Chronic high dose of captopril induces depressive-like behaviors in mice: possible mechanism of regulatory T cell in depression

Abstract

Major depression has various types of symptoms and disease courses with inconsistent response to monoamine-related antidepressants. Thus, monoamine theory may not be the only pathophysiologic pathway relevant to depression. Recently, it has been suggested that regulatory T cell (Treg) is associated with depression. Based on our previous study that showed decreased regulatory T cell (Treg) population following chronic high-dose captopril (CHC, 40 mg/kg/day * 21 days) administration, we examined whether CHC alone can induce depressive-like behaviors in mice even without stressful stimuli. In this study, we found that CHC induced depressive-like behaviors in tail suspension test (TST) and forced swimming test (FST) without systemic illness, while it did not induce anhedonic behavior, anxiety-like behaviors, or sociality-related behavior. The depressive-like behaviors were rescued by either CHC washout or antidepressant. CHC caused reduction in foxp3 and gata3 mRNA expression in the lymph nodes with elevation in plasma IL-1β and IL-6. Interestingly, CHC increased serum angiotensin II level. In the hippocampus, CHC increased TNF-α and IL-6 mRNA expression with microglia activation while reduced glucocorticoid receptor expression. However, CHC did not affect to hippocampal kynurenine pathway, serotonin level, hypothalamic corticotropin-releasing hormone mRNA level, or serum corticosterone level. Consequently, we propose that CHC may induce a specific form of depressive-like behaviors via Treg reduction and microglial activation.

Keywords: angiotensin II; captopril; cytokines; depression; regulatory T cell.

Figure 1. Chronic high-dose captopril (CHC) induced depressive-like behavior in mice

The time-course effect and dose-dependent effect of captopril were examined by observing depression-like behavior in mice by TST and FST. Mice were administrated with captopril (40 mg/kg) for 7, 14, 21 days and their behavior was assessed with TST (A) and FST (B) and n = 10 - 20 in each group. Mice were also administrated with 25 or 40 mg/kg of captopril for more than 21 days and their behavior was assessed with (C) TST and (D) FST and n = 10 - 20 in each group. Chronic high-dose captopril (CHC) is defined as 40 mg/kg of captopril in drinking water for 21 days or more and all behavior tests were performed within a week after CHC administration. Captopril administration was continued during behavioral test. The effect of captopril washout for 7 days after the end of CHC was assessed by TST (E) and FST (F) and n = 10 - 15 in each group. The effect of imipramine co-treatment (Co-Imi) during CHC was assessed by TST (G) and FST (H) and n = 22 – 27 in each group. The effect of imipramine post-treatment (Post-Imi) was assessed by TST (I) and FST (J) and n = 7 - 14 in each group. Sucrose preference test (SP) for 1 % sucrose solution over regular drinking water was examined for 2 days after 2 days of inhabitation to two bottle conditions (K). The data shown are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the controls. # p < 0. 05, ## p < 0.01, #### p < 0.0001 compared with the CHC- treated groups.

Figure 2. CHC-induced depressive-like behavior is associated with altered peripheral immune system

Mice were administrated with CHC (40 mg/ kg/ 21 days or more). Following the termination of CHC treatment, spleens of mice were analyzed with flow cytometry and expressed with percentage of CD4+ CD25+Foxp3+ cells to total lymphocytes (A) and n = 3 in each group. Mesenteric lymph nodes of mice were also dissected for quantitative reverse transcriptase polymerase chain reactions(qPCR) to assess transcription factors of T Helper 1 cells (tbx21), T Helper 2 cells (gata3) and regulatory T cells (foxp3) (B). n= 8 – 15 in each group. Serums of CHC mice and the controls were obtained to measure pro- and anti-inflammatory cytokines, such as TNF-α (C), IL-1β (D), IL-6 (E), IL-17 (F), IL-4 (G), IL-10 (H), IFN-γ(I), using Bio-Rad Bio-Plex® assay and circulating levels of angiotensin II (J) by Enzyme Immunoassay (EIA). n = 8 – 10 in each group. The data shown are mean ± SEM.* p < 0.05, *** p < 0.001 compared with the controls.

Figure 3. CHC increased inflammatory reaction in the hippocampus but not in the hypothalamus

Several markers involved in depression (bdnf, ido, kat, kmo), pro-inflammatory cytokines (tnf-α, il-1β, and il-6), serotonin receptors (5-HT1A and 5-HT2A), angiotensin receptors (AT1bR, AT1bR, and AT2R), and microglial phenotype (cx3cr1, cd200r, and p2ry12) were examined in the hippocampus (A) and n = 7 - 12 in each group. The mRNA expression levels of TNF-α and IL-6 (B) were measured by qPCR to identify the effect of imipramine co-treatment (Co-Imi). The CT values were normalized as a ratio as controls being 1 and RQ value refers to the ratio of respective transcription factors as a percentage of the controls. n = 7 - 12 in each group. An immunohistochemical study was performed to determine Iba-1 immunoreactivity in the hippocampus (C) in 20 X (left), 40 X (right) images and cells that are immunoreactive to Iba-1 were counted in same regions of dentate gyrus. n = 10 - 15 in each group. Scale bar = 100 μm. GR levels of the hippocampus (D) were assessed by western blot analysis and expression of GR was quantified using Image J. n= 5 in each group. The mRNA expressions of immunologic markers (E) such as IL-1β, IL-6, TNF-α in the hypothalamus were measured by qPCR. The CT values were normalized as a ratio as controls being 1 and RQ value refers to the ratio of respective transcription factors as a percentage of the controls. n = 5 in each group. Iba-1 immunoreactivity was also assessed in the hypothalamus (F) in 4 X images. The images were taken from bregma level - 0.46 mm (left) and bregma level - 2.06 mm (right). n = 10 - 15 in each group and shown images are representative images. Scale bar = 50 μm. * p < 0.05, ** p < 0.01 compared with the controls. # p < 0. 05, ## p < 0.01 compared with the CHC- treated groups.

Figure 4. CHC did not affect monoamine system and HPA axis in brain of mice

The levels of serotonin (5- hydroxytryptamine, 5-HT) (A) and hydroxyindoleacetic acid (HIAA) (B) were measured in hippocampus tissue of mice and n = 5 in each group. The mRNA expressions of CRH (corticotropin-releasing hormone) in the hypothalamus (C) were assessed by qPCR and n = 5 in each group. Serum glucocorticoid levels (d) were measured by EIA assay and n= 5 in each group. Data shown are mean ± SEM.