Background
Major depression has various types of symptoms and disease courses with inconsistent response to treatments [
1]. More than 50 % of patients, who recover from a first episode of depression, are likely to suffer additional episode of major depression, resulting in a chronic lifelong illness [
2,
3]. Stress vulnerability constitutes intrinsic brain abnormality and psychosocial stressful stimuli. Thus, identifying and characterization of factors related to stress vulnerability are crucial in management of major depressive disorders [
3].
Involvement of immunology in depression pathophysiology has been a subject of research interest. “Cytokine theory of depression” proposed that cytokines are associated with the pathogenesis of depression by communicating with the central nervous system (CNS) [
4,
5]. Numerous studies have reported that circulating pro-inflammatory cytokines, such as interleukin-1 beta (IL-1β), IL-6, interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α), may be associated with some forms of major depression [
6]. Indoleamine 2,3-dioxygenase (IDO) has been suggested as a factor linking “cytokine theory” and “monoamine theory” in depression, since it is activated by pro-inflammatory cytokines and metabolizes tryptophan with downstream enzymes, leading to serotonin reduction and neurotoxicity [
7,
8]. A tricyclic antidepressant imipramine functions by inhibiting serotonin and norepinephrine reuptake, and it was found to have anti-inflammatory effects [
9]. However, inconsistent findings regarding effects of stress on inflammatory cytokines are reported, and inflammation-focused involvement of immunology in depression remains controversial [
10].
Anti-inflammatory cytokines also have been known to be involved in the pathophysiology of depression and stress response [
4,
11]. Previous studies have suggested that anti-inflammatory cytokines affect depressive behavior as a failure to counterbalance the increased expression of pro-inflammatory cytokines [
12,
13]. The ratio of IFN-γ to IL-10 was elevated in depressed patients, and it was normalized after prolonged antidepressant treatment [
14,
15]. T cells, such as regulatory T cells (T
reg), may communicate with the CNS via anti-inflammatory functions during stressful episodes, and impaired T cell function may directly contribute to the depression pathogenesis [
16]. These findings support and suggest that anti-inflammatory cytokines with T cells may participate in the common pathway in depression and other psychophysiological activities [
13].
As part of the innate immune system within the CNS, microglia was found to be involved in the pathophysiology of depression [
17,
18]. Although classification is oversimplified, microglia can exhibit classically activated M1 phenotype or M2 phenotype with alternate activation [
19]. In vitro, M1 microglia are induced by lipopolysaccharide (LPS) and express pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6 [
19,
20]. M2 microglia express CX3CR1, CD200R, and CD206 to show neuroprotective effect and homeostatic maintenance [
19,
20]. Previous studies found changes in microglial proliferation and activation in mice that were exposed to chronic restraint stress or social defeat stress [
21‐
23]. However, the relationship between peripheral cytokines and microglial phenotypes has not been elucidated in depression until now.
We found for the first time that stress vulnerability could be induced in the mice that were exposed to chronic restraint stress (CRS) with imipramine co-treatment by acute stressor after imipramine discontinuation. Chronic imipramine co-treatment inhibited stress-related behaviors but failed to normalize the decreased peripheral IL-4 and IL-10 and hippocampal M2 microglia factors. In addition, supplement of IL-4 and IL-10 prevented the stress vulnerability with normalization of M2 microglia factors in the hippocampus. Thus, our results propose potential of IL-4 and IL-10 to dampen elevated vulnerability to stress-related events to achieve more comprehensive treatment of depression.
Methods
Experimental animals
Male C57BL/6 mice at age of 7 weeks (Orient Bio Inc. Seoul, Korea) weighing 20–25 g were used for all the experiments. Animals were housed five per cage in a room maintained at 22 ± 0.5 °C with an alternating 12-h light–dark cycle. Food and water were available ad libitum. Animals were allowed to acclimate to the laboratory 1 week before the beginning of the experiments. To reduce variation, all experiments were performed during the light phase of the cycle. All experimental procedures were approved by the Animal Care and Use Committee of the CHA University (IACUC130018).
Drug treatment and stressful stimuli exposure timeline
Experiment 1: restraint stress and imipramine treatment
Animals were randomly assigned to a non-stressed control group or a CRS group, and CRS mice were randomly divided and received either normal saline or imipramine (20 mg/kg) co-treatment (co-Imi+CRS). Imipramine (Sigma-Aldrich, St. Louis, Missouri) was dissolved in physiologic normal saline and was administered intraperitoneally 30 min prior to restraint stress. For restraint stress, the mice were forced into 50 mL Corning tubes with a nose-hole for ventilation, 2 h per day (11:00 AM–1:00 PM). The mice were exposed to restraint stress (2 h/day) for 21 consecutive days (CRS). A series of behavior assays were performed within a week after the discontinuation of CRS and imipramine co-treatment. The hippocampus and the mesenteric lymph nodes were dissected 1 day after behavior assessments for Western blot and qPCR analysis. The whole blood was collected by cardiac puncture.
Experiment 2: post-short-term restraint stress and electrical foot shock
After 21 days of restraint stress and imipramine administration, mice had resting period of 5 days to allow the drug to be completely washed out [
24]. During the washout period, depressive-like behaviors of mice were assessed using sucrose preference (SP) test, elevated plus maze (EPM), and tail suspension test (TST). After 5 days of resting period, foot shock (FS) was delivered in an electrical FS chamber with a grid floor that is connected to an electric shock generator. Mice received a 0.25-mA electrical current of 1-s duration delivered randomly for 10 times over 10 min [
25]. Depressive-like behaviors were assessed again 3 days after electrical FS using SP test, light–dark (LD) box, and forced swim test (FST).
Experiment 3: IL-4 and IL-10 combination treatment
Experiment 2 described above is repeated with cytokine treatment during the 5-day washout period. Co-Imi+CRS group was randomly assigned to saline or a combination of recombinant mouse IL-4 and IL-10 (R&D Systems, Minneapolis, USA). For 5 days, mice were treated by ip injection of saline or combination of cytokines (100 ng of IL-10 and 100 ng of IL-4). Mice were then exposed to electrical FS stress (identical procedure as described above), and their depression-like behavior was assessed 3 days after the additional stress exposure. The hippocampus and the mesenteric lymph nodes were dissected 1 day after behavior assessments for further molecular analysis.
Behavioral testing
Mice were allowed to acclimate to a testing room for at least 30 min before performing the assessments. All assessments were conducted during the light cycle between 9:00 AM and 4:00 PM in a series, one assessment per day. SP test was performed independently. LD test and EPM were done using EthoVision XT9 video tracking system (EthoVision®, Version 9 Noldus, Netherlands). TST and FST were conducted by two observers to minimize error.
Sucrose preference test
Preference for sucrose solution over drinking water was measured in order to assess anhedonia, which is usually altered in depressed mice [
26]. To assess SP, mice were provided with two bottles filled with 1 % sucrose diluted in drinking water or drinking water alone. Animals were acclimatized to two bottle conditions for two consecutive days and were tested for their choice for two additional days. The position of the bottles was interchanged during 4 days of testing. On each test day, the fluid levels were noted. SP was calculated as percentage of sucrose/total fluid consumed.
Light–dark exploration
Anxiety-like behavior was measured using EPM and LD by assessing their tendency to avoid bright light and open spaces [
27,
28]. The apparatus used in this assessment was a box (30 × 30 × 30 cm) consisting of one brightly lit open chamber connected to a darkened enclosed chamber. The chambers were connected by a small square hole (7 × 7 cm). Mice were placed in the corner of the lit chamber, facing away from the dark chamber, and the number of transitions between the chambers and time spent in the dark chamber were manually measured for 10 min. The times spent in each room were recorded using EthoVision XT9 video tracking system (EthoVision
®, Version 9 Noldus, Netherlands).
Elevated plus maze
EPM apparatus and procedure are modified from the original protocol [
28]. The apparatus consisted of four open roof arms (30 × 5 cm) made of white matte Plexiglass. The two opposite arms were enclosed with 20-cm-high walls, and the remaining two opposite arms had no walls. The four arms were placed at 90° to each other around a 5 × 5-cm square in the center. The apparatus was elevated 30 cm above the floor. The mouse was initially placed in the center of the apparatus, facing one of the open arms away from an experimenter, and was allowed to explore the apparatus freely for 5 min. The number of entries to open arms and closed arms was recorded, and the times spent in each arms were recorded using EthoVision XT9 video tracking system (EthoVision
®, Version 9 Noldus, Netherlands).
Tail suspension test
The immobility induced by tail suspension was modified from a method described by Steru et al. [
29]. The apparatus consisted of a cupboard with a hook attached to the top. The mice were suspended by securing the tail to the hook by wrapping adhesive tape around the tail. The tail was suspended carefully not to fold the tail, and the tip of the tail was wrapped 2 cm away from the top of the hook. The data of the mice that climbed their tails were removed from the test. The time spent immobile during a 7-min testing period was measured. The observers were blinded to the groups. The time spent immobile was measured and compared by two observers to minimize the bias.
Forced swimming test
In FST, we assessed the ability of mice to cope with an inescapable stressful situation, which reflects depressive-like behavior [
30]. Mice were individually placed in a 2-L Pyrex beaker (13 cm diameter, 24 cm height), filled with 23 °C water with a depth of 17 cm. All mice were forced to swim for 6 min, and the duration of immobility was measured during the final 5 min of the test. The immobility was defined as the time that the mouse spent floating without struggling and making only the movements necessary to keep its head above the water level. The observers were blinded to the groups. The time spent immobile was measured and compared by two observers to minimize the bias.
Determination of glucocorticoid level
CRS mice were immediately sacrificed after the last restraint stress, and whole blood was collected by cardiac puncture, and serum was isolated and stored at −80 °C until assayed. Serum glucocorticoid level was determined by corticosterone EIA kit according to manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI).
Total protein extraction and Western blot analysis
Hippocampal protein was extracted and expression levels were assessed using Western blotting. After dissecting the hippocampus, the tissue was washed two times with cold Tris-buffered saline (TBS; 20-mM Trizma base and 137 mM NaCl, pH 7.5). Immediately after washing, cells were lysed with SDS lysis buffer (62.5-mM Trizma base, 2 % w/v SDS, 10 % glycerol) containing 0.1 mM Na3VO4, 3 mg/ml aprotinin, and 20 mM NaF. After brief sonication to shear DNA and reduce viscosity, the concentration of protein was determined with a detergent-compatible protein assay reagent (Bio-Rad Laboratories) using bovine serum albumin as the standard. After adding dithiothreitol (5 mM) and bromophenol blue (0.1 % w/v), the proteins were boiled, separated by electrophoresis in 10–16 % polyacrylamide gels (Invitrogen), and transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories). Membranes were blocked on a shaker for 1 h at room temperature. Blocking buffer consisted of TBST (Tris-buffered saline/0.1 % Tween-20) and 5 % skim milk. Primary antibodies were dissolved in the blocking buffer and the membranes were immunoblotted with antibodies against brain-derived neurotrophic factor (BDNF; 1:1000, Abcam), serotonin receptor 1A (5-HT1A; 1:1000, Santa Cruz) and serotonin receptor 2A (5-HT2A; 1:1000, Santa Cruz), IDO (1:500, Abcam), CX3CR1 (1:400, Abcam), and beta-actin (1:1000, Cell Signaling). The membranes were incubated in the anti-rabbit (1:2000, Cell Signaling) or anti-goat IgG secondary antibodies (1:2000, Santa Cruz) dissolved in the blocking buffer at a room temperature for an hour. The membranes were visualized with ECL-plus solution (Amersham Pharmacia Biotech). Then, the membranes were then exposed to chemiluminescence (LAS-4000, Fujifilm) for detection of light emission. Western blot results were scanned on a densitometer and quantified using MultiGauge (Fujifilm).
Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
The whole hippocampus and the mesenteric lymph nodes were dissected a day after behavior assessments for experiments 1 and 3. For RNA extraction, the frozen hippocampus or mesenteric lymph node was homogenized in 1 mL of QIAzol reagent per 100 mg of tissue (Qiagen, Valencia, CA). Chloroform was added to separate the phase that contains RNA, and isopropyl alcohol was added to precipitate RNA. The precipitated RNA pellet was re-dissolved in DEPC-treated water (Bioneer, Seongnam, Korea) after air-drying the pellet. Quantification of RNA concentration was determined by the absorption at 260 nm. One microgram of messenger RNA (mRNA) was reverse-transcribed into cDNA in 20 μL of reaction mix using Maxime RT PreMix Kit (Intron, Seongnam, Korea). Quantitative PCR was performed using AccuPower GreenStar qPCR PreMix (Bioneer, Seongnam, Korea). Primer sequences are listed in Table
1. The cyclic conditions consisted of an initial enzyme activation at 95 °C for 5 min followed by 40 cycles of denaturation at 95 °C for 20 s, annealing, and extension including detection of SYBR Green bound to PCR product at 56 °C for 40 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for normalization. The relative quantities of PCR fragments were calculated using the comparative CT method.
Table 1
List of primers that were used in qRT-PCR
GAPDH | CATGGCCTTCCGTGTTCCTA | GCGGCACGTCAGATCCA |
BDNF | TGCAGGGGCATAGACAAAAGG | CTTATGAATCGCCAGCCAATTCTC |
IDO | GGCTTCTTCCTCGTCTCTCTATTG | TGACGCTCTACTGCACTGGATAC |
KAT2 | GTTCTCCACACACAAGTCTC | GGATCCATCCTGTCAGTCA |
KMO | CCTGTAGAGGACAATATAGGATCAACAA | GCAAGCCCCATCTACTGCAT |
5-HT1A
| CTGTTTATCGCCCTGGATG | ATGAGCCAAGTGAGCGAGAT |
5-HT2A
| CCGCTTCAACTCCAGAACCAAAGC | CTTCGAATCATCCTGTACCCGAA |
GATA3 | AGAACCGGCCCCTTATCAA | AGTTCGCGCAGGATGTCC |
TBX21 | AGGGAACCGCTTATATGTCC | TCTCCATCATTCACCTCCAC |
FOXP3 | GAACCCAATGCCCAACCCTAG | TTCTTGGT TTTGAGGTCAAGGG |
RORγ | CCTGGGCTCCTCGCCTGACC | TCTCTCTGCCCTCAGCCTTGCC |
IL-1β | GGCTGGACTGTTTCTAATGC | ATGGTTTCTTGTGACCCTGA |
IL-4 | ACAGGAGAAGGGACGCCATG | GCAGCTTATCGATGAATCCA |
IL-6 | CCACTTCACAAGTCGGAGGCTTA | GCAAGTGCATCATCGTTGTTCATAC |
IL-10 | CCAGTTTTACCTGGTAGAAGTGATG | TGTCTAGGTCCTGGAGTCCAGCAGACTCAA |
IL-13 | TGGGTCCTGTAGATGGCATTG | AGACCAGACTCCCCTGTGCA |
IL-17 | CTCCAGAAGGCCCTCAGACTAC | GGGTCTTCATTGCGGTGG |
IFN-γ | TGA ACG CTA CAC ACT GCA TCT TGG | CGA CTC CTT TTC CGC TTC CTG AG |
TNF-α | GAGTCCGGGCAGGTCTACTTT | CAGGTCACTGTCCCAGCATCT |
CD200R | AAATGCAAATTGCCAAAATTAGA | GTATAGCTAGCATAAGGCTGCATTT |
CD206 | TCTTTGCCTTTCCCAGTCTCC | TGACACCCAGCGGAATTTC |
CX3CR1 | CAGCATCGACCGGTACCTT | GCTGCACTGTCCGGTTGTT |
NOX2 | GACCCAGATGCAGGAAAGGAA | TCATGGTGCACAGCAAAGTGAT |
Serum cytokine assay
Serum cytokine levels were measured using Bio-Rad Bio-Plex® assay (Bio-Rad, Hercules, CA). The lower detection limits of IL-1β, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ were 9.4, 2.1, 0.2, 1.0, 0.8, 1.2, and 1.4 pg/mL, respectively. The cytokine levels were measured and analyzed according to the manufacturer’s instruction.
Statistical analysis
Data were presented as the mean ± standard mean error (SEM). The statistical significance of differences between groups was assessed with Student’s t test and one-way or two-way analysis of variance (ANOVA) using GraphPad Prism version 5.0 for Mac (GraphPad, La Jolla, CA). Bonferroni’s post hoc analysis was performed when p values were <0.05.
p < 0.05 was considered as statistically significant.
Discussion
Identifying stress vulnerability after antidepressant discontinuation may be useful in treating relapses in depression. In this study, we attempted to find the answer for decreasing vulnerability to additional stressful events in subjects who had previous depressive episode with imipramine discontinuation. We found for the first time that imipramine could not prevent vulnerability to additional stressful stimuli after imipramine discontinuation although imipramine co-treatment to CRS mice inhibited induction of depressive-like behaviors due to its anti-stress effect. The stress vulnerability appears to be related to the decreased serum IL-4 and IL-10 levels despite the imipramine treatment. Thus, we propose that IL-4 and IL-10 may be able to lower the vulnerability to some forms of stress- and depression-related conditions after imipramine discontinuation. In addition, M2 microglia phenotype restoration by IL-4/10 in the hippocampus may be a possible mechanism in lowering the vulnerability towards additional stressful stimuli.
Assuming that the factors that were not recued by imipramine may be involved in the stress vulnerability of co-Imi+CRS mice, we examined the effect of imipramine on alteration of BDNF, serotonin receptors, pro-inflammatory cytokines, components of kynurenine pathway, and microglial phenotype markers in the hippocampus of the mice. BDNF, serotonin receptors, and IDO have been known as main targets of stress and antidepressants in the hippocampus [
35,
36]. Microglia are responsible for homeostasis maintenance, neuron support, and neurogenesis, and reports have shown that microglia and neuroinflammation may be involved in depression pathophysiology [
10,
19,
37]. In accordance to previous studies [
38], BDNF, IDO, and pro-inflammatory cytokine levels did not show significant difference between controls and co-Imi+CRS mice. However, we found that alternative M2 microglia markers, such as CD200R, CX3CR1, and CD206, were reduced in the hippocampus of CRS mice and were not normalized by imipramine treatment. Despite chronic imipramine co-treatment, it could not prevent vulnerability to additional stressful stimuli, and it is speculated to be due to a failure to rescue the M2 microglia phenotype in the hippocampus. This suggests that vulnerability to a subsequent stress may be associated with the changes in M2 phenotypes of microglia, not the main targets of stress and antidepressant in the hippocampus, even though we cannot exclude other brain regions that are also implicated in depression pathophysiology.
Here, imipramine was chosen as an antidepressant, since it has been extensively demonstrated to inhibit the production of pro-inflammatory cytokines and to produce anti-inflammatory cytokines [
14,
15,
39]. However, most of these studies were based on ex vivo or in vitro immunological experiments using co-incubation of the whole blood of depressed patients with imipramine so they bear some limitation [
9,
14,
15,
40]. In the present study, imipramine co-treatment was successful to normalize the IFN-γ and IL-10 production ratio but could not normalize the absolute levels of IL-4 and IL-10. In addition, transcription factors of Th2 and T
reg, which are one of the main producers of IL-4 and IL-10 in T cells [
41], were not restored by imipramine. Furthermore, stress vulnerability to FS was examined in nude mice, which lacked T cell-mediated immunity, but not in their counterpart mice, Balb/c (Additional file
1: Figure S2). These results suggest that stress vulnerability may be related to impaired T cell function, specifically IL-4 and IL-10 production. Although imipramine co-treatment restored peripheral pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, it was not successful to restore the number of leukocytes reduced by CRS in the blood, spleen, and lymph node (Additional file
1: Figure S3). This result suggests that imipramine may exert immunomodulatory effect by targeting certain subsets of leukocytes. Previous studies have reported that progression and consequence of depression may depend on integrity of the peripheral immune system and chronic stress can impair T cell immunity [
42‐
44]. Additionally, IL-4 and IL-10 are suggested to play a significant role in affecting depression-like behaviors [
13,
43,
45,
46]. In the present study, the combination of IL-4 and IL-10 prevented the stress vulnerability in co-Imi+CRS mice that encountered additional stressful stimuli after imipramine discontinuation, even though it did not have an antidepressant effect in the controls or CRS mice. Therefore, we speculated that downregulation of IL-4 and IL-10 may be the impairment in a stress-coping mechanism, which may contribute in developing vulnerability to additional stress-related events.
There are several possible ways that led to changes in microglia by IL-4/10 injection. Chronic physical stress accounts as distress and several reports have reported impaired blood–brain barrier (BBB) permeability in major depressive disorders [
47,
48]. Previous study has suggested peripheral immune imbalance is often accompanied by impairment of the BBB, which may allow the entry of IL-4 and IL-10 into the parenchyma [
49]. Previous study has found that bone marrow-derived monocytes infiltrated into the paraventricular nucleus upon exposure to chronic psychological stress, which may be associated to regional neuronal and microglial activation [
50,
51]. Whether peripheral cytokines reach to parenchyma through the BBB, peripheral cytokine signaling can induce behavioral modification through receptors on the endothelial cells [
47,
51,
52]. IL-4 and IL-10 inside the CNS were found to exert its beneficial effect on cognition and memory by alternatively activating microglia (M2) upon interaction with the respective receptors on the microglial cells [
19,
53‐
55]. Previous study has established that CNS microenvironment affects microglial cell function and suggested that microglia’s auto-regulating ability was one of mechanisms for the CNS immunosuppressive state maintenance [
53]. The choroid plexus and the cerebrospinal fluid (CSF) environment was found to be an important immunomodulatory gate that can skew immune cells towards alternately activated state due to high levels of the Th2 cells and M2 macrophage skewing cytokines in the CSF [
56]. Activated peripheral T cells can shape the CNS by entering the endothelium of the choroid plexus into the cerebrospinal fluid and communicate with the microglia and by modulating and altering the microglial phenotypes [
37,
57,
58]. Furthermore, previous studies reported that the peripheral immune system plays a key role in deciding microglial activation and phenotype and can lead to microglial response [
54,
59]. Taken together, it is speculated that peripheral administration of IL-4/10 directly or indirectly may affect microglial phenotypes across loosen BBB or through the choroid plexus and blood–CSF barrier (BCSF) or by modulating peripheral immune cells in CRS mice.
Previous research has found a decrease in pro-inflammatory cytokine release and an increase in IL-10 production from LPS stimulated-microglia with imipramine treatment, which partially agrees with what we observed in the hippocampus with CRS [
60]. Although several reports examined microglial activation using Iba-1 as target of stress and antidepressants [
10,
21‐
23], normalization of morphological activation marker Iba-1 alone appears to be insufficient to represent and to explain microglial effector function. Recent studies have demonstrated that microglial markers, such as CX3CR1, CD200R, and CD206, have a close relationship in their effector functions [
19,
37]. Disturbance of these factors may cause microglia to be non-functional; these factors are suggested to be responsible for pathology of depression [
10,
61]. CX3CR1 plays a crucial role in regulating neurotoxicity of microglia, and its stimulation was found to be anti-inflammatory [
62,
63]. CX3CR1-deficienct mice showed prolonged depression-like behavior in response to LPS, which suggests that microglial activity may cause a change in stress-related behaviors [
62‐
64]. Especially, the hippocampus was known to have the highest expression of ligand of CX3CR1 and was vulnerable to the loss of CX3CR1 [
10,
63]. In our study, CX3CR1 was decreased in CRS, co-Imi+CRS, and (co-Imi+CRS)+FS mice despite chronic imipramine co-treatment in the hippocampus while it was normalized by IL-4/10. Thus, our results suggest that M2 markers may be involved in the mechanism of peripheral immunity affecting CNS. This might be associated with restoring the impairment in stress-coping response and ameliorating the vulnerability to an additional stressor in co-imi+CRS mice after the imipramine discontinuation.
In this study, we examined increased ratio of IFN-γ to IL-10 and decrease in serum TNF-α, IL-1β, and IL-6 levels in CRS mice, and these results appear to be in contrast to most of the previous studies. Many studies have reported increased level of circulating pro-inflammatory cytokines in depression-like conditions [
6,
9,
39]. However, there are several studies that reported a decrease or no change in pro-inflammatory cytokines in depressive-like conditions, and they explained this phenomena with hypothalamic–pituitary–adrenal (HPA) axis hyperactivation, which inhibits the secretion of pro-inflammatory cytokines [
42,
45,
65‐
67]. In addition, previous studies suggested that pro-inflammatory cytokines might be a mere characteristic or a nonparticipating factor of depression [
46,
65]. In previous studies, dynamic alterations were observed in inflammatory cytokines and microglial function depending on parameters of the stress model [
10,
51]. Chronic unrelenting stress (prolonged restraint stress) was found to induce immunosuppression and anti-inflammatory phenotype [
68]. Therefore, what we observed in this study may be one of many possible immunological manifestations in major depression. Taken together, it could be postulated that stress vulnerability may be sufficiently induced by reduction in anti-inflammatory cytokines and M2 microglia markers without an increase in pro-inflammatory cytokines. However, the further study will be required to elucidate this hypothesis.
Competing interests
There is no conflict of interest.
Authors’ contributions
Han A wrote the first draft and was involved in most experiments. She contributed to conception and experimental design (Figs.
1,
2,
3,
4, and
5). Yeo HL analyzed qPCR and behavior data (Figs.
1,
2, and
3). Park MJ contributed to Western blot data and behavioral data analysis (Figs.
2 and
5). Kim SH contributed to conception and experimental design. Kim SH and Choi HJ drafted the article and revised it. Choi HJ contributed to Western blot data analysis (Fig.
3). Hong CW revised the article, focusing on immune system. Kwon MS designed this study and gave final approval of the revision. All authors commented on the manuscript and have approved the final version.