Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: scRNA-seq reveals irradiation-induced Trem2 expression in skin macrophages. (A) tSNE plots showing cell type annotation based on CellMarker 2.0, and distribution of cell clusters in control and irradiated groups. (B) Proportion of each cell population comparing control and irradiation groups. (C) Annotation of macrophage-related clusters using marker genes. (D) Quantification of macrophage cluster ratios between control and irradiated groups. (E) Dotplot showing the expression of representative genes in macrophage subclusters, highlighting Trem2 -specific enrichment. (F) irGSEA of macrophage clusters, including a gene expression heatmap and GSEA plot. (G) AUCell analysis indicating high activity of the PI3K-Akt pathway in MAC2 macrophage subclusters. (H) Separation of macrophage clusters based on Trem2 expression. (I) Comparison of the ratio of Trem2 -positive and Trem2 -negative clusters between control and irradiated samples. (J) Pseudotime trajectory analysis illustrating the developmental progression of Trem2 -positive and Trem2-negative macrophages.

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Irradiation, Expressing, Control, Marker, Gene Expression, Activity Assay, Comparison

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Radiation-induced ROS–Nrf2–ADAM17 axis promotes Trem2 shedding. (A) Quantification of sTREM2 in culture supernatants of RAW264.7 cells under control and irradiated conditions ( n = 3). (B) ADAM10 transcript levels from RNA-seq and relative mRNA expression measured by qPCR ( n = 3). (C) ADAM17 transcript levels from RNA-seq and relative mRNA expression measured by qPCR ( n = 3). (D and E) Western blotting and analysis of ADAM10 and ADAM17 ( n = 3). (F) Pearson correlation analysis between Trem2 , ADAM10 , ADAM17 , and Nrf2 based on RNA-seq data. (G) GSEA showing enrichment of the “Positive Regulation of Reactive Oxygen Species Metabolic Process” pathway after irradiation. (H) (a) ROS levels detected by fluorescence staining at 2 and 6 h post-irradiation (green), shown with brightfield (BF) (scale bar, 200 μm); (b) quantification of integrated fluorescence intensity (IntDen) ( n = 3). (I) ROS quantification based on fluorescence intensity at excitation/emission wavelengths of 488/525 nm ( n = 3). (J) (a and b) IF detection and quantification of NRF2 (green) with DAPI staining ( n = 3; scale bar, 200 μm); (c and d) Western blotting and quantification of NRF2 ( n = 3). (K and L) Western blotting and quantification of TREM2, BAX, and BCL2 protein levels in control, DMSO, and GW280264X cells with or without irradiation ( n = 3). (M) ELISA detection of sTREM2 in various treatment groups ( n = 3). (N) Cell viability assay using CCK-8 in control (Ctr), DMSO, and GW280264X (ADAM17/ADAM10 inhibitor) groups with or without irradiation ( n = 3). (O) ELISA detection of proinflammatory cytokines IL-1β, TNF-α, and IL-6 in various treatment groups ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Control, Irradiation, RNA Sequencing, Expressing, Western Blot, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, Viability Assay, CCK-8 Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Trem2 overexpression improves radiation resistance of RAW264.7 cells by reducing apoptosis. (A and B) Live/dead staining using Calcein-AM (live, green) and EthD-1 (dead, red); quantification of dead cells based on integrated fluorescence intensity ( n = 3; scale bar, 200 μm). (C) Cell viability assessed by CCK-8 assay in Blank, Vector, OE-Trem2, siNC, and si-Trem2 groups with or without irradiation ( n = 3). (D and E) Scratch assay and distance closure rate quantified at 6, 12, and 24 h relative to 0 h ( n = 3; scale bar, 200 μm). (F) Apoptosis rates determined by flow cytometry across different treatment groups ( n = 3). (G to J) Western blotting and analysis of CD206, CD86, CASPASE-9 (CAS9), total and cleaved CASPASE-3 (pro-CAS3, cleaved CAS3), BCL2, BAX, and cleaved CASPASE-8 (cleaved CAS8) ( n = 3). (K) ELISA analysis of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and the anti-inflammatory cytokine IL-10 in different groups ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; only statistically significant comparisons are shown in panel.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Over Expression, Staining, Fluorescence, CCK-8 Assay, Plasmid Preparation, Irradiation, Wound Healing Assay, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Trem2 deficiency exacerbates radiation-induced apoptosis by suppressing ERK phosphorylation. (A and B) Western blotting and quantification of TREM2 expression in LysM Cr e Trem2 flox/flox (Trem2-Cko) and LysM − Trem2 flox/flox (Trem2-Ctr) BMDM, with or without irradiation ( n = 3). (C) Flow cytometry analysis of macrophage polarization based on M1 markers (CD86 + CD206 − ) and M2 markers (CD86 − CD206 + ) ( n = 3). (D) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis based on RNA-seq data. (E) Apoptosis rate assessed by Annexin V/PI flow cytometry in Trem2-Ctr and Trem2-Cko macrophages after irradiation ( n = 3). (F and G) Western blotting and analysis of phosphorylated ERK1/2 (p-ERK), total ERK1/2 (t-ERK), CASPASE-9 (CAS9), total and cleaved CASPASE-3 (pro-CAS3, cleaved CAS3), BCL2, BAX, and cleaved CASPASE-8 (cleaved CAS8) ( n = 3). (H) ELISA quantification of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and anti-inflammatory cytokine IL-10 in Trem2-Ctr and Trem2-Cko groups ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Phospho-proteomics, Western Blot, Expressing, Irradiation, Flow Cytometry, RNA Sequencing, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Radiation delayed wound healing in radiation-induced skin injury (RISI) and induced TREM2 expression. (A) The RISI mouse model: WT mice received 5-Gy total body x-ray irradiation followed by full-thickness excisional skin wounding (black circle, 12-mm inner diameter). (B) Representative wound area measurements ( n = 6). (C) Quantification of wound closure rates at various time points relative to post-injury day 0 (PID0) ( n = 6). (D and E) H&E and Masson staining of wound tissue sections at PID0, 3, 7, and 14, showing regions close to the wound margin; IF costaining of TREM2 (gold), F4/80 (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) to identify TREM2 + macrophages in control and irradiated groups (scale bar was shown on panel). (F) Quantification of collagen deposition based on Masson staining ( n = 6). (G) Total macrophage ratio calculated as the proportion of F4/80 + DAPI + cells among total DAPI + cells by immunohistochemistry ( n = 6). (H) TREM2 + macrophage ratio calculated as the proportion of TREM2 + F4/80 + DAPI + cells among total F4/80 + DAPI + macrophages ( n = 6). (I) qPCR analysis of Trem2 mRNA expression at different time points ( n = 6). For within-group comparisons, expression was normalized to day 0 of each group. For between-group comparisons, expression levels in the irradiation group were normalized to the corresponding control group at each time point ( n = 6). (J and K) Western blotting and analysis of TREM2 protein expression across time points in control and irradiated groups ( n = 6). (L) ELISA measurement of soluble TREM2 (sTREM2) levels in skin tissue at different time points ( n = 6). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Expressing, Irradiation, Staining, Control, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Radiation reduced RAW264.7 cell viability and induced Trem2 expression. (A) Volcano plot showing DEGs between irradiation and control RAW264.7 cells based on RNA-seq ( n = 3). (B) GSEA identifying Trem2 -associated pathways enriched or suppressed following irradiation. (C) Heatmap of representative genes involved in macrophage differentiation, proliferation, migration, and apoptosis from GSEA-identified pathways. Significantly up-regulated and down-regulated genes are marked with upward (↑) and downward (↓) arrows, respectively. (D and E) Flow cytometry analysis of apoptosis using Annexin V and propidium iodide (PI) staining. Early and late apoptotic cells were quantified from Q2 and Q3 quadrants ( n = 3). (F and G) Live (green)/dead (red) staining; dead cell proportion calculated based on integrated fluorescence density ( n = 3; scale bar, 200 μm). (H) CCK-8 assay shown relative cell viability at 24 h post-irradiation compared to 0 h ( n = 3 biological replicates with 3 technical repeats each). (I) Trem2 transcript level from RNA-seq data. (J) qPCR analysis of Trem2 mRNA expression across different treatment groups ( n = 3). (K and L) Western blot and analysis of TREM2, BAX, and BCL2 protein expression ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Expressing, Irradiation, Control, RNA Sequencing, Migration, Flow Cytometry, Staining, Fluorescence, CCK-8 Assay, Western Blot

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Trem2 deficiency impairs wound healing in RISI. (A) RISI mouse model: Trem2-Ctr and Trem2-Cko mice were subjected to 5-Gy total body x-ray irradiation followed by full-thickness excisional wounding (black circle, 12-mm inner diameter). (B) Representative images of wound areas in each group ( n = 6). (C) Quantification of wound closure rates at indicated time points relative to PID0 ( n = 6). (D) H&E and Masson staining of wound tissues at PID0, 3, 7, and 14 ( n = 6). (E) IF costaining of iNOS (red), CD206 (green), and DAPI (blue) to identify M1- or M2-type macrophages in Trem2-Ctr and Trem2-Cko groups (scale bars shown in panels). (F) Collagen deposition quantified from Masson staining ( n = 6). (G) Quantification of macrophage subsets, calculated as the proportion of iNOS + CD206 − DAPI + cells (M1) or iNOS − CD206 + DAPI + cells (M2) among total DAPI + cells ( n = 6). (H) ELISA quantification of proinflammatory cytokines (IL-1β, TNF-α, and IL-6), anti-inflammatory cytokine IL-10, and total CASPASE3 in Trem2-Ctr and Trem2-Cko groups ( n = 6).

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Irradiation, Staining, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Schematic of TREM2 regulates macrophage apoptosis and impairs wound healing following RISI.

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: GM1 Oligosaccharide Modulates Microglial Activation and α-Synuclein Clearance in a Human In Vitro Model

doi: 10.3390/ijms26157634

Figure Lengend Snippet: Effects of GM1 oligosaccharide (OligoGM1) application on Human embryonic microglial (HCM3) cells. After 2 days of culture (80% of confluence), HCM3 cells were incubated or not incubated (control, CTRL) with OligoGM1 (100 µM). After 48 h, immunofluorescence staining [(against nuclei, ionized calcium-binding adaptor molecule 1 (Iba1) and triggered receptor expressed on myeloid cells 2 (TREM2)] and ELISA for quantification of released cytokines to medium were performed, as described in the : ( a ) Representative immunofluorescence images (20× magnification, scale bar 100 µm) and number of DAPI-positive cells; ( b ) Representative immunofluorescence images (20× magnification, scale bar 100 µm) of Iba1 expression, quantification of Iba1 accumulation (Iba1 area normalized by the number of cells), and area of Iba1-positive cells (area of microglia cells normalized by the µm 2 of Iba1); ( c ) Representative immunofluorescence images (20× magnification, scale bar 100 µm) of TREM2 expression, quantification of TREM2-positive cells (TREM2 area normalized by the number of cells), and area of TREM2-positive cells (area of microglia cells normalized by the µm 2 of TREM2); ( d ) Quantification of released cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). All values are represented as percentage versus CTRL and expressed as mean ± standard error of the mean (SEM, n = 6; ns = not significant, Mann–Whitney test).

Article Snippet: For immunofluorescence analyses, the following antibodies were used: primary mouse monoclonal anti-TREM2 antibody [Cat. 11084-MM08, research resource identifier (RRID):AB_2860315], purchased from Sino Biological Europe GmbH, Europe (Düsseldorfer, Germany); primary rabbit polyclonal anti-αSyn antibody (Cat. 2642S, RRID not available), purchased from Ozyme, Paris, France; and primary goat polyclonal anti-Iba1 antibody (Cat. Ab5076, RRID:AB_2224402), purchased from Abcam, Cambridge, UK.

Techniques: Incubation, Control, Immunofluorescence, Staining, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: GM1 Oligosaccharide Modulates Microglial Activation and α-Synuclein Clearance in a Human In Vitro Model

doi: 10.3390/ijms26157634

Figure Lengend Snippet: OligoGM1 modulation of αSyn-induced activation of HMC3 cells. After 2 days of culture (80% of confluence), HCM3 cells were incubated or not incubated (CTRL) with OligoGM1 (100 µM). After 4 h, αSyn (1 µM) was added to the culture medium for 48 h. At the end of treatment, immunofluorescence staining (against nuclei, Iba1, TREM2, and αSyn) and IL-6/TNF-α quantification from medium were performed as described in the . ( a ) Representative immunofluorescence images (20× magnification, scale bar 100 µm) and number of DAPI-positive cells; ( b ) Representative immunofluorescence images (20× magnification, scale bar 50 µm) of Iba1 expression, quantification of Iba1 accumulation (Iba1 area normalized by the number of cells), and area of Iba1-positive cells (area of microglia cells normalized by the µm 2 of Iba1); ( c ) Representative immunofluorescence images (20× magnification, scale bar 25 µm) of TREM2 and αSyn expressions, quantification of TREM2-positive cells (TREM2 area normalized by the number of cells), area of TREM2-positive cells (area of microglia cells normalized by the µm 2 of TREM2), and quantification of αSyn area accumulated within TREM(+) cells (area of αSyn normalized by the µm 2 of TREM2); ( d ) Quantification of released cytokines TNF-α and IL-6. All values are represented as percentage versus CTRL and expressed as mean ± SEM ( n = 6; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant by one-way ANOVA followed by Fisher’s LSD). * p < 0.05 was considered significant.

Article Snippet: For immunofluorescence analyses, the following antibodies were used: primary mouse monoclonal anti-TREM2 antibody [Cat. 11084-MM08, research resource identifier (RRID):AB_2860315], purchased from Sino Biological Europe GmbH, Europe (Düsseldorfer, Germany); primary rabbit polyclonal anti-αSyn antibody (Cat. 2642S, RRID not available), purchased from Ozyme, Paris, France; and primary goat polyclonal anti-Iba1 antibody (Cat. Ab5076, RRID:AB_2224402), purchased from Abcam, Cambridge, UK.

Techniques: Activation Assay, Incubation, Immunofluorescence, Staining, Expressing