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 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: 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

Journal: The EMBO journal

Article Title: The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism.

doi: 10.15252/embj.201796516

Figure Lengend Snippet: Figure 1. Trem2 p.T66M knock-in mice recapitulate biochemical alterations observed in FTD-like patients.

Article Snippet: Plates were washed three times with washing buffer before incubation for 1 h at RT with 1 lg/ml rat monoclonal anti-Trem2 antibody (R&D Systems, MAB1729) diluted in assay diluent.

Techniques: Knock-In

Journal: The EMBO journal

Article Title: The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism.

doi: 10.15252/embj.201796516

Figure Lengend Snippet: Figure 2. Decreased proliferation and increased apoptosis in Trem2 p.T66M bone marrow-derived macrophages.

Article Snippet: Plates were washed three times with washing buffer before incubation for 1 h at RT with 1 lg/ml rat monoclonal anti-Trem2 antibody (R&D Systems, MAB1729) diluted in assay diluent.

Techniques: Derivative Assay

Journal: The EMBO journal

Article Title: The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism.

doi: 10.15252/embj.201796516

Figure Lengend Snippet: Figure 4. Exaggerated immune response to lipopolysaccharide infection in Trem2 p.T66M mice.

Article Snippet: Plates were washed three times with washing buffer before incubation for 1 h at RT with 1 lg/ml rat monoclonal anti-Trem2 antibody (R&D Systems, MAB1729) diluted in assay diluent.

Techniques: Infection

Journal: The EMBO journal

Article Title: The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism.

doi: 10.15252/embj.201796516

Figure Lengend Snippet: Figure 5. Decreased age-dependent microglial activity in Trem2 p.T66M mice.

Article Snippet: Plates were washed three times with washing buffer before incubation for 1 h at RT with 1 lg/ml rat monoclonal anti-Trem2 antibody (R&D Systems, MAB1729) diluted in assay diluent.

Techniques: Activity Assay

Journal: The EMBO journal

Article Title: The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism.

doi: 10.15252/embj.201796516

Figure Lengend Snippet: Figure 7. Reduced cerebral glucose metabolism in Trem2 p.T66M knock-in mice.

Article Snippet: Plates were washed three times with washing buffer before incubation for 1 h at RT with 1 lg/ml rat monoclonal anti-Trem2 antibody (R&D Systems, MAB1729) diluted in assay diluent.

Techniques: Knock-In