Journal: Brain, behavior, and immunity

Article Title: HX600, a synthetic agonist for RXR-Nurr1 heterodimer complex, prevents ischemia-induced neuronal damage

doi: 10.1016/j.bbi.2018.07.021

Figure Lengend Snippet: HX600 reduces Iba-1, phospho-p38 and TREM2 immunoreactivities in the ischemic brain. Quantitative analysis of the Iba-1, phospho-p38 and TREM2 immunoreactivities (A–C), and typical representative images of group receiving vehicle (D–F) or HX600 treatment (G–I) at time point 1 day after the insult. Immunoreactivities of Iba-1 and phospho-p38 were analyzed from the peri-ischemic area, and TREM2 at the lesion site. Scale bar 100 μm. Data presented as mean ± SD. Unpaired two-tailed t-test, n = 8 in each group. *p < 0.05 ***p < 0.001.

Article Snippet: For phospho-p38 and TREM2 stainings biotinylated secondary antibody (1:200 dilution, Vector Laboratories, Burlingame, CA, USA) was used, followed by reaction with avidin-biotin complex reagent (1:200 dilution, Vector Laboratories, Burlingame, CA, USA) according to the instructions provided by the manufacturer.

Techniques: Two Tailed Test

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 1. Chronic TREM2 activation increases DAM around plaques. (A) Schematic of experimental design. 6-mo-old 5XFAD mice were injected with AD- tau in the HC (bregma: −2.5 mm; lateral: −2.0 mm; depth: −2.2 mm) and overlying cortex (bregma: −2.5 mm; lateral: −2.0 mm; depth: −1.0 mm) and sacrificed 3 mo later to evaluate peri-plaque pathologies. 1 wk before AD-tau injection and every week following until sacrifice, 5XFAD mice were given i.p. injections of 80 mg/ml of the AL002a mouse TREM2 antibody (n = 13 female, n = 14 male) or the IgG control antibody (n = 14 female, n = 12 male). (B) Quantification of TREM2 antibody levels in terminal plasma for 5XFAD mice either chronically treated with the IgG control antibody or the TREM2 antibody. (C) Quantification of TREM2 antibody levels in plasma for 5XFAD mice either acutely treated with the IgG control antibody or the TREM2 antibody. (D) Quantification of TREM2 antibody levels in cortical tissue for 5XFAD mice either acutely treated with the IgG control antibody or the TREM2 antibody. For acute treatment, there were n = 8 IgG control and n = 9 Trem2 antibody–treated age- and sex-matched mice, unless otherwise specified on graphs. (E) Representative images of ipsilateral hemisphere stained with CLEC7A+ microglia, P2RY12+ microglia, and Aβ from IgG control and TREM2 antibody–treated groups. Scale bars, 15 µm. (F) Quantification of mouse sTREM2 levels in plasma for 5XFAD mice either acutely treated with the IgG control antibody or the TREM2 antibody. (G) Quantification

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Injection, Control, Clinical Proteomics, Staining

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 2. Chronic TREM2 activation with a TREM2 antibody results in no changes in Aβ plaque burden. (A) Representative images of Aβ plaques in 5XFAD mice either treated with the IgG control antibody or the TREM2 antibody. Scale bar, 100 µm. (B–E) Quantification of Aβ staining in the ipsi- and contralateral cortices (B and D) and hippocampi (C and E) in female mice. (F–I) Quantification of Aβ staining in the ipsi- and contralateral cortices (F and H) and hippocampi (G and I) in male mice. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14 male). Data are presented as mean ± SEM. Scale bar, 100 µm. Significance was determined using a Student’s t test. ns, P > 0.05.

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Control, Staining

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 3. Chronic TREM2 activation with a TREM2 antibody increases NP-tau pathology. (A) Representative images of ipsi- and contralateral hemi- spheres stained with AT8+ NP-tau pathology in AD-tau injected 5XFAD mice either treated with the IgG control antibody or the TREM2 antibody. Repre- sentative images are from female mice. Scale bars, 100 µm. (B–E) Quantification of p-tau (AT8+) staining in the ipsi- and contralateral cortices (B and D) and hippocampi (C and E) in female mice. (F–I) Quantification of p-tau (AT8+) staining in the ipsi- and contralateral cortices (F and H) and hippocampi (G and I) in male mice. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14 male). Data are presented as mean ± SEM. Significance was determined using a Student’s t test. ns, P > 0.05; *, P < 0.05.

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Staining, Injection, Control

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 4. Chronic TREM2 activation with a TREM2 antibody increases peri-plaque NP-tau pathology and plaque-associated neuritic dystrophy, and acute TREM2 activation results in no changes in AD-tau uptake and degradation. (A) Representative images of BACE1+ and X34+ staining in ipsilateral HC. Scale bars, 20 µm. (B) Representative images of AT8+ and X34+ staining in ipsilateral HC. (C–F) Quantification of the number of BACE1+ staining within 15 µm of plaques in the ipsi- and contra- cortices (C and E) and hippocampi (D and F). (G–J) Quantification of the number of AT8+ staining within 15 µm of plaques in the ipsi- and contra- cortices (G and I) and hippocampi (H and J). (K) AD-tau uptake assay in TREM2 WT BMDMs. Results represent two independent ex- periments. (L) AD-tau degradation assay in TREM2 WT BMDMs. Results represent two independent experiments. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Staining, Degradation Assay, Control

Journal: The Journal of experimental medicine

Article Title: Chronic TREM2 activation exacerbates Aβ-associated tau seeding and spreading.

doi: 10.1084/jem.20220654

Figure Lengend Snippet: Figure 5. Chronic TREM2 activation with a TREM2 antibody increases peri-plaque loss of synaptic marker synapsin. (A) Representative images of Synapsin+ and X34+ staining in ipsilateral HC. Scale bar, 7 µm. (B) Quantification of the number of Synapsin+ puncta within 15 µm of plaques in the ipsi-HC. (C–E) Quantification of the number of Synapsin+ puncta within 15 µm of plaques in the ipsi-cortex, contra-cortex, and HC. Triangle symbol represents female mice, and square symbol represents male mice either treated with the IgG control antibody (n = 14 female, n = 12 male) or the TREM2 antibody (n = 13 female, n = 14 male). Data are presented as mean ± SEM. Significance was determined using a linear regression with sex as a covariate. ns, P > 0.05; *, P < 0.05.

Article Snippet: Biotinylated rat anti-TREM2 antibody (R&D Systems) was added at 1:10,000 dilution in binding buffer and incubated for 1 h at RT.

Techniques: Activation Assay, Marker, Staining, Control

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for alveolar macrophages and not shared with interstitial macrophages and classical monocytes. Data reanalyzed from previously published scRNA-seq data . (b) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for interstitial macrophages and not shared with alveolar macrophages and classical monocytes. Data reanalyzed from previously published scRNA-seq data . (c) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for classical monocytes and not shared with interstitial macrophages and alveolar macrophages. Data reanalyzed from previously published scRNA-seq data . (d) Venn diagram showing the overlap between hyperoxia-induced genes (FC > 2; FDR < 0.05) in 3 different myeloid cell subsets; alveolar macrophages (n = 281), interstitial macrophages (n = 275), classical monocytes (n = 215) (left panel). Venn diagram showing the overlap between hyperoxia-induced genes in the three myeloid cell subsets (n = 18) and whole lung tissues (n = 253 in ) (right panel). (e) SNPs in the Trem2 gene between B6 and DBA mice. (f) Body weight in hyperoxia-exposed WT (B6) (same samples as ) and T2KO mice. Data are mean ± SEM (n = 6 for each group).

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques:

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Single-cell RNA-seq analysis of whole lung tissues from B6 mice exposed to 95% oxygen from P0 to P5 . (b) Trem2 mRNA expression in the lungs of B6 and DBA mice. Bar graphs show mean ± SEM (n = 3-4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p -adj < 0.01 and *** p -adj < 0.001. (c) Immunoblots for TREM2 protein in whole lung tissues collected on P14 from B6 and DBA mice. Bar graphs show mean ± SEM (n = 3 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01. Uncropped images of the blots are shown in . (d) Soluble TREM2 levels in the plasma of B6 and DBA mice on P14 after neonatal hyperoxia exposure. Bar graphs show mean ± SEM (n = 3-8 per group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. (e) H&E staining was performed to assess the alveolar complexity at P14 in WT (B6) mice (left panels) and T2KO mice (right panels) with scale bars denoting 100 μm. The bar graph represents the results of the quantification of alveolar simplification using mean linear intercept (same WT (B6) samples as ). Data are mean ± SEM (n = 3-4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01 and *** p < 0.001. (f) Bronchoalveolar lavage (BAL) was performed at P14 in WT (B6) and T2KO hyperoxia-exposed and normoxic control mice (n = 14 for WT (B6) and n = 11-13 for T2KO mice). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01 and *** p < 0.001. n.s.: not significant. (g) Matrix ultrastructural analysis of lung tissues of neonatal hyperoxia-exposed B6 (WT), DBA and T2KO mice on P14. Manifold of hyperoxia-induced pathological architecture with higher pseudotime representing increasingly disrupted architecture and alveolar simplification. Tissue images show representative tiles along the pseudotime trajectory. (h) Architecture-defining parameters. Identification of top 5 individual matrix features associated with low pseudotime (normal-like interstitium) and high pseudotime (more aberrant interstitium), based on Pearson correlations. Parameters are displayed as the absolute value of the Pearson coefficient (i.e. magnitude of correlation, as shown by height of arrows on y-axis), with all p < 0.001. (i) Visualization of hyperoxia-exposed and normoxic control lung tissues of B6 (WT), DBA and T2KO mice. Hyperoxia-exposed lungs from DBA and T2KO mice with less lung matrix remodeling localize near the root point of the pseudotime trajectory. (j) Bar graphs of matrix pseudotime for hyperoxia-exposed and normoxic control lung tissues of B6 (WT), DBA and T2KO mice. The difference in pseudotime normalized to normoxic control lungs is shown. Data are mean ± SEM (n = 3 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. n.s.: not significant. Each dot represents one mouse (b, d, e, f). See also .

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques: RNA Sequencing, Expressing, Comparison, Western Blot, Clinical Proteomics, Staining, Control

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) BMDMs were obtained from B6 (WT), DBA and T2KO mice and exposed to hyperoxia (95%) for 24 h. (b) Immunoblots of TREM2 protein in whole cell lysates of hyperoxia-exposed BMDMs obtained from B6 (WT), DBA and T2KO mice and the normoxic controls. Uncropped images of the blots are shown in . (c) RNA-seq of hyperoxia-exposed BMDMs from B6 (WT), DBA and T2KO mice and the normoxic controls. (d) Venn diagram showing the overlap between hyperoxia-induced genes in B6 (WT) BMDMs (n = 173) and hyperoxia-suppressed genes in T2KO (n = 570) and DBA (n = 905) BMDMs. Gene Ontology analysis of 34 genes induced by hyperoxia in B6 (WT) BMDMs and suppressed in T2KO and DBA BMDMs. (e) Bar plots for expression of representative genes belonging to the p53 signaling pathway. Data are mean ± SEM. DSeq2 was used for comparisons. ** p -adj < 0.01 and *** p -adj < 0.001. (f) Caspase 3 activity measured in cytosolic fractions from BMDMs from B6 (WT), DBA and T2KO mice. Data are mean ± SEM. (n = 4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. Each dot represents one mouse (e, f). See also .

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques: Western Blot, RNA Sequencing, Expressing, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Immunoblots of TREM2 protein in whole cell lysates of hyperoxia-exposed (1, 2, 4 or 24 h) BMDMs obtained from B6 (WT) mice and normoxic controls (0 h). Uncropped images of the blots are shown in .

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques: Western Blot

Journal: The FASEB Journal

Article Title: Meprin β cleaves TREM2 and controls its phagocytic activity on macrophages

doi: 10.1096/fj.201902183r

Figure Lengend Snippet: FIGURE 1 TREM2 is shed by meprin β in HEK293_ADAM10/17−/− cells. (A) Illustration of membrane bound TREM2 and DAP12. Upon shedding by ADAM10, the remaining C-terminal fragment (CTF) of TREM2 is cleaved within the membrane by γ-secretase, thereby releasing the intracellular domain (ICD). (B) Co-expression of TREM2 with meprin β or ADAM10 in HEK293_ADAM10/17−/− cells and Western blot analyses of TREM2 fragments in membrane-enriched fractions and cell supernatants. Anti-TFR antibody was used as loading control. (C) Conditioned media from TREM2 and ADAM10 transfected HEK293_ADAM10/17−/− cells were incubated with the recombinant ectodomains of meprin β and ADAM10 at 37°C for 4 hours. sTREM2 levels were analyzed by Western blot. (D) HEK293_ADAM10/17−/− cells transfected with TREM2 were incubated with recombinant meprin β and proteins from membrane-enriched fractions and cell supernatants were analyzed by Western blot. (E) Time series showing the cleavage of membrane bound TREM2 in HEK293_ADAM10/17−/− cells by recombinant soluble meprin β analyzed by Western blot. (F) Incubation of TREM2 overexpressing HEK293_ADAM10/17−/− cells with recombinant meprin α or meprin β and analysis of TREM2 by Western blot. All transient cell transfections and Western blot analyses were at least performed in biological triplicates

Article Snippet: On the next day, the plate was incubated for 90 minutes at RT with 0.125 μg/mL of biotinylated polyclonal goat antimouse TREM2 capture antibody (BAF1729; R&D Systems) diluted in blocking buffer.

Techniques: Membrane, Expressing, Western Blot, Control, Transfection, Incubation, Recombinant

Journal: The FASEB Journal

Article Title: Meprin β cleaves TREM2 and controls its phagocytic activity on macrophages

doi: 10.1096/fj.201902183r

Figure Lengend Snippet: FIGURE 2 Meprin β cleaves TREM2 between arginine 136 and aspartate 137. (A) Incubation of sTREM2 with recombinant soluble meprin β (smeprin β) led to the generation of TREM2 fragments analyzed by Western blot (AF1828; R&D Systems; 1:1.000). (B) sTREM2 was incubated with smeprin β for 2 hours at 37°C and subsequently digested with PNGaseF. The meprin β generated TREM2 fragment is about 10 kDa smaller than the uncleaved TREM2 as analyzed by SDS-PAGE. (C) Coomassie stained gel of sTREM2 incubated with meprin β (2 hours at 37°C) that was used for determination of cleavage sites. Gel bands were excised, split into two samples and digested with either chymotrypsin or LysargiNase in heavy water. (E) Illustration of the TREM2 ectodomain construct used for cleavage site identification (sTREM2). While TREM2 is cleaved by ADAM10 and ADAM17 between histidine 157 and serine 158, the major meprin β cleavage site is further N-terminal between arginine 136 and aspartate 137. (F) Co-expression of TREM2 WT and AD-associated variants with meprin β in HEK293_ADAM10/17−/− cells and Western blot analyses of TREM2 fragments in membrane-enriched fractions and cell supernatants. Anti-GAPDH antibody was used as loading control. All transient cell transfections and Western blot analyses were at least performed in biological triplicates

Article Snippet: On the next day, the plate was incubated for 90 minutes at RT with 0.125 μg/mL of biotinylated polyclonal goat antimouse TREM2 capture antibody (BAF1729; R&D Systems) diluted in blocking buffer.

Techniques: Incubation, Recombinant, Western Blot, Generated, SDS Page, Staining, Construct, Expressing, Membrane, Control, Transfection

Journal: The FASEB Journal

Article Title: Meprin β cleaves TREM2 and controls its phagocytic activity on macrophages

doi: 10.1096/fj.201902183r

Figure Lengend Snippet: FIGURE 3 Impaired phagocytosis of fluorogenic E coli particles in cells co-expressing meprin β and TREM2. (A) Illustration of the TREM2/DAP12 fusion construct used in the phagocytosis assays. (B) Proteolytic cleavage of the overexpressed fusion constructs in HEK293_ ADAM10/17−/− cells transfected with meprin β or ADAM10. Proteins from cell supernatants and membrane-enriched fractions were analyzed by Western blot. (C) Phagocytosis assay with transfected HEK293_ADAM10/17−/− cells. Relative particle uptake analyzed by FACS is indicated for differentially transfected cells. Data are represented as means of median fluorescence intensity ± SEM from two independent experiments and expressed relative to empty vector transfected control (n = 8 and 9, respectively). (D) Relative particle uptake of THP-1 cells transfected with wild- type TREM2 and meprin β (n = 10) was analyzed using a Tecan fluorescent reader measuring mean fluorescence intensity of a defined number of cells. (E) Relative particle uptake of U937 cells transfected with wild-type TREM2 and meprin β (n = 6) was analyzed using a Tecan fluorescent reader measuring mean fluorescence intensity of a defined number of cells. All transient cell transfections and Western blot analyses were at least performed in biological triplicates. Statistical differences were calculated by One-way ANOVA followed by a Newman-Keuls multiple comparison test. Comparison of two test groups was performed using an unpaired two-tailed t test (*P < .05; **P < .01; ****P < .0001)

Article Snippet: On the next day, the plate was incubated for 90 minutes at RT with 0.125 μg/mL of biotinylated polyclonal goat antimouse TREM2 capture antibody (BAF1729; R&D Systems) diluted in blocking buffer.

Techniques: Expressing, Construct, Transfection, Membrane, Western Blot, Phagocytosis Assay, Fluorescence, Plasmid Preparation, Control, Comparison, Two Tailed Test

Journal: The FASEB Journal

Article Title: Meprin β cleaves TREM2 and controls its phagocytic activity on macrophages

doi: 10.1096/fj.201902183r

Figure Lengend Snippet: FIGURE 4 Cleavage of TREM2 on macrophages by meprin β. (A) Membrane-enriched fractions from BMDMs of Mep1b−/− mice showed higher levels of mature TREM2 than the wild-type controls visualized by Western blot. Enhanced levels of mature TREM2 at 45 kDa were detected (murine-specific anti-TREM2, clone 5F4) in samples of Mep1b−/− mice, which was decreased upon LPS stimulation. Anti-STAT3 was used as loading control. (B) Densitometric analysis of TREM2 band intensities in non LPS stimulated BMDMs from Mep1b−/− and wild-type mice as seen in (A). The comparison of two test groups (n = 5) was performed using an unpaired two-tailed t test. (*P < .05). (C) Phagocytosis assay with BMDMs from wild-type and Mep1b−/− mice. Cytochalasin D (CytoD) was used to block endocytosis. Relative particle uptake measured by FACS is indicated for different cells. Data are represented as means of median fluorescence intensity ± SD (n = 3). Statistical differences were calculated by One-way ANOVA followed by a Newman-Keuls multiple comparison test (*P < .05). (D) Determination of sTREM2 levels in serum reveals significantly lower levels in Mep1b−/− (n = 7, all male) than in wild-type mice (n = 5, all male). Data are represented as means ± SEM. (E) Cartoon illustrating the interaction of TREM2 with soluble and membrane bound meprin β resulting in the degradation of the TREM2 ectodomain

Article Snippet: On the next day, the plate was incubated for 90 minutes at RT with 0.125 μg/mL of biotinylated polyclonal goat antimouse TREM2 capture antibody (BAF1729; R&D Systems) diluted in blocking buffer.

Techniques: Membrane, Western Blot, Control, Comparison, Two Tailed Test, Phagocytosis Assay, Blocking Assay, Fluorescence

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A, B) Metabolic analysis of oxygen consumption rate (OCR) and (F, G) proton efflux rate (PER) in Common variant (Cv), TREM2 R47H and TREM2 −/− iPS-Mg after 24 h treatment with succinate (10 mM) or citrate (10 mM) . (C) Basal respiration, (D) maximal respiration, (E) spare respiratory capacity , (H, J) basal glycolysis, (I, K) compensatory glycolysis, and (L) mitoOCR/GlycoPER ratio in basal conditions and upon LPS/IFNγ stimulation in iPS-Mg expressing Cv, TREM2 R47H or TREM2 −/− after incubations with citrate or succinate. Data are presented as mean ± SEM. Statistical significance was addressed using One-way ANOVA or Two-way ANOVA with Tukey post-hoc analysis to compare controls and treated groups respectively; *p<0.05 (n=5-6)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Variant Assay, Expressing

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Representative images and ( B, C ) quantification of LipidSpot TM 488 staining of lipid droplets in Common variant (Cv), TREM2 R47H and TREM2 −/− variant iPS-Mg after medium supplementation with citrate (10mM) or succinate (10mM) for 24h. (D) Basal lactate production and (E) lactate production upon LPS/IFN-γ stimulation were determined in the iPS-Mg cell culture medium after supplementation with citrate or succinate. (F) Representative histogram and (G) mitochondrial superoxide (MitoSOX) production as determined by FACS in Cv, TREM2 R47H and TREM2 −/− variant iPS-Mg after succinate or citrate supplementation or rotenone treatment (positive control). Data are presented as mean ± SEM. Statistical significance was addressed using One-way ANOVA or Two-way ANOVA with Tukey post-hoc analysis to compare controls and treated groups respectively; *p<0.05 (n=5-6)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Staining, Variant Assay, Cell Culture, Positive Control

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Metabolic analysis of oxygen consumption rate (OCR) (B) Maximal respiration, and (C) spare respiratory capacity in Common variant (Cv), TREM2 R47H and TREM2 −/− iPS-Mg after 24 h LPS/IFN-γ stimulation and treatment with succinate (10 mM) or citrate (10 mM). Data are presented as mean ± SEM. Statistical significance was addressed using One-way ANOVA or Two-way ANOVA with Tukey post-hoc analysis to compare controls and treated groups respectively; *p<0.05 (n=5-6)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Variant Assay

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Representative histograms of Aβ 1-42 uptake in iPS-Mg upon citrate supplementation. (B) The ability of common variant (Cv), TREM2 R47H and/or TREM2 −/− variant iPS-Mg to phagocytose Aβ 1-42 oligomers was assessed after citrate and succinate supplementation in basal conditions and (C) upon inhibition of glycolysis by 2-Deoxyglucose (2-DG). (D, E) LPS/IFN-γ evoked pro-inflammatory release of TNFα and (F, G) IL-1β were assessed by ELISA in the culture medium of iPS-Mg treated with citrate or succinate in basal conditions and after glycolytic inhibition by 2-DG. Data are presented as mean ± SEM. Statistical significance was addressed using One-way ANOVA or Two-way ANOVA with Tukey post-hoc analysis to compare controls and treated groups respectively; *p<0.05 (n=2-12)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Variant Assay, Inhibition, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Basal and LPS/IFN-γ evoked pro-inflammatory release of TNFα and (B) IL-1β were assessed by ELISA in the culture medium of iPS-Mg expressing common variant TREM2, TREM2 R47H and TREM2 −/− . Data are presented as mean ± SEM. Statistical significance was addressed using two-way ANOVA with Tukey post-hoc analysis *p<0.05 (n=4-10)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Variant Assay

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Shed soluble TREM2 (sTREM2) was determined in the culture medium of unstimulated common variant (Cv), TREM2 R47H and TREM2 −/− iPS-Mg after treatments with citrate or succinate and/or (B) 2-DG mediated inhibition of glycolysis in basal conditions and upon LPS/IFNγ stimulation. Data are presented as mean ± SEM. Statistical significance was addressed using Two-way ANOVA with Tukey post-hoc analysis; *p<0.05 (n=3-8)

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Variant Assay, Inhibition

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: (A) Representative cellular stress proteome array dot blots and heatmaps from (B) Common variant (Cv), (C) TREM2 R47H , (D) TREM2 −/− variant iPS-Mg lysates after 24h incubations with citrate (10mM) or succinate (10mM). (I) Representative western blots and quantifications for HSP70 (E) at basal and (G) upon LPS/IFN-γ stimulation, and ratio p-NFκB/NFκB (F) at basal and (H) following LPS/IFN-γ treatment, in Cv, TREM2 R47H and TREM2 −/− variant iPS-Mg after treatments with citrate or succinate. Citrate supplemented Cv iPS-Mg group is not included in the selected representative blots (I ). Data are presented as mean ± SEM. Statistical significance was addressed using Two-way ANOVA with Tukey post-hoc analysis; *p<0.05.

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques: Variant Assay, Western Blot

Journal: bioRxiv

Article Title: Amelioration of signalling deficits underlying metabolic shortfall in TREM2 R47H human iPSC-derived microglia

doi: 10.1101/2024.02.12.579896

Figure Lengend Snippet: Graphical summary of changes in TREM2 R47H iPS-Mg after supplementation with citrate or succinate

Article Snippet: After blocking supernatant samples from different groups and standards (recombinant human-TREM2-His; Life Technologies) were incubated for 2 h at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/ml; AF1828, R&D Systems).

Techniques:

Journal: bioRxiv

Article Title: The MS4A gene cluster is a key regulator of soluble TREM2 and Alzheimer disease risk

doi: 10.1101/352179

Figure Lengend Snippet: (A) Levels of sTREM2 measured by ELISA in the cell media of human macrophages on day 7 in culture with or without stimulation with IL-4. (B) Effect on levels of sTREM2 in cell culture media by treatment with antibodies against MS4A4A and MS4A6A for 48 h. Two different anti-MS4A4 antibodies were tested, antibody A (Abcam) directed against the N-Terminal cytoplasmic domain or antibody B (Biolegend) directed against the extracellular domain of the protein. These analyses were done without or with IL-4 in culture. Bars are means ± SD. *p<0.05; **p<0.005; ***p<0.005. Pvalues were calculated by Mann-Whitney test or Kruskal Wallis H test for multigroup comparison. (C) Confocal images of human macrophages treated or not with IL-4 for 48 hours and stained for TREM2 (red) and MS4A4A (green) antibodies. Arrowheads (white) indicate colocalizing signal. Scale bar 10 μm.

Article Snippet: The assay consists of a Streptavidin-coated 96-well plates (MSD Streptavidin Gold Plates, cat. no. L15SA-1); a biotinylated polyclonal goat IgG anti-human TREM2 antibody (R&D Systems, cat. no. BAF1828; 0.25 μg/mL, 25 μL/well) as capture antibody, which is raised against amino acids 19174 of human TREM2; a monoclonal mouse IgG anti-human TREM2 antibody (Santa Cruz Biotechnology, B-3, cat. no. sc373828; 1 μg/mL, 50 μL/well) as a detection antibody, which is raised against amino acids 1-160 of human TREM2; and a SULFO-TAG-labeled goat polyclonal anti-mouse IgG secondary antibody (MSD, cat. no. R32AC; 0.5 μg/mL, 25 μL/well).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, MANN-WHITNEY, Staining

Journal: eLife

Article Title: Fibrillar Aβ triggers microglial proteome alterations and dysfunction in Alzheimer mouse models

doi: 10.7554/eLife.54083

Figure Lengend Snippet: ( A ) The comparison of relatively quantified proteins of APPPS1 (yellow) and APP-KI (green) mice versus WT at 12 months shows a modest overlap with a single cell transcriptomics study (blue) . An amount of 3348 proteins (57.2% of all quantified proteins) and their related transcripts were quantified in all three data sets. ( B ) The Venn diagram shows the overlap of significantly regulated proteins in APPPS1 (yellow) and APP-KI (green) mice versus WT at 12 months with unidirectionally regulated microglial transcripts (blue) . Signatures which are specific either for the proteome or transcriptome dataset are separately depicted at the top or right, respectively. The indicated percentage values are based on all relatively quantified proteins. ( C ) Biochemical validation of proteomic data was performed using western blot analysis of microglial lysates from 12 month old mice. This analysis revealed an increased abundance of the up-regulated MARPs APOE, TREM2, CD68 and FABP5 as well as a decreased abundance of the down-regulated MARP CSF1R in APPPS1 and APP-KI compared to WT microglia. Arrows indicate antibody-detected specific bands and asterisk indicates unspecific bands.

Article Snippet: Briefly, free-floating sections were permeabilized with 0.5% Triton-PBS (PBS-T) for 30 min, blocked either in 5% normal goat Serum or 5% donkey serum in PBS-T for 1 hr and incubated overnight at 4°C in blocking solution with the following primary antibodies: IBA1 (1:500, 019–19741,Wako), IBA1 (1:500, ab5076, Abcam), NAB228 (1:2000, sc-32277, Santa Cruz), CD68 (1:500, MCA1957GA, Bio-Rad), TREM2 (1:50, AF1729, R and DSystems), APP-Y188 (1:2000, ab32136, Abcam), CLEC7a (1:50, mabg-mdect, Invivogen), TMEM119 (1:200, ab209064, Abcam), APOE-biotinylated (HJ6.3, 1:100, [ ]), 3552 (1:5000, [ ]) and pE3-Aβ (J8, 1:500, [ ]). pE3-Aβ immunostaining required heat antigen retrieval (25 min at 95°C) in citrate buffer (10 mM, pH 6.0) prior blocking.

Techniques: Single-cell Transcriptomics, Western Blot

Journal: eLife

Article Title: Fibrillar Aβ triggers microglial proteome alterations and dysfunction in Alzheimer mouse models

doi: 10.7554/eLife.54083

Figure Lengend Snippet: Immunohistochemical analysis of TREM2 (red) reveals an increased abundance in IBA1 positive (green) APPPS1 microglia surrounding Aβ plaques (magenta) that is less prominent in APP-KI microglia. Hoechst (HOE, blue) was used for nuclear staining. Boxed regions in upper panels (scale bar: 50 µm) are shown with a higher magnification in lower panels (scale bar: 10 µm).

Article Snippet: Briefly, free-floating sections were permeabilized with 0.5% Triton-PBS (PBS-T) for 30 min, blocked either in 5% normal goat Serum or 5% donkey serum in PBS-T for 1 hr and incubated overnight at 4°C in blocking solution with the following primary antibodies: IBA1 (1:500, 019–19741,Wako), IBA1 (1:500, ab5076, Abcam), NAB228 (1:2000, sc-32277, Santa Cruz), CD68 (1:500, MCA1957GA, Bio-Rad), TREM2 (1:50, AF1729, R and DSystems), APP-Y188 (1:2000, ab32136, Abcam), CLEC7a (1:50, mabg-mdect, Invivogen), TMEM119 (1:200, ab209064, Abcam), APOE-biotinylated (HJ6.3, 1:100, [ ]), 3552 (1:5000, [ ]) and pE3-Aβ (J8, 1:500, [ ]). pE3-Aβ immunostaining required heat antigen retrieval (25 min at 95°C) in citrate buffer (10 mM, pH 6.0) prior blocking.

Techniques: Immunohistochemical staining, Staining

Journal: eLife

Article Title: Fibrillar Aβ triggers microglial proteome alterations and dysfunction in Alzheimer mouse models

doi: 10.7554/eLife.54083

Figure Lengend Snippet:

Article Snippet: Briefly, free-floating sections were permeabilized with 0.5% Triton-PBS (PBS-T) for 30 min, blocked either in 5% normal goat Serum or 5% donkey serum in PBS-T for 1 hr and incubated overnight at 4°C in blocking solution with the following primary antibodies: IBA1 (1:500, 019–19741,Wako), IBA1 (1:500, ab5076, Abcam), NAB228 (1:2000, sc-32277, Santa Cruz), CD68 (1:500, MCA1957GA, Bio-Rad), TREM2 (1:50, AF1729, R and DSystems), APP-Y188 (1:2000, ab32136, Abcam), CLEC7a (1:50, mabg-mdect, Invivogen), TMEM119 (1:200, ab209064, Abcam), APOE-biotinylated (HJ6.3, 1:100, [ ]), 3552 (1:5000, [ ]) and pE3-Aβ (J8, 1:500, [ ]). pE3-Aβ immunostaining required heat antigen retrieval (25 min at 95°C) in citrate buffer (10 mM, pH 6.0) prior blocking.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DAP12 Is Required for Macrophage Recruitment to the Lung in Response to Cigarette Smoke and Chemotaxis toward CCL2

doi: 10.4049/jimmunol.0901171

Figure Lengend Snippet: Habitual cigarette smoking leads to a dose-related increase in expression of DAP12-associated receptors in alveolar macrophages. A and B, Alveolar macrophages were isolated by bronchoscopy and purified by flow cytometry (12). Total RNA was isolated and mRNA transcript levels for CLEC5A (MDL-1) and TREM2 were measured by quantitative RT-PCR. Transcript levels for both receptors were increased in smokers’ macrophages compared with those of nonsmokers (A, B, left panel) and increased in proportion to the numbers of packs per day smoked (A, B, right panel). *p < 0.0001; *p < 0.001 using nonparametric trend test, which tests stepwise increase in each group. Data are expressed as normalized transcript copy number; bars represent means. n = 15 for NS and 12 for S. C, Lung tissue sections were stained for TREM2 protein using anti-TREM2 Ab or isotype-matched control Ig, and macrophage staining intensity was independently scored by two blinded investigators. Data are expressed as staining intensity ± SE. n = 8 healthy subjects and 27 former smokers with COPD. We did not find significant associations of staining intensity with COPD severity (as reported by the Lung Tissue Research Consortium). *p < 0.005. NS, nonsmokers; S, smokers.

Article Snippet: Cells were analyzed by flow cytometry using biotinylated isotype-matched controls or biotinylated goat anti-mouse myeloid DAP12-associated lectin-1 (MDL-1) (BAF1639; R&D Systems, Minneapolis, MN) and biotinylated sheep anti-mouse TREM2 (BAF1729; R&D Systems) with PE-conjugated streptavidin (eBioscience, San Diego, CA) as a secondary Ab.

Techniques: Expressing, Isolation, Purification, Flow Cytometry, Quantitative RT-PCR, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DAP12 Is Required for Macrophage Recruitment to the Lung in Response to Cigarette Smoke and Chemotaxis toward CCL2

doi: 10.4049/jimmunol.0901171

Figure Lengend Snippet: Constituents of cigarette smoke increased MDL-1 and TREM2 surface expression on macrophages and phosphorylated DAP12 in vitro. RAW264.7 cells were exposed to either control media or 50 μg/ml cigarette smoke-conditioned media for 24 h. A and B, Cells were analyzed by flow cytometry for (A) MDL-1 and (B) TREM2 surface expression. Data are displayed as flow cytometric histogram plots with gray line representing isotype-matched control Ab, black filled histogram representing MDL-1 or TREM2 Ab, and black line representing smoke media plus MDL-1 or TREM2 Ab. Data are representative of three independent experiments. C and D, RAW264.7 cells expressing an HA epitope-tagged DAP12 were exposed to C media or 250 μg/ml CS-conditioned media for 60 min. Lysates were immunoprecipitated with anti-HA Ab, and immunoprecipitates were analyzed by SDS-PAGE. Membranes were blotted with PY, then stripped and reblotted with (C) anti-DAP12 Ab as a loading control or (D) anti-SYK Ab (showing increased coimmunoprecipitation of SYK with phospho-DAP12). Data are representative of three independent experiments. C, control; CS, cigarette smoke; PY, phosphotyrosine Ab.

Article Snippet: Cells were analyzed by flow cytometry using biotinylated isotype-matched controls or biotinylated goat anti-mouse myeloid DAP12-associated lectin-1 (MDL-1) (BAF1639; R&D Systems, Minneapolis, MN) and biotinylated sheep anti-mouse TREM2 (BAF1729; R&D Systems) with PE-conjugated streptavidin (eBioscience, San Diego, CA) as a secondary Ab.

Techniques: Expressing, In Vitro, Flow Cytometry, Immunoprecipitation, SDS Page