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R&D Systems anti trem2 neutralizing antibody

B. abortus infection upregulates <t>TREM2</t> 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.

Anti Trem2 Neutralizing Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti trem2 neutralizing antibody - by Bioz Stars, 2026-03

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1) Product Images from "Upregulation of TREM2 expression in M2 macrophages promotes Brucella abortus chronic infection"

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

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2024.1466520

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.

Figure Legend 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.

Techniques Used: 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.

Figure Legend 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.

Techniques Used: 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.

Figure Legend 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.

Techniques Used: 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 .

Figure Legend 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 .

Techniques Used: 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.

Figure Legend 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.

Techniques Used: 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.

Figure Legend 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.

Techniques Used: 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).

Figure Legend 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).

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

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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

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

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

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

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

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

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

Csfr1 + cells (clusters C8-C11 from global UMAP in ) from scRNA-seq analysis of CD45 + cells were re-clustered to obtain higher resolution clustering of myeloid cells. ( A ) Integrated UMAP plot of myeloid cells from 4M (n= 5,473 cells), 12M (n= 4,244 cells) and 20M (n= 5,148 cells) tumors showing 9 sub-clusters. ( B ) Dot plots showing scaled expression of marker genes used to identify myeloid cell sub-clusters. ( C ) Bar graph showing the percentages of various myeloid cell sub-clusters of for each cluster from 4M, 12M and 20M old mice. ( D ) Dot plots showing scaled expression of indicated genes used to identify specific functions. ( E ) Frequencies of CD11b + F480 low among CD11b + cells from CD45 + cells of tumors from 4M and 20M old mice. ( F ) Frequencies of TREM1 + among CD11b + F480 low cells and representative flow cytometry plots showing TREM1 + expression by a gated subpopulation (CD11b + cells) of CD45.2 + cells from 4M and 20M old mice. ( G ) Frequencies of F480 + among CD45 + cells in tumors from 4M and 20M old mice. ( H ) Frequencies of TREM2 + among F480 + cells and representative flow cytometry plots showing TREM2 + expression by a gated subpopulation (F480 + cells) of CD45.2 + cells from 4M and 20M old mice. n = 7 for 4M and n = 6 for 20M for panels E-H. Data in panels D-H were analyzed by unpaired t -test.

Journal: bioRxiv

Article Title: Age-associated modulation of TREM1/2- expressing macrophages promotes melanoma progression and metastasis

doi: 10.1101/2024.11.20.624563

Figure Lengend Snippet: Csfr1 + cells (clusters C8-C11 from global UMAP in ) from scRNA-seq analysis of CD45 + cells were re-clustered to obtain higher resolution clustering of myeloid cells. ( A ) Integrated UMAP plot of myeloid cells from 4M (n= 5,473 cells), 12M (n= 4,244 cells) and 20M (n= 5,148 cells) tumors showing 9 sub-clusters. ( B ) Dot plots showing scaled expression of marker genes used to identify myeloid cell sub-clusters. ( C ) Bar graph showing the percentages of various myeloid cell sub-clusters of for each cluster from 4M, 12M and 20M old mice. ( D ) Dot plots showing scaled expression of indicated genes used to identify specific functions. ( E ) Frequencies of CD11b + F480 low among CD11b + cells from CD45 + cells of tumors from 4M and 20M old mice. ( F ) Frequencies of TREM1 + among CD11b + F480 low cells and representative flow cytometry plots showing TREM1 + expression by a gated subpopulation (CD11b + cells) of CD45.2 + cells from 4M and 20M old mice. ( G ) Frequencies of F480 + among CD45 + cells in tumors from 4M and 20M old mice. ( H ) Frequencies of TREM2 + among F480 + cells and representative flow cytometry plots showing TREM2 + expression by a gated subpopulation (F480 + cells) of CD45.2 + cells from 4M and 20M old mice. n = 7 for 4M and n = 6 for 20M for panels E-H. Data in panels D-H were analyzed by unpaired t -test.

Article Snippet: Anti-TREM2 neutralizing antibody was purchased from Leinco Technologies.

Techniques: Expressing, Marker, Flow Cytometry

( A-B ) YUMM1.7 melanoma cells (500,000) were injected s.c. into the flanks of 4M (young), and 20 months (old). Four days later mice were injected i.p. with either TREM1 inhibitor VJDT or vehicle (control) (A) and injections were repeated every other day until day 20. A separate group of melanoma-bearing mice were treated with anti-TREM2 antibody (B) starting at day 5 after melanoma cell injection and repeated every three days for a total of 4 treatments. Isotype control served as control (B). n = 5 for 4M control and TREM1 treated, n = 4 for 20M in panel A; n = 8 for 4M control, n = 5 for 4M TREM2 treated, n = 7 for 20M control, n = 5 for 20M TREM2 treated in panel B. ( C ) Representative H&E staining of lung tissues from 20M melanoma tumor-bearing mice treated either with isotype control (left) or anti-TREM2 antibody (right). Lung tissues were analyzed 22 days after melanoma cell injection. ( D ) Quantification of lung metastases following treatment of mice with control or anti-TREM2 antibody as described in (B). Tissues were subjected to IHC staining for S100 to detect metastatic melanoma cells (more than 10 S100+ cells per lesion). n = 5 for each group. Scale bar, 300 μM. ( E ) Frequencies of CD206 + and MHCI + among CD11b + and F480 + cells, and expression of CD11c + cells on F480 + cells of tumors from 4M and 20M old mice treated with isotype control or anti-TREM2 antibodies. ( F ) Frequencies of CD4 + and CD8 + among CD45.2 + cells, and expression of CD44 + cells on CD4 + or CD8 + cells of tumors from 4M and 20M old mice treated with isotype control or anti-TREM2 antibodies. n = 8 for 4M control, n = 4 for 4M TREM2 treated, n = 7 for 20M control, n = 5 for 20M TREM2 treated in panels E and F. Data in panels A, B, D, E and F are presented as means + SEM. Data in panel A were analyzed by two-way ANOVA. Data in panel D, E and F were analyzed by unpaired t -test and compared to controls of the same age group.

Journal: bioRxiv

Article Title: Age-associated modulation of TREM1/2- expressing macrophages promotes melanoma progression and metastasis

doi: 10.1101/2024.11.20.624563

Figure Lengend Snippet: ( A-B ) YUMM1.7 melanoma cells (500,000) were injected s.c. into the flanks of 4M (young), and 20 months (old). Four days later mice were injected i.p. with either TREM1 inhibitor VJDT or vehicle (control) (A) and injections were repeated every other day until day 20. A separate group of melanoma-bearing mice were treated with anti-TREM2 antibody (B) starting at day 5 after melanoma cell injection and repeated every three days for a total of 4 treatments. Isotype control served as control (B). n = 5 for 4M control and TREM1 treated, n = 4 for 20M in panel A; n = 8 for 4M control, n = 5 for 4M TREM2 treated, n = 7 for 20M control, n = 5 for 20M TREM2 treated in panel B. ( C ) Representative H&E staining of lung tissues from 20M melanoma tumor-bearing mice treated either with isotype control (left) or anti-TREM2 antibody (right). Lung tissues were analyzed 22 days after melanoma cell injection. ( D ) Quantification of lung metastases following treatment of mice with control or anti-TREM2 antibody as described in (B). Tissues were subjected to IHC staining for S100 to detect metastatic melanoma cells (more than 10 S100+ cells per lesion). n = 5 for each group. Scale bar, 300 μM. ( E ) Frequencies of CD206 + and MHCI + among CD11b + and F480 + cells, and expression of CD11c + cells on F480 + cells of tumors from 4M and 20M old mice treated with isotype control or anti-TREM2 antibodies. ( F ) Frequencies of CD4 + and CD8 + among CD45.2 + cells, and expression of CD44 + cells on CD4 + or CD8 + cells of tumors from 4M and 20M old mice treated with isotype control or anti-TREM2 antibodies. n = 8 for 4M control, n = 4 for 4M TREM2 treated, n = 7 for 20M control, n = 5 for 20M TREM2 treated in panels E and F. Data in panels A, B, D, E and F are presented as means + SEM. Data in panel A were analyzed by two-way ANOVA. Data in panel D, E and F were analyzed by unpaired t -test and compared to controls of the same age group.

Article Snippet: Anti-TREM2 neutralizing antibody was purchased from Leinco Technologies.

Techniques: Injection, Control, Staining, Immunohistochemistry, Expressing

( A-B ) YUMM1.7 melanoma cells (500,000) were injected s.c. into the flank of 20M old mice treated either with isotype control or anti-TREM2 antibody and after 22 days lungs were collected and processed to obtain 5 serial sections per lung. (A) Representative H&E staining of lung tissues from indicated. Scale bar, 4 mM. (B) IHC analysis of S100 staining used to detect lung metastasis in samples described above. Scale bar, 100 μM. ( C ) Frequencies of CD11b + and F480 + among CD45.2 + cells in tumors from 4M and 20M old mice treated with isotype control or anti-TREM2 antibodies. n = 8 for 4M control, n = 4 for 4M TREM2 treated, n = 7 for 20M control, n = 5 for 20M TREM2 treated in panel C. Data in panel C were analyzed by unpaired t -test and compared to controls of the same age group.

Journal: bioRxiv

Article Title: Age-associated modulation of TREM1/2- expressing macrophages promotes melanoma progression and metastasis

doi: 10.1101/2024.11.20.624563

Figure Lengend Snippet: ( A-B ) YUMM1.7 melanoma cells (500,000) were injected s.c. into the flank of 20M old mice treated either with isotype control or anti-TREM2 antibody and after 22 days lungs were collected and processed to obtain 5 serial sections per lung. (A) Representative H&E staining of lung tissues from indicated. Scale bar, 4 mM. (B) IHC analysis of S100 staining used to detect lung metastasis in samples described above. Scale bar, 100 μM. ( C ) Frequencies of CD11b + and F480 + among CD45.2 + cells in tumors from 4M and 20M old mice treated with isotype control or anti-TREM2 antibodies. n = 8 for 4M control, n = 4 for 4M TREM2 treated, n = 7 for 20M control, n = 5 for 20M TREM2 treated in panel C. Data in panel C were analyzed by unpaired t -test and compared to controls of the same age group.

Article Snippet: Anti-TREM2 neutralizing antibody was purchased from Leinco Technologies.

Techniques: Injection, Control, Staining

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