human il8 cxcl8  (R&D Systems)

Journal: Advanced Science

Article Title: Single‐Cell Transcriptome Profiling Reveals Conserved IFNγ‐IL8 Signaling‐Induced Antibacterial Neutrophil States during Bacterial Infection

doi: 10.1002/advs.202504840

Figure Lengend Snippet: Type II interferon signaling pathway enriched in neutrophils for antibacterial responses to S. agalactiae . (A) Volcano plot showing differential gene expression for inflammatory genes at five‐time points post‐infection in neutrophils compared with the control group (0 DPI). DPI: days post‐infection. Genes significantly upregulated (red) or downregulated (blue) at each time point were identified by a two‐sided Wilcoxon rank‐sum test (Bonferroni‐adjusted P ‐value < 0.05). DEGs: differentially expressed gene. DPI: days post‐infection. (B) Heatmap showing the expression of interferon‐related genes across six‐time points in neutrophils, with Z‐score normalized expression levels color‐coded. DPI: days post‐infection. (C) GO enrichment analysis of DEGs in neutrophils at 1 DPI. The right side shows significantly enriched GO terms, and the left side shows significantly depleted GO terms. The significance of enrichment is determined by a hypergeometric test. P ‐value was adjusted by FDR method. DPI: days post‐infection. (D) Violin plot of type II interferon‐mediated signaling pathway scores (GO:00 60333) for neutrophils at six‐time points. For the box plot within each violin plot, middle lines indicate median values, boxes range from the 25th to 75th percentiles, and upper/lower whiskers extend from the hinge to the largest/smallest value no further than 1.5 times the interquartile range (IQR) from the hinge. Significance was determined by a two‐sided Wilcoxon rank‐sum test. DPI: days post‐infection. (E) Cell type specificity of inferred TFs by SCENIC. Regulon Specificity Score (range from 0 to 1) was estimated to order the TFs in neutrophils at 1 DPI. The top5 of TFs were highlighted, with binding motifs of stat1a and stat5 shown on the right. DPI: days post‐infection. (F) UMAP visualization of neutrophil subsets (left) and the expression pattern of genes involved in the type II interferon‐mediated signaling pathway (GO:00 60333) (right) in neutrophils, Neu‐il8 subset was circled by the dashed box, Colors represent the expression of genes in each cluster.

Article Snippet: Similarly, dHL‐60 cells were treated with RPMI 1640 medium (Gibco,11 875 093) containing 10% FBS (Gibco, A3161002C) with 1 and 10 ng mL −1 purified IL8 protein (R&D systems, 208‐IL‐010).

Techniques: Gene Expression, Infection, Control, Expressing, Binding Assay

Journal: Advanced Science

Article Title: Single‐Cell Transcriptome Profiling Reveals Conserved IFNγ‐IL8 Signaling‐Induced Antibacterial Neutrophil States during Bacterial Infection

doi: 10.1002/advs.202504840

Figure Lengend Snippet: IFNγ signaling primes neutrophils state transition to il8 + subtype with antibacterial effect in teleost and mammals. (A) SDS‐PAGE assay showing the purification of recombinant tilapia IFNγ protein in Escherichia coli . The lanes from left to right represent protein markers, unpurified IFNγ protein induced by IPTG, and purified IFNγ protein. (B) Gated neutrophils by flow cytometry from tilapia ( n = 3–4), plated in replicates and cultured under unstimulated, LPS‐stimulated, or LPS + IFNγ‐stimulated conditions for 4 h, followed by scRNA‐seq. After quality control, 7,015 cells from control, 11,814 cells from LPS‐stimulated, and 13,523 cells from LPS + IFNγ‐stimulated conditions were retained. (C) UMAP visualization of neutrophil subtype transitions by RNA velocity analyses. Arrows indicate the directionality of the cell‐cell transition matrix computed by CellRank. Cells are color‐coded by subtypes. A total of 32,352 cells from three groups, including 7,015 cells from control, 11,814 cells from LPS‐stimulated, and 13,523 cells from LPS + IFNγ‐stimulated conditions were analyzed. (D) UMAP visualization of Neu‐il8 subtype density by Mellon analysis across control, LPS‐stimulated, and LPS + IFNγ‐stimulated groups. Neu‐il8 subset was circled by the dashed boxes. UMAPs colored by Mellon log density. Density was computed in high‐dimensional cell‐state space (diffusion maps) (E) Bar plot showing the proportions of neutrophil subtypes across unstimulated, LPS‐stimulated, and LPS + IFNγ‐stimulated conditions. Red boxes highlight Neu‐il8 subset. Cell subtypes are color‐coded. (F) UMAP visualization of neutrophils (top panel) overlaid with RNA velocity maps (bottom panel) in humans (unstimulated, IFNγ‐stimulated) and Nile tilapia (unstimulated, LPS‐stimulated, and LPS + IFNγ‐stimulated), analyzed independently. The expression level of IL8 is highlighted in the left‐bottom corner. Arrows indicate the directionality of the cell‐cell transition matrix computed by CellRank. Cell types are color‐coded. (G) Bar plot showing the proportions of neutrophil subtypes in humans and tilapia. Cell types are color‐coded. (H) Scatter plot showing the S. agalactiae CFU recovered from control and Il8 ‐stimulated immune cells (left), and from control and IL8 ‐stimulated dHL‐60 cells (right) (Methods). S. agalactiae was incubated with control and Il8 ‐stimulated immune cells for 5 h, and then plated on BHI agar for CFUs enumeration.

Article Snippet: Similarly, dHL‐60 cells were treated with RPMI 1640 medium (Gibco,11 875 093) containing 10% FBS (Gibco, A3161002C) with 1 and 10 ng mL −1 purified IL8 protein (R&D systems, 208‐IL‐010).

Techniques: SDS Page, Purification, Recombinant, Flow Cytometry, Cell Culture, Control, Diffusion-based Assay, Expressing, Incubation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Personalized Vascularized Models of Breast Cancer Desmoplasia Reveal Biomechanical Determinants of Drug Delivery to the Tumor.

doi: 10.1002/advs.202402757

Figure Lengend Snippet: Figure 3. Proinflammatory cytokines in the tumoroid microenvironment contribute to loss of vascular glycocalyx. a) Quantification of pro-inflammatory cytokines in 1 mm biopsies centered at tumoroids in the MVN devices; n = 3. b) MVN permeability after short (15 min) and long (12 h) exposure to proinflammatory cytokines, normalized to untreated MVN permeability; n = 3. c) Confocal images of HA in MVNs after long exposure to the cytokines and d) quantification of vascular HA concentration after long exposure to different concentrations of IL8; n = 3. e) Gene expression of IL8 in TCs cultured in 2D or collected from MVN devices; n = 3. f) Quantification of HA protein concentration in 1 mm biopsies centered at MDA-MB-468 tumoroids after intervention with an IL8 blocking antibody (“IL8 block”), and dexamethasone (“Dexa”). g) Vascular concentration of dextran near the tumoroids normalized to concentration at a distance > 5 mm, as measured by proxy of fluorescence intensity, and h) representative images of perfused MVNs in the vicinity of MDA-MB-468 tumoroids (tumoroid cores in circles) subjected to interventions targeting IL8. The scale bar is 500 μm. i) Effective permeability of MDA-MB-468 tumoroid MVNs after different interventions as a function of intravascular pressure; n = 3. Significance assessed by one-way ANOVA after confirming a normal distribution of the data; in (e), significance is plotted only between 2D and MVNs for each TC type; p < 0.05 *, p < 0.01 **, p < 0.001 ***.

Article Snippet: Recombinant human IL8 (#208-IL-010, R&D Systems), IL12 (#219-IL-005, R&D Systems), TNFα (#210-TA-005, R&D Systems), CCL2 (#279-MC-010, R&D Systems), CCL4 (#271-BME-010, R&D Systems) were perfused at a concentration of 5 ng mL−1 together with dextran for short-exposure permeability testing (<15 min) and for 12 h for long-exposure testing.

Techniques: Permeability, Concentration Assay, Gene Expression, Cell Culture, Protein Concentration, Blocking Assay

Journal: Clinical Cancer Research

Article Title: Redirecting B7-H3.CAR T Cells to Chemokines Expressed in Osteosarcoma Enhances Homing and Antitumor Activity in Preclinical Models

doi: 10.1158/1078-0432.CCR-23-3298

Figure Lengend Snippet: RNA-seq and multiplex analyses identify chemokines produced by primary OS samples. A, Expression of 28 chemokines by RNA-seq (FPKM shown; n = 85 OS samples); screen cutoff shown at 1 FPKM. B, Expression of 28 chemokines by protein secretion ( n = 8 patient samples); screen cutoff shown at 3 × 10 3 integrated pixel density. Green bars in A and B denote ligands deemed positive in both RNA and protein screens; error bars indicate SEM. C, Chemokine receptors (ligands in parentheses) CXCR2 (IL8), CXCR3 (CXCL10), CXCR6 (CXCL16), CCR7 (CCL21), and CCR8 (CCL18) as expressed endogenously and detected by flow cytometry on activated T cells ( n = 5 healthy donors; error bars indicate SEM). D, ELISA demonstrating detection of IL8 and CXCL16 in patient transport media specimens ( n = 6 patient tumor samples, 1 normal bone sample; error bars indicate SEM).

Article Snippet: At time point 0, the transwell was placed into a fresh V-shaped lower-chamber plate with 100 μL of RPMI supplemented with 1% FBS and 0, 10, or 30 ng/mL of recombinant human IL8 (R&D Systems) or recombinant human CXCL16 (R&D Systems).

Techniques: RNA Sequencing, Multiplex Assay, Produced, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay