Experimental design
Thirty inpatients with MDD were recruited from the psychiatric ward of Kaohsiung Chang Gung Memorial Hospital, Taiwan, from August 2020 to Sep 2022 and received follow-up after antidepressants treatment for 4 weeks. Twenty-eight healthy controls were enrolled from the community. Institutional Review Board approval was obtained from the hospital ethics committee (201901894A3). Blood samples were obtained for the chromatin immunoprecipitation (ChIP) assay. All participants provided informed consent and received verbal and written information prior to participating in the study.
Participants
The enrollment process was described in our previous work [13]. Patients with MDD were screened with a structured clinical interview based on the Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition) (DSM-V), and detailed assessments of current psychiatric symptoms were recorded. Past medications and scores on the 17-item Hamilton Depression Rating Scale (HAMD-17) were recorded at the same time. The exclusion criteria included having a psychotic disorder, substance dependence (including alcohol), severe obesity (body mass index [BMI] > 34 kg/m2), systemic inflammatory/infection disease and/or the taking of an anti-inflammatory or immune-modulating drug. All patients received blood pressure measurements, chest X-rays, electrocardiographic examinations, and routine blood tests after hospitalization to exclude any possible chronic systemic physical illness. Enrolled patients received no antidepressant for at least 1 week before the first blood sample was collected. Healthy controls recruited from the community had neither a personal history of nor a first-degree relative with a psychiatric disorder. The same psychiatrist who assessed the MDD group assessed the healthy control group using criteria of the DSM-V to exclude psychiatric disease. Following the above clinical examinations, blood samples were collected.
Chromatin immunoprecipitation (ChIP)
PBMCs were isolated from 20-ml blood samples using the Ficoll gradient method. For each sample, collected PBMCs (1 x 107) were transferred to a new tube, PBS was added to 7 ml, 1 ml of 8% formaldehyde was added, and the tube was shaken for 15 min at room temperature. Thereafter, 0.9 ml 10X glycine solution (1.25M glycine) was added and the tube was shaken for 10 min at room temperature. The cells were centrifuged at 800xg for 5 min at 4℃, the supernatant was removed, the pellet was resuspended in 10 ml 1X PBS, and the tube was centrifuged at 800xg for 5 min at 4℃. The resulting pellet was resuspended with 500 ml ChIP cell lysis buffer (Santa Cruz Biotechnology, #SC45000) with protease inhibitors, and the suspension was transferred to a 1.5-ml microtube, incubated on ice for 15 min, and vortexed for 5 min. The sample was centrifuged at 800xg for 5 min at 4℃, the supernatant was carefully removed, and the pellet was resuspended in 130 µl nuclear lysis buffer (50 mM Tris, 10 mM EDTA, 1% SDS). The sample was then sonicated three times for 10 min each time on ice water. In a new microtube, 100 µl sonicated sample was mixed with 900 µl CHIP dilution buffer (0.01% SDS, 1.1% Triton X- 100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl), and then with 40 µl ChIP A/G magnetic beads (Millipore, #16–663) and 4 µl protease inhibitor cocktail II (Millipore, #539132). The sample was vortexed and separated into three microtubes, and each tube was loaded with ChIP-H3K4me3 rabbit monoclonal antibodies (Millipore, #17–614), anti-normal rabbit IgG antibodies (Millipore,#CS200581), or CHIP dilution buffer. The samples were incubated overnight at 4℃ with rotation, the magnetic beads were pelleted with a magnetic separator, and the supernatant was carefully removed. The beads were sequentially washed with 500 µl low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), 500 µl high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), 500 µl LiCl buffer (0.25 M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris, pH 8.1), and 500 µl TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The beads were then mixed with 100 µl ChIP elution buffer (1% SDS, 0.1 M NaHCO3, pH 8.0) and 0.5 µl protease K (Zymoresearch, #D3001-2-20), and incubated at 55℃ for 3 hours. The DNA was purified with a DNA clean-up kit (Zymoresearch, cat.#D5205) following the manufacture's protocol, and the purified DNA was stored at -20℃ for further analysis
Quantification of CHIP DNA
Quantitative real-time PCR was used to quantify the amount of target DNA fragments bearing H3K4me3. A 7500 Fast Real-Time PCR System (Applied Biosystems) was used. The primers used for PCR reactions were as follows:
TNFAIP3-ChIP-P3-F 5'-cagggtggtttttagggttttct-3'
TNFAIP3-ChIP-P3-R 5-taggttctttttcgcagttgttttt-3'
TLR4-ChIP-P3-F 5'-AGGGGAGATTAGAATTCAGACACA-3'
TLR4-ChIP-P3-R 5'-TGCCATAAGGAAATACCACAGACT-3'
TNIP2-ChIP-P5-F 5'-CTGAGCAGCTTCTTGACTTTCTTC-3'
TNIP2-ChIP-P5-R 5'-CTGGCTTTGCTTATAGGCTTGTCT-3'
miR-146a-P3-F 5'-TGGGTTTTTGGACAGAACTGGCT-3'
miR-146a-P3-R 5'-ACTCCAAACAACCGGCACGA-3'
miR-155-F3 5'-GCCGAGCGGTTGCCTTTCTTTAC-3'
miR-155-R3 5'-GTCATCCCAATATACCTGCTTTAG-3'
GAPDH-ChIP-F 5'-TAC TAG CGG TTT TAC GGG CG-3'
GAPDH-ChIP-R 5'-TCG AAC AGG AGG AGC AGA GAG CGA-3'
Statistics
All results are expressed as the mean ± standard deviation. The Chi-square test was used to compare differences in demographic data (e.g., sex and smoking) and Student’s t-test was used to compare differences in age and BMI between groups. An analysis of covariance (ANCOVA) adjusted for age, sex, and body mass index (BMI) was used to compare differences in the H3K4me3 levels at the promoters of TNFAIP3, TLR4, miR-146a, miR-155, and TNIP2 between healthy controls and MDD patients. A paired t-test was used to compare differences in the expression levels of H3K4me3 before and after a 4-week antidepressant treatment. Multiple linear regression was used to analyze factors that correlated with the HAMD-17 scores. All statistical analyses were performed using the Statistical Product and Service Solutions (SPSS) version 22 statistical software. P-values < 0.05 were taken as indicating statistical significance.