Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A-B. Proteomic hiSPECS analysis comparing BV2 iRhom2 -/- (A) or ADAM17 -/- (B) microglia to BV2 NTC microglia. Volcano plots depict the log2 abundance change in the secretome and the negative log10 p-value for each protein (two-sample t-test, N = 12). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. For ease of visualization, the secretome data of the two iRhom2 (iR2 -/- : KO #1 and #2) and ADAM17 (A17 -/- : KO #1 and #2) clones were pooled and compared to the pooled secretome of the two NTC clones (NTC #1 and NTC #2) non-targeting control clones. C. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using the web tool Quantitative Analysis of Regulated Intramembrane Proteolysis (QARIP) ( Ivankov et al , 2013 ). D. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in BV2 cells. The supernatant prepared for the hiSPECS analysis in (A and B) was subjected to ELISA. sTREM2 levels were quantified in conditioned media of the indicated BV2 KO cell lines (N ≥5). NTC (N = 10) contains the combined data of NTC #1 (N = 5) and NTC #2 (N = 5) clones. NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. E. Immunoblot analysis of TREM2 in BV2 KO cell lines. Highly glycosylated, mature species of TREM2 were enriched in the ADAM17 and iRhom2 KO lines. Calnexin served as loading control. F. Surface abundance of TREM2 in BV2 KO cells. Cells were labeled with an antibody against TREM2 or isotype control to assess the geometric mean intensity of indicated BV2 KO cell lines using flow cytometry (N ≥ 3). NTC (N = 6) contains the combined data of NTC #1 (N = 3) and NTC #2 (N = 3) clones. BV2 NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. Data information: Data (D, F) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 μl of a biotinylated anti-TREM2 antibody (R&D systems, BAF1729) at 0.125 μg/ml for 90 minutes at room temperature.

Techniques: Clone Assay, Control, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Western Blot, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Proteomic hiSPECS analysis comparing the secretome of primary microglia isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) pups. The volcano plot displays protein abundance changes between WT and iR2 KO supernatants. The log2 fold change is plotted against the p-value (-log10). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. B. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using QARIP (see ). C. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in primary iRhom2 -/- microglia. The supernatant prepared for the hiSPECS analysis in (A) was subjected to ELISA. sTREM2 levels were quantified in supernatants of primary microglia (N ≥ 7). Two-sided independent Student’s t-test was performed. D. Immunoblot analysis of TREM2 levels in primary microglia lysates derived from wild-type or iRhom2 -/- pups. Three biological replicates of either genotype are shown. Highly glycosylated, mature species of TREM2 were enriched in the lysates of iRhom2-deficient microglia. Actin served as loading control. Data information: Data (C) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 μl of a biotinylated anti-TREM2 antibody (R&D systems, BAF1729) at 0.125 μg/ml for 90 minutes at room temperature.

Techniques: Isolation, Quantitative Proteomics, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Control

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Immunoblot analysis of TREM2 signaling in cell lysates isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) BMDMs. Three biological replicates of either genotype are shown. The membranes were probed with antibodies against iRhom2, ADAM17, TREM2, or antibodies against SYK or phospho-SYK-Y519/520. Calnexin and Actin served as loading control. B. Quantification of phospho-SYK-Y519/520 levels in immunoblots as shown in (A). iRhom2 -/- BMDMs showed stronger SYK phosphorylation than wild-type BMDMs (N = 6). The phospho-SYK/total-SYK ratio was normalized to wild-type. Two-sided independent Student’s t-test was performed. C. iRhom2/ADAM17-mediated TREM2 ectodomain shedding in iRhom2 -/- BMDMs. The sTREM2 levels were quantified in supernatants from cultures in (A) by ELISA (N = 6). Two-sided independent Student’s t-test. Data information: Data (B, C) are represented as means ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After discarding the blocking buffer, plates were incubated with 25 μl of a biotinylated anti-TREM2 antibody (R&D systems, BAF1729) at 0.125 μg/ml for 90 minutes at room temperature.

Techniques: Western Blot, Isolation, Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: TREM2 is down-regulated by HSV1 in microglia and involved in antiviral defense in the brain.

doi: 10.1126/sciadv.adf5808

Figure Lengend Snippet: Fig. 2. HSV1 down-regulates microglial TREM2 expression. (A to C) hiPSC-derived microglia were infected with HSV1 (MOI 1) for 24 hours. (A) Representative immu- noblot of full-length TREM2 (TREM2) and C-terminal fragment (CTF). (B) Quantification of full-length TREM2 in uninfected (UI) microglia versus microglia infected with HSV1 (MOI 1) for 24 hours. (C) sTREM2 levels in cell supernatants of UI versus HSV1-infected microglia 24 hours after infection. (D) TREM2 mRNA levels 24 hours after infection with HSV1 at different MOIs as indicated. (E) TREM2 mRNA levels 8 and 24 hours after infection (p.i.) with HSV1 (MOI 1). (F) TREM2 mRNA levels 24 hours after infection with HSV1 MOI 1 with and without 50 μM acyclovir. (G) TREM2 mRNA 24 hours after incubation with and without UV-inactivated HSV1 (MOI 1). (H) TYROBP (DAP12) mRNA levels 24 hours after infection with HSV1 at different MOIs as indicated. (I) DAP12 protein levels 24 hours after infection with HSV1 (MOI 1). A representative immunoblot is shown. (J) IFNB, IL6, TNFA, and MX1 mRNA levels in UI microglia versus HSV1-infected microglia 24 hours after infection (MOI 1). (K and L) cGAS was knocked down in hiPSC-derived microglia using siRNA. (K) Control and cGAS KD microglia were infected with HSV1 (MOI 3) for 5 hours. Representative immunoblots for the cGAS-STING signaling pathway and cGAS are shown. (L) IFNB, IL6, TNFA, and MX1 mRNA levels were analyzed in control versus cGAS KD microglia 24 hours after infection with HSV1 (MOI 1). All figures represent at least two to three independent experiments; data are presented as mean ± SEM; P values were calculated by one-way ANOVA with Tukey’s multiple comparisons test (F) and Mann-Whitney U test (B to E, G, H, J, and L). **P < 0.001; ***P < 0.0005; ****P < 0.0001.

Article Snippet: Samples were incubated for 48 hours at 4°C with the following primary antibodies: HSV-1 (1:500; B0116, Dako, Cytomation), CC3 (1:300; CS9661, Cell Signaling Technology), Iba-1 (1:400; ab5076, Abcam), and Trem2 (1:50; AF1729, R&D Systems).

Techniques: Expressing, Derivative Assay, Infection, Incubation, Western Blot, Control, MANN-WHITNEY

Journal: Science advances

Article Title: TREM2 is down-regulated by HSV1 in microglia and involved in antiviral defense in the brain.

doi: 10.1126/sciadv.adf5808

Figure Lengend Snippet: Fig. 3. Depletion of TREM2 expression in microglia impairs innate immune responses to HSV1 in vitro. (A to J) TREM2 was knocked down in hiPSC-derived microglia using siRNA. Cells were used for experiments 4 days after transfection. (A) A representative immunoblot of full-length TREM2 (TREM2) of control and TREM2 KD microglia is shown. (B) Quantification of full-length TREM2 protein in control versus TREM2 KD microglia. (C) Quantification of TREM2 mRNA in control and TREM2 KD microglia. (D) Apoptotic cells in control and TREM2 KD cultures were stained using an annexin V antibody and quantified by flow cytometry. (E) IFNB, IL6, TNFA, and MX1 mRNA levels were analyzed in control versus TREM2 KD microglia 24 hours after infection with HSV1 (MOI 1). (F) Control and TREM2 KD microglia were infected with HSV1 (MOI 3) for 5 hours. Representative immunoblots are shown. (G and H) hiPSC-derived neurons were infected with HSV1 (MOI 1) for 24 hours, stained with a fluorescent cell tracker, and added to control and TREM2 KD microglia for 15 hours. (G) Cells were fixed and imaged using a confocal microscope (red, HSV1-infected neurons; blue, DAPI). The negative control were untreated cells. Scale bar, 20 μm. (H) Cells that had phagocytosed HSV1-infected neurons were quantified by flow cytometry. Cytochalasin D (Cyto D; 5 μM) was added to block phagocytosis. (I) Control and TREM2 KD microglia were incubated with fluorescent cell tracker–stained apoptotic neurons for 15 hours. Cells that had phagocytosed apoptotic neurons were quantified by flow cytometry. (J) Control and TREM2 KD microglia were incubated with pHrodo–E. coli beads for 3 hours. Phagocytosis of pHrodo–E. coli beads was quantified using a microplate reader. All figures represent two to three independent experiments; data are presented as mean ± SEM; P values were calculated by Mann-Whitney U test. *P < 0.05; **P < 0.001; ***P < 0.0005; ****P < 0.0001.

Article Snippet: Samples were incubated for 48 hours at 4°C with the following primary antibodies: HSV-1 (1:500; B0116, Dako, Cytomation), CC3 (1:300; CS9661, Cell Signaling Technology), Iba-1 (1:400; ab5076, Abcam), and Trem2 (1:50; AF1729, R&D Systems).

Techniques: Expressing, In Vitro, Derivative Assay, Transfection, Western Blot, Control, Staining, Flow Cytometry, Infection, Microscopy, Negative Control, Blocking Assay, Incubation, MANN-WHITNEY

Journal: Science advances

Article Title: TREM2 is down-regulated by HSV1 in microglia and involved in antiviral defense in the brain.

doi: 10.1126/sciadv.adf5808

Figure Lengend Snippet: Fig. 4. TREM2 is essential for control of HSV1 infection in vitro and in vivo. (A) TREM2 mRNA levels were analyzed 24 hours after infection of hiPSC-derived cocultures of microglia and neurons with HSV1 (MOI 1). (B) sTREM2 levels were measured in supernatants of cocultures 24 hours after infection with HSV1 (MOI 1). (C) Viral titers in coculture supernatants 24 hours after infection with HSV1 (MOI 1) were quantified by viral plaque assay. (D) Cell viability was measured in cocultures 24 hours after infection with HSV1 (MOI 1) using the CyQUANT XTT assay. (E) Trem2 mRNA levels were analyzed in primary murine microglia, neurons, and astrocytes 6 hours after infection with HSV1 (McKrae, MOI 3). (F) Trem2 mRNA levels were analyzed in WT mouse brainstem 4 days after infection with HSV1 via the corneal route [2 × 106 plaque- forming units (PFU)/cornea]. (G and H) WT mice were infected with HSV1 (2 × 106 PFU/cornea), and brainstems were isolated and stained 5 days after infection. (G) Tissue sections were stained for Iba-1 (red) and CC3 (green). Scale bar, 50 μm. (H) Tissue sections were stained for HSV1 (red) and CD68 (green). Scale bar, 20 μm. (I and K) Iba1 and gB mRNA levels were quantified in the brainstem of WT and Trem2−/−mice 4 days after infection with HSV1 via the corneal route (2 × 106 PFU/cornea). (J) Viral titers of isolated brainstems 4 days after infection with HSV1 via the corneal route (2 × 106 PFU/cornea) were quantified by plaque assay. All figures represent two to three independent experiments; n = 15 to 18 per group of mice; data are presented as mean ± SEM; P values were calculated by one-way ANOVA with Tukey’s multiple com- parisons test (A, D, and K) and Mann-Whitney U test (B, C, E, F, I, and J). *P < 0.05; **P < 0.001; ****P < 0.0001.

Article Snippet: Samples were incubated for 48 hours at 4°C with the following primary antibodies: HSV-1 (1:500; B0116, Dako, Cytomation), CC3 (1:300; CS9661, Cell Signaling Technology), Iba-1 (1:400; ab5076, Abcam), and Trem2 (1:50; AF1729, R&D Systems).

Techniques: Control, Infection, In Vitro, In Vivo, Derivative Assay, Viral Plaque Assay, CyQUANT Assay, XTT Assay, Isolation, Staining, Plaque Assay, MANN-WHITNEY

Journal: Science advances

Article Title: TREM2 is down-regulated by HSV1 in microglia and involved in antiviral defense in the brain.

doi: 10.1126/sciadv.adf5808

Figure Lengend Snippet: Fig. 5. The antiviral program is not impaired in response to cGAMP in TREM2-depleted hiPSC-derived microglia. (A and B) Control and TREM2 KD microglia were stimulated with 100 μM cGAMP for 5 hours. (A) IFNB, IL6, TNFA, and MX1 mRNA levels. (B) Representative immunoblots of the cGAS-STING axis and TREM2. (C) cGAS mRNA levels are shown in control and TREM2 KD microglia. (D) Quantification of cGAS protein levels in control and TREM2 KD microglia. (E) Quantification of cGAMP levels in HSV1-infected control versus TREM2 KD microglia 24 hours after infection. cGAMP was not detectable (nd) in UI cells; instead, the detection limit is shown. A.U., arbitrary units. All figures represent two to three independent experiments; data are presented as mean ± SEM; P values were calculated by Mann-Whitney U test.

Article Snippet: Samples were incubated for 48 hours at 4°C with the following primary antibodies: HSV-1 (1:500; B0116, Dako, Cytomation), CC3 (1:300; CS9661, Cell Signaling Technology), Iba-1 (1:400; ab5076, Abcam), and Trem2 (1:50; AF1729, R&D Systems).

Techniques: Derivative Assay, Control, Western Blot, Infection, MANN-WHITNEY

Journal: Journal of Neuroscience

Article Title: Nuclear Receptors License Phagocytosis by Trem2+ Myeloid Cells in Mouse Models of Alzheimer's Disease

doi: 10.1523/jneurosci.4586-14.2015

Figure Lengend Snippet: Figure 6. MerTK and TREM2 are expressed by plaque-associated macrophages. MerTK (green) colocalizes with TREM2 (red) and surround 6e10 plaques (blue) in 12-month-old APP/PS1e9 mice treated with vehicle (A) or bexarotene (A) for 7 d. MerTK (green) also colocalizes with TREM2 (red) in 4-month-old 5XFAD mice treated with vehicle (B), bexarotene (B), or GW0742 (B). Scale bar, 20 m.

Article Snippet: The following antibodies were used: anti-mouse TREM2 (R&D Systems, clone 237920), anti-mouse MerTK (R&D Systems, clone 108928), anti-mouse CD45 (eBioscience, clone 30-F11), and anti-mouse CD11b (eBioscience, clone M1/70).

Techniques:

Journal: Journal of Neuroscience

Article Title: Nuclear Receptors License Phagocytosis by Trem2+ Myeloid Cells in Mouse Models of Alzheimer's Disease

doi: 10.1523/jneurosci.4586-14.2015

Figure Lengend Snippet: Figure 7. MerTKandTREM2areexpressedbyperipherallyderivedmyeloidcells.Microgliawere isolated from 8-month-old 5XFAD mice (4–6 per group) and gated on CD11b/CD45lo and CD11b/CD45hi cell populations (A, A). B, TREM2 expression in the CD11b/CD45hi cells was quantitated. C, Mean fluorescence intensity of MerTK was measured in CD11b/CD45lo and CD11b/CD45hipopulationsinbothtransgenicandnontransgenicmice.Meanfluorescenceinten- sity of MerTK was measured in the TREM2 expressing population (D, quantified in E). *p 0.05. **p 0.01.***p 0.001.n.s.,Notsignificant.

Article Snippet: The following antibodies were used: anti-mouse TREM2 (R&D Systems, clone 237920), anti-mouse MerTK (R&D Systems, clone 108928), anti-mouse CD45 (eBioscience, clone 30-F11), and anti-mouse CD11b (eBioscience, clone M1/70).

Techniques: Isolation, Expressing, Fluorescence

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: High-fat diet feeding increases cleaved TREM2 and TREM2 levels in gonadal white adipose tissue (A) Immunoblot analysis of TREM2 and cleaved TREM2 levels in gonadal white adipose tissue (GWAT), inguinal white adipose tissue (IWAT), and brown adipose tissue (BAT) of mice fed a high-fat diet (HFD) and normal chow diet (NCD) for 4 and 8 weeks ( n = 6 mice per group). (B) ELISA-based quantification of soluble TREM2 (sTREM2) levels in mouse serum ( n = 3 mice per group). (C) Schematic diagram illustrating cleaved membrane-bound TREM2 and soluble TREM2 forms. Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Membrane

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: HFD feeding upregulates Adam10 and Adam17 expression in monocyte/macrophage populations of GWAT (A) Immunoblot analysis of ADAM10 and ADAM17 protein expression in gonadal white adipose tissue (GWAT) of mice after 8 weeks of NCD or HFD feeding ( n = 6 mice per group). (B) Umap plot of Adam10 , Adam17 , and total nuclei isolated from GWAT of NCD and HFD-fed mice (PRJNA942977). Clusters are colored by cell types: adipocyte, mesothelial cell, lymphatic endothelial cell, vascular endothelial cell, adipocyte progenitor cell, smooth muscle cell (SMC), monocyte/macrophage (mono/mac), dendritic cell (DC), B cell, and T cell. (C) Violin plot of Adam10 and Adam17 gene expression levels in total cell types from GWAT of NCD- and HFD-fed mice. (D) Umap plot of Adam10 , Adam17 from monocyte/macrophage population in GWAT of NCD- and HFD-fed mice (PRJNA942977). Clusters are colored by cell types: Lyve1 +macrophage (Mac. Lyve1 ), Trem2 +macrophage (Mac. Trem2 ), Prg4 +macrophage (Mac. Prg4 ), and monocyte. (E) Bubble plot and violin plot of Adam10 and Adam17 expression levels in distinct cell populations from mouse GWAT, determined by single-nucleus RNA sequencing (PRJNA942977). (F) qPCR analysis of Adam10 and Adam17 mRNA expression in isolated F4/80+ macrophages and adipocytes from GWAT after 8 weeks of NCD or HFD feeding ( n = 3 mice per group). Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Expressing, Western Blot, Isolation, Gene Expression, RNA Sequencing

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: Obesity increases ADAM10 and ADAM17 expression in macrophage populations of WAT (A) ADAM10 and ADAM17 expression levels in total cell types of human visceral adipose tissues ( GSE176171 ). Clusters are colored by cell types: Adipocyte, adipose stem and progenitor cells (ASPC), mesothelium, endothelial cell, lymphatic endothelial cell (LEC), pericyte, smooth muscle cell (SMC), macrophage, monocyte, dendritic cell (DC), mast cell, neutrophil, B cell, natural killer cell (NK cell), T cell, and endometrium cell. (B) Bubble plot and violin plot of ADAM10 and ADAM17 gene expressions and representative markers in adipocyte and macrophage populations with body mass index (BMI) in human subcutaneous adipose tissue ( GSE176171 ). (C) Violin plot of TREM2 , ADAM10 , and ADAM17 gene expression levels in human macrophage sub-populations (hMac1, hMac2, hMac3). (D) Correlation analysis of ADAM10 and ADAM17 gene expression levels with BMI in human subcutaneous adipose tissue ( n = 12 patients per group). Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Expressing, Gene Expression

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: Apoptotic adipocytes fail to induce TREM2 shedding in RAW264.7 cells and bone marrow-derived macrophages (BMDMs) (A) Schematic diagram illustrating the experimental method used for co-culturing dying/dead adipocytes with RAW264.7 cells or BMDMs. Apoptotic adipocytes (aAC) were generated by treating differentiated 3T3-L1 adipocytes with brefeldin A (BFA; 5 μg/mL for 24 h). Apoptotic adipocytes (1 × 10 5 cells/well) were directly co-cultured with RAW264.7 cells or BMDMs (5 × 10 5 cells/well) in growth medium. After 24 h of co-culture, non-engulfed or floating apoptotic adipocytes were removed by PBS washing, and RAW264.7 cells or BMDMs were subsequently harvested for immunoblot analysis. (B) Immunoblot analysis of caspase-3 expression levels in 3T3-L1 adipocytes after BFA for 24 h ( n = 3 cells per group). (C) and (D) Immunoblot analysis of TREM2, ADAM10, and ADAM17 protein expression levels in RAW264.7 cells (C) and BMDMs (D) co-cultured with apoptotic adipocytes ( n = 3 cells per group). Black arrow and red arrow indicate each precursor form of ADAM10/17 and active form of ADAM10/17. Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Derivative Assay, Generated, Cell Culture, Co-Culture Assay, Western Blot, Expressing

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: GM6001 blocks TREM2 shedding in RAW264.7 cells induced by pyroptotic adipocytes (A) A schematic diagram illustrating the experimental method used for co-culturing pyroptotic adipocytes with macrophages. Pyroptotic adipocytes were generated by treating differentiated 3T3L1 adipocytes with lipopolysaccharide (LPS; 100 μg/μL, 48 h) followed by ATP (2 mM, 24 h). Pyroptotic adipocytes (1 × 10 5 cells/well) were directly co-cultured with RAW264.7 cells (5 × 10 5 cells/well) or BMDMs for 24 h in growth medium. After co-culture, non-engulfed or floating pyroptotic adipocytes were removed by PBS washing, and RAW264.7 cells and BMDMs were subsequently harvested for immunoblot analysis. (B) Immunoblot analysis of NLRP3, caspase-1, and GSDMD (gasdermin D) expression levels in 3T3-L1 adipocytes after LPS priming for 48 h and ATP treatment for 24 h ( n = 3 cells per group). Black arrow and red arrow indicate each total form of GSDMD and cleaved GSDMD. (C) Immunoblot analysis of TREM2, ADAM10, and ADAM17 expression levels in RAW264.7 cells co-cultured with pyroptotic adipocytes for 24 h in the presence or absence of GM6001 ( n = 4 cells per group). Black arrow and red arrow indicate each precursor form of ADAM10/17 and active form of ADAM10/17. (D) Immunoblot analysis of P-STING and STING expression levels in RAW264.7 cells co-cultured with pyroptotic adipocytes ( n = 3 cells per group). (E) Immunoblot analysis of P-syk, syk, P-PI3K, PI3K, P-PLCγ1, PLCγ1, P-AKT, and AKT protein levels in RAW264.7 cells co-cultured with pyroptotic adipocytes ( n = 3 cells per group). (F) Phagocytosis analysis of RAW264.7 cells co-cultured with pyroptotic adipocytes for 18 h in the presence of GM6001. Adipocytes were tagged with C12-BODIPY (red), and macrophages were stained with DiO (green). Representative images from three independent experiments are shown, with quantification provided in the right panel. Scale bars, 100 μm. Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Generated, Cell Culture, Co-Culture Assay, Western Blot, Expressing, Staining

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: GM6001 prevents the cleavage of TREM2 on BMDMs induced by pyroptotic adipocytes (A) Immunoblot analysis of TREM2, ADAM10, and ADAM17 expression levels in BMDMs co-cultured with pyroptotic adipocytes for 24 h in the presence or absence of GM6001 ( n = 3 cells per group). Black arrow and red arrow indicate each precursor form of ADAM10/17 and active form of ADAM10/17. (B) and (C) Phagocytosis analysis of BMDMs, (B) TREM2 knockdown BMDMs, and negative control (NC). (C) co-cultured with pyroptotic adipocytes for 18 h in the presence of GM6001. Adipocytes were tagged with C12-BODIPY (red), and macrophages were stained with DiO (green). Representative images from three independent experiments are shown, with quantification provided in the right panel. The yellow boxes indicate the regions shown in the magnified images. Scale bars, 200 μm. Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Western Blot, Expressing, Cell Culture, Knockdown, Negative Control, Staining

Journal: iScience

Article Title: Obesity-induced pyroptotic adipocyte death leads to TREM2-dependent macrophage dysfunction and adipose tissue inflammation

doi: 10.1016/j.isci.2025.114358

Figure Lengend Snippet: GM6001 treatment reduces TREM2 shedding and HFD-induced inflammation in GWAT (A) Schematic illustration of the experimental strategy for GM6001 administration (7.5 mg/kg/2 days) in mice fed a high-fat diet (HFD) for 10 weeks. (B) Immunoblot analysis of TREM2, ADAM10, and ADAM17 protein levels in gonadal white adipose tissue (GWAT) of HFD-fed mice treated with or without GM6001 ( n = 4 mice per group). (C) Measurement of soluble TREM2 (sTREM2) levels in the serum of NCD- or HFD-fed mice treated with GM6001 for 10 weeks ( n = 3 mice per group). (D–F) Representative flow profile and quantification of (D) CD11B + CD45 + cells (E) M2/M1 macrophage ratio (CD206 + CD11C − CD45 + CD11B + /CD11C + CD206 − CD45 + CD11B + ), and (F) TREM2 + CD11C + and TREM2 + CD206 + macrophage populations in GWAT of GM6001-treated mice after feeding HFD for 10 weeks ( n = 4 mice per group). (G) Immunofluorescence staining of F4/80 (green) with DAPI (blue) counterstaining in paraffin-embedded GWAT sections from GM6001-treated and control mice ( n = 4 mice per group, scale bars, 100 μm). (H) Immunoblot analysis of P-STING, STING, NLRP3, F4/80, and caspase-1 expression levels in GWAT of HFD-fed mice treated with or without GM6001 ( n = 4 mice per group). Data are presented as mean values ± SEM. p values were determined by the unpaired two-sided Student’s t test and were annotated directly in the figure at the corresponding comparisons.

Article Snippet: Human/Mouse TREM2 Allophycocyanin Mab (FAB17291A, 1:50) were purchased from R&D systems.

Techniques: Western Blot, Immunofluorescence, Staining, Control, Expressing

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A , a diagram of the ex vivo mtROS and phagocytosis assay. B , Total amount of aortic F4/80 + /Trem2 + macrophages were quantified and shown in the bar graph; n=4-7 individual mice per group. C , MitoNeoD MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5 individual mice per group. D , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-6 individual mice per group. E , a diagram of the in vivo phagocytosis assay. F , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-7 individual mice per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Ex Vivo, Phagocytosis Assay, In Vivo

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A-D , Mouse scRNA-seq data were re-analyzed from a previous publication . Uniform manifold approximation and projection (UMAP) representation of 11 aortic CD45 + immune cell clusters were shown in A . Trem2 gene expression pattern ( B ) and Pkm gene expression pattern ( C ) were shown in the UMAP. D , Violin plots show the Pkm and Trem2 expression distribution among aortic macrophage subpopulations. ResMac: resident macrophages; InflaMac: inflammatory macrophages. E , HMDMs transfected with PKM siRNA were treated with 50 μg/ml oxLDL for 24 h before subjected to mtROS assay (left panel) or phagocytosis assay (right panel). MFI was quantified and shown in the bar graph; n=4-5 per group. F , Representative confocal images of macrophages immunostained for PKM2 (green) and Tom20 (red). Nuclei were stained by DAPI (blue). Scale bar: 5 μm. G , HMDMs treated with 20 μg/ml LDL (control) or oxLDL for 3 h were lysed, subjected to cell fractionation into mitochondrial and cytosol fractions. PKM2 and ATP5A (mitochondria fraction loading control) blot images from mitochondrial fractions were shown on the left. PKM2 and β-actin (cytosol fraction loading control) blot images from cytosol fractions were shown on the right. Images were quantified, normalized to each loading control, and expressed as fold change of control. n=4 per group. H , WT or Cd36 -null peritoneal macrophages treated with 20 μg/ml LDL (control) or oxLDL for 3 h and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and ATP5A and blot images were shown. Images were quantified, normalized, and expressed as fold change of control. n=4 per group. I , WT macrophages treated with 20 μg/ml oxLDL or pre-treated 1 or 5μM shikonin before addition of oxLDL, incubating for 3 h, and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and Tom20 and blot images were shown. Images were quantified and expressed as fold change of control. n=3 per group. J , WT macrophages pre-treated with 20 μg/ml oxLDL or in combination with 1 μM shikonin for 24 h before mtROS or phagocytosis assay. The MitoNeoD (left) or pHrodo (right) MFI was quantified and shown in the bar graph; n=3-4 per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Gene Expression, Expressing, Transfection, Phagocytosis Assay, Staining, Control, Cell Fractionation

Journal: Cell reports

Article Title: Negative regulation of TREM2-mediated C9orf72 poly-GA clearance by the NLRP3 inflammasome.

doi: 10.1016/j.celrep.2023.112133

Figure Lengend Snippet: Figure 1. Microglial TREM2 is required for the reduction in poly-GA proteins (A) GO analysis of the biological processes and molecular functions of upregulated differentially expressed genes in poly-GA-expressing cortices. Three bio- logical repeats were performed.

Article Snippet: A 96-well Nunc MaxiSorp plate (44-2404-21; Thermo Fisher) was coated with anti-TREM2 antibody (1:1000, MAB17291; R&D system) in coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) overnight at 4 C. After blocking (1% BSA in PBS), 100 mL of diluted mouse cerebrospinal fluid (in 0.2%BSA/0.05% Tween 20 in PBS) was incubated for 2 h at 4 C. Plates were washed six times (0.05% Tween 20 in PBS) before incubation with biotinylated anti-TREM2 antibody (1:3000; BAF1729; R&D Systems) for 1 h. After washing, the plate was incubated with Streptavidin-HRP (SH1001; MultiSciences) for 1 h in the dark.

Techniques: Expressing

Journal: Cell reports

Article Title: Negative regulation of TREM2-mediated C9orf72 poly-GA clearance by the NLRP3 inflammasome.

doi: 10.1016/j.celrep.2023.112133

Figure Lengend Snippet: Figure 3. Activation of the NLRP3 inflammasome inhibits poly-GA clearance by promoting TREM2 cleavage (A) Fluorescence images of poly-GA proteins in NLRP3 and TREM2 double- or single-knockout motor cortices. (B) Quantification of the area of GFP-GA signal in (A). n = 3, 3, 3, and 5 mice in each group; at least 3 cortex sections per mouse for statistics. (C) Immunoblot of mature TREM2 levels. BV2 cells were treated with IL-1b (10 ng/mL) for 12 h. (D) Immunoblot of cleaved TREM2 (sTREM2) levels in the cell medium of BV2 cells. (E) Immunoblotting showed that GI 254023X inhibited sTREM2 production. BV2 cells were pretreated with GI 254023X (10 mM, 12 h) before IL-1b treatment (10 ng/ mL, 12 h). (F) ELISA of sTREM2 levels in cerebrospinal fluid of control and poly-GA mice. n = 3 mice per group. (G) Immunoblot of p-DAP12 levels. BV2 cells were pretreated with GI 254023X (10 mM, 12 h) before IL-1b treatment (10 ng/mL, 12 h). Cell lysates were immu- noprecipitated by the anti-DAP12 antibody and immunoblotted with the antibody 4G10. (H and I) Quantification of immunofluorescent area of poly-GA. Wild-type or TREM2/ primary microglia were pretreated with GI 254023X (10 mM) and IL-1b (10 ng/mL) and incubated with FLAG-GA-His aggregates for 12 h totally. n = 3 independent experiments. (J) Immunoblot of active ADAM10. ATP1A1 indicated the loading control of BV2 cell membrane. Unless otherwise specified, at least three independent experiments were performed. t test in (F), (H), and (I). One-way ANOVA in (B).

Article Snippet: A 96-well Nunc MaxiSorp plate (44-2404-21; Thermo Fisher) was coated with anti-TREM2 antibody (1:1000, MAB17291; R&D system) in coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) overnight at 4 C. After blocking (1% BSA in PBS), 100 mL of diluted mouse cerebrospinal fluid (in 0.2%BSA/0.05% Tween 20 in PBS) was incubated for 2 h at 4 C. Plates were washed six times (0.05% Tween 20 in PBS) before incubation with biotinylated anti-TREM2 antibody (1:3000; BAF1729; R&D Systems) for 1 h. After washing, the plate was incubated with Streptavidin-HRP (SH1001; MultiSciences) for 1 h in the dark.

Techniques: Activation Assay, Fluorescence, Knock-Out, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Incubation, Membrane