Scientific Papers

FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury | Military Medical Research

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TBI patients

Twenty-two subjects (11 healthy donors and 11 TBI patients) were involved and had no previous history of brain injury. Peripheral blood was collected from healthy donors and TBI patients in ethylene diamine tetraacetic acid-coated tubes. The TBI-injured brain tissues were obtained from TBI patients during treatment according to the planned surgical procedures for removing the damaged brain tissue. The health donors were from the Physical Examination Center, while the TBI patients were from the Department of Neurosurgery, Daping Hospital (Army Special Medical Center). The patients met specific criteria for inclusion, which included being three male individuals aged between 40 and 70 years, diagnosed with moderate or severe TBI and having a Glasgow Coma Score ranging from 7 to 12 within 48 h of the injury. All procedures were approved by the Institutional Research Ethics Committee of Army Medical University, and written informed consent was given by the relatives of each patient in the Department of Neurosurgery, Daping Hospital (Army Special Medical Center). All samples were deidentified and coded by the attending surgeon before transport to the laboratory. The specimens were acquired and processed based on Clinical Investigation and Ethical Approval from the Daping Hospital (Army Special Medical Center) and stored according to the Principles of Human Sample Preservation in China.

Mice and TBI model

C57BL/6 WT mice (n = 400) and FOXO1Lyz2 mice (n = 100) used were 8–10 weeks old and housed in a pathogen-free environment at the animal care center of Army Medical University, China. All procedures used in this study were performed under the ethical approval of the Army Medical University Institutional Animal Care and Use Committee (AMUWEC2019085). C57BL/6 FOXO1fl/fl mice were a kind gift from Pro. Deng Laboratory at the Institute of Materia Medica, College of Pharmacy in Army Medical University. By crossing FOXO1-floxed mice with Lyz2-Cre mice purchased from Cyagen Biosciences of China, which carried the Cre recombinase inserted in the Lysozyme-M (Lyz2) gene locus, we specifically deleted FOXO1 in myeloid cells including neutrophils (named as FOXO1Lyz2) [18].

The mouse model of TBI was constructed as described previously using an automatic impact machine (LinTech, Monrovia, CA, USA) and a downstroke (velocity, 2.5 m/s; deformation depth, 3.5 mm; duration, 150 ms) to create a degree of severe TBI [19, 20]. This reproducible and consistent model is generally associated with 30% mortality within the first 5 min after injury. The sham group underwent the same surgical procedures without impact. The anesthetic recovery time of mice was also assayed to exclude the possibility of anesthesia-induced neurological deficit. To investigate the antagonistic effect of FOXO1 on TBI, we used the selective small molecule inhibitor, 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (AS1842856, HY-100596, MCE, USA), which specifically targets FOXO1. The mice were administered intraperitoneal injections of AS1842856 (10 mg/kg) either 1 h before TBI or on day 30 post-TBI to assess its impact on both acute or chronic stages of TBI.

The experimental design and main detection indexes are shown in Additional file 1: Materials and methods and Fig. S1.

Neutrophil isolation and transfection

Neutrophils from bone marrow and peripheral blood were isolated from mice and humans by Tianjin Haoyang Biological Manufacture Company, China (Lot: LZS11131, LZS1100, TBD2013NR), according to the manufacturer’s protocol. The purity of neutrophils was > 85%, which was confirmed by the fluorescence-activated cell sorting (FACS) with a specific marker [mouse: CD45+ (1:200, 103112), CD11b+ (1:200, 163702), LY6G+ (1:200, 127626); human: CD16+ (1:200, 302006), CD66b+ (1:200, 305118), CD177+ (1:200, 315810)] (Biolegend, USA). The attained neutrophils were diluted in RPMI-1640 media (SH30027.01, HyClone, USA) with 10% (v/v) foetal bovine serum (FBS) (10,091,148, Gibco, USA) to a final concentration and cultured at 37 °C in an atmosphere of 5% (v/v) CO2. All siRNAs were obtained from Wuhan GeneCreate Biological Engineering Co., Ltd. The siRNA sequences for the target genes were listed in Additional file 1: Table S1. The different plasmid was transfected by 4D-Nucleofector system (LONZA company, Swiss) using HL-60 mode. After transfection, the neutrophils were quickly diluted in RPMI-1640 media with 20% (v/v) FBS.

Primary cell culture and co-culture system

Mouse oligodendrocyte progenitor cells (OPCs) were isolated by immunostaining from wild-type (WT) mice as Niu et al. [21] described. The purified OPCs were cultured in poly-D-lysine-coated dishes with OPC mediums in the presence of platelet-derived growth factor AA (PDGF-AA, 10 ng/ml, 100-13A, Peprotech, USA). About 5 d later, the medium was replaced by PDGF-AA free medium to induce OPC differentiates into oligodendrocytes.

Primary neurons were isolated from E18−20 embryos in pregnant mice. The brain cortices were micro-dissected, trypsin digested at 37 ℃, filtered through a 70 µm cell strainer, and cultured onto 6-well plates over poly-D-lysine the surfaces with neurobasal mediums contained with B27 and GlutaMAX for the next 7 d.

On day 7, neutrophils were cultured with primary oligodendrocytes or neurons in 6-well plates for co-culture. The oligodendrocyte-neutrophil medium consisted of 80% OPC medium without PDGF-AA and 20% neutrophil medium. The neuron-neutrophil medium comprised 80% neuron medium and 2% neutrophil medium.

scRNA-seq and neutrophil subclustering

Immune cells were isolated from peripheral blood using a high-density Ficoll gradient. Briefly, peripheral blood was diluted tenfold with FACS buffer [2% FBS in phosphate-buffered saline (PBS)], carefully layered on a Ficoll gradient (11191, Sigma, Germany) and centrifuged at 400 g for 30 min at room temperature. The buffy coat was carefully removed, diluted fivefold with FACS buffer, pelleted (300 g, 5 min, 4 °C), and incubated in cold FACS buffer containing DNase I (LS006344, Worthington Biochemical Corporation, USA) for 10 min at 4 °C. Clumps were dispersed into a single-cell suspension by gentle pipetting. Single-cell suspensions were prepared and loaded on a Chromium Controller (10 × Genomics, Pleasanton, CA, USA) following the manufacturer’s specifications. The Chromium™ Controller and Chromium™ Single Cell 3′ Reagent Version 2 Kit (10 × Genomics, Pleasanton, CA, USA) were used to construct the single-cell library and sequenced using 10 × scRNA-seq by the DNBSEQ platform (BGI-Shenzhen, China).

The gene count matrix generated from Cell Ranger v7.1.0 was converted into a data format (AnnData) compatible with the Scanpy v1.9.3 [22] platform. Doublets were removed by Scrublet v0.2.1 [23, 24] using the default parameters. The principal component analysis was performed for dimensionality reduction using the function (svd_solver = “arpack”). Uniform manifold approximation and projection were then used for two-dimensional visualization of the resulting clusters. Marker genes of each cluster were identified using the function (method = “Wilcoxon”) and the clusters were annotated by cell typist [25]. Differential genes were annotated using Gene Ontology (GO) enrichment analyses.

To determine the subcluster in the neutrophils, Leiden clustering was performed with a resolution of 0.1, and marker genes of each cluster were identified with the Wilcoxon rank-sum test. The measurement of RNA velocity in neutrophils was conducted using scVelo to elucidate the dynamic processes [26]. To determine the biological activities between the subclusters of neutrophils, we employed the decoupler to analyze their biological processes [27]. Then, we used cell phone DB with the default parameters to identify the crosstalk between the subclusters of neutrophil cells [28].

Liquid chromatography-mass spectrometry (LC‑MS) analysis of cell metabolites

A liquid–liquid extraction and LC-MS method was applied to determine the metabolites in isolated neutrophils. Primarily isolated neutrophils were collected by high-speed centrifugation (13,000 g, 20 min) and lyophilized for the following steps by Shanghai Luming Biological Technology Company, China. Each group contained 8 individual samples. In brief, isolated neutrophils were analyzed using an Agilent 7890B gas chromatography system (Agilent Technologies Inc., CA, USA) coupled to an Agilent 5977A Mass Selection Detector system (Agilent Technologies Inc., CA, USA). The data preprocessing and statistical analysis were performed using the Analysis Base File Converter software. Metabolites exhibiting a ford change greater than 1.0 and a P-value less than 0.05 were considered as differential metabolites.

Tandem mass tag proteomics analysis

The isolated neutrophils were transferred to a 1.5 ml centrifuge tube, and subsequently lysed with lysis buffer containing 8 mol/L urea, 100 mmol/L triethylammonium bicarbonate at pH 8.5. Following that, the sample was ultrasonicated for 5 min on ice. The lysate was centrifuged at 12,000 g for 15 min at 4 ℃, and the supernatant was subsequently reduced with 10 mmol/L dl-dithiothreitol for 1 h at 56 ℃. This was followed by alkylation using sufficient iodoacetamide for 1 h at room temperature in the dark. Each protein sample underwent labeling with tandem mass tag and subsequent desalting and lyophilization. The sample was fractionated on a Rigol L3000 HPLC system (RIGOL TECHNOLOGIES CO., LTD, China) using a C18 column (Waters BEH C18, 4.6 mm × 250 mm, 5 μm) with the column chamber maintained at a temperature of 45 °C. For the construction of transition libraries, we conducted shotgun proteomic analyses using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher, USA) and a Q ExactiveTM series mass spectrometer (Thermo Fisher, USA) in the data-dependent acquisition mode. The statistical analysis of protein quantitation results was performed using t-test. Proteins showing significant different were considered differentially expressed if they exhibited fold changes greater than 1.5 and a P-value less than 0.05 [29].

Western blotting

To investigate the associated protein activation, Western blotting analysis was performed. The protein samples were mixed with sample loading buffer (P0015F, Beyotime, China) and subjected to heat at 95 °C for 5 min before being separated on 10% SDS-PAGE gels, and subsequently transferred onto polyvinylidene fluoride membranes. The used antibodies were: FOXO1 (1:1000, 2880 s, Cell Signaling Technology, USA), VCAN (1:500, ET7107-09, Huabio, China), BAX (1:3000, ET1603-34, Huabio, China), BCL-2 (1:3000, ER0602, Huabio, China), neuronal nuclear antigen (NeuN; 1:1000, 36,662, Cell Signaling Technology, USA), MBP (1:1000, PA1-10,008, Thermo Scientific, USA). Finally, the blots were scanned and analyzed by ImageJ software. The normalized band intensities against the corresponding housekeeping protein β-actin (1:1000, 3700 s, Cell Signaling Technology, USA) were calculated for precise comparison.

Quantitative real‑time PCR (qRT‑PCR)

Tissues or cells were washed with ice-cold PBS and then lysed directly with TRIzol (15596026, Thermo Fisher, USA). Total RNA was isolated by the RNeasy Plus Mini Kit (73404, Qiagen, Germany), and reverse transcription was carried out using the Reverse Transcriptase kit (DRR047S, Takara, Japan), all according to the manufacturer’s protocol. qPCR was carried out using FastStart Universal SYBR Green Master Mix (04707516001, Roche, Switzerland) with a Real-Time PCR Detection System (Roche, Switzerland). The relative expression of target genes was calculated using the 2−ΔΔCt method normalized to the housekeeping gene β-actin. The results are presented as a relative change compared to the controls. All the primers for qRT‑PCR were listed in Additional file 1: Table S2.

Molecular docking assay

The VCAN crystal structure was predicted by AlphaFold2, and the BAX crystal structure was based on PDB code: 5w62. The Pymol software was further used to pretreat the proteins. Molecular docking assays were performed by using the GRAMM-X server. After that GROMCS 2020 was used for molecular dynamics simulation, and the AMBER99SB-ILDN force field was selected for 50 ns simulation. Finally, the gmx_MMPBSA package was used to calculate the binding interactions between the two proteins and Pymol software visualizes the results.

Co-immunoprecipitation (Co-IP)

Co-IP was performed using the Pierce Crosstalk Magnetic IP/Co-IP Kit (88,805, Thermo Fisher Scientific, USA). Neutrophils were isolated at a total of 1 × 108 cells and lysed in lysis buffer supplemented with a protease/phosphatase inhibitor cocktail (Pierce) for 30 min on ice. The lysate was then collected by high-speed centrifugation (13,000 g, 10 min) at 4 ℃. The supernatant was incubated with BAX antibody (1:200, ET1603-34, Huabio, China), cross-linked to protein A/G magnetic beads, and incubated overnight in a refrigerator at 4 ℃. After washing with IP lysis buffer, the products underwent Western blotting analysis.

Measurement of intracellular iron and lipid hydroperoxide (LPO) concentrations

Neutrophils were isolated at a total of 1 × 106 cells. Then the iron concentration was quantified using the Cell Total Iron Colorimetric Assay Kit (E-BC-K880-M, Elabscience, China). The LPO concentration was quantified using a Lipid Peroxide Content Assay Kit (BC5245, Solarbio Life sciences, China). All the procedures are according to the manufacturer’s protocol. The reduction of Fe3+ to Fe2+ in the presence of the chromogen was quantified by measuring the absorbance at 590 nm and the LPO was measured at 532 nm and 600 nm using a spectrophotometer (Thermo Fisher, USA).

Cell respiration measurements

Ten thousand neutrophils were prepared to measure the oxygen consumption rate by using the Seahorse XFp Real-Time ATP Rate Assay Kit (Kit 103591-100, Agilent, USA). All the procedures are according to the manufacturer-recommended protocols.

Statistical analysis

All data were analyzed and presented by using GraphPad Prism Software (version 8). Unpaired two-tailed Student’s t-test was applied to compare treated groups with vehicle controls. To analyze parameters depending on two or more factors, two-way ANOVA/multivariate analysis of variance was used with Bonferroni correction. P < 0.05 was considered statistically significant. Data are represented as the mean ± SEM with *P < 0.05, **P < 0.01, ***P < 0.001.

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