Journal: Nature Neuroscience

Article Title: Proinflammatory immune cells disrupt angiogenesis and promote germinal matrix hemorrhage in prenatal human brain

doi: 10.1038/s41593-024-01769-2

Figure Lengend Snippet: a , Schematic diagrams showing the strategy to isolate CD45 + ;CD11b + immune cells from the CTX and GE of prenatal human brain from GW15–23. These CD45 + ;CD11b + cells are subjected to bulk RNA-seq and scRNA-seq, followed by bioinformatics analyses. The transcriptomic data are validated using immunohistochemistry (IHC), immunofluorescence microscopy (IF) and RNAscope-based in situ hybridization. Finally, CD45 + ;CD11b + cells are further characterized using high-dimensional flow cytometry and 3D Matrigel HUVEC assays. b , A volcano plot showing the genes enriched in CD45 + cells from GW20–23 (right) and those enriched in cells from GW14–19 (left). Adjusted P values and fold changes were calculated using DESeq2. By default in DESeq2, the P values attained by the Wald test are corrected for multiple testing using the Benjamini–Hochberg method. The genes shown were filtered to be below the adjusted P value of 0.05 and above a fold change of 1.2 between GW14–19 and GW20–23 comparisons (highlighted by the dashed lines). c , GSEA reveals GO terms enriched in CD45 + cells from GW14–19 and GW20–23. The data in b and c are from 21 independent biological samples. NES, normalized enrichment score; FDR, false discovery rate. d , Images taken from InCucyte S3 Live Imaging Device of HUVEC in Matrigel-based branching morphogenesis at 3, 6, 12, 24 and 48 h after plating. The conditions include 20,000 HUVEC alone and 20,000 HUVEC cocultured with 20,000 GW14–19 or GW20–23 CD45 + cells from prenatal human brain. e , Quantification of average and total endothelial branch lengths formed by HUVEC. Statistics use a two-tailed, unpaired Student’s t -test, and the data represent the mean ± standard error of the mean. The P values represent comparisons between HUVEC coincubated with CD45 + cells versus HUVEC only. Not significant comparisons are not shown. n indicates the number of independent biological samples used for quantification. For each biological sample, at least three technical replicates are used.

Article Snippet: From 0 to 48 h, vascular tube formations were imaged every 5–10 min using a 4× objective on Incucyte S3 Live-Cell Analysis Systems (Essen BioScience).

Techniques: RNA Sequencing Assay, Immunohistochemistry, Immunofluorescence, Microscopy, RNAscope, In Situ Hybridization, Flow Cytometry, Imaging, Two Tailed Test

Journal: Nature Neuroscience

Article Title: Proinflammatory immune cells disrupt angiogenesis and promote germinal matrix hemorrhage in prenatal human brain

doi: 10.1038/s41593-024-01769-2

Figure Lengend Snippet: ( a ) Fluorescence-activated cell sorting (FACS) gating strategy to select for CD45 + ;CD11b + cells. ( b ) Isotype controls used in FACS. ( c ) Heatmap of critical differentially expressed genes in CD45 − and CD45 + cells used in bulk RNA-seq. ( d ) Volcano plot shows the genes enriched in CD45 + cells (right). Genes shown were filtered to be below adjusted p-value of 0.05 and above fold change of 1.2 between CD45 − and CD45 + cells. ( e ) Gene set enrichment analysis (GSEA) reveals gene ontology (GO) terms enriched in CD45 − and CD45 + cells. Data from panels c-e are from 3 independent biological samples of CD45 − cells, and 21 independent biological samples of CD45 + cells. ( f ) Images taken from InCucyte S3 Live Imaging Device of HUVEC and AAV-CMV-GFP transfected CD45 + cells from prenatal human brain samples at 18 and 40 hrs after plating. ( g ) Images taken from InCucyte S3 Live Imaging Device of HUVEC at 3, 6, 12, 24 and 48 hrs after plating. The conditions include 20,000 HUVEC alone, and 20,000 HUVEC co-cultured with 10,000 GW14-19 or GW20-23 CD45 + cells from prenatal human brain. ( h, i ) Quantification of average and total endothelial branch lengths formed by HUVEC that are co-cultured with CD45 + cells ( h ) or CD45 − cells ( i ). Statistics in panels h and i use two-tailed, unpaired Student’s t-test, data represent mean ± SEM. The P values represent comparisons between HUVECs co-incubated with CD45 + cells vs HUVECs only. Not significant comparisons are not shown. n indicates the number of independent biological samples used for quantification.

Article Snippet: From 0 to 48 h, vascular tube formations were imaged every 5–10 min using a 4× objective on Incucyte S3 Live-Cell Analysis Systems (Essen BioScience).

Techniques: Fluorescence, FACS, RNA Sequencing Assay, Imaging, Transfection, Cell Culture, Two Tailed Test, Incubation

Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A ) Overlay histograms of LFA-1 diffusion on monocytes (4578 trajectories), resting mDCs (26756 trajectories) and CCL21 activated mDCs (4213 trajectories). ( B ) Percentage of total mobile LFA-1 population (normalized to 100%) displaying slow and fast diffusion on monocytes, mDCs and 2 min CCL21 activated mDCs. ( C ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on monocytes, mDCs and 2 min CCL21 activated mDCs. ( D ) Stationary fraction of LFA-1 on monocytes, mDCs and CCL21 activated mDCs, displayed as the difference from the total stationary fraction on mDCs, which serve here as the default. Data from A–D on CCL21 activated mDCs is based on 22 cells in independent experiments from 3 different donors. ( E ) Percentage of the stationary, slow and fast diffusing LFA-1 molecules at different time points after CCL21 activation. ( F ) D values for the total mobile, and slow and fast fractions of LFA-1 at different time points after CCL21 activation. Data from E and F is based on 11 cells, 11 independent samples and around 2000 trajectories per time point. A – F Means ± SEM are depicted. The One-way ANOVA followed by the Tukey multiple comparison test were used to determine significant differences between means. The resulting P values are indicated as follows: ns (P >0.05); * (P<0.05) and *** (P<0.0001).

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Diffusion-based Assay, Activation Assay, Comparison

Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A,B ) ICAM-1 (either monomeric: ICAMm, or as nano-aggregates: ICAMagg) was added to resting mDCs and mobility was measured before, and between 1 and 5 min after addition. ( A ) Stationary fraction of LFA-1 molecules on resting mDCs and mDCs + either monomeric ICAM-1 or ICAM-1 nano-aggregates, displayed as the difference with respect to the total stationary fraction on resting mDCs, which serve here as the default. ( B ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on resting mDCs and after addition of either monomeric ICAM-1 or ICAM-1 nano-aggregates. 30 cells divided over 2 independent experiments (3393 trajectories) were imaged for the ICAMm condition and 10 cells (684 trajectories) for ICAMagg. ( C, D ) ICAM-1 (either monomeric or nano-aggregates) was added together with CCL21 to mDCs and mobility was measured before, and 2 minutes after addition. ( C ) Stationary fraction of LFA-1 molecules on resting mDCs (serving as reference control), CCL21 activated mDCs and CCL21 activated mDCs + either monomeric ICAM-1 or ICAM-1 nano-aggregates, displayed as the difference with respect to the total stationary fraction on resting mDCs. ( D ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on resting mDCs, CCL21 activated mDCs and after simultaneous addition of CCL21 and either monomeric ICAM-1 or ICAM-1 nano-aggregates. 16 cells (8423 trajectories) from 2 different donors divided over 5 independent samples (ICAMm) and 7 cells (314 trajectories) from 2 different donors divided over 7 independent samples (ICAMagg) were imaged. ( E ) Quantification of fluorescent ICAM-1 dimers (monomers bound together due to antibody labelling) and nano-aggregates binding in resting and CCL21 activated mDCs, normalized to the area quantified and to the background signal outside of the cell. For this, regions of the cell in between the obvious fluorescent ICAM-1 aggregates were selected, the fluorescent intensity was measured using ImageJ, and used to compare the baseline fluorescent signal across all 4 conditions. 20 cells per condition were imaged. ( A–E ) Means ± SEM are depicted. The One-way ANOVA followed by the Tukey multiple comparison test were used to determine significant differences between means. The resulting P values are indicated as follows: ns ( P>0.05); * (P<0.05), ** (P<0.001) and *** (P<0.0001). ( F ) Quantification of bound ICAM-1 nano-aggregates to resting and CCL21 activated mDCs. After applying a threshold of 25% of the fluorescent signal, all visible fluorescent spots per cell were counted. 20 cells per donor and 3 different donors were imaged. Each data point represents the mean value for 1 donor. Means ± SEM and individual data points are depicted, and dotted lines connecting datapoint of the same experiment indicate that not just in average, but in each individual experiment using a different donor, an increase of ICAM-1 nano-aggregate binding is observed after CCL21 activation. The paired two-tailed Student T-test was used to determine significant differences between means. ( G–I ) Representative examples of confocal images of ICAM-1 binding to mDCs: ( G ) dimeric ICAM-1 to resting cells, ( H ) nano-aggregates of ICAM-1 to resting cells and ( I ) nano-aggregates to CCL21 activated cells. Arrows in H and I point to the binding of individual ICAM-1 nano-aggregates to LFA-1.

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Diffusion-based Assay, Control, Binding Assay, Comparison, Activation Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A, B ) Representative images of ( A ) a monocyte seeded on a TS2/4 pattern, ( B ) a resting mDC on irrelevant IgG1 pattern, ( C ) a resting mDC seeded on a TS2/4 pattern and ( D ) a CCL21 activated mDC seeded on a ICAM-1 pattern. Green corresponds to Talin1 and red to the location of IgG1, TS2/4 or ICAM-1 positive squares. Cells are delineated by white lines. ( E ) Quantification of the degree of Talin1 enhancement to the positive areas in monocytes in different conditions (see Methods). Mean enhancement factor is displayed in red per condition. ( F ) Percentage of positive squares per experiment (n = 3, each a different donor) that showed significantly enhanced Talin1 signal per condition in monocytes. An enhancement factor of ≥1.5 was considered significantly enhanced, since 95% of the control sample (monocytes on IgG1) showed an enhancement factor below this value. ( G ) Quantification of the degree of Talin1 enhancement to the positive areas in mDCs in different conditions (see Methods). Mean enhancement factor is displayed in red per condition. ( H ) Percentage of positive squares per experiment (n = 3, each a different donor) that showed significantly enhanced Talin1 signal per condition in mDCs. Around 60 cells of 3 different donors were analyzed per condition. Monocytes contained 10 positive areas on average per cell, while mDCs contained around 50 positive areas. Means ± SEM are depicted. The Kruskal-Wallis test, followed by Dunn’s multiple comparison test was used to determine significant differences between means in E and G. The One-way ANOVA followed by the Tukey’s multiple comparison test were used to determine significant differences between means in F and H. The resulting P values are indicated as follows: ns ( P>0.05); * (P<0.05) and *** (P<0.0001).

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Control, Comparison