Int J Clin Exp Med 2018;11(3):2812-2820www.ijcem.com /ISSN:1940-5901/IJCEM0060771

Original ArticleFlow cytometric analysis of peripheral blood related immune cells and cytokines in patients with depression

Gen Shen Zhong1,2*, Ru Yang1*, Rui Yan Cai1, Zhi Shan Xu1, Jing Ya Li1, Tong Li1, Min Na Wu3

1Henan Key Laboratory of Neural Regeneration and Repairment, The First Affiliated Hospital of Xinxiang Medi-cal University, Weihui 453100, Henan, China; 2Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang 453003, Henan Province, China; 3College of Basic Medicine, Xinxiang Medical University, Xinxiang 453003, Henan, China. *Equal contributors.

Received July 4, 2017; Accepted December 24, 2017; Epub March 15, 2018; Published March 30, 2018

Abstract: Purposes: Profiling the peripheral blood lymphocyte subpopulations (PBLS), regulatory T cells, immune checkpoints and Th1/Th2/IL-17A cytokines to investigate the immunological variations in patients with depres-sion. Methods: A total of 51 patients diagnosed with depression and 50 healthy persons as normal control were recruited. The profiles of PBLS, regulatory T cells, immune checkpoints (PD-1, TIM-3, LAG-3) on T cells were labeled with specific antibodies, and detected by flow cytometry. Th1/Th2/IL-17A cytokines were determined by the CBA assay. Results: Compared to that of the control group, the percentages of CD3+/CD45+ T cells, CD3+CD4+/CD45+ T cells, CD3+CD8+/CD45+ T cells and CD19+/CD45+ B cells in patients with depression had no significant differences (P>0.05), however, the percentage of CD16+56+/CD45+ NK cells was significantly lower in patients with depression (P<0.05). Moreover, the percentage of CD4+CD25+FoxP3+ Treg cells was very significantly lower in patients with de-pression (P<0.01). As compared to that of the control group, the percentages of CD4+PD-1+ and CD8+PD-1+ T cells were very significantly higher (P<0.01), CD4+TIM-3+ T cells were significantly higher (P<0.05) and CD4+LAG-3+ T cells were very significantly lower (P<0.01) in patients with depression, however, there were no significant differences in percentages of CD8+TIM-3+ and CD8+LAG-3+ T cells (P>0.05). The levels of IL-6 and TNF in patients with depression were significantly higher than that in the control group (P<0.05), but there were no significant differences in IL-2, IL-4, IL-10, IL-17A and IFN-γ concentrations (P>0.05). Conclusion: Our study showed the potential influences of im-munological factors in the development of depression, which could facilitate its diagnosis and treatment.

Keywords: Flow cytometry, depression, lymphocyte subpopulations, treg, checkpoint

Introduction

Depression is a common affective disorder characterized by persistent mood depression. The continuous and rapid development of the society, has resulted in intense competition and various pressures, thereby increase the incidence of depression year by year. The inci-dence of deaths and disabilities caused by depression has also risen, which resulting in low life quality and causing serious psychologi-cal and economic burden to families and soci-ety. The World Health Organization predicts that by 2020, depression will be the second largest disease burden after ischemic heart disease worldwide [1]. In recent years, studies have found that patients with depression have obvious abnormal immune function [2, 3], ma- inly reflected in the cell and immune imbalance, abnormal cytokine levels [4, 5], immunoglobu-

lin level anomaly, lymphocyte subset changes (including Th1/Th2 imbalance, cytotoxic T lym-phocyte increase and regulatory T cells loss, etc.) and other aspects [3, 6]. However, the eti-ology and pathogenesis of depression remains unknown, and its diagnosis mainly relies on psychological counseling. In this study, we pro-filed the lymphocyte subsets, Treg cells, im- mune checkpoints (PD-1, TIM-3, LAG-3) and Th1/Th2/IL-17A cells (IL-2, IL-4, factor IL-6, IL-10, IL-17A, IFN-γ, TNF) immune index in pa- tients with depression, to facilitate effective diagnosis and clinical treatment of depression.

Materials and methods

Participants

A total of 51 cases of depression who visited the Second Affiliated Hospital of Xinxiang Medi- cal College as outpatients and inpatients from

Flow cytometric analysis of the peripheral blood in depression patients

2813 Int J Clin Exp Med 2018;11(3):2812-2820

October 2015 to August 2016, aged 21-55 ye- ars, average age (33.26±5.21), duration 180d-5 years, the average duration (7.06±0.96) mo- nths were included in this study. Inclusion crite-ria: in line with the “Chinese classification and diagnostic criteria for mental disorders (CCMD-3)” single phase depression diagnostic criteria, two deputy chief physicians above the rank of psychiatric specialist performed the diagnosis. Exclusion criteria: patients who did not meet the inclusion criteria or were unwilling to co- operate; patients who took antidepressants, nerve blocks and hormone drugs, contracep-tives, or any drugs that inhibit the immune func-tion in the past two weeks; other psychiatric diagnosis; diagnosed with other serious dis- eases, such as malignant tumor, cardiovas- cular, liver, kidney and hematopoietic system diseases.

The control group included 50 healthy normal people, aged 22-59 years, with an average age of (37.16±5.38) years. There was no difference in age and education level between the depres-sion group and the control group by the t-test and chi-square analysis of the two mean scores (Table 1).

This study was approved by the medical ethics committee of the First Affiliated Hospital of Xinxiang Medical College (batch number: 2015- 074). All participants or their families signed informed consent forms.

Specimen collection and determination

Two ml venous blood was collected from all par-ticipants in EDTA anticoagulant tubes, stored at 4°C, and treated within 2 h.

Detection of lymphocyte subsets by flow cytom-etry

100 μl anticoagulant whole blood was added to two flow tubes, marked A and B, respectively,

within 2 h. The percentage of CD3+/CD45+, CD3+CD4+/CD45+, CD3+CD8+/CD45+, CD16+ 56+/CD45+ and CD19+/CD45+ were counted (accounted for CD45+ cell) and compared be- tween the healthy normal group and depres-sion group.

Detection of treg cells by flow cytometry

Three flow tubes were marked as CD4+ (A tube), CD4+CD25+ (B tube), CD4+CD25+FoxP3+ (C tu- be), respectively. 100 μl anticoagulant whole blood was added to each tube followed by 10 μl of the corresponding labeled antibody [BD Ph- armingenTM anti-human (CD4-FITC/CD25-APC/FoxP3-PE)]. In the C tube, the cells were rup-tured (eBioscience FoxP3 Staining Buffer Set) before adding 10 μl FoxP3-PE antibody. The tubes were incubated at room temperature in the dark for 20 min. Then, 500 μl hemolysin was added to each tube, mixed, and incubat- ed at room temperature in the dark for 10 min. The tubes were incubated for 30 min at room temperature in the dark and detected on flow cytometry within 2 h. The percentage of CD4+

CD25+FoxP3+ positive cells were counted (acc- ounted for CD4+ T cell) and compared between the healthy normal group and depression group using independent sample t-test.

Detection the expression levels of PD-1, TIM-3 and LAG-3 in CD4+ T cells by flow cytometry

Four flow tubes were marked CD4+, CD4+PD-1+, CD4+TIM-3+, and CD4+LAG-3+, respectively. 100 μl anticoagulant blood was added to each tube, followed by 10 μl of the correspond- ing labeled antibody [Biolegend anti-human (CD4-APC/PD-1-FITC/TIM-3-PerCP/LAG-3-PE)], mixed, incubated for 20 min at room tempera-ture. Then, 500 μl hemolysin was added to each tube, mixed, incubated at room tempera-ture in the dark for another 10 min, and detect-ed on flow cytometry within 2 h. The percent-

Table 1. Demographic and clinical characteristics of the study subjects (/x±s)

Healthy controls (n=50)

Depression (n=51) Statistics P

Age (years) 37.16±5.38 33.26±5.21 t=1.262 0.215Education (years) 9.51±3.03 9.46±3.22 t=0.059 0.972Sex, Male% 47% 51% χ2=2.112 0.137Marrige, married% 68% 72% χ2=2.326 0.229Duration (years) - 7.06±0.96 - -

followed by 10 μl CD3/CD4/CD8/CD45 antibody (BD MultitestTM IMK Kit) to the A tube and 10 μl CD3/CD16+56/CD19 antibody (BD Mul- titestTM IMK Kit) to the B tube. The samples were mixed at room tem-perature in the dark and incubated for 20 min. Then, 500 μl hemolysin was added to each tube, mixed, incubated at room temperature in the dark for 10 min, and detected

Flow cytometric analysis of the peripheral blood in depression patients

2814 Int J Clin Exp Med 2018;11(3):2812-2820

age of CD4+PD-1+, CD4+TIM-3+, and CD4+LAG-3+ positive cells (accounted for CD4+ T cell) were counted and compared between the healthy normal group and depression group using inde-pendent sample t-test.

Detection of expression levels of PD-1, TIM-3 and LAG-3 in CD8+ T cells by flow cytometry

Four flow tubes were marked CD8+, CD8+PD-1+, CD8+TIM-3+, CD8+LAG-3+, respectively. 100 μl anticoagulant blood was added to each tube, followed by 10 μl of the corresponding labeled antibody [Biolegend anti-human (CD8-APC/PD- 1-FITC/TIM-3-PerCP/LAG-3-PE)], mixed, and in- cubated at room temperature in the dark for 20 min. Then, 500 μl hemolysin was added to each tube, mixed, incubated at room tempera-ture in the dark for 10 min, and detected on flow cytometry within 2 h. The percentage of CD8+PD-1+, CD8+TIM-3+, and CD8+LAG-3+ posi-

tive cells (accounted for CD8+ T cell) were counted and compared between the healthy normal group and depression group using inde-pendent sample t-test.

Detection of related cytokines by flow cytom-etry

The samples were processed according to the instructions provided by the Cytometric Bead Array (CBA) human Th1/Th2/Th17 cytokine kit of BD Company, and then IL-2, IL-4, IL-6, IL-10, IL-17A, TNF and IFN-γ were detected and ana-lyzed. The concentration of cytokines was cal-culated according to the protocol of CBA kit, which was then compared between the normal control group and depression group.

Statistical analysis

Flowjo 7.6.1 software was used to analyze the flow chart, and SPSS 20 software was used for statistical analysis. All measurement data were expressed by /x±s. The two groups were com-pared using independent sample t-test, and the difference was considered as statistically sig-nificant when P<0.05.

Results

Detection of lymphocyte subsets in the depres-sion and control groups

As compared to that of the control group, the percentages of CD3+/CD45+ T cells, CD3+CD4+/

Figure 1. Flow cytometry of peripheral blood lymphocyte subsets. A. Flow cytometry graph of the healthy control group; B. Flow cytometry graph of the depression group.

Table 2. Comparison of peripheral blood lymphocyte subsets between the two groups (/x±s)

Healthy controls (n=50)

Depression (n=51)

CD3+ (%) 73.20±1.32 72.90±1.45CD3+CD4+ (%) 38.10±1.66 37.80±2.49CD3+CD8+ (%) 28.10±2.08 28.40±1.43CD16+56+ (%) 14.10±0.74 9.70±1.16*CD19+ (%) 9.30±1.70 9.80±1.03Note: *P<0.05.

Flow cytometric analysis of the peripheral blood in depression patients

2815 Int J Clin Exp Med 2018;11(3):2812-2820

Figure 2. Flow cytometry of peripheral blood Treg cells. First, the single staining of CD4 antibody was used to define the quadrant of CD4+CD25+, then the CD4CD25 antibody double staining was used to define the quadrant of CD4+CD25+Foxp3+, finally the CD4CD25Foxp3 antibody triple staining was used to detect the percent-age of CD4+CD25+Foxp3+ positive cell accounting for the CD4 positive T cells.

Flow cytometric analysis of the peripheral blood in depression patients

2816 Int J Clin Exp Med 2018;11(3):2812-2820

CD45+ T cells, CD3+CD8+/CD45+ T cells and CD19+/CD45+ B cells in patients with depres-sion had no significant difference (P>0.05). However, the percentage of CD16+56+/CD45+ NK cells in patients with depression was signifi-cantly lower than that in the control group (t=-10.124, P<0.05) (Figure 1 and Table 2).

Detection of Treg cells in the depression and control groups

The percentage of CD4+CD25+FoxP3+ Treg cells was very significantly lower in the patients with depression than that in the control group (t=6.121, P<0.01) (Figure 2 and Table 3).

Detection of PD-1, TIM-3 and LAG-3 in pe-ripheral blood CD4+ T and CD8+ T cells in the healthy normal group and depression group

As compared to that in the control group, the percentages of CD4+PD-1+ and CD8+PD-1+ T cells were very significantly higher (t=12.306 and 5.059, P<0.01, respectively), CD4+TIM-3+ T cells was significantly higher (t=2.968, P<0.05) and CD4+LAG-3+ T cells was very significantly lower (t=-9.460, P<0.01) in the patients with depression. However, there were no significant differences in the percentages of CD8+TIM-3+ and CD8+LAG-3+ T cells between the two groups (P>0.05) (Figure 3 and Table 4).

Detection of related cytokines

The levels of IL-6 and TNF in patients with depression were significantly higher than that in the control group (t=3.057 and 5.836, P<0.05, respectively). There were no signifi- cant differences in IL-2, IL-4, IL-10, IL-17A and IFN-γ concentrations between the two groups (P>0.05) (Figure 4).

Discussion

Depression was resulted by many factors. Its etiology and pathogenesis are complex and still unclear. The immune system plays an important role in the development and treat-

ment of depression [7]. Based on the surface markers and different functions, lymphocytes can be divided into T lymphocytes, B lympho-cytes and NK cells. T lymphocytes are mainly involved in the cellular immunity, B lympho-cytes in humoral immunity, and NK cells in in- dependent spontaneous antigen stimulation to exert cytotoxic effects. These three types of lymphocytes maintain homeostasis of the hu- man body. Based on the cell surface markers and different functions, T lymphocytes can be divided into CD4 T and CD8 T subsets, CD8 T are the cytotoxic T lymphocytes, responsible for the removal of target cells, while CD4 T are the helper T cells (Th cells) that can be divided into Th1, Th2, Th17 and regulatory T cells (Treg cells) based on the function and secreted fac-tors [8].

Patients with depression have broad immu- nosuppression. CD4+CD25+FoxP3+ regulatory T cells are a crucial subset of T cells, and regula-tory T cells suppress effector T cell function by various ways, such as the secretion of inhibito-ry cytokines and cell contact, in order to main-tain the body’s immune balance [9]. Li Y et al showed that patients with depression had no significant difference in the number of periph-eral blood lymphocyte subsets [10], but their regulatory T cells were significantly lower than those in the control group. Excessive suppres-sion of regulatory T cell function can cause enhanced immune response, as seen in auto-immune diseases, tumors, inflammatory dis-eases and other diseases related to regulatory T cells. This study used flow cytometry assay to detect the peripheral blood lymphocyte sub-sets and CD4+CD25+FoxP3+ regulatory T cells in depression patients, and found that the per-centage of regulatory T cells was significantly decreased in the depression group as com-pared to healthy controls. However, there were no significant difference between the two gro- up among the percentages of lymphocyte sub-sets, CD3 cells, helper T cells (CD3+CD4+), cyto-toxic T cells (CD3+CD8+), and the B cell ratio. But the NK cell ratio was significantly decreas- ed in the depression group as compared to the control group.

Cytokines are heterogeneous proteins secret-ed by various cells (Th1, Th2, Th17, macro-phages and B cells), and are divided into pro-inflammatory cytokines (IL-1, IL-2, IL-6, IL-12, TNF-α, TNF-β, IFN-α, IFN-γ etc.) and anti-inflam-

Table 3. Comparison of peripheral blood Treg cells between the two groups (/x±s)

Healthy controls (n=50)

Depression (n=51)

CD4+CD25+FoxP3+ (%) 3.45±0.33 2.23±0.31**Note: **P<0.01.

Flow cytometric analysis of the peripheral blood in depression patients

2817 Int J Clin Exp Med 2018;11(3):2812-2820

Figure 3. Flow cytometry of PD-1, TIM-3 and LAG-3 in peripheral blood CD4+ T and CD8+ T cells. A. Control group using CD4 or CD8 antibody single staining. B. Healthy control group using CD4 or CD8 antibody combined with PD-1, Tim-3 or LAG-3 double staining; C. Depression group with CD4 or CD8 antibody single staining. D. Depression group which was CD4 or CD8 antibody combined with PD-1, Tim-3 or LAG-3 double staining.

Flow cytometric analysis of the peripheral blood in depression patients

2818 Int J Clin Exp Med 2018;11(3):2812-2820

matory cytokines (IL-4, IL-10, IL-13). In 1991, Smith propos- ed that the excessive release of some pro-inflammatory cy- tokines can cause abnormal activities related to depres-sion [11]. The results of this study showed that the serum levels of IL-6 and TNF were significantly higher in patients with depression than in the control group. Maes12 sugge- sted that depression is an in- flammatory disease, and pro-inflammatory cytokine levels are increased in the etiology of depression index [12]. Gu- pta R et al found that the serum levels of TNF-α were significantly decreased in de- pression patients after treat-ment [13]. Li B et al have shown that TNF-α is involved in the development of depres-sion in SD rats [14]. Liu CX et al found that the plasma lev-els of pro-inflammatory cyto-kines were significantly hig- her in patients with depres-sion than in the normal sub-jects, but the levels of these cytokines decreased after tre- atment with antidepressants [15]. This study found that pa- tients with depression had in- creased pro-inflammatory cy- tokine levels, which is in ag- reement with several domes-tic and international studies [14-16]. However, the levels of IL-2, IL-4, IL-10, IL-17A and IFN-γ were comparable be- tween the two groups, per-haps due to the pathogenesis of depression, complexity of the human immune system, or differences within the gro- up, diagnostic tools, demogra- phic data.

Recent studies have shown that immune checkpoint plays an important role in the re- gulation of T cells [17-19]. This

Table 4. Percentage of PD-1, TIM-3 and LAG-3 positive cells in peripheral blood CD4+ T and CD8+ T cells in the two groups (/x±s)

Healthy controls (n=50) Depression (n=51)CD4+PD-1+ (%) 40.68±3.32 82.82±6.90**CD4+TIM-3+ (%) 1.67±0.26 2.30±0.40*CD4+LAG-3+ (%) 73.36±3.22 23.28±4.77**CD8+PD-1+ (%) 35.30±4.46 54.72±7.33**CD8+TIM-3+ (%) 0.30±0.05 0.31±0.08CD8+LAG-3+ (%) 32.18±8.59 36.24±5.51Note: *P<0.05, **P<0.01.

Figure 4. Comparison of serum cytokines levels in peripheral blood of the depression and control groups. A. Concentration of IL-2 in the control and depression groups; B. Concentration of IL-4 in the control and depression groups; C. Concentration of IL-6 in the control and depression groups; D. Concentration of IL-10 in the control and depression groups; E. Concentra-tion of IL-17A in the control and depression groups; F. Concentration of TNF in the control and depression groups; G. Concentration of IFN-γ in the control and depression groups; The concentrations of IL-6 and TNF in the depres-sion group were significantly higher than those in the healthy control group, *P<0.05.

Flow cytometric analysis of the peripheral blood in depression patients

2819 Int J Clin Exp Med 2018;11(3):2812-2820

study examined the expression of PD-1, TIM-3 and LAG-3 in CD4 T and CD8 T cells, and found that the percentages of CD4+PD-1+, CD8+PD-1+ T cells and CD4+TIM-3+ T cells in peripheral blood was significantly higher in the depression patients than in the healthy control group. Huang YH et al found that after the suppres- sion of PD-1 and TIM-3 with specific antibodies, the tumor killing ability of CTL was enhanced [17]. Zhang L et al showed that the expressions of PD-1, TIM-3 and LAG-3 in cultured CIK were increased by varying degrees, and they were involved in the negative regulation of T cell function [19]. However, this study showed that the percentage of CD4+LAG-3+ T cells in periph-eral blood was significantly lower in the depres-sion patients than the healthy control group, while the CD8+TIM-3+ and CD8+LAG-3+ percent-age of T cells were comparable between the two groups. These results indicated that in pa- tients with depression, PD-1 may play a role in the negative regulation of CD4 T and CD8 T, TIM-3 mainly through the role of CD4 T, while LAG-3 may or may not play a role in depression. However, due to the complexity of the immune system, testing methods adopted, instruments used and other factors, these results need fur-ther validation.

In summary, our results indicated the close relationship between depression and imbal-ance of immune function. This study examined the total lymphocytes, T cells and B cells in patients with depression. Although the abso-lute numbers of total lymphocytes, T cells and B cells were similar, the percentage of CD16+ 56+ NK cells and CD4+CD25+FoxP3+ regulatory T cells were significantly decreased in patients with depression; the serum levels of IL-6, TNF were significantly increased, the expression of PD-1 on the surface of CD4+ T cells was signi- ficantly increased, TIM-3 was significantly in- creased, while only PD-1 expression was sig- nificantly increased on the surface of CD8+ T cells. The expression of TIM-3 and LAG-3 did not change significantly, indicating that CD4+ T cells may play a more important role in the development of depression than CD8+ T cells. Recently, Baruch K reported that PD-1 anti- bodies could delay the onset of Alzheimer’s disease in mice and improve their memory [20]. Combined with the findings in this study, we hypothesized that PD-1 may play an important role in the development of depression, and PD-1 antibodies may be useful in the treatment

of depression. These results could facilitate clinical diagnosis and potential treatment strat-egies for depression.

Acknowledgements

The authors thank Dr. Dong-jun Zhang for the selfless help in the evaluation and screening of patients with depression. This work was sup-ported by the Disciplinary group of Psychology and Neuroscience, Xinxiang Medical University (2016PN-KFKT-12); Henan Key Laboratory of Neural Regeneration and Repairment (HNS- JXF-2016-006; HNSJXF-2016-012 ); the Young scientist project in the First Affiliated Hospital of Xinxiang Medical Unviersity (QN-2017-A010); Key Scientific Research Projects for Higher Education of Henan Province (grant No. 17A3- 10022; 15A320063).

Disclosure of conflict of interest

None

Address correspondence to: Gen Shen Zhong, Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang 453003, Henan Province, China. E-mail: zhonggs@xxmu.edu.cn; Min Na Wu, College of Basic Medicine, Xinxiang Medical University, Jinsui Road 601#, Xinxiang 453003, Henan, China. E-mail: min-nawu@xxmu.edu.cn

References

[1] Moussavi S, Chatterji S, Verdes E, Tandon A, Patel V and Ustun B. Depression, chronic dis-eases, and decrements in health: results from the World Health Surveys. Lancet 2007; 370: 851-858.

[2] Toben C and Baune BT. An act of balance be-tween adaptive and maladaptive immunity in depression: a role for T lymphocytes. J Neuro-immune Pharmacol 2015; 10: 595-609.

[3] Blume J, Douglas SD and Evans DL. Immune suppression and immune activation in depres-sion. Brain Behav Immun 2011; 25: 221-229.

[4] Solis AC, Marques AH, Dominguez WV, Prado EB, Pannuti CM, Lotufo RF and Lotufo-Neto F. Evaluation of periodontitis in hospital outpa-tients with major depressive disorder. A focus on gingival and circulating cytokines. Brain Be-hav Immun 2016; 53: 49-53.

[5] Liu Y, Feng H, Mao H, Mo Y, Yin Y, Liu W, Song M and Wang S. Impact on serum 5-HT and TH1/TH2 in patients of depressive disorder at acute stage treated with acupuncture and

Flow cytometric analysis of the peripheral blood in depression patients

2820 Int J Clin Exp Med 2018;11(3):2812-2820

western medication. Zhongguo Zhen Jiu 2015; 35: 539-43.

[6] Noto C, Ota VK, Santoro ML, Ortiz BB, Rizzo LB, Higuchi CH, Cordeiro Q, Belangero SI, Bressan RA, Gadelha A, Maes M and Brietzke E. Effects of depression on the cytokineprofile in drug naïve first-episode psychosis. Schizophr Res 2015; 164: 51-58.

[7] Young JJ, Bruno D and Pomara N. A review of the relationship between proinflammatory cy-tokines and major depressive disorder. J Affect Disord 2014; 169: 15-20.

[8] Grosse L, Carvalho LA, Birkenhager TK, Hoogendijk WJ, Kushner SA, Drexhage HA and Bergink V. Circulating cytotoxic T cells and natural killer cells as potential predictors for antidepressant response in melancholic de-pression. Restoration of T regulatory cell popu-lations after antidepressant therapy. Psycho-pharmacology (Berl) 2016; 233: 1679-1688.

[9] Li J, Zhao R, Li X, Sun W, Qu M, Tang Q, Yang X and Zhang S. Shen-Qi-Jie-Yu-Fang exerts ef-fects on a rat model of postpartum depres- sion by regulating inflammatory cytokines and CD4(+)CD25(+) regulatory T cells. Neuropsy-chiatr Dis Treat 2016; 12: 883-896.

[10] Li Y, Xiao B, Qiu W, Yang L, Hu B, Tian X and Yang H. Altered expression of CD4(+)CD25(+) regulatory T cells and its 5-HT(1a) receptor in patients with major depression disorder. J Af-fect Disord 2010; 124: 68-75.

[11] Smith BS. The macrophage theory of depres-sion. Med Hypotheses 1991; 35: 298-306.

[12] Maes M, Song C, Lin AH, Bonaccorso S, Kenis G, De Jongh R, Bosmans E and Scharpe S. Negative immunoregulatory effects of antide-pressants: inhibition of interferon-gamma and stimulation of interleukin-10 secretion. Neuro-psychopharmacology 1999; 20: 370-379.

[13] Gupta R, Gupta K, Tripathi AK, Bhatia MS and Gupta LK. Effect of mirtazapine treatment on serum levels of brain-derived neurotrophic fac-tor and tumor necrosis factor-α in patients of major depressive disorder with severe depres-sion. Pharmacology 2016; 97: 184-188.

[14] Li B, Wang B, Chen M, Li G, Fang M and Zhai J. Expression and interaction of TNF-α and VEGF in chronic stress-induced depressive rats. Exp Ther Med 2015; 10: 863-868.

[15] Liu CX, Han LL, Yang ZZ and Yuan Q. An analy-sis of serum levels of cytokines and relative factors in depression patients. Chinese Jour-nal of Behavioral Medicine and Brain Science 2014; 23: 801-804. in Chinese.

[16] Kim YK, Na KS, Shin KH, Jung HY, Choi SH and Kim JB. Cytokine imbalance in the pathophysi-ology of major depressive disorder. Prog Neu-ropsychopharmacol Biol Psychiatry 2007; 31: 1044-1053.

[17] Huang YH, Zhu C, Kondo Y, Anderson AC, Gan-dhi A, Russell A, Dougan SK, Petersen BS, Me-lum E, Pertel T, Clayton KL, Raab M, Chen Q, Beauchemin N, Yazaki PJ, Pyzik M, Ostrowski MA, Glickman JN, Rudd CE, Ploegh HL, Franke A, Petsko GA, Kuchroo VK, Blumberg RS. CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature 2015; 517: 386-390.

[18] Grosso JF, Kelleher CC, Harris TJ, Maris CH, Hipkiss EL, De Marzo A, Anders R, Netto G, Get-net D, Bruno TC, Goldberg MV, Pardoll DM and Drake CG. LAG-3 regulates CD8+ T cell ac- cumulation and effector function in murine self- and tumor-tolerance systems. J Clin Invest 2007; 117: 3383-3392.

[19] Zhang L, Wang J, Wei F, Wang K, Sun Q, Yang F, Jin H, Zheng Y, Zhao H, Wang L, Yu W, Zhang X, An Y, Yang L, Zhang X and Ren X. Profiling the dynamic expression of checkpoint molecules on cytokine-induced killer cells from non-small-cell lung cancer patients. Oncotarget 2016; 7: 43604-43615.

[20] Baruch K, Deczkowska A, Rosenzweig N, Tsit-sou-Kampeli A, Sharif AM, Matcovitch-Natan O, Kertser A, David E, Amit L and Schwartz M. PD-1 immune checkpoint blockage reduces pathology and improves memory in mouse models of Alzheimer’s disease. Nat Med 2016; 22: 135-137.