and anti-inflammatory cytokines in patients with unipolar and bipolar ...

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 unipolar and bipolar depression

T

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 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 b

A R T I C LE I N FO

A B S T R A C T

Keywords: Major depressive disorder Bipolar disorder Depressive episode Cytokines

Background: Immune system dysregulation is critical in the physiopathology of major depressive disorder (MDD) and bipolar disorder (BD). However, it is unclear whether both diseases present the same inflammatory patterns during depressive episodes. We explored the differences in pro- and anti-inflammatory cytokines between unipolar and bipolar depression (BDD) and the trajectory of these cytokines after acute-phase treatment. 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 cytokine 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 fluctuations of cytokines induced by medication may provide a hint regarding the prediction of treatment response.

1. Introduction Major depressive disorder (MDD) and bipolar disorder (BD) are chronic, severe mood disorders, leading to substantial health and economic burdens to patients worldwide (Merikangas et al., 2011; Smith, 2014). In China, the lifetime prevalence rates of MDD and BD are relatively high at approximately 3.3% and 1.5%, respectively (Hou et al., 2016; Huang et al., 2016; Merikangas et al., 2011). Although neurochemical, 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

⁎

disorders, particularly as an aberrant peripheral cytokine network. 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), transmission 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: [email protected] (C. Zhang), [email protected] (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.

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 matched 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 supplementary Table S1). Assessments were conducted independently at baseline and week 12 by two highly trained psychiatrists. We monitored the medication adherence every four weeks through a face-toface interview with the revisiting patients or through telephone followups if they did not have time to come to the clinics. We asked the patients 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).

illustrated that levels of the soluble IL-2 receptor, TNF-α, sIL-6R, and IL4 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 antidepressants 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 interaction between T helper-1/2 lymphocytes and neurotransmitters such as noradrenaline and serotonin, which can be altered by antidepressants (Wiedlocha et al., 2018). Mood stabilizers, such as lithium, can target parts of the arachidonic acid cascade, which includes cyclooxygenase-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 inflammatory 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 consideration 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 episodes 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, including IL-4, IL-13 and IL-10, could induce microglia to antagonize proinflammatory 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 affective state and that the differences may change along with the therapeutic 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.

2.2. 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 Bonferroniadjusted significance tests were used for multiple comparisons. A partial 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.

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 specialized psychiatrist using the Structured Clinical Interview for DSM-IVTR 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 66


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Table 1 Demographic and clinical features of participants. HC (n = 62)

MDD (n = 64)

BDD (n = 61)

F(t)/χ2

P

Demographic features Age(years)a Education(years)a BMIa Gender(Male/Female)b Marriage(single/married)b

30.81 ± 4.67 15.97 ± 2.58 23.10 ± 2.96 29/33 37/25

31.08 ± 5.76 15.80 ± 2.39 22.61 ± 3.49 31/33 37/27

29.52 ± 5.93 15.65 ± 2.38 21.74 ± 3.52 30/31 45/16

1.418 0.235 2.629 0.075 5.065

0.245 0.791 0.075 0.963 0.131

Clinical features Age of first onset with depressionc Duration of current episode (weeks)c

NA NA

28.75 ± 16.98 33.27 ± 45.13

24.23 ± 11.82 28.54 ± 32.63

1.043 0.561

0.309 0.455

Comorbidity(positive/negative) Generalized anxiety disorderb Social phobiab Obsessive-compulsive disorderb HAMD-17c HAMAc YMRSc

NA NA NA NA NA NA

8/56 12/52 3/61 20.63 ± 3.10 17.20 ± 5.84 1.22 ± 1.61

19/42 6/55 9/52 21.75 ± 4.04 16.36 ± 6.72 1.39 ± 1.68

6.413 2.051 3.647 −1.746 0.749 −0.595

0.011 0.156 0.056 0.084 0.455 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

in the initial demographic and clinical data between these 29 BDD patients and the 61 BDD patients enrolled at baseline (see supplementary 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).

3.1. Demographic and clinical features We enrolled sixty-two HCs, 64 MDD and 61 BDD (17 bipolar I disorder 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 levels 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 67


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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.

Baseline IL-4(pg/ml) IL-6(pg/ml) IL-13(pg/ml) TNF-α(pg/ml) 12-week IL-4(pg/ml) IL-6(pg/ml) IL-13(pg/ml) TNF-α(pg/ml)

Pd

HC

MDD

BDD

Mean ± SD

Mean ± SD

Mean ± SD

1.51 ± 1.20 3.63 ± 3.83 16.93 ± 13.14 15.28 ± 16.02

1.35 ± 0.90 3.48 ± 3.53 20.74 ± 13.89 15.62 ± 13.12

2.03 ± 4.123 4.01 ± 4.43 15.87 ± 13.82 14.59 ± 21.69

1.51 ± 1.20 3.63 ± 3.83 16.93 ± 13.14 15.28 ± 16.02

0.54 2.71 8.04 2.83

4.40 ± 6.56 6.34 ± 8.84 11.76 ± 19.66 12.20 ± 23.96

± ± ± ±

0.95 2.13 7.97 2.77

MDD vs HC

BDD vs HC

MDD vs BDD

Z

P

Z

P

Z

P

0.205 0.495 0.037 0.022

−0.622 −0.576 1.600 0.153

1.000 1.000 0.329 0.458

−1.712 −1.181 −0.937 −1.253

0.261 0.714 1.000 0.631

−1.205 −0.627 2.537 2.687

0.684 1.000 0.033 0.022

0.000 0.164 0.000 0.000

−6.751 −0.796 −4.836 −5.482

0.000 1.000 0.000 0.000

0.778 −1.094 −4.543 −1.723

1.000 0.822 0.000 0.255

−6.255 −2.052 0.416 −2.819

0.000 0.120 1.000 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

were differences in cytokines between MDD and BDD patients with depression as well, not only in pro-inflammatory TNF-α but also in antiinflammatory IL-13. While the four cytokine levels did not differ between the patient groups and controls at baseline, which was inconsistent 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

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 significantly 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. Another 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

Table 3 Partial correlation analysis between cytokines and clinical characteristics. HAMD-17

IL-4 R P

e

IL-6 R P

e

IL-13 R P

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

0.151 0.247

−0.012 0.953

0.015 0.910

0.016 0.938

−0.084 0.581

−0.051 0.803

0.139 0.287

−0.208 0.308

−0.160 0.222

−0.083 0.688

−0.115 0.377

−0.030 0.884

0.142 0.276

−0.157 0.445

0.076 0.615

−0.066 0.749

0.080 0.537

−0.193 0.344

−0.037 0.777

−0.063 0.760

0.164 0.207

0.073 0.723

0.133 0.307

−0.093 0.652

−0.042 0.783

−0.034 0.870

0.003 0.979

−0.152 0.458

−0.240 0.065

−0.241 0.236

−0.183 0.371

0.024 0.854

−0.343 0.086

−0.005 0.975

−0.283 0.161

−0.043 0.744

−0.145 0.480

−0.077 0.557

−0.266 0.189

e

TNF-α e R −0.032 P 0.808

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


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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.

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 patients, IL-4 and TNF-α increased in BDD patients who showed a response 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 consideration at the same time. Classical activation of neuro-inflammatory systems in the central nervous system (CNS) is associated with the secretion 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 responses (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 heterodimeric subunit IL-13 receptor alpha 1 has been reported in dopaminergic 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

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-inflammatory 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 monotherapy or a combination with quetiapine in the present study. Petersein 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 results as the pro-inflammatory properties of lithium (Petersein et al., 2015). Another study investigated the effect of mood stabilizers, including 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 lithium or valproate, in the cytokine network has not been identified due to the complex bidirectional interaction among gene expression, cellular signal transduction pathways and immune indicators. Our data

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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 factorα.

anti-inflammatory cytokine release and has been suggested as a potential 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 cytokine 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 neuroplasticity. 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

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 related 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 addressed 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 explore 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 70


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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, TNFα 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 neuroimaging 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 exploring 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.

Conflict of interest

Acknowledgments

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).

We would like to thank all patients who participated in our study.

The authors of this paper do not have any commercial associations 71


R. Mao et al.

Special thanks go out to our colleagues for supporting patient recruitment: Xiao Lin, Chenglei Wang, Lvchun Cui, Bingcong Xu, Yu Wang.

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