Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: Myeloid cells dominate the acute response to ICH and contribute to early damage. (A) Single cells were isolated from the right hemisphere of naïve, sham-operated and collagenase-induced ICH mice 24 h post-surgery and immunophenotyped using flow cytometry. (B) Left: gating strategy used during flow cytometry analysis. Right: cell counts are shown from the following populations: CD45 int CD11b + microglia, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ), CD45 hi CD11b − CD45R + B cells, and CD45 hi CD11b − CD3 + T cells. Data presented as mean+s.e.m., n =5, two independent experiments. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001, **** P <0.0001, determined by one-way ANOVA with Tukey's post-hoc test. (C) Formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (left) and ICH (right) cases were immunostained for the myeloid cell marker CD11b (green) and DAPI (blue), with red blood cell (RBC) autofluorescence shown in red. One representative image from three cases per group shown. Scale bars: 100 µm (50 μm in insets). (D) White blood cells were isolated from whole blood of wild-type (WT) ( n =7) and chemokine receptor 2 ( Ccr2 ) −/− ( n =9) male littermate mice. Top left: CD115 + monocytes and Ly6G + neutrophils as a percentage of all CD45 + immune cells. Bottom left: representative flow plots of the circulating monocyte compartment of each genotype. Right: the amount of Ly6C hi monocytes as a percentage of total circulating monocytes from each genotype. ns, not significant; **** P <0.0001, determined by unpaired two-tailed t -test. (E) Following a 3 day training period on the rotarod assay, WT and Ccr2 −/− littermates were subjected to collagenase-induced ICH and tested on the rotarod again 24 h later. Data presented as net difference in latency to fall from baseline performance, n =7-9, four independent experiments. * P <0.05, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Isolation, Flow Cytometry, Formalin-fixed Paraffin-Embedded, Control, Marker, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: IL-1 produced by mononuclear phagocytes controls acute recruitment of myeloid cells during ICH. (A) Mice were subjected to collagenase-induced ICH and culled at 4 h ( n =5) and 24 h ( n =4) post-ICH. 20 µm coronal brain sections were immunostained for either IL-1α (green, top row) or IL-1β (green, bottom row) together with Iba1 (red) and DAPI (blue). Dashed lines represent haematoma border, arrows define haematomal IL-1β+ cells, and insets are higher-magnification images of respective white boxes. Scale bars: 50 µm. (B) Quantification of IL-1 + cells. (C) 8 µm sections of formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (top) and acute ICH (bottom) cases were immunostained for Iba1 (red), IL-1β (green) and DAPI (blue). Red blood cell (RBC) autofluorescence can be seen in white. One representative image from three patients per group is shown. Scale bars: 100 µm. Insets are higher-magnification images of the areas within the white boxes. (D) Representative flow plot of cells isolated from brains of mice injected with central (10 µg intrastriatal) and peripheral (100 mg kg −1 subcutaneous) IL-1 receptor antagonist (IL-1Ra) ( n =4) or placebo ( n =5) and subjected to ICH, two independent experiments. (E) Cell counts are shown from the following populations: CD45 hi CD11b − lymphocytes, CD45 int CD11b + microglia, CD45 hi CD11b + myeloid cells, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ). Data presented as mean+s.e.m. ns, not significant; * P <0.05, ** P <0.01, *** P <0.001, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Produced, Formalin-fixed Paraffin-Embedded, Control, Isolation, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: C3–C3R interactions mediate developmentally-typical D1r elimination in vivo in males, but not females. Neutrophil inhibitor factor (NIF), a peptide that binds specifically to the CD11b subunit of C3 receptors (C3Rs), was assessed for its efficacy in reducing microglial phagocytic activity ex vivo. a In microglia isolated from whole brain, 60 ng, but not 120 ng NIF inhibited microglial phagocytosis of fluorescent beads (Table ). Representative images precede histograms; scale bar equals 20 µm. n = 4, with 2 replications/condition. b Microglia were isolated from whole brain and incubated with 60 ng NIF, and then immunocytochemically assessed for NIF and CD11b at 30, 60, and 120 min. Closed arrow heads indicate NIF (green) immunoreactivity. Scale bar equals 10 µm. There was no change in c NIF or (Table ) d CD11b (Table ) immunoreactivity over 2 h, suggesting that NIF was not causing the degradation of its receptor. n = 3; 2 replications/condition. To determine if developmental D1r downregulation requires C3–C3R interactions, NIF or Vehicle was microinjected in the NAc at e P30 in males and g P22 in females (both represent sex-specific ages prior to D1r downregulation), and then tissue was assessed at P38 and P30, respectively, an age at which D1rs should be developmentally downregulated. f In males, NIF-treated hemispheres exhibited significantly more (~25%) D1r immunoreactivity than within-animal vehicle-treated hemispheres (Table ). h In females, NIF-treated hemispheres exhibited the same D1r immunoreactivity as within-animal vehicle-treated hemispheres (Table ). Representative images precede histograms; scale bars equal 100 µm. n = 4/sex with counterbalanced injections. For ex vivo experiments, data were analyzed with one-way ANOVAs and Holm-Sidak’s post-hoc comparisons. For in vivo experiments, D1r data from 4–7 different sections per animal were averaged and then calculated as a percentage of within-animal vehicle control levels. Data were analyzed with 2-tailed one-sample t -tests. Histograms portray the mean ± SEM with individual data points overlaid. Significant post-hoc Holm-Sidak t -tests ( a – d ) and one-sample t -tests from 100 ( f , h ) ( p < 0.05) comparisons are delineated with an asterisk. All statistics are in Table

Article Snippet: Resulting cell bodies were incubated with anti-rat CD11b (i.e., microglia-specific) magnetic beads (Miltenyi Biotec #130–105–634; Auburn, CA) and then separated from the CD11b- population via magnetic columns.

Techniques: In Vivo, Activity Assay, Ex Vivo, Isolation, Incubation, Control

Journal: Nature Communications

Article Title: Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats

doi: 10.1038/s41467-018-06118-z

Figure Lengend Snippet: Detailed statistics corresponding with Fig. 3

Article Snippet: Resulting cell bodies were incubated with anti-rat CD11b (i.e., microglia-specific) magnetic beads (Miltenyi Biotec #130–105–634; Auburn, CA) and then separated from the CD11b- population via magnetic columns.

Techniques: Comparison, Ex Vivo, In Vivo, Injection

Journal: bioRxiv

Article Title: Mouse and human microglial phenotypes in Alzheimer’s disease are controlled by amyloid plaque phagocytosis through Hif1α

doi: 10.1101/639054

Figure Lengend Snippet: a , Schematic of the methodology employed in this study . b , Representative immunofluorescence image of the hippocampus 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 (from n =12-19 animals per genotype and age group) showing that XO4 + microglia are present in 6m 5xFAD plaque-affected regions (top panels). d , left, the percentage of XO4 + microglia isolated from plaque-affected regions in 1, 4, and 6m old WT and 5xFAD mice, ( n = 12-19 per genotype and age group; male and female mice pooled) and right, the percentage of XO4 + microglia isolated from plaque-affected and non-affected regions in 6m old male and female WT and 5xFAD mice ( n = 6-8 per genotype), expressed as mean ± SEM. e , PCA of bulk RNA-seq. Cx, Cortex; Cb, Cerebellum f, g Gene cytometry plots showing genes that are differentially expressed between XO4 + and XO4 − microglia and/or genes that are differentially expressed between old (4, 6 month) and young (1 month) microglia. Gene scores are calculated as the product of the log fold change and –log 10 (FDR). Example genes in each quadrant are labelled in red (upregulated over time or phagocytosis) or blue (downregulated). 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 , heat map of targeted LC-SWATH-MS analysis of detected peptides within DEGs in n =3-5 biological replicates of WT (blue), XO4 − 5xFAD (orange) and XO4 + 5xFAD (green) microglia. 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 log fold change compared to WT microglia, normalized relative to peptides in Supplementary table 2. p values in d and j were calculated by one-way ANOVA using Tukey’s multiple comparison test.

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 FACSAria™ III cell sorter.

Techniques: Immunofluorescence, Injection, Staining, Isolation, RNA Sequencing, Cytometry, Expressing, Comparison, Quantitation Assay

Journal: bioRxiv

Article Title: Mouse and human microglial phenotypes in Alzheimer’s disease are controlled by amyloid plaque phagocytosis through Hif1α

doi: 10.1101/639054

Figure Lengend Snippet: a , Dimensionality reduction representation (viSNE, representative of n =3 mice per genotype) of myeloid cells isolated from WT (top) and 5xFAD (bottom) 6m male mice. Microglia (CD11b + CD45 lo CX3CR1 + ) are colored for expression of CD11b, CD45, CX3CR1, Methoxy-XO4, CD115, CD33 and TREM2, whereas remaining myeloid cells are grayscale for clarity. b , PCA of 893 single cells x 1671 feature genes showing the distribution of cells from each FACS-sorted sample. c , PCA plot of single microglia colored by SC3 clusters and composition of automated clusters as a percentage of sequenced FACS-sorted cell populations. d . PCA plots for single microglia colored by expression of selected ageing microglia genes (i-ii), homeostatic (iii) and XO4 + signature genes (iv-v). e, f Diffusion maps pseudotime analysis of microglial populations ordered by their expression of e , ageing DEGs (6m WT v 24m WT, 42 DEGs) or f , phagocytic DEGs (6m 5xFAD XO4 − v 6m 5xFAD XO4 + , 474 DEGs) g , 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. h , Hierarchical clustering and heat map 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 FACSAria™ III cell sorter.

Techniques: Isolation, Expressing, Diffusion-based Assay

Journal: bioRxiv

Article Title: Mouse and human microglial phenotypes in Alzheimer’s disease are controlled by amyloid plaque phagocytosis through Hif1α

doi: 10.1101/639054

Figure Lengend Snippet: a-c UMAP projection of single microglia nuclei from control and AD patient frontal cortex, cases ( n =172 nuclei) and controls ( n =277 nuclei). The microglial population was determined by similarity to known microglial markers . The UMAP projection is colored by a , disease diagnosis b , XO4 + score c , or ageing signature score. Diffusion maps pseudotime analysis of microglial populations ordered by their expression of d , XO4 + DEGs (taking top 10% of respective DE genes regardless of overlap with aging DEGs, 167 DEGs between 5xFAD XO4 + and XO4 − mice) or e , ageing DEGs (top 10% or 167 DEGs between 24M WT and 6M WT mice). f , scatter plot showing the relationship between ageing and XO4 + pseudotime in individual cells and the density of cells at each point during the ageing (left) and XO4 + (bottom) trajectories. g , UMAP projection of single human microglia colored by expression of selected cluster specific-genes. h Hierarchical clustering and heat map showing expression of the overlapping DEGs in each of the 4 mouse microglia clusters with the DEGs between human control and AD microglia. The mouse-human concordance on the direction of change between control and AD (human) or WT and XO4 + populations is shown for each gene ( p =0.000641). i , SCENIC regulon analysis showing that Hif1a and Elf3 are predicted to control the XO4 + gene regulatory network. j , UMAP projection of single human microglia colored by HIF1A regulon activity. k , Fluorescently labeled synaptosome internalization by primary microglia transfected with GFP-tagged inducible HIF1A and/or ELF3 overexpression constructs. The data are presented as mean ± SEM and show the difference in synaptosome internalization between GFP + and GFP − (non-transfected) cells tested from within the same well.

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 FACSAria™ III cell sorter.

Techniques: Control, Diffusion-based Assay, Expressing, Activity Assay, Labeling, Transfection, Over Expression, Construct