, infectious or allergy
diseases and other factors may impact the analysis of plasma cytokines
(Birur et al., 2017; Rethorst et al., 2014). At baseline, we excluded
participants who were alcohol drinkers or smokers and those with re-
lated somatic disorders. Meanwhile, we matched the three groups for
age, gender, education, and BMI. Additionally, compared with other
studies, both the MDD and BDD patients in our study underwent a
wash-out period of more than 4 weeks. Therefore, the results revealed
actual differences in the inflammatory features between these two types
of depressive patients without the effects of these confounders.
However, some limitations in this study should be noted and ad-
dressed in the future. First, the sample size was relatively small, which
may have lessened the power to detect differences in cytokine levels
among the three groups. In addition, subgroups analyses based on the
different drugs could not be performed, which made it difficult to ex-
plore the effects of treatment on the cytokines. The attrition rate in the
BDD group may have been slightly high. Unfortunately, regarding the
patients who dropped out, we did not perform any further investigation
to determine the reason why they failed to continue with the treatment.
Second, exercise exerts anti-inflammatory effects mainly by modifying
anti-inflammatory cytokine release and has been suggested as a po-
tential treatment option for clinical inflammatory conditions
(Euteneuer et al., 2017; Kayambu et al., 2015; Mikkelsen et al., 2017).
However, the physical exercise related information of the participants
was not collected, which made it difficult to control its effects on the
cytokines in our work. Third, it would have been more convincing to
include a reference standard, such as CRP, in the study to estimate the
extent of the inflammatory responses. Impaired neuroplasticity has
been implicated as a pathological mechanism in mood disorders
(Duman, 2004; Duman and Monteggia, 2006; Lin, 2009). In addition,
brain-derived neurotrophic factor (BDNF) is a crucial neurotrophic
factor in neurogenesis and synaptic plasticity that could, in part, be
mediated by cytokines (Koike et al., 2013; Lin, 2009; Patas et al., 2014).
Our team recently reported that mature BDNF and the mature BDNF/
precursor BDNF ratio in plasma were lower in BDD than in unipolar
depression (Zhao et al., 2017). Consequently, it is better to monitor the
behavior of downstream molecules, such as BDNF, in relation to cyto-
kine changes. Determining a relationship between these factors can
further the understanding of the mechanisms of how the immune
system affects emotion and behaviors through underlying neuroplasti-
city. White matter (WM) microstructure can be injured by inducing
oligodendrocytes apoptosis in a pro-inflammatory state. However, the
re-myelination of oligodendrocytes can be promoted by their own

70
R. Mao et al. Journal of Affective Disorders 237 (2018) 65–72

Fig. 3. Comparisons of IL-4, IL-6, TNF-α and IL-13 in remitted patients with unipolar depression, remitted patients with bipolar depression and healthy
control persons at week 12. a: plasma levels of IL-4, b: plasma levels of IL-6, c: plasma levels of IL-13, d: plasma levels of TNF-α; Data was present as mean ± SE; *:
P < 0.05, **: P < 0.01; MDD major depressive disorder, BDD bipolar depression, HC healthy control; IL-4 interleukin-4, IL-6 interleukin-6, IL-13 interleukin-13, TNF-
α tumor necrosis factor-α.

immunomodulatory capacities or by several growth factors secreted by
microglia (Favrais et al., 2011; Peferoen et al., 2014; Zeis et al., 2016).
Associations between structural connectivity in critical cortico-limbic
networks and inflammation-related cytokines have been reported in
patients with BD (Benedetti et al., 2016). A combination of neuroima-
ging and neuro-inflammatory markers analyses could provide more
important information about the differences between MDD and BDD in
terms of immune signaling pathways.
In summary, we investigated pro- and anti-inflammatory cytokines
in Chinese MDD and BDD patients during depressive episodes. We
found that these patients presented different immune features in the
same affective state, and some of the cytokines varied as patients
transitioned from depression to response/remission after treatment.
Currently, although biological criteria are not used for the diagnosis of
mood disorders, the finding that different inflammatory patterns exist
in MDD and BDD may provide a hint for further investigations ex-
ploring candidate biomarkers for distinguishing these two disorders. In
addition, the relationship observed between cytokines and prognoses
may provide some clues for new therapeutic drugs and individual
strategies in the future.
that might pose a conflict of interest in connection with this manuscript.
Contributors
Ruizhi Mao, Chen Zhang and Yiru Fang conceived and designed the
experiments. Ruizhi Mao contributed to the data analysis and wrote the
manuscript under the supervision of Yiru Fang and Chen Zhang. Jun
Chen, Guoqing Zhao, Ruibai Zhou, Fan Wang, Jingjing Xu, Tao Yang,
Yousong Su, Jia Huang, Zhiguo Wu, Lan Cao, Yong Wang, Yingyan Hu,
Chengmei Yuan, Zhenghui Yi, Wu Hong, Zuowei Wang, Daihui Peng
contributed to the clinical aspects of MDD and BDD.
Funding
This work was supported by the National Key Research and
Development Program of China (2016YFC1307100), the National
Natural Science Foundation of China (91232719, 81771465), the
National Key Clinical Disciplines at Shanghai Mental Health Centre
(OMA-MH, 2011-873), the Shanghai Municipal Commission of Health
and Family Planning, Key Developing Disciplines (2015ZB0405).

Conflict of interest Acknowledgments

The authors of this paper do not have any commercial associations We would like to thank all patients who participated in our study.

71
R. Mao et al.
Special thanks go out to our colleagues for supporting patient recruit-
ment: Xiao Lin, Chenglei Wang, Lvchun Cui, Bingcong Xu, Yu Wang.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.jad.2018.04.115.
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