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

Journal: Nature Communications

Article Title: Transcriptional signature in microglia associated with Aβ plaque phagocytosis

doi: 10.1038/s41467-021-23111-1

Figure Lengend Snippet: a Schematic of the methodology employed in this study, created with BioRender.com. M, male, F, female, WT, wild-type, Cx, cortex and subcortical regions, Cb, cerebellum. b Representative immunofluorescence image of the hippocampus (HC) of WT and 5xFAD mice injected with methoxy-XO4 and stained with Iba1 (AlexaFluor 488, n = 6 animals per genotype), scale bar = 250 μm, inset 50 μm. c Representative FACS plot showing that XO4 + microglia are present in 6 m 5xFAD plaque-affected regions (top panels). d Left, the percentage of XO4 + microglia isolated from plaque-affected regions in 1, 4 and 6 m old WT (m, month) and 5xFAD mice (from n = 6 animals per genotype at 1 m; 4 m WT, n = 19 animals; 4 m 5xFAD, n = 22; 6 m WT, n = 14; 6 m 5xFAD n = 14) and right, the percentage of XO4 + microglia isolated from plaque-affected and non-affected regions in 6 m old male and female WT and 5xFAD mice (F, Cx, n = 8 per genotype; M, Cx, n = 6 per genotype; F, Cb, n = 4 per genotype; M, Cb, n = 3 per genotype), expressed as mean ± SEM, *** p = 0.003 and **** p = 4.6 × 10 −5 for 4 m, p = 9 × 10 −6 for 6 m, and p = 5.2 × 10 −5 for F Cx vs Cb by Kruskal-Wallis and Dunn’s multiple comparison tests. e PCA of bulk RNA-seq. Cx, Cortex; Cb, Cerebellum. f , g Gene cytometry plots showing DEGs between XO4 + and XO4 − microglia and/or DEGs expressed between old (4, 6 m) and young (1 m) microglia. Gene scores are calculated as the product of the LFC and –log 10 (FDR). Example genes in each quadrant are labelled in red (upregulated over time or phagocytosis) or blue (downregulated). Gene density low = 0, high = 0.2. h i Venn diagram showing the overlap between genes whose expression levels could be explained by the age, region and XO4 covariate as well as GO and KEGG terms associated with XO4 covariate genes. h ii Table showing the 21 core microglial neurodegeneration signature genes and their direction of differential expression in DAM , CD11c + , MGnD and XO4 + microglia. i Heatmap of targeted LC-SWATH-MS analysis of detected peptides within DEGs in biological replicates of WT (green, n = 4 animals), XO4 − 5xFAD (orange, n = 5) and XO4 + 5xFAD (blue, n = 4) microglia. Colour scale represents log 2 -transformed normalized fold changes compared to WT microglia. clustering method = ward.D2, distance = maximum. j Comparison of RNA and protein expression for selected genes, and quantitation of a tryptic peptide in Aβ in microglia. Data are expressed as mean ± SEM LFC compared to WT microglia, normalized relative to peptides in Supplementary Data . p -Values were calculated by one-way ANOVA using Holm-Sidak’s multiple comparison test. Data are from WT ( n = 4 animals), XO4 − 5xFAD ( n = 5), XO4 + 5xFAD ( n = 4) for protein analyses; WT ( n = 5), XO4 − 5xFAD ( n = 7), XO4 + 5xFAD ( n = 7) for RNA analyses.

Article Snippet: The cell pellet was then stained with antibodies to microglial cell surface markers (CD11b-BV650, 1:200 Biolegend, #141723; CD45-BV786, 1:200, BD Biosciences #564225; CX3CR1-FITC, 1:100, Biolegend, #149019; CD11a, 1:20, BD Biosciences, #558191, TREM2-APC, 1:10, R&D Systems, #FAB17291N; CD33-PE, 1:20, eBioscience, #12-0331-82; CD115-BV711, 1:40, Biolegend, #135515) for isolation using the FACSAriaTM III cell sorter.

Techniques: Immunofluorescence, Injection, Staining, Isolation, Comparison, RNA Sequencing, Cytometry, Expressing, Quantitative Proteomics, Data-independent acquisition, Transformation Assay, Quantitation Assay

Journal: Nature Communications

Article Title: Transcriptional signature in microglia associated with Aβ plaque phagocytosis

doi: 10.1038/s41467-021-23111-1

Figure Lengend Snippet: a PCA of 893 single cells (6 m WT = 243 cells, 24 m WT = 121 cells, 6 m 5xFAD XO4 − = 95 cells, 6 m 5xFAD XO4 + = 434 cells; m, month) and 1671 feature genes showing the distribution of cells from each FACS-sorted sample. PC, principal component. b PCA plot of single microglia coloured by single cell consensus (SC3) clusters and composition of automated clusters as a percentage of sequenced FACS-sorted cell populations. c PCA plots for single microglia coloured by expression of selected ageing microglia genes (i-ii), homeostatic (iii) and signature genes associated with XO4 + microglia (iv-v). min = 0 for all genes, Defa17 max = 4.77 , Defa24 max = 7.41 , Crybb1 max = 4.13 , Cst7 max = 5.47 , Ccl3 max = 4.89. d , e Diffusion maps pseudotime analysis of microglial populations ordered by their expression of ( d ) ageing DEGs (24 m WT vs 6 m WT, 42 DEGs) or ( e ) phagocytic DEGs (6 m 5xFAD XO4 + vs 6 m 5xFAD XO4 − , 474 DEGs). f Scatter plot showing the relationship between ageing and phagocytosing pseudotime in individual cells, and the density of cells at each point during the ageing (bottom) and phagocytosing (left) trajectories. g Hierarchical clustering and heatmap showing expression of the top 50 DEGs across the 4 SC3 clusters.

Article Snippet: The cell pellet was then stained with antibodies to microglial cell surface markers (CD11b-BV650, 1:200 Biolegend, #141723; CD45-BV786, 1:200, BD Biosciences #564225; CX3CR1-FITC, 1:100, Biolegend, #149019; CD11a, 1:20, BD Biosciences, #558191, TREM2-APC, 1:10, R&D Systems, #FAB17291N; CD33-PE, 1:20, eBioscience, #12-0331-82; CD115-BV711, 1:40, Biolegend, #135515) for isolation using the FACSAriaTM III cell sorter.

Techniques: Expressing, Diffusion-based Assay

Journal: Nature Communications

Article Title: Transcriptional signature in microglia associated with Aβ plaque phagocytosis

doi: 10.1038/s41467-021-23111-1

Figure Lengend Snippet: a – c UMAP projection of single microglia nuclei from control and AD patient entorhinal and frontal cortex samples, combined by integrating data from – , comprising 102 patients; AD ( n = 5891 microglia nuclei), mild AD ( n = 1591 microglia nuclei), controls ( n = 2988 microglia nuclei), Other Dementia ( n = 3 microglia nuclei) and TREM2 R62H variant ( n = 1458 microglia nuclei). Clustering and analysis of signature scores is performed using Seurat v3. UMAP projection is coloured by ( a ) study of origin, ( b ) Seurat cluster and ( c ) XO4 + score. d Box plots for gene signature scores in each human microglial cluster for the AD vs Trem2KO AD signature, AD vs WT signature , DAM vs homeostatic, and DAM2 vs DAM1 signatures . The lower, middle and upper hinges represent the lower quartile, median and upper quartile, respectively, while the upper and lower whiskers extend ±1.5 times of the interquartile range from the hinges. For each signature score category, pairwise Wilcoxon test between each cluster and base mean was computed. Multiple testing was corrected for using Bonferroni correction. * p < 0.05, ** p < 0.01; *** p < 0.001, **** p < 0.0001, exact p values are provided in the Source data. e The proportion of cells in Clusters 10 and 11 in patients with any cells in Cluster 10 or Cluster 11, respectively (please see Supplementary Fig. for sample size details), grouped according to disease status and/or TREM2 genotype ( * p = 0.047, Wilcoxon Test with No AD as reference). The lower, middle, and upper hinges represent the lower quartile, median and upper quartile, respectively, while the upper and lower whiskers extend ±1.5 times of the interquartile range from the hinges. f Cluster 10 and Cluster 11 DEGs relative to all other human microglia clusters (adjusted p -value < 0.05). Genes of interest associated with XO4 + microglia are highlighted in red. g Heatmap of enriched KEGG pathways in the human microglial Seurat clusters, coloured by log 2 (-log 10 (adjusted p -value)). h Fluorescently labelled synaptosome internalization by human primary microglia treated with AF647-labelled fAβ. The data are mean ± SEM of 3 independent biological replicates and are expressed as fold change in synaptosome internalization relative to non-treated microglia. Differences are reported between AF488-fAβ + and AF488-fAβ − cells tested from within the same well. i Histograms showing fluorescence intensity of HIF1A intracellular staining in AF488-fAβ + and AF488-fAβ − human primary microglia assayed from within the same well. Secondary antibody control cells are stained with AF647 secondary antibodies alone. j Fluorescently labelled synaptosome internalization by primary microglia transfected with GFP-tagged inducible HIF1A and/or ELF3 overexpression constructs. The data are the mean ± SEM of 5 independent biological replicates and are expressed as fold change in synaptosome internalization between GFP + and GFP − (non-transfected) cells tested from within the same well. * p = 0.0188, *** p = 0.0002 by two-way ANOVA and Sidak’s multiple comparison test on the raw synaptosome internalization percentages.

Article Snippet: The cell pellet was then stained with antibodies to microglial cell surface markers (CD11b-BV650, 1:200 Biolegend, #141723; CD45-BV786, 1:200, BD Biosciences #564225; CX3CR1-FITC, 1:100, Biolegend, #149019; CD11a, 1:20, BD Biosciences, #558191, TREM2-APC, 1:10, R&D Systems, #FAB17291N; CD33-PE, 1:20, eBioscience, #12-0331-82; CD115-BV711, 1:40, Biolegend, #135515) for isolation using the FACSAriaTM III cell sorter.

Techniques: Control, Variant Assay, Fluorescence, Staining, Transfection, Over Expression, Construct, Comparison

Journal: medRxiv

Article Title: Cell autonomous microglia defects in a stem cell model of frontotemporal dementia

doi: 10.1101/2024.05.15.24307444

Figure Lengend Snippet: Pathway analysis of genes differentially expressed between MAPT IVS10+16 and isogenic controls shows an enrichment of genes involved in TREM2 signaling. Red bars, significantly upregulated genes. Blue bars, significantly downregulated genes. Significance defined as FDR-BH≤0.05. Three biological replicates were included for each donor. B. Immunocytochemistry for TREM2 (cyan) and nucleus (NucBlue) Scale bar represents 12µm. C-D. TREM2 on the cell membrane (mTREM2) was measured by flow cytometry. C. Histogram overlay of mTREM2 MFI. D. mTREM2 MFI quantification in both donor pairs. *, p<0.05. E. Soluble TREM2 (sTREM2) levels measured by ELISA in media from both donor pairs. ***, p<0.05; p-values were calculated using an unpaired t-test with Welch’s correction in (D) and (E). F. TREM2 mediates pathways in microglia that regulate actin/cytoskeletal organization, proliferation and survival, cytokines, autophagy, and metabolism. Those genes involved in these pathways that were significantly differentially expressed FDR-BH<0.05 are annotated in red (upregulated in mutant iMGL) or blue (downregulated in mutant iMGL).

Article Snippet: Then, iMGLs were stained with CD11b-PE/Cy7 (Cat#: 101216; Clone: M1/70), CD45-Alexa Fluor 700 (Cat#: 304024; Clone: H130), TREM2-APC (R&D, FAB17291A) for 20 min at 4°C in the dark followed by washing with FACS buffer.

Techniques: Immunocytochemistry, Membrane, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mutagenesis