Chemical dampening of Ly6Chi monocytes in the periphery produces anti-depressant effects in mice

The involvement of systemic immunity in depression pathogenesis promises a periphery-targeting paradigm in novel anti-depressant discovery. However, relatively little is known about druggable targets in the periphery for mental and behavioral control. Here we report that targeting Ly6Chi monocytes in blood can serve as a strategy for anti-depressant purpose. A natural compound, ginsenoside Rg1 (Rg1), was firstly validated as a periphery-restricted chemical probe. Rg1 selectively suppressed Ly6Chi monocytes recruitment to the inflamed mice brain. The proinflammatory potential of Ly6Chi monocytes to activate astrocytes was abrogated by Rg1, which led to a blunted feedback release of CCL2 to recruit the peripheral monocytes. In vitro study demonstrated that Rg1 pretreatment on activated THP-1 monocytes retarded their ability to trigger CCL2 secretion from co-cultured U251 MG astrocytes. CCL2-triggered p38/MAPK and PI3K/Akt activation were involved in the action of Rg1. Importantly, in mice models, we found that dampening Ly6Chi monocytes at the periphery ameliorated depression-like behavior induced by neuroinflammation or chronic social defeat stress. Together, our work unravels that blood Ly6Chi monocytes may serve as the target to enable remote intervention on the depressed brain, and identifies Rg1 as a lead compound for designing drugs targeting peripheral CCL2 signals.

subjected to a 10-day social defeat stress paradigm to induce depression-like behaviors. Rg1 was administered once daily for 28 days before the initiation of a battery of behavioral tests to assess the anxiety and depression-like behaviors.

Immunohistochemistry
Mice were perfused transcardially with about 200 mL of 10% PBS-buffered formalin via the ascending aorta. The brains were carefully removed and fixed overnight in 10% formalin plus 20% sucrose before paraffin embedding and coronal sectioning (8 μm). Antigen retrieval was performed with EDTA. After quenching endogenous peroxidase with 0.3% H 2 O 2 for 10 min, the sections were blocked by 10% goat serum for 1 h at room temperature. Then, sections were incubated overnight at 4 °C with primary anti-GFAP antibody (1:800, Epitomics, USA). After rinsing with PBS for 3 times, the sections were incubated with biotinylated secondary antibody for 30 min at room temperature. Immunoreactivity was visualized by 3, 3-diaminobenzidine (DAB).
Sections were washed thoroughly, mounted onto gelatin-coated slides, dehydrated and coverslipped before being examined by microscopy (Leica, Germany). GFAP staining intensity was assessed using Image J software (Bethesda, MD, USA), and the average optical density was used for the statistical analysis of GFAP intensity (3 sections were quantified per mouse brain). A non-stained region was selected and used as the background. All the sections were examined by pathologists in a blinded manner.

Immune cell isolation
To isolate infiltrated immune cells from mice brain, the hemispheres were carefully minced to homogenates with ice-cold PBS. After resuspension with 100% Percoll medium to generate 30% Percoll system, the mixtures (10 mL) were carefully layered onto the 70% Percoll system (2 mL), centrifuged at 500 × g, 18 ℃ for 30 min. The cell layers at the 30/70% interface were carefully collected, thoroughly washed before further analysis.
To isolate the mononuclear cells from blood and spleen homogenates, the lympholyte-Mammal separation medium was utilized. Briefly, blood was diluted 1:1 with PBS (v/v), precisely layered onto the surface of the Lympholyte-M medium and centrifuged at 1200 × g for 20 min under room temperature. Then, the cell layer at the interface was carefully transferred into a 15-ml centrifuge tube and thoroughly washed with PBS. The remaining red blood cells were removed with lysis buffer.

Flow cytometry
The flow cytometric analysis was carried out with standard methods. Briefly, cells were firstly 9 incubated in anti-CD16/32 blocking solution, and stained at 4°C for 30 min with specific antibody cocktails as follows: anti-CD45-PE, anti-CD45-Percp, anti-Ly6C-FITC, anti-Ly6C-PE, anti-CD11b-APC, anti-Ly6G-Percp, anti-CD4-Percp. After washing, the samples were stained with FITC-conjugated anti-goat secondary antibody for 30 min. After all staining was completed, the samples were washed twice in PBS and fixed in 1% paraformaldehyde before analysis. Data acquisition was performed on a FACSCalibur flow cytometer (BD Biosciences, USA) and analyzed with the FlowJo software (TreeStar).

Quantitative real-time PCR
Total RNA was isolated from the brain and cell samples using 1 mL of Trizol reagent (Takara, Japan). Total RNA (0.5 μg) was reverse-transcribed to cDNA using a PrimeScript RT Master Mix kit (Takara, Japan). Real-time qPCR reactions were carried out in a total volume of 15 μM with SYBR Green I PCR mix kit (Takara, Japan). The mRNA concentrations of all detected genes were normalized to that of β-actin. The determination of the mRNA fold changes was carried out using the 2 −ΔΔCT method. Detailed primer sequences for the targeted genes are listed as follows:

Target genes
Forward (
Following the incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody, the signals were detected using an enhanced chemiluminescence kit (Thermo Fisher Scientific, 10 Waltham, MA, USA) and were captured using a ChemiDoc XRS+ System (Bio-Rad, CA, USA).

LC-MS/MS determination of neurotransmitter and their metabolites
Briefly, the brain cortex (50 mg) was homogenated in ice-cold water (containing 20 mM ascorbic acid and 5.0 μg/mL caffeic acid) and the protein was precipitated with ice-cold acetonitrile The mobile phases were eluted at 0.2 mL/min following the gradient as follows: 30% B maintained for 1.5 min, increased to 65% at 3.5 min and held for 1.5 min, increased to 75% at 8.0 min, then decreased to 30% at 10.0 min followed by 3.0 min for equilibration. The flow was diverted to the waste in the initial 4.5 minutes. The mass spectrometer was operating at the