Journal: Cell Death & Disease

Article Title: Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages

doi: 10.1038/s41419-021-03872-9

Figure Lengend Snippet: A – C Expression of M1 markers CD11c, CD86, and CD9 and D , E M2 markers CD206 and CD301 by ATMs of induced CLS and interstitial macrophages. F , G Expression of CD64 (all macrophages) or TREM2. Targeted adipocytes are marked by an asterisk. M2 ATMs outside of CLS are highlighted by arrows. Scale bars = 50 µm.

Article Snippet: After fixation, the tissue was washed with PBS, blocked with staining buffer (3% bovine serum albumin (BSA) in PBS) for 1 h, and stained with pre-labeled antibodies in staining buffer (1:100 for antibodies from BioLegend, San Diego, USA: CD9 [Cat# 124810], CD36 [Cat# 102610], CD38 [Cat# 102716], CD11c [Cat# 117312], CD64 [Cat# 139332], CD86 [Cat# 105020], CD274 [Cat# 124312], F4/80 [Cat# 123122] and from R&DSystems, Minneapolis, USA: TREM2 [Cat# FAB17291A]; 1:200 for antibodies from AbD serotec, BioRad, Feldkirchen, Germany: CD301 [Cat# MCA2392A647] and CD206 [Cat# MCA2235A647T] at 4 °C overnight.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages

doi: 10.1038/s41419-021-03872-9

Figure Lengend Snippet: Whole mount antibody staining of CLS formed in vivo in lean mice under homeostatic conditions. A – C ATMs in in vivo formed CLS express pro-inflammatory markers CD11c, CD86, and CD9, while interstitial ATMs are negative (highlighted by arrows). D , E Interstitial ATMs express the anti-inflammatory markers CD206 and CD301 (highlighted by arrows), while ATMs in CLS are negative. F , G Expression of CD64 (all macrophages) or TREM2 (highlighted by arrows). Asterisks mark adipocytes inside CLS. Scale bars = 100 µm.

Article Snippet: After fixation, the tissue was washed with PBS, blocked with staining buffer (3% bovine serum albumin (BSA) in PBS) for 1 h, and stained with pre-labeled antibodies in staining buffer (1:100 for antibodies from BioLegend, San Diego, USA: CD9 [Cat# 124810], CD36 [Cat# 102610], CD38 [Cat# 102716], CD11c [Cat# 117312], CD64 [Cat# 139332], CD86 [Cat# 105020], CD274 [Cat# 124312], F4/80 [Cat# 123122] and from R&DSystems, Minneapolis, USA: TREM2 [Cat# FAB17291A]; 1:200 for antibodies from AbD serotec, BioRad, Feldkirchen, Germany: CD301 [Cat# MCA2392A647] and CD206 [Cat# MCA2235A647T] at 4 °C overnight.

Techniques: Staining, In Vivo, Expressing

Journal: The Journal of Clinical Investigation

Article Title: TREM-2 promotes Th1 responses by interacting with the CD3 ζ -ZAP70 complex following Mycobacterium tuberculosis infection

doi: 10.1172/JCI137407

Figure Lengend Snippet: (A and B) Expression levels of TREM-2 in CD4+ T cells, CD8+ T cells, CD14+ monocytes (Mo), or PBMCs from HCs (n = 10) or patients with active TB (n = 10) were analyzed by quantitative real-time PCR (A) and flow cytometry (B). White and shaded histograms, respectively, show staining with TREM-2 Abs and an isotype control. (C) PBMCs from HCs or patients with active TB were double-stained with anti-CD4 (green) and anti–TREM-2 (red) Abs and then observed by fluorescence microscopy. Scale bars: 50 μm; original magnification, ×40 (enlarged insets). (D and E) Flow cytometric analysis of TREM-2 expression in CD4+ T cells from HCs (n = 81) and patients with active TB (n = 116), latent TB (n = 30), cured TB (n = 43), or COPD (n = 22). (F–H) C57BL/6 mice were injected i.p. with 1 × 106 CFU M. bovis BCG (n = 3) or H37Rv (n = 3). The proportion of TREM-2+CD4+ T cells was assessed by flow cytometry in the indicated organs 28 days p.i. (G), or in spleens at the indicated time points (H). Data represent the mean ± SD from at least 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired Student’s t test (A) and 1-way ANOVA (E–H).

Article Snippet: For TCR stimulation, human TREM-2 + CD4 + versus TREM-2 – CD4 + T cells were incubated for the indicated durations with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone OKT3, BD) and anti-CD28 (1 μg/mL, clone CD28.2, BD) Abs plus IL-2 (10 ng/mL) and IFN-γ (10 ng/mL) or IL-4 (20 ng/mL), whereas mouse TREM-2 + CD4 + versus TREM-2 – or WT versus TREM-2 –/– CD4 + T cells were incubated with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone 145-2C11, BioLegend) and anti-CD28 (1 μg/mL, clone 37.51, BioLegend) Abs (1 μg/mL) plus IL-2 and IFN-γ (20 ng/mL).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Staining, Fluorescence, Microscopy, Injection

Journal: The Journal of Clinical Investigation

Article Title: TREM-2 promotes Th1 responses by interacting with the CD3 ζ -ZAP70 complex following Mycobacterium tuberculosis infection

doi: 10.1172/JCI137407

Figure Lengend Snippet: Expression levels of T cell activation markers including CD44 (A), CD69 (B), and CD25 (C) as well as the chemokine receptor CXCR3 (D) were determined by flow cytometry in TREM-2+CD4+ and TREM-2–CD4+ T cells from patients with active TB (n = 30). The percentages of positive cells for each indicated marker in TREM-2+CD4+ versus TREM-2–CD4+ T cells were compared. (E and F) Flow cytometric analysis of CD45RO/CCR7 (n = 40) and CD45RO/CD27 (n = 21) staining to define the T cell subsets in TREM-2+CD4+ versus TREM-2–CD4+ T cells from patients with active TB. The percentages of naive T cells , Tem cells, Tcm cells, and terminally differentiated effector Temra cells were compared between TREM-2+CD4+ and TREM-2–CD4+ T cells. Data represent the mean ± SD from at least 3 independent experiments. Unpaired Student’s t test was performed in A–F. *P < 0.05 and ***P < 0.001.

Article Snippet: For TCR stimulation, human TREM-2 + CD4 + versus TREM-2 – CD4 + T cells were incubated for the indicated durations with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone OKT3, BD) and anti-CD28 (1 μg/mL, clone CD28.2, BD) Abs plus IL-2 (10 ng/mL) and IFN-γ (10 ng/mL) or IL-4 (20 ng/mL), whereas mouse TREM-2 + CD4 + versus TREM-2 – or WT versus TREM-2 –/– CD4 + T cells were incubated with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone 145-2C11, BioLegend) and anti-CD28 (1 μg/mL, clone 37.51, BioLegend) Abs (1 μg/mL) plus IL-2 and IFN-γ (20 ng/mL).

Techniques: Expressing, Activation Assay, Flow Cytometry, Marker, Staining

Journal: The Journal of Clinical Investigation

Article Title: TREM-2 promotes Th1 responses by interacting with the CD3 ζ -ZAP70 complex following Mycobacterium tuberculosis infection

doi: 10.1172/JCI137407

Figure Lengend Snippet: (A) Expression of TREM-2L on F4/80+ macrophages, CD11c+ DCs, CD3+ T cells, and CD19+ B cells in spleens of H37Rv-infected mice. (B and C) Expression levels of TREM-2L on DCs were detected after H37Rv infection or heat-killed H37Rv treatment (n = 4). (D–F) Splenic T cells sorted from WT or TREM-2–/– mice (n = 3) were labeled with CFSE and then cocultured with heat-killed, H37Rv-primed DCs for 5 days in the presence of IgG or mouse TREM-2–Fc protein. The proportion (D) and absolute number (E) of proliferated cells and IFN-γ production (F) were assessed by flow cytometry. (G–I) TREM-2+CD4+ versus TREM-2–CD4+ T cells sorted from PBMCs from HCs (n = 3) were labeled with CFSE and then cocultured with heat-killed, H37Rv-primed DCs for 5 days in the presence of IgG or human TREM-2–Fc protein. The proportion (G) and absolute number (H) of proliferated cells as well as IFN-γ production (I) were assessed by flow cytometry. Data represent the mean ± SD from at least 3 independent experiments. ***P < 0.001, by unpaired Student’s t test (B and C) and 1-way ANOVA (D–I).

Article Snippet: For TCR stimulation, human TREM-2 + CD4 + versus TREM-2 – CD4 + T cells were incubated for the indicated durations with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone OKT3, BD) and anti-CD28 (1 μg/mL, clone CD28.2, BD) Abs plus IL-2 (10 ng/mL) and IFN-γ (10 ng/mL) or IL-4 (20 ng/mL), whereas mouse TREM-2 + CD4 + versus TREM-2 – or WT versus TREM-2 –/– CD4 + T cells were incubated with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone 145-2C11, BioLegend) and anti-CD28 (1 μg/mL, clone 37.51, BioLegend) Abs (1 μg/mL) plus IL-2 and IFN-γ (20 ng/mL).

Techniques: Expressing, Infection, Labeling, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: TREM-2 promotes Th1 responses by interacting with the CD3 ζ -ZAP70 complex following Mycobacterium tuberculosis infection

doi: 10.1172/JCI137407

Figure Lengend Snippet: (A) The correlation between the frequency of CD4+ T cells and TREM-2 expression was analyzed in HCs (n = 81) and patients with active TB (n = 116) using SPSS software. r, correlation coefficient. (B) CFSE-labeled TREM-2+CD4+ or TREM-2–CD4+ T cells from patients with active TB were stimulated with heat-killed, H37Rv-primed DCs, and anti-CD3/anti-CD28 (1 μg/mL) for 3 days, and then cell proliferation was assessed by flow cytometry. (C–E) PBMCs from patients with active TB (n = 30) were stimulated with anti-CD3/anti-CD28 (αCD3/αCD28, 1 μg/mL), M. tuberculosis–specific Ag ESAT-6 (10 μg/mL), or CFP-10 (10 μg/mL) for 12 hours. The percentages of T cells producing IFN-γ (C), TNF (D), and IL-2 (E) in TREM-2+CD4+ or TREM-2–CD4+ T cells were detected by flow cytometry. Data represent the mean ± SD from at least 3 independent experiments. ***P < 0.001, by Spearman’s correlation analysis for analysis of the correlations in A and unpaired Student’s t test (C–E).

Article Snippet: For TCR stimulation, human TREM-2 + CD4 + versus TREM-2 – CD4 + T cells were incubated for the indicated durations with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone OKT3, BD) and anti-CD28 (1 μg/mL, clone CD28.2, BD) Abs plus IL-2 (10 ng/mL) and IFN-γ (10 ng/mL) or IL-4 (20 ng/mL), whereas mouse TREM-2 + CD4 + versus TREM-2 – or WT versus TREM-2 –/– CD4 + T cells were incubated with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone 145-2C11, BioLegend) and anti-CD28 (1 μg/mL, clone 37.51, BioLegend) Abs (1 μg/mL) plus IL-2 and IFN-γ (20 ng/mL).

Techniques: Expressing, Software, Labeling, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: TREM-2 promotes Th1 responses by interacting with the CD3 ζ -ZAP70 complex following Mycobacterium tuberculosis infection

doi: 10.1172/JCI137407

Figure Lengend Snippet: (A and B) Naive CD4+ T cells isolated from WT or TREM-2–/– mice were treated with anti-CD3 mAb (1 μg/mL) for 30 minutes. F4/80+ macrophages treated with LPS (1 μg/mL, 30 min) were used as a control. Cell lysates (input), anti–TREM-2 (A), or anti-CD3ζ immunoprecipitates (B) were analyzed by Western blotting for TREM-2, CD3ζ, and DAP12. (C and D) 293T cells were transfected with a PSG5 vector containing HA-tagged TREM-2, FLAG-tagged CD3ζ, and Myc-tagged ZAP70. Blots of cell lysates (input) or anti-HA (C) and anti-FLAG (D) immunoprecipitates were analyzed by Western blotting for HA, FLAG, and Myc. (E) Human CD4+ T cells or CD11b+ monocytes were sorted from PBMCs from HCs or patients with active TB. Anti–TREM-2 immunoprecipitates were analyzed for CD3ζ and ZAP70. (F) WT versus TREM-2–/– mouse CD4+ T cells were treated with anti-CD3/anti-CD28 Abs (1 μg/mL) and analyzed by Western blotting for CD3ζ (Tyr83) and ZAP70 (Tyr315) phosphorylation. (G and H) PBMCs from patients with active TB (n = 10) were stimulated with anti-CD3/anti-CD28 (1 μg/mL) for 5 minutes. p-CD3ζ (G) and p-ZAP70 (H) in TREM-2+CD4+ versus TREM-2–CD4+ T cells were analyzed by flow cytometry. Data are shown as the MFI of the indicated molecules and represent the mean ± SD from at least 3 independent experiments. ***P < 0.001, by unpaired Student’s t test (G and H). Mϕ, macrophages.

Article Snippet: For TCR stimulation, human TREM-2 + CD4 + versus TREM-2 – CD4 + T cells were incubated for the indicated durations with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone OKT3, BD) and anti-CD28 (1 μg/mL, clone CD28.2, BD) Abs plus IL-2 (10 ng/mL) and IFN-γ (10 ng/mL) or IL-4 (20 ng/mL), whereas mouse TREM-2 + CD4 + versus TREM-2 – or WT versus TREM-2 –/– CD4 + T cells were incubated with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone 145-2C11, BioLegend) and anti-CD28 (1 μg/mL, clone 37.51, BioLegend) Abs (1 μg/mL) plus IL-2 and IFN-γ (20 ng/mL).

Techniques: Isolation, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: TREM-2 promotes Th1 responses by interacting with the CD3 ζ -ZAP70 complex following Mycobacterium tuberculosis infection

doi: 10.1172/JCI137407

Figure Lengend Snippet: (A–D) A PSG5 plasmid encoding the Ig domain, WT (FL), or truncated forms of TREM-2, respectively, deleting Ig (ΔIg), the transmembrane (ΔTM), or the cytosolic domain (ΔCD), as well as 5 mutant forms of TREM-2 (C36A, R47H, C60A, T66M, and K173A) were constructed. (B and D) 293T cells were transfected with truncated or mutant TREM-2 plasmids. A co-IP assay was performed to analyze the interaction among TREM-2, CD3ζ, and ZAP70. (E–I) Sorted CD4+ T cells (n = 4) were transfected with mutant TREM-2–GFP plasmids. CD3ζ (Tyr83) and ZAP70 (Tyr319) phosphorylation was analyzed in GFP+ cells by flow cytometry. Data represent the mean ± SD from at least 3 independent experiments. *P < 0.05 and **P < 0.01, by 1-way ANOVA (G and I).

Article Snippet: For TCR stimulation, human TREM-2 + CD4 + versus TREM-2 – CD4 + T cells were incubated for the indicated durations with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone OKT3, BD) and anti-CD28 (1 μg/mL, clone CD28.2, BD) Abs plus IL-2 (10 ng/mL) and IFN-γ (10 ng/mL) or IL-4 (20 ng/mL), whereas mouse TREM-2 + CD4 + versus TREM-2 – or WT versus TREM-2 –/– CD4 + T cells were incubated with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone 145-2C11, BioLegend) and anti-CD28 (1 μg/mL, clone 37.51, BioLegend) Abs (1 μg/mL) plus IL-2 and IFN-γ (20 ng/mL).

Techniques: Plasmid Preparation, Mutagenesis, Construct, Transfection, Co-Immunoprecipitation Assay, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: TREM-2 promotes Th1 responses by interacting with the CD3 ζ -ZAP70 complex following Mycobacterium tuberculosis infection

doi: 10.1172/JCI137407

Figure Lengend Snippet: (A) Sorted human CD4+ T cells were transfected with PSG5–TREM-2 plasmids for 24 hours and then stimulated with anti-CD3/anti-CD28 Abs. STAT1 and STAT4 phosphorylation was analyzed by Western blotting. (B) Mouse WT versus TREM-2–/– CD4+ T cells were stimulated with anti-CD3/anti-CD28 Abs (1 μg/mL) plus IL-2 (10 ng/mL) and IFN-γ (20 ng/mL) for 30 minutes in the presence of PP2 (1 μM) or DMSO control. STAT1 and STAT4 phosphorylation was determined by Western blotting. (C and D) Primary human CD4+ T cells (n = 4) were transfected with WT or mutant forms of TREM-2–GFP plasmids. STAT1 and STAT4 phosphorylation was analyzed in GFP+ cells by flow cytometry. (E) Human CD4+ T cells from PBMCs from patients with active TB were sorted. Anti-TREM-2 immunoprecipitates were analyzed for IFN-γR, IL-4R, and CD3ζ. (F and G) PBMCs from patients with active TB (n = 16) were stimulated with anti-CD3/anti-CD28 Abs (1 μg/mL) in the presence of anti–IFN-γR Abs (20 μg/mL). STAT1 (Ser727) and STAT4 (Tyr693) phosphorylation in TREM-2+ or TREM-2–CD4+ T cells was analyzed by flow cytometry, and the MFI of p-STAT1 and p-STAT4 was quantified. (H) Human TREM-2+CD4+ versus TREM-2–CD4+ T cells (n = 4) were stimulated with heat-killed, H37Rv-primed DCs for 3 days in the presence of IgG or anti–IFN-γR Abs (20 μg/mL). Data represent the mean ± SD from at least 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1 way ANOVA (C–H).

Article Snippet: For TCR stimulation, human TREM-2 + CD4 + versus TREM-2 – CD4 + T cells were incubated for the indicated durations with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone OKT3, BD) and anti-CD28 (1 μg/mL, clone CD28.2, BD) Abs plus IL-2 (10 ng/mL) and IFN-γ (10 ng/mL) or IL-4 (20 ng/mL), whereas mouse TREM-2 + CD4 + versus TREM-2 – or WT versus TREM-2 –/– CD4 + T cells were incubated with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone 145-2C11, BioLegend) and anti-CD28 (1 μg/mL, clone 37.51, BioLegend) Abs (1 μg/mL) plus IL-2 and IFN-γ (20 ng/mL).

Techniques: Transfection, Western Blot, Mutagenesis, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: TREM-2 promotes Th1 responses by interacting with the CD3 ζ -ZAP70 complex following Mycobacterium tuberculosis infection

doi: 10.1172/JCI137407

Figure Lengend Snippet: (A) Rag2–/– mice (n = 6) were injected i.v. with 5 × 106 WT or TREM-2–/– CD4+ T cells, followed by i.p. injection of 1 × 106 CFU H37Rv. On day 28 p.i., lungs and spleens were collected. (B) The frequency of pulmonary and splenic CD4+ T cells was determined by flow cytometry. (C) CFSE-labeled WT or TREM-2–/– CD4+ T cells were stimulated with heat-killed, H37Rv-primed DCs, and anti-CD3/anti-CD28 Abs (1 μg/mL) for 3 days, and then cell proliferation was assessed by flow cytometry. (D) Splenocytes were stimulated with PMA (50 nM), ionomycin (1 μg/mL), and brefeldin A (BFA) (1 μg/mL) for 6 hours. The percentages of IFN-γ–, TNF-, and IL-2–producing cells were analyzed by flow cytometry. (E) IFN-γ, TNF, and IL-2 concentrations in the lungs, spleens, and peripheral blood were determined by ELISA. (F) The bacterial burden in the lungs was determined by plate count and calculated as CFU per lung. (G) Lung sections were stained with H&E and analyzed by histopathology under the microscope. Scale bars: 50 μm. Data represent the mean ± SD from at least 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired Student’s t test (B–F).

Article Snippet: For TCR stimulation, human TREM-2 + CD4 + versus TREM-2 – CD4 + T cells were incubated for the indicated durations with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone OKT3, BD) and anti-CD28 (1 μg/mL, clone CD28.2, BD) Abs plus IL-2 (10 ng/mL) and IFN-γ (10 ng/mL) or IL-4 (20 ng/mL), whereas mouse TREM-2 + CD4 + versus TREM-2 – or WT versus TREM-2 –/– CD4 + T cells were incubated with heat-killed, H37Rv-primed DCs or anti-CD3 (1 μg/mL, clone 145-2C11, BioLegend) and anti-CD28 (1 μg/mL, clone 37.51, BioLegend) Abs (1 μg/mL) plus IL-2 and IFN-γ (20 ng/mL).

Techniques: Injection, Flow Cytometry, Labeling, Enzyme-linked Immunosorbent Assay, Staining, Histopathology, Microscopy

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Antibodies

Article Snippet: The stimuli used were 0.5 mM ATP (Sigma) and 10 μg/mL TREM2 antibody (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Reduced cell surface localization of R47H TREM2 does not impair antibody-mediated activation of TREM2 in pMac. a Schematic of microglia phenotypes investigated in this study. b , c Reduced cell surface expression of R47H TREM2. Cell surface proteins on pMac were biotinylated and pulled down, the level of TREM2 protein enrichment was measured by Western blotting vs whole cell lysate, probed on separate blots. c Means ± SEM, for N = 5 harvests measured on separate Western blots. 2-tailed paired t-test: ** p = 0.0011 for R47H versus WT. d Increased sTREM2 production from R47H TREM2 pMac. sTREM2 were measured from unstimulated pMac supernatants by ELISA. Means ± SEM, for N = 3 harvests measured on same ELISA plate, data for each harvest was normalised to the average cell count. 1-way ANOVA, with Dunnett’s post hoc test: p = 0.0005 R47H versus WT (***), p = 0.0066 KO versus WT (**). e , f Co-localization of TREM2 with subcellular compartment markers in fixed and permeabilized pMac, images are a confocal slice at 4 μm, taken with Opera Phenix microscope. Inset panels are × 3 magnification of a selected cell. Calnexin used as a marker for ER, TGN46 for TGN, and LAMP1 for lysosomes. f Co-localization expressed as a ratio of TREM2 intensity to compartment marker intensity, in regions automatically segmented by high marker staining. Means ± SEM, for N = 3 harvests, each in triplicate wells. 2-way ANOVA, with Bonferroni’s post hoc test, no significant differences. g , h TREM2-activating antibody stimulation (used at concentration of 2.4 μg/1 × 10 6 cells and 3.84 μg/mL, for 10 min) of both WT and R47H TREM2 pMac caused SYK phosphorylation, measured by Western blotting. No response seen in TREM2 antibody-stimulated TREM2 KO cells, or cells treated with a goat IgG isotype control. h Means ± SEM, for N = 3 harvests measured on separate Western blots. 2-way ANOVA, with Sidak’s post hoc test, pairwise comparisons to WT for each treatment: p < 0.001 for KO stimulated with TREM2 Ab versus WT (****). i Calcium response in response to TREM2 antibody is similar in WT and R47H TREM2 pMac, measured by peak Fluo4-AM fluorescence, normalised to minimum fluorescence and cell number. Means ± SEM, for N = 3 harvests. 2-way ANOVA, with Dunnett’s multiple comparison test, pairwise comparisons to WT for each treatment: p = 0.0049 for KO stimulated with TREM2 Ab versus WT (**)

Article Snippet: The stimuli used were 0.5 mM ATP (Sigma) and 10 μg/mL TREM2 antibody (R&D Systems).

Techniques: Activation Assay, Expressing, Protein Enrichment, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Counting, Microscopy, Marker, Staining, Concentration Assay, Phospho-proteomics, Control, Fluorescence, Comparison

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Phagocytosis of dead SH-SY5Ys and synaptosomes is reduced in TREM2 KO, but not R47H TREM2, relative to WT pMac. a After 3 h of phagocytosis of pHrodo-labelled dead SH-SY5Ys, immunofluorescence staining shows that TREM2 is highly recruited to the phagocytic cup (marked by white arrow) during engulfment of cells expressing the neuronal marker TUJ1, whereas in b TREM2 is lost before maturation to RAB9+ endosomes (marked by white arrow). c – f Phagocytosis is impaired in TREM2 KO pMac only. Representative images of phagocytosis of SH-SY5Ys ( c ) or synaptosomes ( e ) shown in yellow, by pMac (red cytoplasm and blue nucleus), taken at 3 h with INCell 6000. Inset is a section of the image magnified 3-fold. d , f Means were quantified for the parameters: number of spots per cell, sum of spot areas (μm 2 ) per cell, percentage of cells containing phagocytosed particles per field. Data was normalised to mean for each genotype per experiment. Means ± SEM, for N = 3 harvests. Repeated-measures 2-way ANOVA, Dunnett’s post hoc test, pairwise comparisons to the WT for each time: * p < 0.05, ** p < 0.01, *** p < 0.001. g , h Phagocytosis of dead SH-SY5Ys results in SYK phosphorylation, which is unaffected in R47H TREM2 cells but attenuated in TREM2 KO line, measured by Western blotting at 0.5, 1, and 2 h after phagocytosis initiation. h Means ± SEM, for N = 3 harvests. Repeated-measures 2-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to the WT for each time: p = 0.0008 at 0.5 h stimulation for KO versus WT (***)

Article Snippet: The stimuli used were 0.5 mM ATP (Sigma) and 10 μg/mL TREM2 antibody (R&D Systems).

Techniques: Immunofluorescence, Staining, Expressing, Marker, Phospho-proteomics, Western Blot

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Divergent phenotypes in TREM2 KO and R47H TREM2 pMac, regarding cell morphology, migration, survival, and inflammatory responses. a , b TREM2 KO pMac are smaller and rounder than WT. Cell morphology measured by microscopy of pMac stained with CellTracker Deep Red, cells were fixed and imaged on INCell 6000 microscope. Representative images shown ( a ), and mean cell area (μm 2 ) and roundness were automatically quantified from 9 fields per well in triplicate wells using Columbus software ( b ). Means ± SEM, for N = 3 harvests. 1-way ANOVA with Dunnett’s post hoc test: p = 0.019 for cell area in KO versus WT (*), p = 0.007 for roundness in KO versus WT (**). c , d TREM2 KO pMac migrate slower towards C5a, but not ADP, compared with WT. Cell migration measured by transwell assay. Migration of pMac towards 30 μM ADP or 3 nM C5a is compared to unstimulated migration over 6 h ( c ), and inhibitors used to unmask the contribution of purinergic receptors P2RY1 (3 μM MRS2179), P2RY12 (30 μM PSB0739), and P2RY13 (10 μM MRS2211) to ADP-induced migration ( d ). Data is expressed as the percentage of migrated cells and was normalised to average migration for the harvest. Means ± SEM, for N = 4 harvests. 2-way ANOVA with Dunnett’s post hoc test. Black annotations compare stimulation to unstimulated control, or ADP + purinergic inhibitors to the ADP-only control. Grey annotations compare R47H or KO versus WT for each stimulation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, all unannotated comparisons are not significant. e , f TREM2 KO pMac exhibit increased cell death in the absence of M-CSF, whereas WT and R47H pMac remain viable. Survival after M-CSF withdrawal measured at 3, 7, and 10 days, with a comparison between M-CSF-deficient condition ( e ) to full-media controls ( f ) in the same plate. Means ± SEM, for N = 3 harvests. Repeated-measures 2-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to WT for each time: M-CSF-deficient KO versus WT at 3 days p = 0.0051 (**), at 7 days p = 0.003 (**), and at 10 days p = 0.0001 (***). Full media KO versus WT at 3 days p = 0.035 (*). g , h Comparable secretion of TNF and IL-6 by pMac in response to E. coli LPS (100 ng/mL, 4 h) ± priming by interferon-γ (100 ng/mL, 24 h prior to LPS). Concentration of TNF ( g ) and IL-6 ( h ) was measured by separate ELISAs of the same supernatants, normalised to cell number, and normalised to the average pg/mL/cell for the harvest. Means ± SEM, for N = 4 harvests. 2-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to WT for each stimulation: p = 0.0097 for TNF secreted from R47H versus WT with LPS ± IFNγ stimulation (**). As expected, IFNγ-priming enhances TNF and IL-6 secretion upon LPS stimulation, significance is not depicted for clarity, but p < 0.001 for IFNγ+LPS versus LPS, for TNF and IL-6 of all genotypes

Article Snippet: The stimuli used were 0.5 mM ATP (Sigma) and 10 μg/mL TREM2 antibody (R&D Systems).

Techniques: Migration, Microscopy, Staining, Software, Transwell Assay, Control, Comparison, Concentration Assay

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Top DEGs of R47H TREM2 and TREM2 KO pMac overlap; furthermore, the R47H DEGs that are shared with TREM2 KO represent numerous biological processes, distributed between five protein-protein interaction (PPI) modules. a Heatmap of top upregulated and top downregulated DEGs for R47H TREM2 and TREM2 KO, with the relative expression for each gene represented by colours on the corresponding row. A selection of “relevant genes” was also included. Unbiased clustering (dendrogram on the left) shows that genes separate into two major clusters based on whether they are upregulated or downregulated in TREM2 KO or R47H TREM2, and there is no genotype-specific segregation. b Five modules of functionally related DEGs identified by PPI network analysis of TREM2 KO DEGs, with enriched gene ontology (GO) terms shown. R47H DEGs were only included where they were also differentially expressed in TREM2 KO and were overlaid onto clusters identified using TREM2 KO data. Clusters identified numerically on the x -axis, with TREM2 KO on the left and R47H TREM2 on the right, showing a similar pattern of dysregulated cell functions. Number of DEGs represented by circle size, and p value represented by colour

Article Snippet: The stimuli used were 0.5 mM ATP (Sigma) and 10 μg/mL TREM2 antibody (R&D Systems).

Techniques: Expressing, Selection

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Transcriptomics reveal high overlap in dysregulated genes of R47H TREM2 and TREM2 KO pMac, relative to WT. a Validation of pMac “microglial identity” by dendrogram comparison to Abud et al. transcriptomes of iPS cells, iPSC-haematopoietic progenitor cells (iHPC), iPSC-microglia-like cells (iMGL), iMGL without TGFβ1 or without CD200 supplementation, iMGL co-cultured with rat cortical neurons, blood-derived monocytes, blood-derived dendritic cells, and primary foetal and adult human microglia . b Plot of principal component analysis (PCA) with the first two principal components separating the RNA-seq samples by differentiation age and genotype. Ages represented by shapes, and genotypes represented by colour. c Venn diagram of differentially expressed genes (DEGs) identified relative to the WT line, showing the overlap between R47H TREM2 and TREM2 KO DEGs. DEGs are separately categorised as “upregulated” or “downregulated” relative to the WT

Article Snippet: The stimuli used were 0.5 mM ATP (Sigma) and 10 μg/mL TREM2 antibody (R&D Systems).

Techniques: Biomarker Discovery, Comparison, Cell Culture, Derivative Assay, RNA Sequencing

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Extracellular matrix-adhesion modifiers and adhesion to vitronectin are dysregulated in R47H TREM2 and TREM2 KO pMac. TGFβ treatment does not rescue adhesion deficits. a Validation of selected RNA-seq hits by qRT-PCR of unstimulated pMac. Means ± SEM, for N = 7 harvests, including the 3 samples originally used for RNA-seq (open symbols) plus 4 samples harvested independently from a separate differentiation (filled symbols). Repeated-measures 1-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to the WT. b Effect of TGFβ-stimulation (50 ng/mL, 24 h) on mRNA levels of selected RNA-seq hits, measured by qRT-PCR. Means ± SEM, for N = 3 samples harvested independently to Fig. a. Two-way ANOVA, with Sidak’s post hoc test. c TREM2 KO pMac secrete reduced levels of TGFβ1 compared with WT, and TGFβ1 secretion is partly SYK-dependent. Total (inactive and active) TGFβ1 measured from supernatants by ELISA, cells treated ± OXSI-2 (2 μM, 24 h) to inhibit SYK. Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Sidak’s post hoc test. d Fibronectin protein expression is reduced in both R47H TREM2 and TREM2 KO versus WT. Fibronectin measured by Western blotting of pMac ± TGFβ1 stimulation (50 ng/mL, 24 h). Means ± SEM, quantified for N = 3 harvests on separate blots. Two-way ANOVA, with Sidak’s post hoc test. TGFβ1 vs unstimulated was not significant. e αVβ3 complex formation is reduced in both R47H TREM2 and TREM2 KO versus WT. Intact surface integrins αVβ3 and αVβ5 measured by flow cytometry in pMac ± TGFβ1 stimulation (50 ng/mL, 24 h). Data is expressed as the difference in median fluorescence intensity of the specific antibody versus isotype control, normalised (by subtraction) to the average for the harvest. Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Sidak’s post hoc test. f Adhesion to vitronectin is reduced for both R47H TREM2 and TREM2 KO versus WT, and treatment with TGFβ (50 ng/mL, 24 h prior to assay) increases αVβ3/5-dependent adhesion. Adhesion measured after 3 h by crystal violet colorimetric assay, and normalised to BSA-blocked wells (by division, and the result subtracted from 1). αVβ3/5 inhibitor (10 μM cilengitide) was added at the start of the assay to determine αVβ3/5-specific adhesion to vitronectin (striped bars). Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Dunnett’s post hoc test. Black annotations compare stimulations to unstimulated control. Grey annotations compare R47H or KO versus WT for each condition. WT = grey circles, R47H = orange squares, TREM2 KO = burgundy triangles. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, all unannotated comparisons are not significant

Article Snippet: The stimuli used were 0.5 mM ATP (Sigma) and 10 μg/mL TREM2 antibody (R&D Systems).

Techniques: Biomarker Discovery, RNA Sequencing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Flow Cytometry, Fluorescence, Control, Colorimetric Assay

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Top 5 upstream regulators predicted to oppose TREM2 KO (group 1 genes)

Article Snippet: The stimuli used were 0.5 mM ATP (Sigma) and 10 μg/mL TREM2 antibody (R&D Systems).

Techniques: Activation Assay, Recombinant

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Top 5 upstream regulators predicted to mimic TREM2 KO (group 1 genes)

Article Snippet: The stimuli used were 0.5 mM ATP (Sigma) and 10 μg/mL TREM2 antibody (R&D Systems).

Techniques: Activation Assay