Journal: bioRxiv

Article Title: Mapping Endothelial-Macrophage Interactions in Diabetic Vasculature: Role of TREM2 in Vascular Inflammation and Ischemic Response

doi: 10.1101/2024.05.14.594235

Figure Lengend Snippet: (A) Information of human donors included in the spatial transcriptome sequencing (ST-seq). (B) SpatialFeaturePlots of VWF and CD68 expression in the intima as revealed by ST-seq. Left plots show histology-guided annotation. (C) Pie charts showing probability of the presence of specific cell types within ‘EC-dominant’ spots/voxels in ND and T2D mesenteric arteries, computed through integrative analysis of ST-seq and whole-artery scRNA-seq. (D-F) Analysis for EC-MP interactions based on intima scRNA-seq data. (D) Comparison of intercellular interactions between ND vs T2D computed through CellChat. (E) Gene expression of TREM2 ligands in ND vs T2D ECs. (F) CellphoneDB analysis reveals the TREM2-ligand complexes between MPs and/or ECs in ND and T2D arteries. (G) TREM2 expression levels in ST-seq spots/voxels with specific dominant cell types as indicated, determined through integrative analysis of ST- and scRNA-seq. *p<0.05, ***p<0.001 based on Students t-test. (H) Immunohistochemistry of human mesenteric arteries for CD31 and TREM2, from left to right showing higher magnification and zoom-in images. Scale bar = 50μm.

Article Snippet: Intramuscular injections of TREM2-Ab (R&D systems; MAB17291) or rat IgG isotype control (R&D systems; MAB0061)

Techniques: Sequencing, Expressing, Comparison, Immunohistochemistry

Journal: bioRxiv

Article Title: Mapping Endothelial-Macrophage Interactions in Diabetic Vasculature: Role of TREM2 in Vascular Inflammation and Ischemic Response

doi: 10.1101/2024.05.14.594235

Figure Lengend Snippet: (A,B) TREM2 expression in ND and T2D donors (in A) and in 5 MP clusters (C1-C5) (in B) quantified by intima scRNA-seq. (C) Trem2 expression in the aortic MΦs from male C57Bl/6 mice fed chow vs HFHS diet for 4 months (n = 4/group), profiled by scRNA-seq. ( D,E) Relative TREM2 mRNA levels measured by qPCR in human MΦ (THP-1 derived) (in D) and mouse MΦ (Raw 264.7) (in E) treated with 25 mM Glucose plus TNFα (HT) for 72 hr or 25mM mannitol as normal glucose osmolarity control (NM). Graphs represent mean ± SEM . (F) Immunofluorescence staining of TREM2 in THP-1-derived MΦs under NM and HT, with DAPI counterstain. (G-I) Leukocyte adhesion assay with ECs cultured under HT for 72 hr and incubated with CMFDA labeled THP-1 cells with or without TREM2 knockdown (in G, H) or neutralizing antibody against TREM2, with IgG as isotype control (in I). (G) Representative images and (H,I) quantification of adhered monocytes. ( J) Representative SRS images of THP-1-derived MΦ under treatments as indicated. The first and the second column represent single-frequency SRS images targeted at the protein (–CH 3 symmetric stretching, 2940 cm −1 ) and lipid (–CH 2 symmetric stretching, 2845 cm −1 ) channels, respectively. The third column represents the ratiometric images of CH 2 /CH 3 (lipid/protein). Scale bar = 20 µm. (K) Quantification of average lipid/protein ratios from ( J ), plotted with mean ± SEM from 3 independent experiments. *p<0.05, **p<0.01 based on Student’s t test (in D, E, G, H) or one-way ANOVA followed by Dunnets test (in K).

Article Snippet: Intramuscular injections of TREM2-Ab (R&D systems; MAB17291) or rat IgG isotype control (R&D systems; MAB0061)

Techniques: Expressing, Derivative Assay, Immunofluorescence, Staining, Cell Adhesion Assay, Cell Culture, Incubation, Labeling

Journal: bioRxiv

Article Title: Mapping Endothelial-Macrophage Interactions in Diabetic Vasculature: Role of TREM2 in Vascular Inflammation and Ischemic Response

doi: 10.1101/2024.05.14.594235

Figure Lengend Snippet: (A) Schematic of scratch wound assay using HMVECs and conditioned medium (CM) from THP-1-derived MΦ with or without TREM2 knockdown and treated by NM/HT. (B,C) Representative images and quantification of scratch wound assay using ECs incubated with MΦ CM across a 24 h time course. (D) Area under curve at 24 h showing the % wound closure based on (C). (E) qPCR of VCAM1 and ICAM1 expression in ECs treated with MΦ CM. Data represents mean ± SEM of n = 5 (B-D) and n = 3 (E). *p<0.05, **p<0.01 based on ANOVA followed by Dunnets post-hoc test.

Article Snippet: Intramuscular injections of TREM2-Ab (R&D systems; MAB17291) or rat IgG isotype control (R&D systems; MAB0061)

Techniques: Scratch Wound Assay Assay, Derivative Assay, Incubation, Expressing

Journal: bioRxiv

Article Title: Mapping Endothelial-Macrophage Interactions in Diabetic Vasculature: Role of TREM2 in Vascular Inflammation and Ischemic Response

doi: 10.1101/2024.05.14.594235

Figure Lengend Snippet: (A) Laser speckle images of flow perfusion in male mice treated with or without STZ (DM vs ND), 5 days post hindlimb ischemia (HLI). Note the decreased flow in the ligated right limb (white arrow) of DM mouse as compared to ND. ( B) MΦ TREM2 expression in non-ligated (NI) or ligated (I) limb muscles from ND and DM mice, as profiled by scRNA-seq (n = 2/group). (C) IB4-TREM2 co-IF on the non-ligated vs ligated limb muscles from DM mice. (D-F) DM was induced in male and female C57Bl/6 mice followed by HLI and intramuscular injection of TREM2 neutralizing antibody (TREM2-Ab) into the ischemic limbs on post-op Day 1, 4, & 8. IgG was used as an isotype control. ( E) Representative images of flow perfusion on Day 1 and 6. (F) Quantification of perfusion ratio, calculated by comparing the flow perfusion in the ligated vs non-ligated (R/L) limb of the same animals. Data represented as mean ± SEM of n = 8/group (mixed between n = 7 male and n = 9 female). (G) TREM2 mRNA levels determined by bulk RNA-seq of from muscle biopsy of human healthy control (HC/n = 61) and patients with critical limb ischemia (CTLI/n = 64), as published (Ryan et al., 2018). (H-J) scRNA-seq profiled expression of TREM2 (in H) and TREM2 ligand in ECs (in I) in the surgically collected (non-)ischemic (NI/I) muscles from human PAD patients with (n = 1) or without (n = 2) co-existing DM. (J,K) CD31-TREM2 co-IF in (non-)ischemic muscles from human patients with PAD (n = 7). (J) shows representative images and (K) shows quantification of CD31-TREM2 co-staining expressed by Pearson’s R values. *p<0.05 based on paired t-test.

Article Snippet: Intramuscular injections of TREM2-Ab (R&D systems; MAB17291) or rat IgG isotype control (R&D systems; MAB0061)

Techniques: Expressing, Muscles, Injection, RNA Sequencing Assay, Staining

Journal: Nature Communications

Article Title: Deciphering the spatial landscape and plasticity of immunosuppressive fibroblasts in breast cancer

doi: 10.1038/s41467-024-47068-z

Figure Lengend Snippet: A CellChat dominant sender and receiver plot showing incoming and outgoing interaction strength for each cell type identified in the BC atlas (73,426 cells from 43 patients). The size of each circle corresponds to the total number of significant interactions, colored per cell type. B Scatter plots with linear regression lines showing correlations in the content of FAP+ CAF clusters quantified by flow cytometry in BC ( N = 87 patients). p values from two-sided Pearson’s correlation test. C Same as ( B ) analyzing correlations between the content in ECM-myCAF, TGFβ-myCAF, Wound-myCAF and Detox-iCAF with TREM2+ or FOLR2+ macrophages in BC ( N = 25). D Bar plot showing the percentages (%) of migration of CD14+ monocytes after 6 h of transwell co-culture with FAP+ CAF clusters. Data are mean ± SEM ( n = 4 independent experiments). p values from two-sided Student’s t test. E % of CD14+ CD16+ myeloid cells among total CD14+ monocytes after 24 h of co-culture with FAP+ CAF clusters (Detailed gating strategy in Supplementary Fig. ). Data are mean ± SEM ( n = 9). p values from two-sided Student’s t test. F Same as ( E ) for TREM2+ macrophages. p values from two-sided Student’s t test. G Same as ( E ) for FOLR2+ macrophages. p values from two-sided Mann–Whitney test. H % of FOXP3+ regulatory T cells among CD4+ CD25+ T lymphocytes after 16 h of co-culture with FAP+ CAF clusters (Detailed gating strategy in Supplementary Fig. ). Data are mean ± SEM ( n = 8). p values from two-sided Mann–Whitney test. I Same as ( H ) for the % of PD-1+ FOXP3+ T lymphocytes. p values from two-sided Mann–Whitney test. J Same as ( H ) for the % of CTLA-4+ FOXP3+ T lymphocytes. p values from two-sided Mann–Whitney test. K % of Perforin+ among total CD16+ NK cells after 24 h of co-culture with FAP+ CAF clusters (Detailed gating strategy in Supplementary Fig. ). Data are mean ± SEM ( n = 7). p values from two-sided Student’s t test. L Same as ( K ) for Granzyme B+ NK cells. p values from two-sided Student’s t test. M Same as ( K ) for CD16+ CD56 high NK cells. p values from two-sided Student’s t test. N Same as ( K ) for NKG2A+ NK cells. p values from two-sided Student’s t test. Source data are provided as a Source Data file.

Article Snippet: Isotype control antibodies for macrophages subsets were used: iso-anti-CD16-BV650 (BV650 Mouse IgG1 κ Isotype Control, 1:50, BD Biosciences, #563231), iso-anti-CD56-BUV395 (BUV395 Mouse IgG2b κ Isotype Control; 1:50, BD Biosciences, #563558), iso-anti-CD14-Pecy7 (PE-Cy7 Mouse IgG2a κ Isotype Control, 1:50, BD Biosciences, #557907), iso-anti-FOLR2-PEC (PE Mouse IgG1 κ Isotype Ctrl Antibody; 1:100, Biolegend, #400112) and iso-anti-TREM2-APC (Rat IgG2B Isotype Control, 1:50, Novus Biologicals, #MAB0061).

Techniques: Flow Cytometry, Migration, Co-Culture Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Deciphering the spatial landscape and plasticity of immunosuppressive fibroblasts in breast cancer

doi: 10.1038/s41467-024-47068-z

Figure Lengend Snippet: CAF heterogeneity and plasticity shape a structured organization of the tumor micro-environment in BC. In this paper, we describe spatial organization, plasticity and interactions of FAP+ CAF clusters with neighboring cells by combining analysis of single-cell data, spatial transcriptomics and functional assays. We identify spatially organized cellular EcoCellTypes, which are precisely localized within tumors and composed of specific FAP+ myCAF or iCAF clusters. Distances to cancer cells induce a gradient of FAP+ CAF cluster identities. Detox-iCAF are found around blood vessels composed of ap-EC and in close proximity to FOLR2+ TAM. Detox-iCAF serve as a reservoir and can give rise to ECM-myCAF in presence of cancer cells, either directly or indirectly through the Wound-myCAF cluster, by DPP4- and YAP1/TEAD-dependent mechanisms. ECM-myCAF localize close to tumor cells, where they can reach a TGFβ-myCAF phenotype in presence of T lymphocytes. In addition, specific TAM are found in different FAP+ CAF cluster-enriched territories. While FOLR2+ TAM are close to Detox-iCAF, TREM2+ and SPP1+ TAM are enriched in ECM-myCAF, IFNαβ-myCAF and TGFβ-myCAF-enriched niches. Our data show that spatial organization in BC tumors is related to reciprocal interactions of FAP+ CAF clusters with cancer and immune cells in specific spatial domains. Importantly, we identify that the content in Detox-iCAF and TGFβ-myCAF at DCIS diagnosis is a predictive factor for the recurrence of DCIS into invasive breast cancer. The figure was created with Biorender.com.

Article Snippet: Isotype control antibodies for macrophages subsets were used: iso-anti-CD16-BV650 (BV650 Mouse IgG1 κ Isotype Control, 1:50, BD Biosciences, #563231), iso-anti-CD56-BUV395 (BUV395 Mouse IgG2b κ Isotype Control; 1:50, BD Biosciences, #563558), iso-anti-CD14-Pecy7 (PE-Cy7 Mouse IgG2a κ Isotype Control, 1:50, BD Biosciences, #557907), iso-anti-FOLR2-PEC (PE Mouse IgG1 κ Isotype Ctrl Antibody; 1:100, Biolegend, #400112) and iso-anti-TREM2-APC (Rat IgG2B Isotype Control, 1:50, Novus Biologicals, #MAB0061).

Techniques: Functional Assay

Journal: bioRxiv

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

doi: 10.1101/2024.09.13.612888

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

Article Snippet: Plates were washed and incubated with rat anti-TREM2 detection antibody (Sigma Aldrich, clone 5F4, stock: 1 mg/ml) at 1:1000 dilution for 1 hour.

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.13.612888

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

Article Snippet: Plates were washed and incubated with rat anti-TREM2 detection antibody (Sigma Aldrich, clone 5F4, stock: 1 mg/ml) at 1:1000 dilution for 1 hour.

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

Journal: bioRxiv

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

doi: 10.1101/2024.09.13.612888

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

Article Snippet: Plates were washed and incubated with rat anti-TREM2 detection antibody (Sigma Aldrich, clone 5F4, stock: 1 mg/ml) at 1:1000 dilution for 1 hour.

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

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) Clustering of all cell subsets from human atherosclerotic endarterectomy samples (Fernandez et. al., Nat Med 2019). b ) Clustering of monocyte/macrophage populations from human atherosclerotic endarterectomy samples (Fernandez et. al., Nat Med 2019). c ) PTPRC and CD14 expression of clustered monocyte/macrophage populations from 3b. d ) Foamy macrophage gene (FABP5, LGALS3) expression of clustered monocyte/macrophage populations from 3b. e ) Inflammatory macrophage gene (IL1B, NLRP3) expression of clustered monocyte/macrophage populations from 3b. f ) TREM2 expression of clustered monocyte/macrophage populations from 3b. g ) Volcano plot of enrichment of genes from samples from either asymptomatic or symptomatic patients. TREM2 in red. DEGs were determined by Wald test with DESeq2.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Expressing

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , Schematic for CRISPR knockout screening approach for oxLDL uptake. BV2 macrophages were loaded with CRISPR pooled guide library (Gouda). Cells were made foamy by overnight treatment with soluble cholesterol and then challenged for 4 h with DiI-oxLDL and sorted for DiI high and DiI low cells. Guides were sequenced from sorted populations. b , Confocal micrograph showing BV2 DiI uptake after 4-h incubation with DiI-oxLDL. Representative of two independent experiments. c , CRISPR guide enrichment comparing log-normalized enrichment in DiI high ( x axis) versus DiI low ( y axis). Gray error bands delineate guides with log fold change < 1. d , Selected gene enrichments comparing DiI low versus DiI high . e , Peritoneal macrophages were isolated from WT or Trem2 −/− mice and treated with soluble cholesterol to induce foamy cell formation. After overnight culture, cells were analyzed for Trem2 expression by flow cytometry ( n = 5 biologically independent replicates per group). Data are mean ± s.e.m. Student’s t -test, **** P < 0.0001. f , Bodipy staining for total neutral lipid accumulation was performed by flow cytometry on peritoneal macrophages from WT or Trem2 −/− mice, cultured overnight in media alone or in media with soluble cholesterol ( n = 6 biologically independent replicates for untreated and n = 4 biologically independent replicates for foamy). Data are mean ± s.e.m. Student’s t -test. g , Peritoneal macrophages were isolated from WT or Trem2 −/− mice and treated with soluble cholesterol to induce foamy cell formation. After overnight culture, cells were treated with DiI-oxLDL for 4 h and assessed for uptake by flow cytometry ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. h , CD36 expression from peritoneal macrophages isolated from WT or Trem2 −/− mice and treated with soluble cholesterol overnight to induce foamy cell formation ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. NS, not significant.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: CRISPR, Knock-Out, Incubation, Isolation, Expressing, Flow Cytometry, Staining, Cell Culture

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) Time course analysis of DiI-oxLDL uptake in WT BV2 cells differentiated in media with 20 μg/mL of soluble cholesterol overnight prior to addition of DiI-oxLDL (n = 5 biological replicates/group). Data are mean ± S.E.M. b ) CRISPR guide enrichment by rank-order was plotted against P-value for DiI-oxLDL-low compared against DiI-oxLDL-high to identify top enriched guides. Trem2 in red. P-values calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. c ) Two sided P-value vs count and p value vs false discovery rate (FDR) for DiI-oxLDL-low compared against DiI-oxLDL-high to identify top enriched guides. p-values and FDR calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. d ) Top 15 ‘importance index’ genes associated with foamy cell commitment by Trade-seq analysis , were compared for gene rank and enrichment in CRISPR screen. Trem2 highlighted in gray. P-values calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. e) CD36 expression (left) and percent CD36 high (right) of foamy peritoneal macrophages cultured with soluble cholesterol overnight from WT (blue) and Trem2−/− mice (red). Gated on F4/80+ CD11b+ live cells (n = 5 biological replicates/group). Data are mean ± S.E.M. Student’s t-test, P = ** < 0.01. f ) SR-AI expression (left), MFI (middle) and percent SR-AI positive (right) of foamy peritoneal macrophages cultured with soluble cholesterol overnight from WT (blue) and Trem2−/− mice (red). Gated on F4/80+ CD11b+ live cells (n = 5 biological replicates/group). Data are mean ± S.E.M. Student’s t-test

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: CRISPR, Expressing, Cell Culture

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) Cranial artery plaques were stained either for CD68 and Trem2 to identify co−expressing foamy macrophages within human plaques (top) or with CD68 and isotype control with secondary antibody (bottom). Representative image from 3 independent samples. b ) Carotid artery endarterectomy samples from three patients stained for isotype control or Trem2 using DAB (3,3′-Diaminobenzidine) immunohistochemical staining. Representative images from 6 independent samples.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Staining, Expressing, Immunohistochemical staining

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , Schematic for mixed bone marrow chimera experiment. Ldlr −/− mice were lethally irradiated and rescued by donor bone marrow from (50%) LysM cre R26 tdTomato (WT) and (50%) Trem2 −/− mice. Recipient mice were rested for 8 weeks and then fed an HFD for an additional 8 weeks to induce atherosclerosis. b , Flow cytometry gating of blood immune cells (CD45 + ) after 8-week HFD feeding, showing ratio of monocytes derived from WT (tdTomato + ) and Trem2 −/− progenitors. c , Confocal micrograph of whole-mount aorta showing tdTomato labeling (red) and CD45 (white) staining to define cellular contributions to foamy macrophages. Representative image from two independent experiments. d , Quantification of tdTomato + cells in blood compared to foamy macrophages from whole-mount aorta images ( n = 3 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. e , Foamy FACS was performed on CD64 + CD11b + macrophages isolated from mixed bone marrow chimera aorta. Macrophages were separated into tdTomato + and tdTomato − populations and then assessed for foamy representation by SSC and Bodipy (neutral lipid) staining. f , Flow cytometric overlap between tdTomato + (red) and Trem2 −/− (blue) derived macrophages from digested atherosclerotic aorta. g , Quantification derived from flow cytometric foamy FACS comparing relative contribution to foamy macrophages ( n = 4 mice per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. KO, knockout. MACS, macrophage.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Irradiation, Flow Cytometry, Derivative Assay, Labeling, Staining, Isolation, Knock-Out

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , CX3CR1 creER Trem2 flox/flox Ldlr −/− (Trem2 ΔMФ ) or littermate control mice (which included Cre − animals CX3CR1 +/+ Trem2 fl/fl Ldlr −/− and Cre + animals CX3CR1 creER/+ Trem2 fl/+ Ldlr −/− ) were fed TAM-HFD for 8 weeks ( b – e ) or 16 weeks ( f – i ). b , After 8 weeks of TAM-HFD, aortas were analyzed by en face analysis for percentage Oil Red O (ORO) staining on the arch ( n = 17 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. c , Aortic sinus plaque area measured after ORO staining in 8-week TAM-HFD samples (n = 17 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. d , Serum cholesterol levels from 8-week TAM-HFD-fed mice ( n = 11 mice per group for Cntl and n = 8 for Trem2 ΔMФ ). Data are mean ± s.e.m. e , Weight data from 8-week TAM-HFD-fed mice ( n = 9 mice pergroup). Data are mean ± s.e.m. f , En face ORO staining of aorta after 16-week TAM-HFD feeding ( n = 12 mice per group). Data are mean ± s.e.m. Student’s t -test, **** P < 0.0001. g , Aortic sinus plaque area after 16-week TAM-HFD feeding ( n = 11 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, *** P < 0.001. h , Serum cholesterol after 16-week TAM-HFD feeding ( n = 10 mice per group). Data are mean ± s.e.m. i , Weight of mice after 16-week TAM-HFD feeding ( n = 10 mice per group for Cntl and n = 7 for Trem2 ΔMФ ). Data are mean ± s.e.m.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Staining

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) Trem2 expression and quantification from atherosclerotic aortae (n = 2 mice/group for Cntl and n = 3 for Trem2ΔMФ). Briefly, Cntl or Trem2ΔMФ mice were fed TAM-HFD for 16 weeks then aorta were harvested, digested and flow cytometry was run. Histogram was gated on live, CD45 + CD11b + CD64+ cells. b ) Flow cytometric gating strategy for identifying major blood immune cell populations. c ) Blood immune cell profiling by flow cytometry in indicated mice after 16 weeks TAM-HFD feeding (n = 12 mice/group for Cntl and n = 6 for Trem2ΔMФ). Data are mean ± S.E.M. Student’s t-test. d ) Classical monocyte bead uptake in the blood was measured by flow cytometry 24 hours after i.v. bead injection in indicated strains after 16 weeks TAM-HFD feeding (n = 7 mice/group for Cntl and n = 5 for Trem2ΔMФ). Data are mean ± S.E.M. Student’s t-test.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, Injection

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , Following the schematic in , CX3CR1 creER Trem2 flox/flox Ldlr −/− (Trem2 ΔMФ ) or littermate control mice were treated continuously with TAM-HFD for the indicated times. b , Serum from 8-week TAM-HFD-fed mice were assessed for cytokine levels by multiplex assay ( n = 10 mice per group). Data are mean ± s.e.m. c , Serum from 16-week TAM-HFD-fed mice were assessed for cytokine levels by multiplex assay ( n = 9 mice per group). Data are mean ± s.e.m. d , Blood immune cells were assessed after 16 weeks of TAM-HFD by flow cytometry ( n = 12 mice per group for Cntl and n = 6 for Trem2 ΔMФ ). Data are mean ± s.e.m. e , Monocyte recruitment was assessed by bead labeling and recruitment experiment—images from representative histologic and immunofluorescence images with lipid content (red) and beads (green). Representative image from two independent experiments. f , Quantification of plaque-associated beads that were counted per section for 8-week or 16-week TAM-HFD experiments from experiments in ( n = 7 mice per group for Cntl and n = 5 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test. NS, not significant; ORO, Oil Red O.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Multiplex Assay, Flow Cytometry, Labeling, Immunofluorescence

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , Confocal micrograph showing CD68 staining (green) and DAPI (blue) for macrophage area in Cntl or Trem2-deficent mice after 16-week TAM-HFD feeding. Representative image from two independent experiments. b , Quantification of CD68 + macrophage area per section in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01. c , Quantification of the percentage of plaque that is macrophages (CD68 + ) in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test. d , Confocal micrograph showing Ki67 staining (magenta) and CD68 staining (green) for proliferation in Cntl or Trem2-deficient mice after 16-week TAM-HFD feeding. Representative image from two independent experiments. e , Quantification of Ki67 + macrophages (CD68 + ) per section in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group for 8-week TAM-HFD, n = 7 mice per group for Cntl 16-week TAM-HFD and n = 6 mice per group for 16-week TAM-HFD Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01. f , Confocal micrograph of TUNEL staining (magenta) and CD68 staining (green) for detection of dying cells within atherosclerotic lesions after 16-week TAM-HFD feeding. Representative image from two independent experiments. g , Quantification of TUNEL + macrophages (CD68 + ) per section in 8-week or 16-week TAM-HFD samples ( n = 6 mice per group for Cntl 8-week TAM-HFD, n = 7 mice per group for Trem2 ΔMФ 8-week TAM-HFD, n = 7 mice per group for Cntl 16-week TAM-HFD and n = 7 mice per group for Trem2 ΔMФ 16-week TAM-HFD). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Staining, TUNEL Assay

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , Schematic for intervention study where mice were fed an HFD for 8 weeks and then switched to TAM/HFD for an additional 8 weeks before being killed. b , En face aorta analysis of plaque area after 16 weeks of diet-switch intervention study. Representative image from two independent experiments. c , Quantification of plaque area in aorta and aortic sinus ( n = 8 mice per group for Cntl and n = 9 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. d , Blood immune population analysis after 16-week diet-switch intervention model ( n = 5 mice per group). Data are mean ± s.e.m. e , Total serum cholesterol levels after 16-week diet-switch model ( n = 4 mice per group for Cntl and n = 5 for Trem2 ΔMФ ). Data are mean ± s.e.m. f , Quantification of plaque macrophage proliferation analysis by Ki67 + macrophages (CD68 + ) ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. g , Quantification of TUNEL + macrophages (CD68 + ) in plaques after 16-week diet-switch model ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. ORO, Oil Red O.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: TUNEL Assay

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , WT and Trem2 −/− BV2s assessed for Trem2 expression by flow cytometry. WT ( b ) or Trem2 −/− ( c ) BV2 macrophages DEGs from bulk RNA-seq determined by Wald test with DESeq2. d , GSEA plot of cholesterol biosynthesis pathways. ES, enrichment score. e , Pathway analysis of RNA-seq data comparing WT and Trem2 −/− non-foamy BV2 cells. f , Pathway analysis of RNA-seq data comparing WT and Trem2 −/− foamy BV2 cells. e , f , Significant pathways determined using weighted Kolmogorov–Smirnov test. g , WT or Trem2 −/− cell supernatant assessed for cytotoxicity by LDH assay after 16 h ( n = 6 biological replicates per group). Foamy: 20 μg ml −1 cholesterol; foamy hi : 80 μg ml −1 cholesterol. Data are mean ± s.e.m. Two-tailed ANOVA, *** P < 0.001. h , DiI-oxLDL uptake for WT or Trem2 −/− non-foamy and foamy BV2 macrophages ( n = 6 for non-foamy WT and Trem2 −/− and n = 4 foamy WT and Trem2 −/− biological replicates). Data are mean ± s.e.m. Student’s t -test, *** P < 0.001. i , WT or Trem2 −/− non-foamy and foamy BV2 macrophage efferocytosis. Efferocytotic cells were determined by the percent of BV2s that were positive for CTV-labeled splenocytes ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01 and **** P < 0.0001. j , WT or Trem2 −/− non-foamy and foamy BV2 macrophage sXBP1 expression. Tunicamycin was used as a positive control ( n = 6 biological replicates per group). FMO (fluorescence minus one) shows unstained control. Data are mean ± s.e.m. Two-tailed ANOVA, ** P < 0.01. k , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) plus 10 μM PBA. Cell supernatant was assessed for cytotoxicity by LDH assay after 16 h ( n = 6 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. l , GSEA plot of cholesterol efflux pathways from RNA-seq. m , GSEA plot of NR1H2 and NR1H3 gene target pathways from RNA-seq. n , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) ± T0901317 percent cytotoxicity ( n = 5 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. o , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) ± 10 μM T0901317, assessed for sXBP1 levels by flow cytometry. Tunicamycin was used as a positive control ( n = 5 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. KO, knockout; NS, not significant.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, RNA Sequencing Assay, Lactate Dehydrogenase Assay, Two Tailed Test, Labeling, Positive Control, Fluorescence, Knock-Out

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) Heat map of nonfoamy macrophages comparing top enriched WT and Trem2−/− genes. b ) Heat map of foamy macrophages comparing top enriched WT and Trem2−/− genes. c ) Normalized enrichment scores for top pathways associated with WT BV2 compared to Trem2−/− BV2 in media alone (nonfoamy) or following foamy differentiation. Significant pathways were determined using Weighted-Kolmogorov-Smirnov (WKS) test. d ) Heat map of nonfoamy and foamy macrophages comparing matrix metalloprotease genes from WT and Trem2−/− BV2. e ) Cytokine supernatant analysis from cultured WT or Trem2−/− cells cultured with either media alone or media with 20 mg/ml soluble cholesterol overnight (n = 3 biological replicates/group). Data are mean ±S.E.M.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Cell Culture

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) WT or Trem2−/− peritoneal macrophages were differentiated in media control, media with 20 μg/mL or 80 μg/mL soluble cholesterol to induce foamy macrophage formation. Cell supernatant was assessed for cytotoxicity by LDH assay after 16 hours (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = * < 0.05. b ) WT or Trem2−/− peritoneal macrophages were differentiated in media control or media with 20 μg/mL soluble cholesterol, then cultured with irradiated, cell trace violet (CTV) labeled splenocytes for 2 hours. Percentage of efferocytotic cells were determined by the % of peritoneal macrophages that were positive for CTV labeled splenocytes (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = *** < 0.001, ****<0.0001. c ) WT or Trem2−/− peritoneal macrophages were differentiated in media control or media with 20 μg/mL soluble cholesterol, then assessed for activation of ER stress response by sXBP1 levels by flow cytometry. Tunicamycin was used as a positive control (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = *** < 0.001.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Lactate Dehydrogenase Assay, Two Tailed Test, Cell Culture, Irradiation, Labeling, Activation Assay, Flow Cytometry, Positive Control

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) Clustering of all cell subsets from human atherosclerotic endarterectomy samples (Fernandez et. al., Nat Med 2019). b ) Clustering of monocyte/macrophage populations from human atherosclerotic endarterectomy samples (Fernandez et. al., Nat Med 2019). c ) PTPRC and CD14 expression of clustered monocyte/macrophage populations from 3b. d ) Foamy macrophage gene (FABP5, LGALS3) expression of clustered monocyte/macrophage populations from 3b. e ) Inflammatory macrophage gene (IL1B, NLRP3) expression of clustered monocyte/macrophage populations from 3b. f ) TREM2 expression of clustered monocyte/macrophage populations from 3b. g ) Volcano plot of enrichment of genes from samples from either asymptomatic or symptomatic patients. TREM2 in red. DEGs were determined by Wald test with DESeq2.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Expressing

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , Schematic for CRISPR knockout screening approach for oxLDL uptake. BV2 macrophages were loaded with CRISPR pooled guide library (Gouda). Cells were made foamy by overnight treatment with soluble cholesterol and then challenged for 4 h with DiI-oxLDL and sorted for DiI high and DiI low cells. Guides were sequenced from sorted populations. b , Confocal micrograph showing BV2 DiI uptake after 4-h incubation with DiI-oxLDL. Representative of two independent experiments. c , CRISPR guide enrichment comparing log-normalized enrichment in DiI high ( x axis) versus DiI low ( y axis). Gray error bands delineate guides with log fold change < 1. d , Selected gene enrichments comparing DiI low versus DiI high . e , Peritoneal macrophages were isolated from WT or Trem2 −/− mice and treated with soluble cholesterol to induce foamy cell formation. After overnight culture, cells were analyzed for Trem2 expression by flow cytometry ( n = 5 biologically independent replicates per group). Data are mean ± s.e.m. Student’s t -test, **** P < 0.0001. f , Bodipy staining for total neutral lipid accumulation was performed by flow cytometry on peritoneal macrophages from WT or Trem2 −/− mice, cultured overnight in media alone or in media with soluble cholesterol ( n = 6 biologically independent replicates for untreated and n = 4 biologically independent replicates for foamy). Data are mean ± s.e.m. Student’s t -test. g , Peritoneal macrophages were isolated from WT or Trem2 −/− mice and treated with soluble cholesterol to induce foamy cell formation. After overnight culture, cells were treated with DiI-oxLDL for 4 h and assessed for uptake by flow cytometry ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. h , CD36 expression from peritoneal macrophages isolated from WT or Trem2 −/− mice and treated with soluble cholesterol overnight to induce foamy cell formation ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. NS, not significant.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: CRISPR, Knock-Out, Incubation, Isolation, Expressing, Flow Cytometry, Staining, Cell Culture

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) Time course analysis of DiI-oxLDL uptake in WT BV2 cells differentiated in media with 20 μg/mL of soluble cholesterol overnight prior to addition of DiI-oxLDL (n = 5 biological replicates/group). Data are mean ± S.E.M. b ) CRISPR guide enrichment by rank-order was plotted against P-value for DiI-oxLDL-low compared against DiI-oxLDL-high to identify top enriched guides. Trem2 in red. P-values calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. c ) Two sided P-value vs count and p value vs false discovery rate (FDR) for DiI-oxLDL-low compared against DiI-oxLDL-high to identify top enriched guides. p-values and FDR calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. d ) Top 15 ‘importance index’ genes associated with foamy cell commitment by Trade-seq analysis , were compared for gene rank and enrichment in CRISPR screen. Trem2 highlighted in gray. P-values calculated using the negative-binomial model from MAGeCK package and adjusted using Benjamini-Hochberg procedure. e) CD36 expression (left) and percent CD36 high (right) of foamy peritoneal macrophages cultured with soluble cholesterol overnight from WT (blue) and Trem2−/− mice (red). Gated on F4/80+ CD11b+ live cells (n = 5 biological replicates/group). Data are mean ± S.E.M. Student’s t-test, P = ** < 0.01. f ) SR-AI expression (left), MFI (middle) and percent SR-AI positive (right) of foamy peritoneal macrophages cultured with soluble cholesterol overnight from WT (blue) and Trem2−/− mice (red). Gated on F4/80+ CD11b+ live cells (n = 5 biological replicates/group). Data are mean ± S.E.M. Student’s t-test

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: CRISPR, Expressing, Cell Culture

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) Cranial artery plaques were stained either for CD68 and Trem2 to identify co−expressing foamy macrophages within human plaques (top) or with CD68 and isotype control with secondary antibody (bottom). Representative image from 3 independent samples. b ) Carotid artery endarterectomy samples from three patients stained for isotype control or Trem2 using DAB (3,3′-Diaminobenzidine) immunohistochemical staining. Representative images from 6 independent samples.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Staining, Expressing, Immunohistochemical staining

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , Schematic for mixed bone marrow chimera experiment. Ldlr −/− mice were lethally irradiated and rescued by donor bone marrow from (50%) LysM cre R26 tdTomato (WT) and (50%) Trem2 −/− mice. Recipient mice were rested for 8 weeks and then fed an HFD for an additional 8 weeks to induce atherosclerosis. b , Flow cytometry gating of blood immune cells (CD45 + ) after 8-week HFD feeding, showing ratio of monocytes derived from WT (tdTomato + ) and Trem2 −/− progenitors. c , Confocal micrograph of whole-mount aorta showing tdTomato labeling (red) and CD45 (white) staining to define cellular contributions to foamy macrophages. Representative image from two independent experiments. d , Quantification of tdTomato + cells in blood compared to foamy macrophages from whole-mount aorta images ( n = 3 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. e , Foamy FACS was performed on CD64 + CD11b + macrophages isolated from mixed bone marrow chimera aorta. Macrophages were separated into tdTomato + and tdTomato − populations and then assessed for foamy representation by SSC and Bodipy (neutral lipid) staining. f , Flow cytometric overlap between tdTomato + (red) and Trem2 −/− (blue) derived macrophages from digested atherosclerotic aorta. g , Quantification derived from flow cytometric foamy FACS comparing relative contribution to foamy macrophages ( n = 4 mice per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. KO, knockout. MACS, macrophage.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Irradiation, Flow Cytometry, Derivative Assay, Labeling, Staining, Isolation, Knock-Out

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , CX3CR1 creER Trem2 flox/flox Ldlr −/− (Trem2 ΔMФ ) or littermate control mice (which included Cre − animals CX3CR1 +/+ Trem2 fl/fl Ldlr −/− and Cre + animals CX3CR1 creER/+ Trem2 fl/+ Ldlr −/− ) were fed TAM-HFD for 8 weeks ( b – e ) or 16 weeks ( f – i ). b , After 8 weeks of TAM-HFD, aortas were analyzed by en face analysis for percentage Oil Red O (ORO) staining on the arch ( n = 17 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. c , Aortic sinus plaque area measured after ORO staining in 8-week TAM-HFD samples (n = 17 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. d , Serum cholesterol levels from 8-week TAM-HFD-fed mice ( n = 11 mice per group for Cntl and n = 8 for Trem2 ΔMФ ). Data are mean ± s.e.m. e , Weight data from 8-week TAM-HFD-fed mice ( n = 9 mice pergroup). Data are mean ± s.e.m. f , En face ORO staining of aorta after 16-week TAM-HFD feeding ( n = 12 mice per group). Data are mean ± s.e.m. Student’s t -test, **** P < 0.0001. g , Aortic sinus plaque area after 16-week TAM-HFD feeding ( n = 11 mice per group for Cntl and n = 12 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, *** P < 0.001. h , Serum cholesterol after 16-week TAM-HFD feeding ( n = 10 mice per group). Data are mean ± s.e.m. i , Weight of mice after 16-week TAM-HFD feeding ( n = 10 mice per group for Cntl and n = 7 for Trem2 ΔMФ ). Data are mean ± s.e.m.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Staining

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) Trem2 expression and quantification from atherosclerotic aortae (n = 2 mice/group for Cntl and n = 3 for Trem2ΔMФ). Briefly, Cntl or Trem2ΔMФ mice were fed TAM-HFD for 16 weeks then aorta were harvested, digested and flow cytometry was run. Histogram was gated on live, CD45 + CD11b + CD64+ cells. b ) Flow cytometric gating strategy for identifying major blood immune cell populations. c ) Blood immune cell profiling by flow cytometry in indicated mice after 16 weeks TAM-HFD feeding (n = 12 mice/group for Cntl and n = 6 for Trem2ΔMФ). Data are mean ± S.E.M. Student’s t-test. d ) Classical monocyte bead uptake in the blood was measured by flow cytometry 24 hours after i.v. bead injection in indicated strains after 16 weeks TAM-HFD feeding (n = 7 mice/group for Cntl and n = 5 for Trem2ΔMФ). Data are mean ± S.E.M. Student’s t-test.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, Injection

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , Following the schematic in , CX3CR1 creER Trem2 flox/flox Ldlr −/− (Trem2 ΔMФ ) or littermate control mice were treated continuously with TAM-HFD for the indicated times. b , Serum from 8-week TAM-HFD-fed mice were assessed for cytokine levels by multiplex assay ( n = 10 mice per group). Data are mean ± s.e.m. c , Serum from 16-week TAM-HFD-fed mice were assessed for cytokine levels by multiplex assay ( n = 9 mice per group). Data are mean ± s.e.m. d , Blood immune cells were assessed after 16 weeks of TAM-HFD by flow cytometry ( n = 12 mice per group for Cntl and n = 6 for Trem2 ΔMФ ). Data are mean ± s.e.m. e , Monocyte recruitment was assessed by bead labeling and recruitment experiment—images from representative histologic and immunofluorescence images with lipid content (red) and beads (green). Representative image from two independent experiments. f , Quantification of plaque-associated beads that were counted per section for 8-week or 16-week TAM-HFD experiments from experiments in ( n = 7 mice per group for Cntl and n = 5 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test. NS, not significant; ORO, Oil Red O.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Multiplex Assay, Flow Cytometry, Labeling, Immunofluorescence

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , Confocal micrograph showing CD68 staining (green) and DAPI (blue) for macrophage area in Cntl or Trem2-deficent mice after 16-week TAM-HFD feeding. Representative image from two independent experiments. b , Quantification of CD68 + macrophage area per section in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01. c , Quantification of the percentage of plaque that is macrophages (CD68 + ) in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test. d , Confocal micrograph showing Ki67 staining (magenta) and CD68 staining (green) for proliferation in Cntl or Trem2-deficient mice after 16-week TAM-HFD feeding. Representative image from two independent experiments. e , Quantification of Ki67 + macrophages (CD68 + ) per section in 8-week or 16-week TAM-HFD samples ( n = 5 mice per group for 8-week TAM-HFD, n = 7 mice per group for Cntl 16-week TAM-HFD and n = 6 mice per group for 16-week TAM-HFD Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01. f , Confocal micrograph of TUNEL staining (magenta) and CD68 staining (green) for detection of dying cells within atherosclerotic lesions after 16-week TAM-HFD feeding. Representative image from two independent experiments. g , Quantification of TUNEL + macrophages (CD68 + ) per section in 8-week or 16-week TAM-HFD samples ( n = 6 mice per group for Cntl 8-week TAM-HFD, n = 7 mice per group for Trem2 ΔMФ 8-week TAM-HFD, n = 7 mice per group for Cntl 16-week TAM-HFD and n = 7 mice per group for Trem2 ΔMФ 16-week TAM-HFD). Data are mean ± s.e.m. Student’s t -test, * P < 0.05 and ** P < 0.01.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Staining, TUNEL Assay

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , Schematic for intervention study where mice were fed an HFD for 8 weeks and then switched to TAM/HFD for an additional 8 weeks before being killed. b , En face aorta analysis of plaque area after 16 weeks of diet-switch intervention study. Representative image from two independent experiments. c , Quantification of plaque area in aorta and aortic sinus ( n = 8 mice per group for Cntl and n = 9 for Trem2 ΔMФ ). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01. d , Blood immune population analysis after 16-week diet-switch intervention model ( n = 5 mice per group). Data are mean ± s.e.m. e , Total serum cholesterol levels after 16-week diet-switch model ( n = 4 mice per group for Cntl and n = 5 for Trem2 ΔMФ ). Data are mean ± s.e.m. f , Quantification of plaque macrophage proliferation analysis by Ki67 + macrophages (CD68 + ) ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. g , Quantification of TUNEL + macrophages (CD68 + ) in plaques after 16-week diet-switch model ( n = 5 mice per group). Data are mean ± s.e.m. Student’s t -test, * P < 0.05. ORO, Oil Red O.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: TUNEL Assay

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a , WT and Trem2 −/− BV2s assessed for Trem2 expression by flow cytometry. WT ( b ) or Trem2 −/− ( c ) BV2 macrophages DEGs from bulk RNA-seq determined by Wald test with DESeq2. d , GSEA plot of cholesterol biosynthesis pathways. ES, enrichment score. e , Pathway analysis of RNA-seq data comparing WT and Trem2 −/− non-foamy BV2 cells. f , Pathway analysis of RNA-seq data comparing WT and Trem2 −/− foamy BV2 cells. e , f , Significant pathways determined using weighted Kolmogorov–Smirnov test. g , WT or Trem2 −/− cell supernatant assessed for cytotoxicity by LDH assay after 16 h ( n = 6 biological replicates per group). Foamy: 20 μg ml −1 cholesterol; foamy hi : 80 μg ml −1 cholesterol. Data are mean ± s.e.m. Two-tailed ANOVA, *** P < 0.001. h , DiI-oxLDL uptake for WT or Trem2 −/− non-foamy and foamy BV2 macrophages ( n = 6 for non-foamy WT and Trem2 −/− and n = 4 foamy WT and Trem2 −/− biological replicates). Data are mean ± s.e.m. Student’s t -test, *** P < 0.001. i , WT or Trem2 −/− non-foamy and foamy BV2 macrophage efferocytosis. Efferocytotic cells were determined by the percent of BV2s that were positive for CTV-labeled splenocytes ( n = 5 biological replicates per group). Data are mean ± s.e.m. Student’s t -test, ** P < 0.01 and **** P < 0.0001. j , WT or Trem2 −/− non-foamy and foamy BV2 macrophage sXBP1 expression. Tunicamycin was used as a positive control ( n = 6 biological replicates per group). FMO (fluorescence minus one) shows unstained control. Data are mean ± s.e.m. Two-tailed ANOVA, ** P < 0.01. k , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) plus 10 μM PBA. Cell supernatant was assessed for cytotoxicity by LDH assay after 16 h ( n = 6 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. l , GSEA plot of cholesterol efflux pathways from RNA-seq. m , GSEA plot of NR1H2 and NR1H3 gene target pathways from RNA-seq. n , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) ± T0901317 percent cytotoxicity ( n = 5 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. o , WT or Trem2 −/− foamy BV2 macrophages (80 μg ml −1 cholesterol) ± 10 μM T0901317, assessed for sXBP1 levels by flow cytometry. Tunicamycin was used as a positive control ( n = 5 biological replicates per group). Data are mean ± s.e.m. Two-tailed ANOVA, **** P < 0.0001. KO, knockout; NS, not significant.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, RNA Sequencing Assay, Lactate Dehydrogenase Assay, Two Tailed Test, Labeling, Positive Control, Fluorescence, Knock-Out

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) Heat map of nonfoamy macrophages comparing top enriched WT and Trem2−/− genes. b ) Heat map of foamy macrophages comparing top enriched WT and Trem2−/− genes. c ) Normalized enrichment scores for top pathways associated with WT BV2 compared to Trem2−/− BV2 in media alone (nonfoamy) or following foamy differentiation. Significant pathways were determined using Weighted-Kolmogorov-Smirnov (WKS) test. d ) Heat map of nonfoamy and foamy macrophages comparing matrix metalloprotease genes from WT and Trem2−/− BV2. e ) Cytokine supernatant analysis from cultured WT or Trem2−/− cells cultured with either media alone or media with 20 mg/ml soluble cholesterol overnight (n = 3 biological replicates/group). Data are mean ±S.E.M.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Cell Culture

Journal: Nature cardiovascular research

Article Title: Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis

doi: 10.1038/s44161-023-00354-3

Figure Lengend Snippet: a ) WT or Trem2−/− peritoneal macrophages were differentiated in media control, media with 20 μg/mL or 80 μg/mL soluble cholesterol to induce foamy macrophage formation. Cell supernatant was assessed for cytotoxicity by LDH assay after 16 hours (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = * < 0.05. b ) WT or Trem2−/− peritoneal macrophages were differentiated in media control or media with 20 μg/mL soluble cholesterol, then cultured with irradiated, cell trace violet (CTV) labeled splenocytes for 2 hours. Percentage of efferocytotic cells were determined by the % of peritoneal macrophages that were positive for CTV labeled splenocytes (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = *** < 0.001, ****<0.0001. c ) WT or Trem2−/− peritoneal macrophages were differentiated in media control or media with 20 μg/mL soluble cholesterol, then assessed for activation of ER stress response by sXBP1 levels by flow cytometry. Tunicamycin was used as a positive control (n = 5 biological replicates/group). Data are mean ± S.E.M. Two-tailed ANOVA, P = *** < 0.001.

Article Snippet: The following antibodies were used: Trem2 APC rat anti-mouse (clone 237920, R&D Systems); Trem2 FITC rat anti-mouse (clone 78.18, eBioscience); CD68 rat anti-mouse (clone FA-11, BioLegend); Ki67 rabbit anti-mouse (clone SP6, Abcam); CD45 BV480 rat anti-mouse (clone 30-F11, BioLegend); CD11b BV605 rat anti-mouse (clone M1/70, BioLegend); Ly6G BV785 rat anti-mouse (clone 1A8, BioLegend); Ly6C BV421 rat anti-mouse (clone HK1.4, BioLegend); CD115 PerCPCy5.5 rat anti-mouse (clone AFS98, BioLegend); TCRβ APC hamster anti-mouse (clone H57–597, BioLegend); CD19 FITC rat anti-mouse (clone 1D3, BioLegend); and sXBP1 AF647 rat anti-mouse (clone E9V3E, Cell Signaling Technology).

Techniques: Lactate Dehydrogenase Assay, Two Tailed Test, Cell Culture, Irradiation, Labeling, Activation Assay, Flow Cytometry, Positive Control

Journal: Neural Regeneration Research

Article Title: A novel phenotype of B cells associated with enhanced phagocytic capability and chemotactic function after ischemic stroke

doi: 10.4103/1673-5374.371365

Figure Lengend Snippet: Subsets of infiltrating B cells in ischemic stroke model mice revealed by scRNA-seq. (A) t-SNE plot of all B cells from ischemic mouse brains at 5 and 14 days after tMCAO; each color represents one subcluster. (B) Violin plots of several marker genes for the three B cell subclusters identified in this profile. (C) Heatmap visualization of the expression level of the top 10 marker genes in each B cell subcluster. (D) Box plot showing the expression level of 10 macrophage markers and 4 B-cell markers in the C2 subcluster compared with all other B cell subclusters. ****Bonferroni adjusted P -value < 0.0001, C2 vs . others. (E) Dot plot displaying the representative macrophage marker genes of the three B cell subclusters. (F) Representative gating strategy for lymphocytes/single cells/CD45 high /CD19 + B cells (green) in the ischemic mouse brain. MLBs (purple) were identified by the high expression levels of C1Q, TREM2, APOE, CSF1R, and CX3CR1. (G) Representative mIHC images of mouse brain sections at 14 days after tMCAO, showing coronal topography of infarct (dashed line). The sample was stained for CD19 (green, Opal 520), C1Q (orange, Opal620), TREM2 (cyan, Opal 480), APOE (red, Opal 690), CSF1R (white, Opal 780), and CX3CR1 (yellow, Opal 570). Nuclei were labeled with DAPI (blue). White arrows, CD19 + C1Q + TREM2 + APOE + CSF1R + CX3CR1 + ; white triangle, expressing CD19 and at least another macrophage-associated marker; white asterisk, only CD19 + . Scale bars: 1000 µm (overview) and 20 µm (inset). APOE: Apolipoprotein E; C0–2: clusters 0–2; C1Q: complement component 1q; CSF1R: colony-stimulating factor 1 receptor; CX3CR1: C-X3-C motif chemokine receptor 1; DAPI: 4′,6-diamidino-2-phenylindole; mIHC: multiplex immunohistochemistry; MLBs: macrophage-like B cells; tMCAO: transient middle cerebral artery occlusion; scRNA-seq: single-cell RNA sequencing; t-SNE: t-distributed stochastic neighbor embedding; TREM2: triggering receptor expressed on myeloid cells-2.

Article Snippet: After blocking, the sections were incubated for 1 hour at room temperature (25–30°C) with the following primary antibodies: rabbit anti-CD19 (1:30, Abcam, Cambridge, UK, Cat# ab245235, RRID: AB_2895109), rabbit anti-C1Q (1:100, Abcam, Cat# ab182451, RRID: AB_2732849), rat anti-TREM2 (1:50, Abcam, Cat# ab86491; RRID: AB_1925525), rabbit anti-APOE (1:100, Abcam, Cat# ab183596, RRID: AB_2832971), rabbit anti-CSF1R (1:100, Abcam, Cat# ab254357, RRID: AB_2927559), rabbit anti-CX3CR1 (1:100, Abcam, Cat# ab8020, RRID: AB_306202), rat anti-myelin basic protein (MBP; 1:400, Abcam, Cat# ab7349, RRID: AB_305869), and rabbit anti-CC motif chemokine ligand 3 (CCL3; 1:200, Abcam, Cat# ab179638, RRID: AB_2927558).

Techniques: Marker, Expressing, Staining, Labeling, Multiplex Assay, Immunohistochemistry, RNA Sequencing Assay

Journal: Neural Regeneration Research

Article Title: A novel phenotype of B cells associated with enhanced phagocytic capability and chemotactic function after ischemic stroke

doi: 10.4103/1673-5374.371365

Figure Lengend Snippet: Transcriptomic and functional changes in MLBs involving immune responses and phagocytic activity. (A) Volcano plot showing 378 upregulated (purple) and 9 downregulated (red) DEGs (|log2(fold change)| > 0.1 and Bonferroni adjusted P -value < 0.01) of MLBs compared with other B cells. (B) GO enrichment analysis of DEGs identified key BP, CC, and MF in MLBs compared with other B cells; results are displayed in bubble plots in accordance with Z-score and significance (-log10[adjusted P -value]), and the sizes of the bubbles reflect the number of genes under each term. (C) Bar plot showing the major upregulated GO terms of BP. (D) Dot plot showing the score comparison of typical functions between MLBs and other B cells, including inflammatory capacity, chemokine production, migration capacity (trafficking score), and phagocytic capacity. (E) Heatmap showing the relationship between the GO terms associated with phagocytic functions and the most involved genes. The top blue bars show the log-transformed fold change of the involved genes, and the bars on the right show the Z-score of the involved GO terms. (F) Violin plots showing the expression level of phagocytosis/endocytosis-related genes ( Fcer1g , Fcgr1 , and Trem2 ) and lysosome-related genes ( Cd63 , Cd68 , and Ifitm3 ) from (E). (G) Positively enriched gene sets identified by GSEA. The NES, nominal P -value, and adjusted P-value of each gene set are shown. (H) Pathview analysis of the phagosome (mmu04145) signaling pathway from the KEGG database. (I) Representative mIHC images of brain sections at 14 days after tMCAO. Sections were stained for CD19 (white, Opal 780), TREM2 (green, Opal 520), CSF1R (cyan, Opal 480), C1Q (orange, Opal 620), CX3CR1 (yellow, Opal 570), and MBP (red, Opal 690). Nuclei were labeled with DAPI (blue). (J) Representative 3D-rendering images by Imaris software of brain sections stained with CD19 (gray, Alexa Fluor 647), TREM2 (green, Alexa Fluor 488), and MBP (red, Alexa Fluor 594). Nuclei were labeled with DAPI (blue). (J1) Representative image showing the phagocytosis of MBP (red) by CD19 + B cells (gray). (J2) Representative image showing the contact between MBP (red) and TREM2 (green) staining. Scale bars: 5 µm. BP: Biological processes; C0-2: clusters 0-2; C1Q: complement component 1q; CC: cellular components; CSF1R: colony-stimulating factor 1 receptor; CX3CR1: C-X3-C motif chemokine receptor 1; DAPI: 4′,6-diamidino-2-phenylindole; DEGs: differentially expressed genes; GO: Gene Ontology; GSEA: gene set enrichment analysis; KEGG: Kyoto encyclopedia of genes and genomes; MBP: myelin basic protein; MF: molecular functions; mIHC: multiplex immunohistochemistry; MLBs: macrophage-like B cells; NES: normalized enrichment scores; tMCAO: transient middle cerebral artery occlusion; TREM2: triggering receptor expressed on myeloid cells-2.

Article Snippet: After blocking, the sections were incubated for 1 hour at room temperature (25–30°C) with the following primary antibodies: rabbit anti-CD19 (1:30, Abcam, Cambridge, UK, Cat# ab245235, RRID: AB_2895109), rabbit anti-C1Q (1:100, Abcam, Cat# ab182451, RRID: AB_2732849), rat anti-TREM2 (1:50, Abcam, Cat# ab86491; RRID: AB_1925525), rabbit anti-APOE (1:100, Abcam, Cat# ab183596, RRID: AB_2832971), rabbit anti-CSF1R (1:100, Abcam, Cat# ab254357, RRID: AB_2927559), rabbit anti-CX3CR1 (1:100, Abcam, Cat# ab8020, RRID: AB_306202), rat anti-myelin basic protein (MBP; 1:400, Abcam, Cat# ab7349, RRID: AB_305869), and rabbit anti-CC motif chemokine ligand 3 (CCL3; 1:200, Abcam, Cat# ab179638, RRID: AB_2927558).

Techniques: Functional Assay, Activity Assay, Migration, Transformation Assay, Expressing, Staining, Labeling, Software, Multiplex Assay, Immunohistochemistry

Journal: Neural Regeneration Research

Article Title: A novel phenotype of B cells associated with enhanced phagocytic capability and chemotactic function after ischemic stroke

doi: 10.4103/1673-5374.371365

Figure Lengend Snippet: MLBs are a key regulator in the recruitment of immune cells by releasing chemokines. (A) Heatmap showing the relationship between GO terms and chemokine and cytokine genes. The top blue bars show the log-transformed fold change of the involved genes, and the bars on the right show the Z-score of the involved GO terms. (B) Bar plot showing the GO terms involved in the recruitment of peripheral immune cells. (C) Circle plots displaying the inferred CCL signaling networks between B cells and other immune cells from ischemic brains. Edge width represents the communication probability. (D) Summary of selected ligand-receptor interactions between different cell clusters in the CCL signaling pathway from MLBs to other immune cells. The size of each circle reflects the P -values. The color gradient indicates the level of interaction. (E) Violin plot showing the expression patterns of genes involved in the CCL signaling pathway. (F) Circle plots displaying the inferred CXCL signaling networks between B cells and other immune cells from ischemic brains. (G) Summary of selected ligand-receptor interactions between different cell clusters in the CXCL signaling pathway from MLBs to other immune cells. (H) Violin plot showing the expression pattern of genes involved in the CXCL signaling pathway. (I) Representative mIHC images of brain sections at 14 days after tMCAO. The sample was stained for CD19 (green, Opal 520), TREM2 (red, Opal 690), and CCL3 (yellow, Opal 570). Nuclei were labeled with DAPI (blue). White arrows, CD19 + TREM2 + CCL3 + ; white asterisk, CD19 + TREM2 + . Scale bar: 20 µm. C0–2: Clusters 0–2; CCL3: CC motif chemokine ligand 3; DAPI: 4′,6-diamidino-2-phenylindole; GO: Gene Ontology; mIHC: multiplex immunohistochemistry; MLBs: macrophage-like B cells; tMCAO: transient middle cerebral artery occlusion; TREM2: triggering receptor expressed on myeloid cells-2.

Article Snippet: After blocking, the sections were incubated for 1 hour at room temperature (25–30°C) with the following primary antibodies: rabbit anti-CD19 (1:30, Abcam, Cambridge, UK, Cat# ab245235, RRID: AB_2895109), rabbit anti-C1Q (1:100, Abcam, Cat# ab182451, RRID: AB_2732849), rat anti-TREM2 (1:50, Abcam, Cat# ab86491; RRID: AB_1925525), rabbit anti-APOE (1:100, Abcam, Cat# ab183596, RRID: AB_2832971), rabbit anti-CSF1R (1:100, Abcam, Cat# ab254357, RRID: AB_2927559), rabbit anti-CX3CR1 (1:100, Abcam, Cat# ab8020, RRID: AB_306202), rat anti-myelin basic protein (MBP; 1:400, Abcam, Cat# ab7349, RRID: AB_305869), and rabbit anti-CC motif chemokine ligand 3 (CCL3; 1:200, Abcam, Cat# ab179638, RRID: AB_2927558).

Techniques: Transformation Assay, Expressing, Staining, Labeling, Multiplex Assay, Immunohistochemistry

Journal: Neural Regeneration Research

Article Title: A novel phenotype of B cells associated with enhanced phagocytic capability and chemotactic function after ischemic stroke

doi: 10.4103/1673-5374.371365

Figure Lengend Snippet: The dynamic functional plasticity and quantitative changes of MLBs at different stages of stroke. (A) Heatmap showing the comparison of the log-transformed fold change of DEGs at 5 and 14 days after tMCAO. DEGs were divided into groups (#1–3) on the basis of adjusted P values and fold changes. Group 1 included DEGs that were significantly upregulated in C2 at both 5 and 14 days after tMCAO; groups 2 and 3 included upregulated DEGs at either 5 or 14 days. (B) Metascape enrichment analysis for DEGs in groups 1, 2, and 3. The GO terms were grouped into several clusters on the basis of their membership similarity. (C, D) Bar plot showing activated (Z-score ≥ 2) GO terms of biological process in MLBs with different levels of activation state at 5 and 14 days after tMCAO. (E) Flow cytometry analysis of B cell infiltration. The percentages of CD19 + B cells among lymphocytes at 5 and 14 days after tMCAO are shown. (F) The percentages of CD45 high CD19 + C1Q + TREM2 + CSF1R + B cells among CD19 + B cells at 5 and 14 days after tMCAO are shown. n = 4–5 mice per group. ** P ≤ 0.01 (Student’s t -test). C1Q: Complement component 1q; CSF1R: colony-stimulating factor 1 receptor; DEGs: differentially expressed genes; GO: Gene Ontology; MLBs: macrophage-like B cells; tMCAO: transient middle cerebral artery occlusion; TREM2: triggering receptor expressed on myeloid cells-2.

Article Snippet: After blocking, the sections were incubated for 1 hour at room temperature (25–30°C) with the following primary antibodies: rabbit anti-CD19 (1:30, Abcam, Cambridge, UK, Cat# ab245235, RRID: AB_2895109), rabbit anti-C1Q (1:100, Abcam, Cat# ab182451, RRID: AB_2732849), rat anti-TREM2 (1:50, Abcam, Cat# ab86491; RRID: AB_1925525), rabbit anti-APOE (1:100, Abcam, Cat# ab183596, RRID: AB_2832971), rabbit anti-CSF1R (1:100, Abcam, Cat# ab254357, RRID: AB_2927559), rabbit anti-CX3CR1 (1:100, Abcam, Cat# ab8020, RRID: AB_306202), rat anti-myelin basic protein (MBP; 1:400, Abcam, Cat# ab7349, RRID: AB_305869), and rabbit anti-CC motif chemokine ligand 3 (CCL3; 1:200, Abcam, Cat# ab179638, RRID: AB_2927558).

Techniques: Functional Assay, Transformation Assay, Activation Assay, Flow Cytometry

Journal: Neural Regeneration Research

Article Title: A novel phenotype of B cells associated with enhanced phagocytic capability and chemotactic function after ischemic stroke

doi: 10.4103/1673-5374.371365

Figure Lengend Snippet: Subsets of infiltrating B cells in ischemic stroke model mice revealed by scRNA-seq. (A) t-SNE plot of all B cells from ischemic mouse brains at 5 and 14 days after tMCAO; each color represents one subcluster. (B) Violin plots of several marker genes for the three B cell subclusters identified in this profile. (C) Heatmap visualization of the expression level of the top 10 marker genes in each B cell subcluster. (D) Box plot showing the expression level of 10 macrophage markers and 4 B-cell markers in the C2 subcluster compared with all other B cell subclusters. ****Bonferroni adjusted P -value < 0.0001, C2 vs . others. (E) Dot plot displaying the representative macrophage marker genes of the three B cell subclusters. (F) Representative gating strategy for lymphocytes/single cells/CD45 high /CD19 + B cells (green) in the ischemic mouse brain. MLBs (purple) were identified by the high expression levels of C1Q, TREM2, APOE, CSF1R, and CX3CR1. (G) Representative mIHC images of mouse brain sections at 14 days after tMCAO, showing coronal topography of infarct (dashed line). The sample was stained for CD19 (green, Opal 520), C1Q (orange, Opal620), TREM2 (cyan, Opal 480), APOE (red, Opal 690), CSF1R (white, Opal 780), and CX3CR1 (yellow, Opal 570). Nuclei were labeled with DAPI (blue). White arrows, CD19 + C1Q + TREM2 + APOE + CSF1R + CX3CR1 + ; white triangle, expressing CD19 and at least another macrophage-associated marker; white asterisk, only CD19 + . Scale bars: 1000 µm (overview) and 20 µm (inset). APOE: Apolipoprotein E; C0–2: clusters 0–2; C1Q: complement component 1q; CSF1R: colony-stimulating factor 1 receptor; CX3CR1: C-X3-C motif chemokine receptor 1; DAPI: 4′,6-diamidino-2-phenylindole; mIHC: multiplex immunohistochemistry; MLBs: macrophage-like B cells; tMCAO: transient middle cerebral artery occlusion; scRNA-seq: single-cell RNA sequencing; t-SNE: t-distributed stochastic neighbor embedding; TREM2: triggering receptor expressed on myeloid cells-2.

Article Snippet: Single-cell suspensions were incubated with the following primary antibodies for 30 minutes on ice at 4°C in the dark: rat anti-CD45 (1:200, Pacific Blue [PB], Biolegend, San Diego, CA, USA, Cat# 103126, RRID: AB_493535), rat anti-CD19 (1:200, phycoerythrin [PE], Biolegend, Cat# 115508, RRID: AB_313643), rat anti-CD19 (1:200, Brilliant Violet 785 [BV785], Biolegend, Cat# 115543, RRID: AB_11218994), rabbit anti-C1Q (1:200, fluorescein isothiocyanate [FITC], Abcam, Cat# ab4223, RRID: AB_304387), rat anti-TREM2 (1:200, allophycocyanin [APC], R&D Systems, Cat# FAB17291A, RRID: AB_884527), mouse anti-APOE (1:200, PE, Biolegend, Cat# 803405, RRID: AB_2801140), rat anti-CSF1R (1:200, BV605, Biolegend; Cat# 135517, RRID: AB_2562760), mouse anti-CX3CR1 (1:200, PE/Cyanine7, Biolegend, Cat# 149016, RRID: AB_2565700), rat anti-CD5 (1:200, peridinin chlorophyll protein [PerCP]/Cyanine5.5, Biolegend, Cat# 100623, RRID: AB_2563432), rat anti-F4/80 (1:200, FITC, Biolegend, Cat# 123107, RRID: AB_893500), and rat anti-CD23 (1:200, PE/Cyanine7, Biolegend, Cat# 101613, RRID: AB_2103037).

Techniques: Marker, Expressing, Staining, Labeling, Multiplex Assay, Immunohistochemistry, RNA Sequencing Assay

Journal: Neural Regeneration Research

Article Title: A novel phenotype of B cells associated with enhanced phagocytic capability and chemotactic function after ischemic stroke

doi: 10.4103/1673-5374.371365

Figure Lengend Snippet: Transcriptomic and functional changes in MLBs involving immune responses and phagocytic activity. (A) Volcano plot showing 378 upregulated (purple) and 9 downregulated (red) DEGs (|log2(fold change)| > 0.1 and Bonferroni adjusted P -value < 0.01) of MLBs compared with other B cells. (B) GO enrichment analysis of DEGs identified key BP, CC, and MF in MLBs compared with other B cells; results are displayed in bubble plots in accordance with Z-score and significance (-log10[adjusted P -value]), and the sizes of the bubbles reflect the number of genes under each term. (C) Bar plot showing the major upregulated GO terms of BP. (D) Dot plot showing the score comparison of typical functions between MLBs and other B cells, including inflammatory capacity, chemokine production, migration capacity (trafficking score), and phagocytic capacity. (E) Heatmap showing the relationship between the GO terms associated with phagocytic functions and the most involved genes. The top blue bars show the log-transformed fold change of the involved genes, and the bars on the right show the Z-score of the involved GO terms. (F) Violin plots showing the expression level of phagocytosis/endocytosis-related genes ( Fcer1g , Fcgr1 , and Trem2 ) and lysosome-related genes ( Cd63 , Cd68 , and Ifitm3 ) from (E). (G) Positively enriched gene sets identified by GSEA. The NES, nominal P -value, and adjusted P-value of each gene set are shown. (H) Pathview analysis of the phagosome (mmu04145) signaling pathway from the KEGG database. (I) Representative mIHC images of brain sections at 14 days after tMCAO. Sections were stained for CD19 (white, Opal 780), TREM2 (green, Opal 520), CSF1R (cyan, Opal 480), C1Q (orange, Opal 620), CX3CR1 (yellow, Opal 570), and MBP (red, Opal 690). Nuclei were labeled with DAPI (blue). (J) Representative 3D-rendering images by Imaris software of brain sections stained with CD19 (gray, Alexa Fluor 647), TREM2 (green, Alexa Fluor 488), and MBP (red, Alexa Fluor 594). Nuclei were labeled with DAPI (blue). (J1) Representative image showing the phagocytosis of MBP (red) by CD19 + B cells (gray). (J2) Representative image showing the contact between MBP (red) and TREM2 (green) staining. Scale bars: 5 µm. BP: Biological processes; C0-2: clusters 0-2; C1Q: complement component 1q; CC: cellular components; CSF1R: colony-stimulating factor 1 receptor; CX3CR1: C-X3-C motif chemokine receptor 1; DAPI: 4′,6-diamidino-2-phenylindole; DEGs: differentially expressed genes; GO: Gene Ontology; GSEA: gene set enrichment analysis; KEGG: Kyoto encyclopedia of genes and genomes; MBP: myelin basic protein; MF: molecular functions; mIHC: multiplex immunohistochemistry; MLBs: macrophage-like B cells; NES: normalized enrichment scores; tMCAO: transient middle cerebral artery occlusion; TREM2: triggering receptor expressed on myeloid cells-2.

Article Snippet: Single-cell suspensions were incubated with the following primary antibodies for 30 minutes on ice at 4°C in the dark: rat anti-CD45 (1:200, Pacific Blue [PB], Biolegend, San Diego, CA, USA, Cat# 103126, RRID: AB_493535), rat anti-CD19 (1:200, phycoerythrin [PE], Biolegend, Cat# 115508, RRID: AB_313643), rat anti-CD19 (1:200, Brilliant Violet 785 [BV785], Biolegend, Cat# 115543, RRID: AB_11218994), rabbit anti-C1Q (1:200, fluorescein isothiocyanate [FITC], Abcam, Cat# ab4223, RRID: AB_304387), rat anti-TREM2 (1:200, allophycocyanin [APC], R&D Systems, Cat# FAB17291A, RRID: AB_884527), mouse anti-APOE (1:200, PE, Biolegend, Cat# 803405, RRID: AB_2801140), rat anti-CSF1R (1:200, BV605, Biolegend; Cat# 135517, RRID: AB_2562760), mouse anti-CX3CR1 (1:200, PE/Cyanine7, Biolegend, Cat# 149016, RRID: AB_2565700), rat anti-CD5 (1:200, peridinin chlorophyll protein [PerCP]/Cyanine5.5, Biolegend, Cat# 100623, RRID: AB_2563432), rat anti-F4/80 (1:200, FITC, Biolegend, Cat# 123107, RRID: AB_893500), and rat anti-CD23 (1:200, PE/Cyanine7, Biolegend, Cat# 101613, RRID: AB_2103037).

Techniques: Functional Assay, Activity Assay, Comparison, Migration, Transformation Assay, Expressing, Staining, Labeling, Software, Multiplex Assay, Immunohistochemistry

Journal: Neural Regeneration Research

Article Title: A novel phenotype of B cells associated with enhanced phagocytic capability and chemotactic function after ischemic stroke

doi: 10.4103/1673-5374.371365

Figure Lengend Snippet: MLBs are a key regulator in the recruitment of immune cells by releasing chemokines. (A) Heatmap showing the relationship between GO terms and chemokine and cytokine genes. The top blue bars show the log-transformed fold change of the involved genes, and the bars on the right show the Z-score of the involved GO terms. (B) Bar plot showing the GO terms involved in the recruitment of peripheral immune cells. (C) Circle plots displaying the inferred CCL signaling networks between B cells and other immune cells from ischemic brains. Edge width represents the communication probability. (D) Summary of selected ligand-receptor interactions between different cell clusters in the CCL signaling pathway from MLBs to other immune cells. The size of each circle reflects the P -values. The color gradient indicates the level of interaction. (E) Violin plot showing the expression patterns of genes involved in the CCL signaling pathway. (F) Circle plots displaying the inferred CXCL signaling networks between B cells and other immune cells from ischemic brains. (G) Summary of selected ligand-receptor interactions between different cell clusters in the CXCL signaling pathway from MLBs to other immune cells. (H) Violin plot showing the expression pattern of genes involved in the CXCL signaling pathway. (I) Representative mIHC images of brain sections at 14 days after tMCAO. The sample was stained for CD19 (green, Opal 520), TREM2 (red, Opal 690), and CCL3 (yellow, Opal 570). Nuclei were labeled with DAPI (blue). White arrows, CD19 + TREM2 + CCL3 + ; white asterisk, CD19 + TREM2 + . Scale bar: 20 µm. C0–2: Clusters 0–2; CCL3: CC motif chemokine ligand 3; DAPI: 4′,6-diamidino-2-phenylindole; GO: Gene Ontology; mIHC: multiplex immunohistochemistry; MLBs: macrophage-like B cells; tMCAO: transient middle cerebral artery occlusion; TREM2: triggering receptor expressed on myeloid cells-2.

Article Snippet: Single-cell suspensions were incubated with the following primary antibodies for 30 minutes on ice at 4°C in the dark: rat anti-CD45 (1:200, Pacific Blue [PB], Biolegend, San Diego, CA, USA, Cat# 103126, RRID: AB_493535), rat anti-CD19 (1:200, phycoerythrin [PE], Biolegend, Cat# 115508, RRID: AB_313643), rat anti-CD19 (1:200, Brilliant Violet 785 [BV785], Biolegend, Cat# 115543, RRID: AB_11218994), rabbit anti-C1Q (1:200, fluorescein isothiocyanate [FITC], Abcam, Cat# ab4223, RRID: AB_304387), rat anti-TREM2 (1:200, allophycocyanin [APC], R&D Systems, Cat# FAB17291A, RRID: AB_884527), mouse anti-APOE (1:200, PE, Biolegend, Cat# 803405, RRID: AB_2801140), rat anti-CSF1R (1:200, BV605, Biolegend; Cat# 135517, RRID: AB_2562760), mouse anti-CX3CR1 (1:200, PE/Cyanine7, Biolegend, Cat# 149016, RRID: AB_2565700), rat anti-CD5 (1:200, peridinin chlorophyll protein [PerCP]/Cyanine5.5, Biolegend, Cat# 100623, RRID: AB_2563432), rat anti-F4/80 (1:200, FITC, Biolegend, Cat# 123107, RRID: AB_893500), and rat anti-CD23 (1:200, PE/Cyanine7, Biolegend, Cat# 101613, RRID: AB_2103037).

Techniques: Transformation Assay, Expressing, Staining, Labeling, Multiplex Assay, Immunohistochemistry

Journal: Neural Regeneration Research

Article Title: A novel phenotype of B cells associated with enhanced phagocytic capability and chemotactic function after ischemic stroke

doi: 10.4103/1673-5374.371365

Figure Lengend Snippet: The dynamic functional plasticity and quantitative changes of MLBs at different stages of stroke. (A) Heatmap showing the comparison of the log-transformed fold change of DEGs at 5 and 14 days after tMCAO. DEGs were divided into groups (#1–3) on the basis of adjusted P values and fold changes. Group 1 included DEGs that were significantly upregulated in C2 at both 5 and 14 days after tMCAO; groups 2 and 3 included upregulated DEGs at either 5 or 14 days. (B) Metascape enrichment analysis for DEGs in groups 1, 2, and 3. The GO terms were grouped into several clusters on the basis of their membership similarity. (C, D) Bar plot showing activated (Z-score ≥ 2) GO terms of biological process in MLBs with different levels of activation state at 5 and 14 days after tMCAO. (E) Flow cytometry analysis of B cell infiltration. The percentages of CD19 + B cells among lymphocytes at 5 and 14 days after tMCAO are shown. (F) The percentages of CD45 high CD19 + C1Q + TREM2 + CSF1R + B cells among CD19 + B cells at 5 and 14 days after tMCAO are shown. n = 4–5 mice per group. ** P ≤ 0.01 (Student’s t -test). C1Q: Complement component 1q; CSF1R: colony-stimulating factor 1 receptor; DEGs: differentially expressed genes; GO: Gene Ontology; MLBs: macrophage-like B cells; tMCAO: transient middle cerebral artery occlusion; TREM2: triggering receptor expressed on myeloid cells-2.

Article Snippet: Single-cell suspensions were incubated with the following primary antibodies for 30 minutes on ice at 4°C in the dark: rat anti-CD45 (1:200, Pacific Blue [PB], Biolegend, San Diego, CA, USA, Cat# 103126, RRID: AB_493535), rat anti-CD19 (1:200, phycoerythrin [PE], Biolegend, Cat# 115508, RRID: AB_313643), rat anti-CD19 (1:200, Brilliant Violet 785 [BV785], Biolegend, Cat# 115543, RRID: AB_11218994), rabbit anti-C1Q (1:200, fluorescein isothiocyanate [FITC], Abcam, Cat# ab4223, RRID: AB_304387), rat anti-TREM2 (1:200, allophycocyanin [APC], R&D Systems, Cat# FAB17291A, RRID: AB_884527), mouse anti-APOE (1:200, PE, Biolegend, Cat# 803405, RRID: AB_2801140), rat anti-CSF1R (1:200, BV605, Biolegend; Cat# 135517, RRID: AB_2562760), mouse anti-CX3CR1 (1:200, PE/Cyanine7, Biolegend, Cat# 149016, RRID: AB_2565700), rat anti-CD5 (1:200, peridinin chlorophyll protein [PerCP]/Cyanine5.5, Biolegend, Cat# 100623, RRID: AB_2563432), rat anti-F4/80 (1:200, FITC, Biolegend, Cat# 123107, RRID: AB_893500), and rat anti-CD23 (1:200, PE/Cyanine7, Biolegend, Cat# 101613, RRID: AB_2103037).

Techniques: Functional Assay, Comparison, Transformation Assay, Activation Assay, Flow Cytometry