ccr10 specific blocker bi6901  (MedChemExpress)

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: Surface expression of ( A, C ) CCR3 or ( B, D ) CCR10 on murine neutrophils obtained from ( A, B ) the gut, blood, and bone marrow (BM) 3 dpi with STm, or ( C, D ) the bronchoalveolar lavage (BAL), blood, and bone marrow 1 dpi with Ab, analyzed by flow cytometry. Left panels show representative histograms of ( A, C ) CCR3 or ( B, D ) CCR10 expression on the surface of neutrophils (gated on live, CD45 + CD11b + Ly6G + cells) from ( A, B ) the gut (blue), blood (red), and bone marrow (BM; black) or ( C, D ) BAL (blue), blood (red), and bone marrow (BM; black). Right panels show the percentage of ( A, C ) CCR3 + or ( B, D ) CCR10 + neutrophils obtained from ( A, B ) gut, blood, and BM or ( C, D ) BAL, blood, and BM. Data are from six independent experiments. ( E–H ) Uninfected bone marrow neutrophils were unstimulated or treated with the indicated stimuli for 4 hr. Surface expression of ( E, G ) CCR3 and ( F, H ) CCR10 on neutrophils was determined by flow cytometry. Left panels show representative histograms of ( E, G ) CCR3 or ( F, H ) CCR10 surface expression after stimulation with: ( E, F ) cytokines IFNγ + TNFɑ + GM-CSF (blue); fMLP (magenta); phorbol 12-myristate 13-acetate (PMA) (purple); lipopolysaccharide (LPS) (red); ( G, H ) cytokines IFNγ + TNFɑ + Granulocyte-macrophage colony stimulating factor (GM-CSF, blue); beads alone (magenta); cytokines plus beads (red). Right panels show the percentage of ( E, G ) CCR3 + or ( F, H ) CCR10 + neutrophils following stimulation with the indicated stimuli. US = unstimulated. Data shown are pooled from two independent experiments. ( I, J ) Bone marrow cells enriched for neutrophils were infected with opsonized STm at a multiplicity of infection (MOI) = 10 for 1 hr with (violet) or without (red) pretreatment with cytochalasin D for 30 min before infection. Surface expression of ( I ) CCR3 or ( J ) CCR10 was determined by flow cytometry. Data are from two independent experiments. Left panels show representative histograms of surface receptor staining on neutrophils, and right panels show the percentages. ( A–J , right panels) Bars represent the mean ± standard deviation (SD). ( A–D ) Data were analyzed by one-way analysis of variance (ANOVA) for paired samples (non-parametric Friedman test), assuming non-normal distribution and non-equal SD given the differences in the variance among the groups, followed by Dunn’s multiple comparisons test. ( E–J ) Data were analyzed by one-way ANOVA for paired samples, applying the Greenhouse–Geisser correction given the differences in variance among the groups; Bonferroni’s multiple comparison test was performed to compare between relevant stimulation conditions. Significant changes are indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, not significant.

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques: Expressing, Flow Cytometry, Infection, Staining, Standard Deviation, Comparison

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: ( A ) Surface expression of CCR3 and CCR10 on neutrophils obtained from the gut of WT mice ( n = 19, pooled from six independent experiments) infected with STm for 3 days, analyzed by flow cytometry. ( B ) Percentage of CCR3 + and CCR10 + neutrophils obtained from the gut, blood, and bone marrow of Ccl28 +/+ ( n = 19) and Ccl28 −/− mice ( n = 14) infected with STm for 3 days, analyzed by flow cytometry. ( C ) Surface expression of CCR3 and CCR10 on neutrophils obtained from the bronchoalveolar lavage (BAL) of WT mice ( n = 8, pooled from two independent experiments) infected with Ab for 1 day, analyzed by flow cytometry. ( D ) Percentage of CCR3 + neutrophils (WT n = 9; Ccl28 −/− n = 8) and CCR10 + neutrophils (WT n = 4; Ccl28 −/− n = 4) obtained from the BAL, lung, blood, and bone marrow of WT and Ccl28 −/− littermates infected with Ab for 1 day, analyzed by flow cytometry. ( A, C ) Left panels show representative contour plots, and right panels show the percentages of neutrophils expressing the indicated receptor on their surface. Symbols represent data from individual mice, bars represent the geometric means.

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques: Expressing, Infection, Flow Cytometry

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: Neutrophils enriched from wild-type mouse bone marrow were infected at multiplicity of infection (MOI) = for 5 min to 4 hr with ( A, B ) opsonized Salmonella enterica serovar Typhimurium (STm) or ( C, D ) Acinetobacter baumannii (Ab). ( A, C ) Surface CCR10 or ( B, D ) intracellular CCR10 staining was detected by flow cytometry. Neutrophils were obtained from ( E ) the gut, blood, and bone marrow 3 dpi with STm or ( F ) bronchoalveolar lavage (BAL), blood, and bone marrow 1 dpi with Ab. Surface (clear histograms) and intracellular (filled histograms) CCR10 expression was analyzed by flow cytometry. ( A–F ) Left panels show representative histograms, and right panels show the percentage of neutrophils expressing CCR10 on their surface (clear bars) or intracellularly (filled bars). Bars represent the mean. Data were analyzed by paired t test ( A–D ) or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test ( E, F ) on log-transformed data. Significant changes are indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns, not significant.

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques: Infection, Staining, Flow Cytometry, Expressing, Comparison, Transformation Assay

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: ( A ) Murine bone marrow neutrophils were stimulated with IFNγ + TNFɑ + GM-CSF for 4 hr before adding 1 × 10 6 cells to the upper compartment of a transwell chamber for chemotaxis assays. Each of the chemokines (CCL28, CCL11, or CXCL1), or no chemokine (NC), was placed in separate lower compartments. The transwell plate was incubated for 2 hr at 37°C. Cells that migrated to the lower compartment were enumerated by flow cytometry. Neutrophil chemotaxis index was calculated by taking the number of cells that migrated in response to a chemokine and dividing it by the number of cells that migrated in the absence of a chemokine. Data are from four independent experiments. ( B, C ) Infection of bone marrow neutrophils. ( B ) Opsonized STm (1 × 10 7 CFU) or ( C ) opsonized Ab (1 × 10 7 CFU) were cultured alone, or added to bone marrow neutrophils (1 × 10 6 cells) stimulated with CCL28, CCL11, or no chemokine, for 2.5 hr (STm) or 4.5 hr (Ab) at 37°C. Neutrophils were lysed with 1% Triton-X and surviving bacteria were enumerated by plating serial dilutions. Percentage of bacterial survival was calculated for each condition by taking the CFU from bacteria incubated with neutrophils and dividing it by the CFU from bacteria incubated without neutrophils, multiplied by 100. Data shown for each infection comprise three independent experiments. Bars represent the mean ± standard deviation (SD). ( D ) The effect of the CCR3 antagonist SB328437 on neutrophil-mediated STm killing was evaluated by performing the experiment as described in panel ( B ), with or without the antagonist. Data shown comprise three independent experiments. ( E–G ) Reactive oxygen species (ROS) production (2′,7′-dichlorodihydrofluorescein diacetate [H 2 DCFDA] conversion to fluorescent DCF) detected by flow cytometry in bone marrow neutrophils infected with STm as described in panel ( B ). In ( F, G ), cells were stimulated with CCL28 in the presence of an anti-CCR3 antibody, an anti-CCR10 antibody, or isotype controls. Left panels show representative histograms, and right panels show the percentage of ROS + neutrophils in the indicated treatment groups. ( H, I ) Neutrophil extracellular trap (NET) formation detected by fluorescence microscopy using Helix dye in human neutrophils activated with platelets. Cells were unstimulated (no chemokine, NC), stimulated with CCL28 alone, or with CCL28 and the CCR3 agonist SB328737 and/or the CCR10 agonist BI-6901, as indicated. ( H ) Representative images of fluorescence microscopy with DAPI (blue) and Helix (green). ( I ) Quantification of NETs represented as percentage of cells forming NETs based on observed morphology. Connected circles represent NET abundance in cell populations from the same donor following different indicated treatments. ( A–E ) Bars represent the mean ± SD. ( A–C ) Data were analyzed by non-parametric analysis of variance (ANOVA) (Kruskal–Wallis’s test), assuming non-equal SD given the differences in the variance among the groups, followed by Dunn’s multiple comparisons test. ( D, I ) Data were analyzed by ratio paired t test. ( E–G ) Log-transformed data were analyzed by one-way ANOVA for paired samples. Greenhouse–Geisser correction was applied in F and G given the differences in variance among the groups. Tukey’s multiple comparison test was performed to compare all conditions to each other. ( I ) Ratio paired t tests were used to compare NET levels in samples from the same donor. Significant changes are indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, not significant.

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques: Chemotaxis Assay, Incubation, Flow Cytometry, Infection, Cell Culture, Bacteria, Standard Deviation, Fluorescence, Microscopy, Transformation Assay, Comparison

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet: As indicated, cells were unstimulated (NC), stimulated with CCL28 alone, or with CCL28 and the CCR3 antagonist SB328437 and/or the CCR10 antagonist BI-6901 (as in ). ( A ) Representative contour plots, and ( B ) percentage of Helix + MPO + neutrophils in the indicated treatment groups. Connected circles represent NET abundance in cell populations from the same donor following different indicated treatments. Ratio paired t tests were used to compare NET levels in samples from the same donor. Significant changes are indicated by *p ≤ 0.05; ns, not significant.

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques:

Journal: eLife

Article Title: CCL28 modulates neutrophil responses during infection with mucosal pathogens

doi: 10.7554/eLife.78206

Figure Lengend Snippet:

Article Snippet: For fluorescence microscopy analysis, neutrophils were incubated with autologous activated platelets (1:10 ratio) ( ) for 3.5 hr in a 24-well plate with a poly- L -lysine-treated coverslip and stimulated with human recombinant CCL28 (50 nM) (BioLegend), the CCR3 antagonist SB328437 (10 mM, Tocris Bioscience), and/or the CCR10 antagonist BI-6901 (20 mM, Boehringer-Ingelheim).

Techniques: Sequencing, Generated, CRISPR, Isolation, Blocking Assay, In Vitro, Control, Recombinant, Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, cDNA Synthesis, Incubation, Selection, Software, Infection, RNA Extraction, Staining, Immunofluorescence, Bacteria, Protease Inhibitor, Inhibition, Preserving, Cell Isolation, Extraction

Journal: Discovery Medicine

Article Title: Blocking CCR10 Expression Activates m6A Methylation and Alleviates Vascular Endothelial Cell Injury

doi: 10.24976/discov.med.202335174.5

Figure Lengend Snippet: Fig. 4. Impact of CCR10 on m6A methylation. (A) Comparison of CCR10 expression among the BI6901 group, injury group and

Article Snippet: The BI6901 group was first treated with endotoxin to establish the injury model and then intervened by the CCR10-specific blocker BI6901 (HY-116835, MCE, Trenton, NJ, USA, 50 ng/mL). qRTPCR verified the blocking effect, and changes in m6A methylation were determined.

Techniques: Methylation, Comparison, Expressing

Journal: bioRxiv

Article Title: Langerhans islets induce anti-tumor immunity at the expense of glycemic control and predict chemotherapy response in pancreatic cancer

doi: 10.1101/2021.03.13.435100

Figure Lengend Snippet: CCR10 is expressed on CD4+ FoxP3-T cells and selective inhibition of CCR10 abrogates the TH1-type cytokine profile (A) Immunofluorescence of serial human PDA tissue samples as indicated. Scale bars, 100 μm. (B) Schematic overview of workflow as indicated. (C) Cytokine alterations within the explant model after 24 hr using human PDA tissue samples. Data from five different patients is presented before and after treatment with the small molecule inhibitor of CCR10 (Anti-CCR10). (D) Alteration of the intratumoral CD4+, CD8+ and CD163+ immune cell infiltration within the explant model using human PDA tissue samples. Data from seven different patients is presented before and after treatment with the small molecule inhibitor of CCR10 (Anti-CCR10). The two tissues with the highest absolute number of intratumoral CD4+ cells are indicated in green. PDA= pancreatic ductal adenocarcinoma See also Figure S3.

Article Snippet: The explants were treated with a small molecule inhibitor of CCR10 (BI-6901) from (Boehringer Ingelheim, Germany).

Techniques: Inhibition, Immunofluorescence