Journal: Brain, behavior, and immunity

Article Title: Early life stress impairs synaptic pruning in the developing hippocampus

doi: 10.1016/j.bbi.2022.09.014

Figure Lengend Snippet: ELS reduces expression of TREM2 and CD11b while increasing CX3CR1 in P17 microglia. (A) Scatter plots for quantifying Trem2 positive microglia in CTL, LB and UPS. (B) Percentage of TREM2-positive microglia. Median fluorescence intensity (MFI) plots and surface expression of CD11b (C-D), CD45 (E-F) and CX3CR1 (G-H). CTL-males: n = 16, CTL-females: n = 14, LB males: n = 10, LB-females: n = 9, UPS-males: n = 12, UPS-females: n = 16. Error bars represent mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Microglia were isolated from the hippocampus as described above and blocked with rat- anti CD16/CD32 Fc antibodies 1:100 (Cat# 553142, BD Biosciences) and then stained with rat-anti CD11b-PeCy7 1:100 (Cat# 25-0112-82, ebiosciences), rat-anti CD45-PeCPCy5.5 antibodies 1:100 (Cat# 45-0451-82, ebiosciences), APC-rat anti Trem2 antibodies 1:10 (Cat# FAB17291A R & D systems), or APC-mouse anti CX3CR1 antibodies 1:100 (Cat# 149008) for 25 min on ice.

Techniques: Expressing, Fluorescence

Journal: Brain, behavior, and immunity

Article Title: Early life stress impairs synaptic pruning in the developing hippocampus

doi: 10.1016/j.bbi.2022.09.014

Figure Lengend Snippet: Trem2 is essential for normal phagocytic activity. (A-B) Effects of Trem2 genotype (WT, Hets, Ko) on ex vivo phagocytic activity in microglia isolated from the hippocampus of 17-day old pups (n = 10–17 pups per group, 50 % females). (C) Representative confocal images (top row) and Imaris models (middle and lower rows) of microglia from Trem2 wildtype (WT), heterozygous (Hets) and knockout P17 littermates. Staining for Iba1 (green), CD68 (blue), and PSD95 (red). Middle row: reconstruction of Iba1 & CD68 staining. Lower row: reconstruction of CD68 and PSD95 staining inside microglia. Effects of Trem2 genotype on microglial volume (D), CD68 volume inside microglia (E), number of PSD95 puncta in microglia (F) and PSD95 puncta inside CD68 (G), n = 5 cells per mouse and 4–5 mice per group, 50 % females). Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Microglia were isolated from the hippocampus as described above and blocked with rat- anti CD16/CD32 Fc antibodies 1:100 (Cat# 553142, BD Biosciences) and then stained with rat-anti CD11b-PeCy7 1:100 (Cat# 25-0112-82, ebiosciences), rat-anti CD45-PeCPCy5.5 antibodies 1:100 (Cat# 45-0451-82, ebiosciences), APC-rat anti Trem2 antibodies 1:10 (Cat# FAB17291A R & D systems), or APC-mouse anti CX3CR1 antibodies 1:100 (Cat# 149008) for 25 min on ice.

Techniques: Activity Assay, Ex Vivo, Isolation, Knock-Out, Staining

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: Characterization of BMDM from wild-type, TREM2-IPD, TREM2-sol and TREM2-KO mice. a Schematic representation of the TREM2 receptor. Black, red and yellow asterisks indicate the cleavage site in WT, the mutated cleavage site in TREM2-IPD and in TREM2-sol, respectively. b Flow cytometry analysis of murine cell surface TREM2 on BMDM. MFI: median fluorescence intensity. Sheddase inhibitor: DCP333 (DPC), sheddase activator PMA. c Analysis of supernatants from b of murine soluble TREM2 from BMDM. d ATP-based cell survival assay of BMDM upon M-CSF deprivation for 2 and 3.5 days. ATP levels of cells cultured with M-CSF ( n = 7) were set as 100% survival and compared to the ATP concentration after 2 ( n = 4) and 3.5 days ( n = 3) without M-CSF for each genotype. Statistics: Holm–Sidak’s two-way ANOVA multiple comparisons (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). e In vitro phagocytosis capacity of BMDM over 12 h (area-under-the curve) with 5 µg pHrodo-myelin per well ( n = 3). Fluorescence measurements in wells without prey were used as controls (data not shown). f Representative images of the Cathepsin B activity assay taken by the In-Cell Analyzer. The nuclei are stained with DAPI (blue), and the red fluorescence signals are derived from cleaved Magic red. g Quantification of the Cathepsin B assay images. The fluorescence integrated density of the Magic red signal was measured and normalized to the nuclei count. A significant ( p < 0.05) increase in normalized fluorescence between the DMSO control and K-18 within one genotype is marked by #. Statistics for d and g : Holm–Sidak’s two-way ANOVA with multiple comparisons (* p ≤ 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001). Statistics for e : One-way ANOVA test with Holm–Sidak’s multiple comparisons test (*** p < 0.001; **** p < 0.0001). All data are presented as means ± SEM

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Flow Cytometry, Fluorescence, Clonogenic Cell Survival Assay, Cell Culture, Concentration Assay, In Vitro, Activity Assay, Staining, Derivative Assay, Control

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: MRI indicated myelination deficits in TREM2-sol and TREM2-KO in the acute cuprizone model. a Schematic diagram of the experimental setup for the cuprizone treatment and recovery. Groups consisted of mice treated for 5 weeks with control food or 0.2% cuprizone in food and then switched back to control food (normal food) for the 4-week recovery. MRI measurements were performed at week 0 (baseline), week 3 (except for TREM2-KO and wt2) and week 5 of cuprizone intoxication, at week 7 (2 weeks of recovery on control food, except for TREM2-KO and wt2) and at week 9 (4 weeks of recovery on control food). Mice were culled at week 9 immediately after the last MRI measurement. b Representative MRI images acquired from three mice at baseline, at maximal pathology (5 weeks of receiving 0.2% cuprizone) and at recovery (4 weeks after switching to control food) for the different genotypes. c Corresponding T2-weighted MRI signal intensity (relative to the signal intensity at baseline) in external capsule (EC). Group sizes: n = 7–9 for all genotypes and timepoints. Data are shown as means ± SEM. Statistics: ANOVA with random effects comparisons indicated significant differences with respect to WT mice: *0.01 < p < 0.05, ***0.0001 < p < 0.001, **** p < 0.0001. For each group examined, T2-weighted signals were significantly increased with respect to baseline values (significances not shown). d Analysis of TREM2 levels in the brain for mice receiving control food (ctrl), at peak of cuprizone intoxication (week 5, cpz) and after 4-week recovery (rec). n.d. not detected. Statistics: Holm–Sidak’s multiple comparison test one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, ++++ p < 0.0001 to the respective wt group). Wt1, as well as wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, wt2 is the wild-type group for the TREM2-KO study

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Control, Comparison

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: TREM2-sol and TREM2-KO display myelin debris, lack of remyelination and axonal pathology in the EC. Representative pictures for the different genotypes and timepoints from histological stainings detecting a myelin with Luxol Fast Blue (LFB) and corresponding quantitative optical density (OD) analysis of LFB in the EC (normalized to WT at control food), b mature oligodendrocytes (GST-π) and corresponding image analysis in EC (GST-π soma area in %), c myelin basic protein debris (dMBP) and corresponding image analysis in EC (dMBP-stained area in %), d neurofilament (SMI312) and corresponding image analysis in EC (SMI312-stained area in %). Group sizes: n = 3-7 for all genotypes and timepoints. Data shown as means ± SEM. wt: wild-type, TREM2-IPD: TREM2 cleavage-reduced, TREM2-sol: TREM2 soluble-only, TREM2-KO: TREM2 knockout. Ctrl: control food, cpz: cuprizone food for 5 weeks, rec: recovery on control food for 4 weeks. EC: external capsule, CC: corpus callosum. Scale bars: 300 µm (overview), 50 µm (close-up). Statistics: Holm–Sidak`s multiple comparison test one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Comparisons not indicated are non-significant. Wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, Wt ctrl2, wt cpz2 and wt rec2 are the wild-type groups for the TREM2-KO study. Only statistical analysis within a study was performed. For c the analysis of the respective wt group for TREM2-KO was omitted as no dMBP signal was observed

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Control, Staining, Knock-Out, Comparison

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: Increase of NF-L in plasma from TREM2-sol and TREM2-KO in the acute cuprizone model. NF-L measurements in plasma of WT, TREM2-IPD, TREM2-sol and TREM2-KO mice receiving control food (ctrl), at 5 weeks of cuprizone intoxication (cpz) and at 4-week recovery on normal food (rec). Group sizes: wt ctrl ( n = 4), wt cpz ( n = 4), wt rec ( n = 4), TREM2-IPD ctrl ( n = 3), TREM2-IPD cpz ( n = 7), TREM2-IPD rec ( n = 7), TREM2-sol ctrl ( n = 4), TREM2-sol cpz ( n = 7), TREM2-sol rec ( n = 4), TREM2-KO cpz ( n = 4). One-way ANOVA Holm–Šídák's multiple comparisons test, * p < 0.05, *** p < 0.001, **** p < 0.0001. Comparisons not indicated are non-significant. Wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, wt ctrl2, wt cpz2 and wt rec2 are the wild-type groups for the TREM2-KO study. Only statistical analysis within a study was performed

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Clinical Proteomics, Control

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: TREM2-IPD and TREM2-sol mice show both sustained microglia/astrocyte activation and enhanced LAMP-1 in the EC. Representative images for the different genotypes and timepoints from histological stainings detecting a Iba1 and corresponding image analysis of Iba1-positive soma numbers (normalized to WT at week 5 cuprizone), b astrocytes (GFAP) and corresponding image analysis (GFAP-stained area in %), c LAMP-1 (lysosomal-associated membrane protein 1) and corresponding image analysis (LAMP1-stained area in %), as well as d TMEM119 (homeostatic marker) and corresponding image analysis (TMEM119-stained area in %). Group sizes: n = 2-7 for all genotypes and timepoints. Data are shown as means ± SEM. WT: wild-type, TREM2-IPD: TREM2 cleavage-reduced, TREM2-sol: TREM2 soluble-only, TREM2-KO: TREM2 knockout. Ctrl: control food, cpz: cuprizone food for 5 weeks, rec: recovery on control food for 4 weeks. EC: external capsule. CC: corpus callosum. Scale bars: 300 µm (overview), 50 µm (close-up). Statistics: Holm–Sidak’s multiple comparison test one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Comparisons not indicated are non-significant. wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, wt ctrl2, wt cpz2 and wt rec2 are the wild-type groups for the TREM2-KO study. Only statistical analysis within a study was performed. For d the analysis of the respective wt group for TREM2-KO was omitted as no relevant TMEM119 signal was observed

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Activation Assay, Staining, Membrane, Marker, Knock-Out, Control, Comparison

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: Brain cytokine/chemokine response is reduced in TREM2-sol and TREM2-KO, but enhanced in TREM2-IPD. Cytokine/chemokine measurements in brain detecting MIP-1a, MIP-1b, IP-10 and MCP-1 in WT, TREM2-IPD, TREM2-sol and TREM2-KO with control food (ctrl), at 5 weeks of cuprizone intoxication (cpz) and at 4-week recovery on normal food (rec). Measurements are normalized to wt cpz. Group sizes: wt ctrl ( n = 4), wt cpz ( n = 4), wt rec ( n = 4), TREM2-IPD ctrl ( n = 4), TREM2-IPD cpz ( n = 7), TREM2-IPD rec ( n = 7), TREM2-sol ctrl ( n = 4), TREM2-sol cpz ( n = 7), TREM2-sol rec ( n = 4), TREM2-KO ctrl ( n = 7), TREM2-KO cpz ( n = 4), TREM2-KO rec ( n = 7). Statistics: ordinary one-way ANOVA Holm–Šídák's multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Comparisons not indicated are non-significant. wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, wt ctrl2, wt cpz2 and wt rec2 are the wild-type groups for the TREM2-KO study. Only statistical analysis within a study was performed

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Control

Journal: Journal of Neuroinflammation

Article Title: Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model

doi: 10.1186/s12974-022-02671-z

Figure Lengend Snippet: TREM2-IPD showed enhanced myelination in the chronic cuprizone model. a Schematic diagram of the experimental setup for the cuprizone treatment and recovery. Groups consisted of mice treated for 12 weeks with control food (normal food) or 0.2% cuprizone in food and then switched back to control food for the 3-week recovery. MRI measurements were performed at timepoints indicated. Mice were culled at week 15 immediately after the last MRI measurement. b T2-weighted signals in the CC and EC during the 12-week intoxication period and the recovery phase were significantly increased with respect to baseline values and compared to analyses for mice receiving control diet throughout the experiment. The significance levels # 0.01 < p < 0.05 and ### p < 0.001 correspond to ANOVA with random effects comparisons between WT and TREM2-IPD animals. Representative images for the different genotypes and at week 15 from histological stainings detecting c myelin with Luxol Fast Blue (LFB) and corresponding quantitative optical density analysis of LFB in the EC and CC, and d mature oligodendrocytes (GST-π) and corresponding image analysis in EC and CC (GST-π soma area in %). Group sizes: n = 5–7 for all genotypes and timepoints. Male mice were used for the cuprizone groups. Data are shown as means ± SEM. WT: wild-type, TREM2-IPD: TREM2 cleavage-reduced. Ctrl: control food, rec: recovery on control food for 3 weeks. Control refers to TREM2-IPD mice receiving normal food throughout the study. EC: external capsule, CC: corpus callosum. Scale bars: 500 µm. Statistics: ordinary one-way ANOVA Holm–Šídák’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant

Article Snippet: Next, the cells were transferred to a 96-well clear V-Bottom microplate and stained with a human/mouse TREM2 Alexa Fluor 488 antibody (R&D Systems FAB17291G) or a rat IgG2A Alexa Fluor ® 488-conjugated isotype control (R&D Sytems, IC006G), for 30 min at 4 °C.

Techniques: Control

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: 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: 96-well plate was coated overnight at 4 °C with TREM2 coating antibody (MAB17291-100, R&D Systems) in ELISA Coating Buffer (421701, Biolegend).

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: 96-well plate was coated overnight at 4 °C with TREM2 coating antibody (MAB17291-100, R&D Systems) in ELISA Coating Buffer (421701, Biolegend).

Techniques: RNA Sequencing Assay, Expressing, Comparison, Western Blot, 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: 96-well plate was coated overnight at 4 °C with TREM2 coating antibody (MAB17291-100, R&D Systems) in ELISA Coating Buffer (421701, Biolegend).

Techniques: Western Blot, RNA Sequencing Assay, 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: 96-well plate was coated overnight at 4 °C with TREM2 coating antibody (MAB17291-100, R&D Systems) in ELISA Coating Buffer (421701, Biolegend).

Techniques: Western Blot