Journal: Military Medical Research

Article Title: FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury

doi: 10.1186/s40779-024-00523-w

Figure Lengend Snippet: High expression of FOXO1 in neutrophils aggravates neuronal damage in the acute stage of TBI. a Mortality of mice within 48 h after from TBI ( n = 50). b Behavioural recovery of mice was assessed using the Longa score and mNSS at 48 h post-TBI (sham group, n = 6; WT TBI mice, n = 7; FOXO1 △Lyz2 TBI mice, n = 8). c Immunostaining of the neutrophil marker CD177 (green), FOXO1 (red), and nuclei (DAPI, blue) in brain tissue from 3 TBI patients. Scale bar = 5 μm. d Immunostaining of the neutrophil markers LY6G (green), FOXO1 (red), and nuclei (DAPI, blue) in brain tissue from the TBI mouse model ( n = 5). Scale bar = 10 μm. e Flow cytometry analysis of the percentage of FOXO1 high neutrophils among leukocytes in peripheral blood of sham group and TBI mice. The experiment was repeated three times and n = 8 mice per group each time. f Western blotting showing the expression of NeuN in brain injury areas of mice. g Immunostaining of the neuron marker NeuN (red) and nuclei (DAPI, blue) in brain tissue from mice. Scale bar = 50 μm. In f and g , n = 6 per group for assay while here showed 3 biologically independent samples to represent the average status. Data are represented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns non-significant. FOXO1 forkhead box protein O1, TBI traumatic brain injury, WT wild-type, mNSS modified neurological severity score, DAPI 4,6-diamidino-2-phenylindole dihydrochloride, NeuN neuronal nuclear antigen

Article Snippet: 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.

Techniques: Expressing, Immunostaining, Marker, Flow Cytometry, Western Blot, Modification

Journal: Military Medical Research

Article Title: FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury

doi: 10.1186/s40779-024-00523-w

Figure Lengend Snippet: FOXO1 enhances the anti-apoptosis and IL-6 release abilities of neutrophils. a Flow cytometry showing the percentage of apoptosis in neutrophil infiltrate to brain injury in TBI mice ( n = 5). b ELISA analysis (left) and flow cytometry (right) of IL-6 levels in neutrophils of mice collected after TBI ( n = 5). c Western blotting showing the expression of VCAN in neutrophils in both TBI patients and TBI mouse models. Three biologically independent samples of each group represented the average status here. d Flow cytometry showing the percentage of apoptosis in neutrophils transfected with different over/si-expression plasmids. Three biologically independent samples to represent the average status. e ELISA analysis of IL-6 in neutrophils of mice. The experiment was repeated three times. f Putative FOXO1 binding sequence of the mouse VCAN promoter gene. VCAN mut 1: deletion of one FOXO1 binding site at − 1874 to − 1885 bp; VCAN mut 2: deletion of two FOXO1 binding sites at − 1874 to − 1885 bp and − 1281 to − 1292 bp; VCAN mut 3: deletion of three FOXO1 binding sites at − 1874 to − 1885 bp, − 1281 to − 1292 bp and − 802 to − 813 bp. g Luciferase activity of FOXO1 co-transfected with mutated reporters under specific conditions. All transfected cells were treated under the indicated conditions for 6 h and lysed for dual-luciferase measurements. h ChIP of the FOXO1 binding sequence from the murine VCAN promoter gene. After treating neutrophils with the indicated conditions for 6 h, the total chromatin was collected and amplified as input (positive control). Antibodies against FOXO1 were used to pull down the binding segments, of which IgG was introduced as a negative control. i The RMSD showed by the backbone atoms of the VCAN-BAX system during the molecular docking. j Pymol MOE software was used to predict the binding between VCAN and BAX. k Co-IP of VCAN and BAX in neutrophils from bone marrow in a mouse model. β-actin was detected in the supernatant after IP. l Immunostaining showing the expression, location, and binding between BAX and VCAN in neutrophils from bone marrow in the mouse model of TBI. Scale bar = 1 μm. m Immunostaining showing the expression of the neuron marker NeuN (green) and nuclei (DAPI, blue) in neurons co-cultured with neutrophils treated as described above. Scale bar = 50 μm. The experiment was repeated 5 times. Data are represented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns non-significant. FOXO1 forkhead box protein O1, TBI traumatic brain injury, ELISA enzyme‐linked immunosorbent assay, ORF open reading frame, mut mutant, ChIP chromatin immunoprecipitation, VCAN Versican, RMSD root mean square deviation, BAX BCL-2-associated X protein, DAPI 4,6-diamidino-2-phenylindole dihydrochloride, Co-IP co-immunoprecipitation

Article Snippet: 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.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Transfection, Binding Assay, Sequencing, Luciferase, Activity Assay, Amplification, Positive Control, Negative Control, Software, Co-Immunoprecipitation Assay, Immunostaining, Marker, Cell Culture, Mutagenesis, Chromatin Immunoprecipitation, Immunoprecipitation

Journal: Military Medical Research

Article Title: FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury

doi: 10.1186/s40779-024-00523-w

Figure Lengend Snippet: High expression of FOXO1 in neutrophils reduces the incidence of depression after TBI. FOXO1 △Lyz2 mice and their WT littermate were randomly divided into a sham group and a TBI group. a Body weight and behavioral tests, including the sucrose preference and TST in mice, n = 30 in the sham group, n = 100 in WT TBI mice, n = 30 in FOXO1 △Lyz2 sham mice, and n = 30 in FOXO1 △Lyz2 TBI mice. b The behavioral tests of sucrose preference and TST were assayed in WT TBI mice for the identification of depression after TBI. Sham group, n = 15; TBI with depression group, n = 37; TBI without depression group, n = 55. c The behavioral tests of sucrose preference and TST were assayed in FOXO1 △Lyz2 TBI mice for the identification of depression after TBI. Sham group, n = 15; TBI with depression group, n = 6; TBI without depression group, n = 22. d Immunostaining of fibrinogen (green) and CD31 (red) in the brain tissue of TBI mice (scale bar = 20 μm). e Immunostaining of FOXO1 (red), LY6G (green), and nuclei (DAPI, blue) in brain injury tissue of the TBI mouse model (scale bar = 20 μm). In d and e , in each group, n = 5 for data collection. Data are represented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns non-significant. FOXO1 forkhead box protein O1, TBI traumatic brain injury, WT wild-type, TST tail suspension test, DAPI 4,6-diamidino-2-phenylindole dihydrochloride

Article Snippet: 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.

Techniques: Expressing, Immunostaining, Suspension

Journal: Military Medical Research

Article Title: FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury

doi: 10.1186/s40779-024-00523-w

Figure Lengend Snippet: High expression of FOXO1 in neutrophils increases myelin damage. a Proteomic analysis of brain tissues from mice. The top 30 proteins are enriched terms by GO analysis (group of TBI with depression vs. group of TBI without depression in WT mice). b Immunostaining of MBP (green) and nuclei (DAPI, blue) in the brain tissue of different groups ( n = 5). Scale bar = 50 μm. c Electron microscopy image of corpus callosum sections and quantification of myelinated axon number and g-ratio ( n = 6). Scale bar = 1 μm. d Western blotting showing the expression of MBP in neutrophils in the TBI mouse model ( n = 3). In each group, three biologically independent samples were represented. Data are represented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns non-significant. FOXO1 forkhead box protein O1, GO Gene Ontology, TBI traumatic brain injury, MBP myelin basic protein, DAPI 4,6-diamidino-2-phenylindole dihydrochloride

Article Snippet: 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.

Techniques: Expressing, Immunostaining, Electron Microscopy, Western Blot

Journal: Military Medical Research

Article Title: FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury

doi: 10.1186/s40779-024-00523-w

Figure Lengend Snippet: FOXO1 disrupts iron homeostasis in neutrophils and oligodendrocytes. a Volcano plot showing variation in protein expression in brain-insured tissues between the group of TBI with depression and the group of TBI without depression in WT mice. The fold change (FC) log (base 2) is on the X-axis, and the negative false log discovery rate ( P- value) (base 10) is on the Y-axis. Higher expression levels are indicated by red and lower by green. b Volcano plot showing variation in protein expression of neutrophils between the TBI-induced depression group and the insusceptible group. The FC log (base 2) is on the X-axis, and the negative false log discovery rate ( P -value) (base 10) is on the Y-axis. Higher expression levels are indicated by red and lower by green. c Western blotting showing the expression of FOXO1, FTL11, TFRC, and xCT in neutrophils from the TBI mice with and without depression ( n = 6). The sham group served as the control. In each group, three biologically independent samples were represented here to show the average status. d Intracellular iron colorimetric assay showing the concentration of total Fe in neutrophils from bone marrow of the TBI mice with and without depression ( n = 5). The sham group served as the control. e LPO assay showing the concentration of LPO in neutrophils from the bone marrow of the TBI mice with and without depression ( n = 5). The sham group served as the control. f Putative FOXO1 binding sequence of the mouse TFRC promoter gene. TFRC wt1: the reporter plasmid containing the two FOXO1 binding sites at − 1070 to − 1080 bp and − 1085 to − 1095 bp; TFRC mut1: the reporter plasmid containing the two mutated FOXO1 binding sites at − 1070 to − 1080 bp and − 1085 to − 1095 bp; TFRC wt2: the reporter plasmid containing the FOXO1 binding site at − 526 to − 536 bp; TFRC mut2: the reporter plasmid containing the mutated FOXO1 binding site at − 526 to − 536 bp. g Luciferase activity of FOXO1 co-transfected with mutated reporters under specific conditions. All transfected cells were treated under the indicated conditions for 6 h and lysed for dual-luciferase measurements. h ChIP of the FOXO1 binding sequence from the murine TFRC promoter gene. After treating neutrophils with the indicated conditions for 6 h, the total chromatin was collected and amplified as input (positive control). Antibodies against FOXO1 were used to pull down the binding segments, of which IgG was introduced as a negative control. i Electron microscopy image of the form of mitochondria in neutrophils. j Intracellular iron colorimetric assay showing the concentration of total Fe in neutrophils. k LPO assay showing the concentration of LPO in neutrophils from bone marrow. l Immunostaining of MBP (green) and nuclei (DAPI, blue) in oligodendrocytes (scale bar = 50 μm). Each experiment was repeated three times. Data are represented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. FOXO1 forkhead box protein O1, TBI traumatic brain injury, wt wild-type, FTH1 ferritin heavy chain 1, FTL1 ferritin heavy chain 1, TFRC transferrin receptor, xCT system xc(−) cystine/glutamate antiporter, mut mutant, LPO lipid hydroperoxide, ChIP chromatin immunoprecipitation, MBP myelin basic protein, DAPI 4,6-diamidino-2-phenylindole dihydrochloride

Article Snippet: 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.

Techniques: Expressing, Western Blot, Control, Colorimetric Assay, Concentration Assay, Binding Assay, Sequencing, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Amplification, Positive Control, Negative Control, Electron Microscopy, Immunostaining, Mutagenesis, Chromatin Immunoprecipitation

Journal: Military Medical Research

Article Title: FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury

doi: 10.1186/s40779-024-00523-w

Figure Lengend Snippet: The effects and mechanism of FOXO1 high neutrophils in the acute and chronic stages of TBI. FOXO1 forkhead box protein O1, TBI traumatic brain injury, VCAN Versican, TFRC transferrin receptor, MBP myelin basic protein, BAX B-cell lymphoma-2 (BCL-2)-associated X protein, BCL-2 B-cell lymphoma-2, IL-6 interleukin-6

Article Snippet: 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.

Techniques: