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R&D Systems anti trem2
Anti Trem2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews

anti trem2 - by Bioz Stars, 2026-04

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B. abortus infection upregulates TREM2 expression in M2. TREM2 mRNA and protein levels of B. abortus -infected BMDMs, BMDM-M1, or BMDM-M2 (MOI = 100) were measured by RT-PCR (A–C) and Western blotting (D) at the indicated time points, respectively. TREM2 gene expression was detected by RT-PCR with normalization to β-actin. RT-PCR data represents the result of three technical replicates (mean ± SD). (E) BMDM-M2 were left uninfected (NI) or infected with B. abortus (MOI = 100), heat-killed B. abortus (HK), or the ΔvirB2 mutant B. abortus (MOI = 100) for 24 h prior to harvesting for TREM2 protein levels assay by Western blotting. (F) BMDM-M2 were infected with B. abortus (MOI = 100) in the presence of DMSO or Baf A1(100 nM) for the indicated times. TREM2 expression in M2 were analyzed by western blotting. Immunoblots (D–F) were representative of three independent experiments. Detection of cellular β-actin was used as loading control. The intensity of the bands was quantified using the Bio-Rad densitometry. The 0 time point mean ratio of protein (TREM2): β-actin was defined as 100%. Quantitative data are depicted under the Western image. Western blotting and RT-PCR results were analyzed using a one-way ANOVA followed by Bonferroni correction. The uninfected (uni) cells were designed as control. P-values <0.05 were considered significant (*, P <0.05). NS indicates no significance.

Journal: Frontiers in Immunology

Article Title: Upregulation of TREM2 expression in M2 macrophages promotes Brucella abortus chronic infection

doi: 10.3389/fimmu.2024.1466520

Figure Lengend Snippet: B. abortus infection upregulates TREM2 expression in M2. TREM2 mRNA and protein levels of B. abortus -infected BMDMs, BMDM-M1, or BMDM-M2 (MOI = 100) were measured by RT-PCR (A–C) and Western blotting (D) at the indicated time points, respectively. TREM2 gene expression was detected by RT-PCR with normalization to β-actin. RT-PCR data represents the result of three technical replicates (mean ± SD). (E) BMDM-M2 were left uninfected (NI) or infected with B. abortus (MOI = 100), heat-killed B. abortus (HK), or the ΔvirB2 mutant B. abortus (MOI = 100) for 24 h prior to harvesting for TREM2 protein levels assay by Western blotting. (F) BMDM-M2 were infected with B. abortus (MOI = 100) in the presence of DMSO or Baf A1(100 nM) for the indicated times. TREM2 expression in M2 were analyzed by western blotting. Immunoblots (D–F) were representative of three independent experiments. Detection of cellular β-actin was used as loading control. The intensity of the bands was quantified using the Bio-Rad densitometry. The 0 time point mean ratio of protein (TREM2): β-actin was defined as 100%. Quantitative data are depicted under the Western image. Western blotting and RT-PCR results were analyzed using a one-way ANOVA followed by Bonferroni correction. The uninfected (uni) cells were designed as control. P-values <0.05 were considered significant (*, P <0.05). NS indicates no significance.

Article Snippet: Anti-TREM2 neutralizing antibody (FAB17291A), mouse rIFN- γ (Cat: NP_032363), and mouse rIL-4 (Cat: P07750) were purchased from R&D Systems (Shanghai, China).

Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mutagenesis, Control

TREM2 contributes to B. abortus phagocytosis in M2. (A) TREM2 protein levels of B. abortus -infected RAW-M2(MOI = 100) were measured by Western blotting at the indicated time points. (B) RAW-M2-NT, RAW-M2-ΔTREM2, RAW-M2-Vector, or RAW-M2-TREM2+ cells were assessed for TREM2 protein expression by Western blotting. B. abortus (expressing GFP) were opsonized with mouse serum (C) or unopsonized (D) for 20 min, and then these bacteria were used to infect Raw-M2 derivative cell lines for another 4 h Phagocytosis was analyzed by flow cytometry through qualification of GFP + cells (top panel). Representative images of phagocytosis of GFP-expressing Brucella in RAW-M2-NT and RAW-M2-ΔTREM2 (bottom panel) Scale bar = 50 µm (E) BMDM-M2 were pretreated with TREM2 antibody (1 μg/mL to 10 μg/mL) or isotype antibody treated for 20 min, and then infected with B. abortus (expressing GFP) for 4 h Cells were analyzed by flow cytometry to quantify levels of phagocytosis. Immunoblots (A, B) were representative of three independent experiments. Detection of cellular β-actin was used as loading control. The intensity of the bands was quantified using the Bio-Rad densitometry. The 0 time point (A) or RAW-M2-NT (B) mean ratio of protein (TREM2): β-actin was defined as 100%. Quantitative data are depicted under the Western image. Phagocytosis was expressed as means ± standard deviations from two independent experiments. Phagocytosis results were analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant (*, P <0.05). NS indicates no significance.

Journal: Frontiers in Immunology

Article Title: Upregulation of TREM2 expression in M2 macrophages promotes Brucella abortus chronic infection

doi: 10.3389/fimmu.2024.1466520

Figure Lengend Snippet: TREM2 contributes to B. abortus phagocytosis in M2. (A) TREM2 protein levels of B. abortus -infected RAW-M2(MOI = 100) were measured by Western blotting at the indicated time points. (B) RAW-M2-NT, RAW-M2-ΔTREM2, RAW-M2-Vector, or RAW-M2-TREM2+ cells were assessed for TREM2 protein expression by Western blotting. B. abortus (expressing GFP) were opsonized with mouse serum (C) or unopsonized (D) for 20 min, and then these bacteria were used to infect Raw-M2 derivative cell lines for another 4 h Phagocytosis was analyzed by flow cytometry through qualification of GFP + cells (top panel). Representative images of phagocytosis of GFP-expressing Brucella in RAW-M2-NT and RAW-M2-ΔTREM2 (bottom panel) Scale bar = 50 µm (E) BMDM-M2 were pretreated with TREM2 antibody (1 μg/mL to 10 μg/mL) or isotype antibody treated for 20 min, and then infected with B. abortus (expressing GFP) for 4 h Cells were analyzed by flow cytometry to quantify levels of phagocytosis. Immunoblots (A, B) were representative of three independent experiments. Detection of cellular β-actin was used as loading control. The intensity of the bands was quantified using the Bio-Rad densitometry. The 0 time point (A) or RAW-M2-NT (B) mean ratio of protein (TREM2): β-actin was defined as 100%. Quantitative data are depicted under the Western image. Phagocytosis was expressed as means ± standard deviations from two independent experiments. Phagocytosis results were analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant (*, P <0.05). NS indicates no significance.

Article Snippet: Anti-TREM2 neutralizing antibody (FAB17291A), mouse rIFN- γ (Cat: NP_032363), and mouse rIL-4 (Cat: P07750) were purchased from R&D Systems (Shanghai, China).

Techniques: Infection, Western Blot, Plasmid Preparation, Expressing, Bacteria, Flow Cytometry, Control

TREM2 enhances B. abortus survival by suppressing M2 intracellular ROS production. (A) RAW-M2-NT, RAW-M2-ΔTREM2, RAW-M2-Vector, or RAW-M2-TREM2+ cells were infected with B. abortus (MOI = 100), respectively. Intracellular numbers of B. abortus at the indicated times were analyzed by CFU assay. (B) BMDM-M2 macrophages were infected with B. abortus (MOI = 100) in the presence of DMSO or Baf A1 (100 nM) or mock treatment. The intracellular numbers of B. abortus were analyzed by CFU assay at the indicated time points. CFU was expressed as means ± standard deviations from two independent experiments. *P <0.05 vs. Baf A1. BMDM-M2 were infected with B. abortus (MOI = 100) for 8 h, 24 h, or 48 h in the presence of DMSO or Baf A1 (100 nM) or mock treatment. The concentration of nitric oxide (NO) in the culture supernatant collected was measured using Griess reagent (C) . The intracellular ROS production was detected by DCFH-DADHE. The graph demonstrates the SEM and mean of changes in production of intracellular ROS compared with the uninfected cells (as 100%) (D) . The mitochondria ROS (mROS) concentrations were detected by Mito-SOX. The graph demonstrates the SEM and mean of changes in production of mROS compared with the uninfected cells (as 100%) (top panel), representative images of Mito-SOX red (mitochondrial ROS marker) staining of Mock, DMSO and Baf A1 (48 h) (bottom panel). Scale bar = 50 µm (E) . DMSO-pretreated BMDM-M2 were infected with B. abortus (MOI = 100), or Baf A1 (100 nM)-pretreated BMDM-M2 were infected with B. abortus (MOI = 100) in the presence or absence of 10 μM DPI (F) or in the presence or absence of 10 μM Mito-TEMPO (G) , and then bacterial survival was analyzed at the indicated times by CFU assay. *P <0.05 vs. Baf A1 alone. There is no significance (NS) between Baf A1 treatment and Baf A1 plus Mito-TEMPO. (H) Isotype pretreated or anti-TREM2 antibody (5 ug/ml) pretreated BMDM-M2 macrophages were infected with B. abortus (MOI = 100) in the presence of Mock or 10 μM Mito-TEMPO, and then bacterial survival at 48 h post-infection was analyzed by CFU assay. All results were analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant (*, P <0.05). NS indicates no significance.

Journal: Frontiers in Immunology

Article Title: Upregulation of TREM2 expression in M2 macrophages promotes Brucella abortus chronic infection

doi: 10.3389/fimmu.2024.1466520

Figure Lengend Snippet: TREM2 enhances B. abortus survival by suppressing M2 intracellular ROS production. (A) RAW-M2-NT, RAW-M2-ΔTREM2, RAW-M2-Vector, or RAW-M2-TREM2+ cells were infected with B. abortus (MOI = 100), respectively. Intracellular numbers of B. abortus at the indicated times were analyzed by CFU assay. (B) BMDM-M2 macrophages were infected with B. abortus (MOI = 100) in the presence of DMSO or Baf A1 (100 nM) or mock treatment. The intracellular numbers of B. abortus were analyzed by CFU assay at the indicated time points. CFU was expressed as means ± standard deviations from two independent experiments. *P <0.05 vs. Baf A1. BMDM-M2 were infected with B. abortus (MOI = 100) for 8 h, 24 h, or 48 h in the presence of DMSO or Baf A1 (100 nM) or mock treatment. The concentration of nitric oxide (NO) in the culture supernatant collected was measured using Griess reagent (C) . The intracellular ROS production was detected by DCFH-DADHE. The graph demonstrates the SEM and mean of changes in production of intracellular ROS compared with the uninfected cells (as 100%) (D) . The mitochondria ROS (mROS) concentrations were detected by Mito-SOX. The graph demonstrates the SEM and mean of changes in production of mROS compared with the uninfected cells (as 100%) (top panel), representative images of Mito-SOX red (mitochondrial ROS marker) staining of Mock, DMSO and Baf A1 (48 h) (bottom panel). Scale bar = 50 µm (E) . DMSO-pretreated BMDM-M2 were infected with B. abortus (MOI = 100), or Baf A1 (100 nM)-pretreated BMDM-M2 were infected with B. abortus (MOI = 100) in the presence or absence of 10 μM DPI (F) or in the presence or absence of 10 μM Mito-TEMPO (G) , and then bacterial survival was analyzed at the indicated times by CFU assay. *P <0.05 vs. Baf A1 alone. There is no significance (NS) between Baf A1 treatment and Baf A1 plus Mito-TEMPO. (H) Isotype pretreated or anti-TREM2 antibody (5 ug/ml) pretreated BMDM-M2 macrophages were infected with B. abortus (MOI = 100) in the presence of Mock or 10 μM Mito-TEMPO, and then bacterial survival at 48 h post-infection was analyzed by CFU assay. All results were analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant (*, P <0.05). NS indicates no significance.

Article Snippet: Anti-TREM2 neutralizing antibody (FAB17291A), mouse rIFN- γ (Cat: NP_032363), and mouse rIL-4 (Cat: P07750) were purchased from R&D Systems (Shanghai, China).

Techniques: Plasmid Preparation, Infection, Colony-forming Unit Assay, Concentration Assay, Marker, Staining

TREM2-mediated mitochondrial ROS production is required for M2 pyroptosis in response to B. abortus . BMDM-M2 were infected with B. abortus . BMDM-M2 were primed with E. coli LPS (1 μg/ml) for 4 h, followed by infection with opsonized B. abortus (MOI:100) for 24 h in the presence of DMSO, 10 μM Mito-TEMPO + Baf A1 (100 nM), 10 μM DPI + Baf A1 (100 nM) or Baf A1 (100 nM). Immunoblot showing GSDMD and GSDMD-N in lysates of BMDM-M2. Immunoblots are representative of three independent experiments (A) . LDH release was measured by LDH-release kit in the supernatant of cells. The graph demonstrates the SEM and mean of changes in LDH release compared with cells lysed with Triton X-100 (as 100%) (B) . The viability of BMDM-M2 was examined by MTT assay. The graph demonstrates the SEM and mean of changes of cell viability compared with uninfected BMDM-M2 (as 1) (C) . LDH and viability results were analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant t (*, P <0.05). NI means uninfected. B.a means B. abortus .

Journal: Frontiers in Immunology

Article Title: Upregulation of TREM2 expression in M2 macrophages promotes Brucella abortus chronic infection

doi: 10.3389/fimmu.2024.1466520

Figure Lengend Snippet: TREM2-mediated mitochondrial ROS production is required for M2 pyroptosis in response to B. abortus . BMDM-M2 were infected with B. abortus . BMDM-M2 were primed with E. coli LPS (1 μg/ml) for 4 h, followed by infection with opsonized B. abortus (MOI:100) for 24 h in the presence of DMSO, 10 μM Mito-TEMPO + Baf A1 (100 nM), 10 μM DPI + Baf A1 (100 nM) or Baf A1 (100 nM). Immunoblot showing GSDMD and GSDMD-N in lysates of BMDM-M2. Immunoblots are representative of three independent experiments (A) . LDH release was measured by LDH-release kit in the supernatant of cells. The graph demonstrates the SEM and mean of changes in LDH release compared with cells lysed with Triton X-100 (as 100%) (B) . The viability of BMDM-M2 was examined by MTT assay. The graph demonstrates the SEM and mean of changes of cell viability compared with uninfected BMDM-M2 (as 1) (C) . LDH and viability results were analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant t (*, P <0.05). NI means uninfected. B.a means B. abortus .

Article Snippet: Anti-TREM2 neutralizing antibody (FAB17291A), mouse rIFN- γ (Cat: NP_032363), and mouse rIL-4 (Cat: P07750) were purchased from R&D Systems (Shanghai, China).

Techniques: Infection, Western Blot, MTT Assay

B. abortus induces M2 macrophages proliferation in a TREM2-dependent fashion. BMDM-M2 was uninfected (NI), or infected with B. abortus (MOI:100) for 48 h in the presence of DMSO, 10 μM Mito-TEMPO + Baf A1 (100 nM), or Baf A1 (100 nM), followed by using BrdU incorporation assay to measure the proliferation of BMDM-M2. The graph demonstrates the SEM and mean of changes of cell proliferation compared with uninfected BMDM-M2 (as 1) (A) . Immunoblot showing β-catenin in lysates of the above BMDM-M2 at 48 h post-infection (B) . Anti-TREM2 or isotype antibody-pretreated BMDM-M2 was uninfected (NI), or infected with B. abortus (MOI:100) for 48 h, followed by using BrdU incorporation assay to measure proliferation of BMDM-M2. The graph demonstrates the SEM and mean of changes of cell proliferation compared with uninfected BMDM-M2 (as 1) (C) . Immunoblot showing β-catenin in lysates of anti-TREM2 or isotype antibody-pretreated BMDM-M2 at 48 h post-infection (D) . Immunoblots are representative of three independent experiments. Cell proliferation were analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant t (*P <0.05). ns, not significant.

Journal: Frontiers in Immunology

Article Title: Upregulation of TREM2 expression in M2 macrophages promotes Brucella abortus chronic infection

doi: 10.3389/fimmu.2024.1466520

Figure Lengend Snippet: B. abortus induces M2 macrophages proliferation in a TREM2-dependent fashion. BMDM-M2 was uninfected (NI), or infected with B. abortus (MOI:100) for 48 h in the presence of DMSO, 10 μM Mito-TEMPO + Baf A1 (100 nM), or Baf A1 (100 nM), followed by using BrdU incorporation assay to measure the proliferation of BMDM-M2. The graph demonstrates the SEM and mean of changes of cell proliferation compared with uninfected BMDM-M2 (as 1) (A) . Immunoblot showing β-catenin in lysates of the above BMDM-M2 at 48 h post-infection (B) . Anti-TREM2 or isotype antibody-pretreated BMDM-M2 was uninfected (NI), or infected with B. abortus (MOI:100) for 48 h, followed by using BrdU incorporation assay to measure proliferation of BMDM-M2. The graph demonstrates the SEM and mean of changes of cell proliferation compared with uninfected BMDM-M2 (as 1) (C) . Immunoblot showing β-catenin in lysates of anti-TREM2 or isotype antibody-pretreated BMDM-M2 at 48 h post-infection (D) . Immunoblots are representative of three independent experiments. Cell proliferation were analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant t (*P <0.05). ns, not significant.

Article Snippet: Anti-TREM2 neutralizing antibody (FAB17291A), mouse rIFN- γ (Cat: NP_032363), and mouse rIL-4 (Cat: P07750) were purchased from R&D Systems (Shanghai, China).

Techniques: Infection, BrdU Incorporation Assay, Western Blot

TREM2 promotes M2 proliferation during the chronic brucellosis. C57BL/6 mice were uninfected (NI) or infected intraperitoneally with 1 × 10 6 CFU of B. abortus. Uninfected mice were sacrificed, and infected mice were sacrificed at 3, 9 and 30 days postinfection (dpi). RT-PCR gene expression analysis of TREM2 (A) or Arg1 (B) in CD11b + splenocytes from B. abortus -infected or -uninfected C57BL/6 mice at the indicated times. Data are mean ± SD of five mice/group. (C) Spleen cells from infected C57BL/6 mice were stained for flow cytometry analysis. Cells were assessed for CD11b + CD206 + . Data are mean ± SD of five mice/group. (D) C57BL/6 mice were infected intraperitoneally (i.p) with 1 × 10 6 CFU of B. abortus . Mice were mock treated or treated i.p. with DMSO or Baf A1 1mg per kg body weight daily from 18 dpi to 30 dpi. RT-PCR gene expression analysis of TREM2 in CD11b + splenocytes from C57BL/6 mice after 30 days. Data are mean ± SD of five mice/group. (E) CD11b + CD206 + number were assessed by flow cytometry analysis at NI and 30 dpi in spleen of mice. Data are mean ± SD of five mice/group. (F) Representative images of IHC staining of spleen red pulp with anti-CD206 antibody at 30 dpi. Data was analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant t (*P <0.05). Bar is 100 μm. ns, not significant.

Journal: Frontiers in Immunology

Article Title: Upregulation of TREM2 expression in M2 macrophages promotes Brucella abortus chronic infection

doi: 10.3389/fimmu.2024.1466520

Figure Lengend Snippet: TREM2 promotes M2 proliferation during the chronic brucellosis. C57BL/6 mice were uninfected (NI) or infected intraperitoneally with 1 × 10 6 CFU of B. abortus. Uninfected mice were sacrificed, and infected mice were sacrificed at 3, 9 and 30 days postinfection (dpi). RT-PCR gene expression analysis of TREM2 (A) or Arg1 (B) in CD11b + splenocytes from B. abortus -infected or -uninfected C57BL/6 mice at the indicated times. Data are mean ± SD of five mice/group. (C) Spleen cells from infected C57BL/6 mice were stained for flow cytometry analysis. Cells were assessed for CD11b + CD206 + . Data are mean ± SD of five mice/group. (D) C57BL/6 mice were infected intraperitoneally (i.p) with 1 × 10 6 CFU of B. abortus . Mice were mock treated or treated i.p. with DMSO or Baf A1 1mg per kg body weight daily from 18 dpi to 30 dpi. RT-PCR gene expression analysis of TREM2 in CD11b + splenocytes from C57BL/6 mice after 30 days. Data are mean ± SD of five mice/group. (E) CD11b + CD206 + number were assessed by flow cytometry analysis at NI and 30 dpi in spleen of mice. Data are mean ± SD of five mice/group. (F) Representative images of IHC staining of spleen red pulp with anti-CD206 antibody at 30 dpi. Data was analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant t (*P <0.05). Bar is 100 μm. ns, not significant.

Article Snippet: Anti-TREM2 neutralizing antibody (FAB17291A), mouse rIFN- γ (Cat: NP_032363), and mouse rIL-4 (Cat: P07750) were purchased from R&D Systems (Shanghai, China).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Flow Cytometry, Immunohistochemistry

TREM2 is essential to control B. abortus infection and mediates inflammatory response during the chronic brucellosis. C57BL/6 mice were infected intraperitoneally with 1 × 10 6 CFU of B. abortus . Mice were mock treated or treated i.p. with DMSO or Baf A1 1 mg per kg body weight daily from 18 dpi to 30 dpi. (A) Mice were sacrificed after 30 days, and diluted spleen homogenates were added to agar plates for CFU determination. Each symbol represents an animal and the median values are marked by horizontal bold lines. IL-10 (B) and TNFα (C) in serum were measured with ELISA kits. Data are mean ± SD of five mice/group. TNFα (D) , IL-6 (E) , and INFγ (F) gene expression in spleen were analyzed by RT-PCR with normalization to β-actin. Data are mean ± SD of five mice/group. Spleen cells were stained for flow cytometry analysis of CD11b + F4/80 + (G) , CD11b + CD11c + (H) , and CD11b + Ly6G + (I) . Splenic homogenates were submitted to a myeloperoxidase (MPO) activity assay (J) . Data are mean ± SD of five mice/group. Data was analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant t (*P <0.05).

Journal: Frontiers in Immunology

Article Title: Upregulation of TREM2 expression in M2 macrophages promotes Brucella abortus chronic infection

doi: 10.3389/fimmu.2024.1466520

Figure Lengend Snippet: TREM2 is essential to control B. abortus infection and mediates inflammatory response during the chronic brucellosis. C57BL/6 mice were infected intraperitoneally with 1 × 10 6 CFU of B. abortus . Mice were mock treated or treated i.p. with DMSO or Baf A1 1 mg per kg body weight daily from 18 dpi to 30 dpi. (A) Mice were sacrificed after 30 days, and diluted spleen homogenates were added to agar plates for CFU determination. Each symbol represents an animal and the median values are marked by horizontal bold lines. IL-10 (B) and TNFα (C) in serum were measured with ELISA kits. Data are mean ± SD of five mice/group. TNFα (D) , IL-6 (E) , and INFγ (F) gene expression in spleen were analyzed by RT-PCR with normalization to β-actin. Data are mean ± SD of five mice/group. Spleen cells were stained for flow cytometry analysis of CD11b + F4/80 + (G) , CD11b + CD11c + (H) , and CD11b + Ly6G + (I) . Splenic homogenates were submitted to a myeloperoxidase (MPO) activity assay (J) . Data are mean ± SD of five mice/group. Data was analyzed using a one-way ANOVA followed by Bonferroni correction. P-values <0.05 were considered significant t (*P <0.05).

Article Snippet: Anti-TREM2 neutralizing antibody (FAB17291A), mouse rIFN- γ (Cat: NP_032363), and mouse rIL-4 (Cat: P07750) were purchased from R&D Systems (Shanghai, China).

Techniques: Control, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Flow Cytometry, Activity Assay

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.

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

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.

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.

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

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

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

Techniques: CRISPR, Expressing, Cell Culture

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.

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.

Techniques: Staining, Expressing, Control, Immunohistochemical staining

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.

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.

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

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.

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.

Techniques: Control, Staining

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.

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.

Techniques: Expressing, Flow Cytometry, Injection

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.

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.

Techniques: Control, Multiplex Assay, Flow Cytometry, Labeling, Immunofluorescence

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.

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.

Techniques: Staining, TUNEL Assay

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.

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.

Techniques: TUNEL Assay

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.

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.

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

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.

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.

Techniques: Cell Culture

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.

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.

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

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