Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet: ( A ) For the colitis model, wild-type (WT) mice were gavaged with streptomycin 24 hr prior to oral infection with approximately 1 × 10 9 CFU S. enterica serovar Typhimurium (STm). At 4 days post-infection (dpi), CCL28 in feces was quantified by ELISA. Data shown comprise two independent experiments (uninfected, n = 10; STm, n = 10). Bars represent the mean ± standard deviation (SD). ( B ) STm CFU in the fecal content collected 1–3 dpi, and in the cecal content 3 dpi from WT (filled circles) and Ccl28 −/− (white circles) littermate mice. ( C ) CFU recovered from the Peyer’s patches, mesenteric lymph nodes, spleen, bone marrow, and blood at 3 dpi. Data shown comprise eight independent experiments (WT, n = 24; Ccl28 −/− , n = 18). Some of the spleen data points were published as a preliminary characterization in and are combined with the new dataset. Bars represent the geometric mean, dotted lines represent the limit of detection. ( D ) Representative pseudocolor dot plots of neutrophils (CD11b + Ly6G + cells; gated on live, CD45 + cells) obtained from the gut tissues of uninfected (Naive) and STm-infected WT or Ccl28 −/− mice 2 or 3 dpi, as determined by flow cytometry. ( E ) Frequency of neutrophils in the live CD45 + cells obtained from the gut mucosa of WT (filled circles) or Ccl28 −/− mice (white circles). Naive mouse data shown comprise four independent experiments (WT, n = 14; Ccl28 −/ − , n = 9); 2 dpi data comprise four independent experiments (WT, n = 14; Ccl28 −/− , n = 14); 3 dpi data comprise eight independent experiments (WT, n = 24; Ccl28 −/− , n = 18). Bars represent the geometric mean. ( F ) Relative expression levels (qPCR) of Cxcl1 (CXCL1), Tnfa (TNFα), Ifng (IFNγ), Csf3 (G-CSF), Il1b (IL-1β), and Il17a (IL-17A) in the cecal tissue of STm-infected WT (filled circles, n = 13) or Ccl28 −/− mice (white circles, n = 8), 3 dpi, relative to uninfected control mice. Bars represent the geometric mean. Data shown comprise four independent experiments. ( G–I ) Histopathological analysis of the cecum collected from STm-infected WT or Ccl28 −/− mice, 3 dpi (WT, n = 11; Ccl28 −/− , n = 7). Scale bars indicate 100 µm. ( G ) Sum of the total histopathology score (bars represent the mean; symbols represent individual mice), ( H ) histopathology scores showing the individual analyzed parameters of each mouse (stacked bar height represents the overall score), and ( I ) hematoxylin and eosin (H&E)-stained sections from one representative animal for each group (×200 magnification). For ( B ) and ( C ), CFU data were log-normalized before statistical analysis by Welch’s t test. Mann–Whitney U was used for all other datasets where statistical analysis was performed. A significant difference relative to WT controls is indicated by *p ≤ 0.05, **p ≤ 0.01; ns, not significant.

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation, Flow Cytometry, Expressing, Control, Histopathology, Staining, MANN-WHITNEY

Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet: ( A ) STm CFU in the fecal content collected 1 and 2 dpi, and in the cecal content 2 dpi from wild-type (WT, filled circles) and Ccl28 −/− (white circles) littermate mice. ( B ) STm CFU recovered from the Peyer’s patches, mesenteric lymph nodes, spleen, bone marrow, and blood at 2 dpi. Data shown comprise four independent experiments (WT, n = 14; Ccl28 −/− , n = 13). Bars represent the geometric mean; dotted lines represent the limit of detection. CFU data were log-normalized before statistical analysis by Welch’s t test. A significant difference relative to WT controls is indicated by *p ≤ 0.05, ns, not significant.

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet: ( A, B ) For the bacteremia model, mice were infected by intraperitoneal injection with S . Typhimurium (STm, 1 × 10 3 CFU) or sterile PBS (uninfected control). ( A ) At 4 days post-infection, CCL28 in serum was quantified by ELISA of wild-type (WT) mice (uninfected, n = 7; STm, n = 12). Data shown comprise two independent experiments. Bars represent the mean ± standard deviation (SD). ( B ) STm CFU was determined in the spleen, liver, and blood of WT mice (black squares) and Ccl28 −/− mice (white squares) 4 days after intraperitoneal infection with STm (1 × 10 3 CFU). Data shown comprise two independent experiments (WT, n = 5; Ccl28 −/− , n = 5). Bars represent the geometric mean. ( C, D ) In vitro antimicrobial activity of CCL28 against STm WT, STm ΔphoQ , E. coli K12, and A. baumannii . ( C ) 5 × 10 5 CFU/ml of each strain ( A. baumannii additionally at 5 × 10 8 CFU/ml) was incubated with recombinant murine CCL28 at the indicated concentrations ( n = 4 per group), and CFU were enumerated after 2 hr. ( D ) STm WT (1 × 10 7 CFU/ml) was incubated with recombinant murine CCL28 (50 nM) or CCL11 (25 nM) and CFU were enumerated at 75 min ( n = 4 per group) and 150 min ( n = 6 per group). Bars represent the geometric mean. ( A ) Data were analyzed by Mann–Whitney U relative to uninfected controls. ( B ) CFU data were log-normalized before statistical analysis by Welch’s t test. ( C ) Log-transformed data were analyzed by non-parametric one-way analysis of variance (ANOVA) (Kruskal–Wallis) for independent samples. Dunn’s multiple comparison test was performed to compare bacterial CFU at each time point relative to time zero (control group). Significant changes are indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: Infection, Injection, Sterility, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation, In Vitro, Activity Assay, Incubation, Recombinant, MANN-WHITNEY, Transformation Assay, Comparison

Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet: Flow cytometry quantification of live, CD45 + CD11b − immune cells recovered from WT and Ccl28 −/− mouse gut, blood, and bone marrow, before (Naive) and during STm infection (2 and 3 dpi). Data indicate the relative abundance of B cells ( A ), CD11b − CD3 − CD19 + , CD8 + T cells ( B ), CD11b − CD19 − CD3 + CD8 + CD4 − , and CD4 + T cells ( C ), CD11b − CD19 − CD3 + CD4 + CD8 − as a proportion of total live CD45 + cells profiled from each tissue. Each data point represents measurements from one mouse, with filled points from WT and empty points from Ccl28 −/− mice. Data are derived from the same set of pooled experiments presented in . Bars represent the median. Comparisons between WT and Ccl28 −/− mice were made by Mann–Whitney test on unnormalized data. p-values <0.06 indicated; ns, not significant.

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: Flow Cytometry, Infection, Derivative Assay, MANN-WHITNEY

Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet: Flow cytometry quantification of live, CD45 + CD11b + immune cells recovered from WT and Ccl28 −/− mouse gut, blood, and bone marrow, before (Naive) and during STm infection (2 and 3 dpi). ( A ) Data indicate the relative abundance of neutrophils (CD11b + Ly6G + ) in the blood and bone marrow, as a proportion of total live CD45 + cells profiled. ( B ) Expression of CCR3 by eosinophils isolated from the gut and blood compartments of Naive and STm-infected (3 dpi) mice. The relative abundance of eosinophils ( C , CD11b + Ly6G − SiglecF + side scatter high ), macrophage-like F4/80 + CD11c − cells ( D , CD11b + Ly6G − SiglecF − F4/80 + CD11c − ), and conventional dendritic cell-like CD11c + F4/80 cells ( E , CD11b + Ly6G − SiglecF − CD11c + F4/80 − ), as a proportion of total live CD45 + cells profiled from each tissue. Each data point represents measurements from one mouse, with filled points from WT and empty points from Ccl28 −/− mice. Data are derived from the same set of pooled experiments presented in . Bars represent the median. Comparisons between WT and Ccl28 −/− mice were made by Mann–Whitney test on unnormalized data. ns, not significant.

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: Flow Cytometry, Infection, Expressing, Isolation, Derivative Assay, MANN-WHITNEY

Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet: The levels of myeloperoxidase (MPO; A ) neutrophil elastase ( B ), and S100A9 (a component of the antimicrobial calcium-binding protein calprotectin; C ), were measured by ELISA from the fecal and cecal supernatant of STm-infected WT and Ccl28 −/− littermate mice. Statistical comparisons on data from WT and Ccl28 −/− mice were made by Mann–Whitney test on unnormalized data, with p-values indicated.

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Infection, MANN-WHITNEY

Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet: ( A ) Wild-type (WT) mice (solid black line) and Ccl28 −/− mice (dashed magenta line) were intratracheally infected with approximately 1 × 10 8 CFU Acinetobacter baumannii (Ab) and their survival was determined for 10 days. Data shown comprise two independent experiments (WT, n = 8; Ccl28 −/− , n = 8). ( B–H ) WT mice ( n = 9) and Ccl28 −/− mice ( n = 8) were intratracheally infected with Ab and sacrificed 1 day post-infection (dpi). Data shown comprise three independent experiments. Symbols represent data from individual mice. ( B–D ) Ab CFU were quantified from the BAL (bronchoalveolar lavage) fluid, ( C ) lung tissue, and ( D ) blood in WT (gray symbols) and Ccl28 −/− mice (magenta symbols). Bars represent the geometric mean. ( E ) Representative pseudocolor dot plots of neutrophils (CD11b + Ly6G + cells; gated on live, CD45 + cells) and ( F ) frequency of neutrophils obtained from the BAL, lung, blood, and bone marrow of Ab-infected WT or Ccl28 −/− mice, as determined by flow cytometry. Lines represent the geometric mean. ( G ) The number of live host cells per mL of BAL, determined using an automated cell counter with Trypan Blue counterstain to assess viability, from uninfected WT (Uninf., n = 5), and Ab-infected WT ( n = 9); and Ccl28 −/− mice ( n = 8). Bars represent the geometric mean. ( H ) Relative abundance of different leukocyte populations as a proportion of the live CD45 + cell population was assessed in the BAL. Each bar represents data from one mouse. ( I ) Representative immunofluorescence image of lungs from WT and Ccl28 −/− mice, uninfected or infected with Ab , stained for the neutrophil marker Ly6G (magenta). 4′,6-diamidino-2-phenylindole (DAPI, blue) was used to label nuclei. Scale bars indicate 20 µm. ( J ) Quantification of Ly6G + cells per high-power field (HPF) from immunofluorescence images of lungs from WT mice ( n = 4) and Ccl28 −/− mice ( n = 4). Bars represent the mean ± standard deviation (SD). ( K ) Histopathological analysis of lungs from WT and Ccl28 −/− mice infected with Ab at 1 dpi. Each bar represents an individual mouse. ( L ) Relative expression levels (qPCR) of Cxcl1 (CXCL1), Tnfa (TNFα), Ifng (IFNγ), Csf3 (G-CSF), Il1b (IL-1β), and Il17a (IL-17A) in the lung of WT ( n = 11) or Ccl28 −/− mice ( n = 12) infected with Ab (1 dpi). Bars represent the geometric mean. Data shown comprise three independent experiments. For ( A ), survival curves were statistically compared using a log-rank (Mantel–Cox) test. For ( B–D ), CFU data were log-normalized before analysis by Welch’s t test. For ( F ), ( G ), and ( L ), Mann–Whitney U was used to compare groups with unknown distribution. A significant difference between groups is indicated by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. ns, not significant.

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: Infection, Flow Cytometry, Immunofluorescence, Staining, Marker, Standard Deviation, Expressing, MANN-WHITNEY

Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet: Data indicate the relative abundance of neutrophils ( A , CD11b + Ly6G + ), eosinophils ( B , CD11b + Ly6G − SiglecF + side scatter high ), macrophage-like F4/80 + CD11c − cells ( C ), CD11b + Ly6G − SiglecF − F4/80 + CD11c − , and conventional dendritic cell-like CD11c + F4/80 cells ( D ), CD11b + Ly6G − SiglecF − CD11c + F4/80 − as proportions of total live CD45 + cells in the bronchoalveolar lavage (BAL), lungs, blood, and bone marrow, from uninfected (naive) and 1 day post-inoculation with A. baumannii (Ab) , profiled by flow cytometry. Each data point is a quantification from one mouse, with filled points representing WT and empty points as Ccl28 −/− mice. Data are derived from the same pool of repeated experiments presented in , with additional data from naive mice (blood and BM measurements from naive mice are repeated from and included to ease comparison to Ab infection). Comparisons between WT and Ccl28 −/− mice were made by Mann–Whitney test on unnormalized data. *p ≤ 0.05; ns, not significant.

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: Flow Cytometry, Derivative Assay, Comparison, Infection, MANN-WHITNEY

Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet: Data indicate the relative abundance of B cells ( A , CD11b − CD3 − CD19 + ), CD8 + T cells ( B , CD11b − CD19 − CD3 + CD4 − CD8 + ), and CD4 + T cells ( C ), CD11b − CD19 − CD3 + CD4 + CD8 − , as proportions of total live CD45 + cells in the bronchoalveolar lavage (BAL), lungs, blood, and bone marrow, from uninfected (naive) and 1 dpi with A. baumannii (Ab) , profiled by flow cytometry. Each data point is a quantification from one mouse, with filled points representing WT and empty points as Ccl28 −/− mice. Data are derived from the same pool of repeated experiments presented in , with additional data from naive mice (blood and BM measurements from naive mice are repeated from and included to ease comparison to Ab infection). Comparisons between WT and Ccl28 −/− mice were made by Mann–Whitney test on unnormalized data. ns, not significant.

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: Flow Cytometry, Derivative Assay, Comparison, Infection, MANN-WHITNEY

Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet: The levels of myeloperoxidase (MPO; A ) neutrophil elastase ( B ), and S100A9 ( C ), were measured by ELISA from the supernatant of the bronchoalveolar lavage fluid (BAL) from uninfected WT and Ab-infected WT and Ccl28 −/− littermates. Statistical comparisons on data from WT and Ccl28 −/− mice were made by Mann–Whitney test on unnormalized data, with p-values indicated.

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, MANN-WHITNEY

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: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

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: ( 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: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

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: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet:

Article Snippet: 50 nM of recombinant mouse CCL28, CCL11 (R&D Systems), or CXCL1 (Peprotech) were placed into the lower compartment of a Transwell chamber.

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

Journal: Molecular medicine reports

Article Title: Knockdown of CCL28 inhibits endometriosis stromal cell proliferation and invasion via ERK signaling pathway inactivation.

doi: 10.3892/mmr.2021.12573

Figure Lengend Snippet: Figure 1. CCL28 and CCR10 are highly expressed in the serum and endometrial tissues of patients with EM. Serum samples of 40 patients with EM and 40 healthy patients were collected to detect CCL28 levels, and 15 endometrial tissue samples from patients with EM and 15 endometrial tissues from healthy patients were also collected. (A) CCL28 levels in the serum of patients with EM were detected using ELISA. mRNA expression levels of (B) CCL28 and (C) CCR10 in endometrial tissues of patients with EM were detected by reverse transcription‑quantitative PCR. (D) Protein expression levels of CCL28 and CCR10 in endometrial tissues of patients with EM were detected using western blotting. (E) Protein expression levels of CCL28 and CCR10 in endo‑ metrial tissues of patients with EM were detected by immunohistochemistry (x200, 50 µm). **P<0.01 vs. normal. CCL28, C‑C motif chemokine ligand 28; CCR10, CC chemokine receptor 10; EM, endometriosis.

Article Snippet: Subsequently, samples were incubated with primary antibodies against ccl28 (1:200; cat. no. 18214‐1‐aP; ProteinTech Group, inc.) and ccr10 (1:100; cat. no. 22071‐1‐aP; ProteinTech Group, inc.) for 1 h at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Immunohistochemistry

Journal: Molecular medicine reports

Article Title: Knockdown of CCL28 inhibits endometriosis stromal cell proliferation and invasion via ERK signaling pathway inactivation.

doi: 10.3892/mmr.2021.12573

Figure Lengend Snippet: Figure 2. Knockdown of CCL28 expression in endometrial stromal cells by lentiviral transduction. (A) endometrial stromal cells were identified via immuno‑ cytochemistry, which was used to analyze CK19 and vimentin expression (x200, 50 µm). (B) CD10 and CD90 were detected using flow cytometry to identify the percentage of EM stromal cells. shCCL28‑1, ‑2 and ‑3 were constructed to transduce endometrial stromal cells, CCL28 (C) mRNA and (D) protein expression levels were detected to determine the knockdown efficiency of the constructs. **P<0.01 and ***P<0.001 vs. shNC. Control, cells cultured with medium; shNC, cells infected with negative control lentivirus; shCCL28‑1, ‑2 and ‑3, cells transduced with shCCL28 lentivirus‑1, ‑2 and ‑3; CCL28, C‑C motif chemokine ligand 28; sh, short hairpin RNA; CK19, cytokeratin‑19.

Article Snippet: Subsequently, samples were incubated with primary antibodies against ccl28 (1:200; cat. no. 18214‐1‐aP; ProteinTech Group, inc.) and ccr10 (1:100; cat. no. 22071‐1‐aP; ProteinTech Group, inc.) for 1 h at room temperature.

Techniques: Knockdown, Expressing, Transduction, Immunocytochemistry, Flow Cytometry, Construct, Control, Cell Culture, Infection, Negative Control, shRNA

Journal: Molecular medicine reports

Article Title: Knockdown of CCL28 inhibits endometriosis stromal cell proliferation and invasion via ERK signaling pathway inactivation.

doi: 10.3892/mmr.2021.12573

Figure Lengend Snippet: Figure 3. Knockdown of CCL28 in endometrial stromal cells significantly suppresses cell proliferation and invasion. (A) Following CCL28 knockdown in endometrial stromal cells, cell proliferation was detected using a Cell Counting Kit‑8 assay at 0, 12, 24 and 48 h. (B) Cell invasion at 48 h was detected using a Transwell invasion assay (x200, 50 µm). (C) CCL28 levels in endometrial stromal cell supernatants were detected using an ELISA. (D) mRNA expression levels of CCL28, CCR10, MMP2, MMP9 and ITGB1 were examined using reverse transcription‑quantitative PCR. (E) Activities of MMP2 and MMP9 were detected using gelatinase zymography. (F) Protein expression levels of CCL28, CCR10, MMP2, MMP9 and ITGB1 were detected via western blotting. (G) Protein expression levels of p‑ERK/ERK ratio were detected via western blotting. **P<0.01 and ***P<0.001 vs. shNC. shNC, cells infected with negative control lentivirus; shCCL28‑1 and ‑2, cells transduced with shCCL28 lentivirus‑1 and ‑2; CCL28, C‑C motif chemokine ligand 28; CCR10, CC chemokine receptor 10; ITGB1, integrin β1; sh, short hairpin RNA; p, phosphorylated; OD, optical density.

Article Snippet: Subsequently, samples were incubated with primary antibodies against ccl28 (1:200; cat. no. 18214‐1‐aP; ProteinTech Group, inc.) and ccr10 (1:100; cat. no. 22071‐1‐aP; ProteinTech Group, inc.) for 1 h at room temperature.

Techniques: Knockdown, CCK-8 Assay, Transwell Invasion Assay, Enzyme-linked Immunosorbent Assay, Expressing, Zymography, Western Blot, Infection, Negative Control, Transduction, shRNA

Journal: Molecular medicine reports

Article Title: Knockdown of CCL28 inhibits endometriosis stromal cell proliferation and invasion via ERK signaling pathway inactivation.

doi: 10.3892/mmr.2021.12573

Figure Lengend Snippet: Figure 4. Treatment with CCL28 recombinant protein increases CCL28 and CCR10 expression in healthy human endometrial stromal cells. (A) Healthy human endometrial stromal cells were identified using immunocytochemistry to analyze CK19 and vimentin expression (x200, 50 µm). (B) CD10 and CD90 were detected via flow cytometry to identify the percentage of healthy endometrial stromal cells. Healthy human endometrial stromal cells were then treated with CCL28 recombinant protein at concentrations of 0, 5, 10, 20 and 40 ng/ml. (C) Cell proliferation was detected using a Cell Counting Kit‑8 assay to determine the effect of CCL28 recombinant protein. Subsequently, at 48 h after CCL28 recombinant protein treatment, the mRNA expression levels of (D) CCL28 and (E) CCR10 were detected using reverse transcription‑quantitative PCR. (F) Relative protein expression levels of CCL28 and CCR10 were analyzed via western blotting at 48 h after CCL28 recombinant protein treatment. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 ng/ml; and #P<0.05 and ##P<0.01 vs. 10 ng/ml. CCL28, C‑C motif chemokine ligand 28; CCR10, CC chemokine receptor 10; CK19, cytokeratin‑19; OD, optical density.

Article Snippet: Subsequently, samples were incubated with primary antibodies against ccl28 (1:200; cat. no. 18214‐1‐aP; ProteinTech Group, inc.) and ccr10 (1:100; cat. no. 22071‐1‐aP; ProteinTech Group, inc.) for 1 h at room temperature.

Techniques: Recombinant, Expressing, Immunocytochemistry, Flow Cytometry, CCK-8 Assay, Western Blot

Journal: Molecular medicine reports

Article Title: Knockdown of CCL28 inhibits endometriosis stromal cell proliferation and invasion via ERK signaling pathway inactivation.

doi: 10.3892/mmr.2021.12573

Figure Lengend Snippet: Figure 5. CCL28 may contribute to endometriosis progression by regulating MMP2, MMP9 and ITGB1 expression via activating the ERK signaling pathway. Healthy endometrial stromal cells were pre‑treated with 10 µmol/l PD98059 (ERK inhibitor) for 30 min, and then treated with 20 ng/ml CCL28 recom‑ binant protein for 48 h. (A) Cell proliferation was detected using the Cell Counting Kit‑8 assay at 0, 12, 24 and 48 h. (B) Cell invasion was detected using the Transwell invasion assay at 48 h (x200, 50 µm). (C) MMP2 and MMP9 activity was detected via gelatinase zymography. Relative protein expression levels of (D) MMP2, MMP9 and ITGB1 and (E) the p‑ERK/ERK ratio were analyzed via western blotting. **P<0.01, ***P<0.001 vs. vehicle + DMSO; and ###P<0.001 vs. CCL28 + vehicle. Vehicle, solvent of CCL28 recombinant proteins; DMSO, solvent of PD98059; CCL28, C‑C motif chemokine ligand 28; ITGB1, integrin β1; p, phosphorylated; OD, optical density.

Article Snippet: Subsequently, samples were incubated with primary antibodies against ccl28 (1:200; cat. no. 18214‐1‐aP; ProteinTech Group, inc.) and ccr10 (1:100; cat. no. 22071‐1‐aP; ProteinTech Group, inc.) for 1 h at room temperature.

Techniques: Expressing, CCK-8 Assay, Transwell Invasion Assay, Activity Assay, Zymography, Western Blot, Solvent, Recombinant

Journal: Biology of reproduction

Article Title: Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

doi: 10.1095/biolreprod.116.141903

Figure Lengend Snippet: FIG. 1. Expression of CCL28 in the porcine uterine endometrium during the estrous cycle and pregnancy. A) Real-time RT-PCR analysis of CCL28 mRNA in the uterine endometrium. Endometrial tissue samples from cyclic and pregnant gilts were analyzed by real-time RT-PCR, and the data are reported as expression relative to that detected on Day 12 of the estrous cycle after normalization of the transcript amount to the endogenous RPL7 control. B) In situ hybridization analysis of CCL28 mRNA in the uterine endometrium. Expression of CCL28 mRNA was localized mainly to endometrial GE cells during the estrous cycle and pregnancy. Representative uterine sections from Day 12 of pregnancy, hybridized with a DIG-labeled sense CCL28 cRNA probe (Sense) as a negative control, are shown. D, day; C, estrous cycle; P, pregnancy; LE, luminal epithelium; GE, glandular epithelium; St, stroma; BV, blood vessels. Bars ¼ 200 lm and 100 lm in inset. C) Immunoblot analysis of the CCL28 protein in uterine flushings on Day 12 of the estrous cycle and pregnancy. Uterine flushings were obtained from Day (D) 12 of the estrous cycle (C) and pregnancy (P) and the presence of the CCL28 protein was determined. We loaded rCCL28 as a positive control.

Article Snippet: Fifty-five micrograms of protein from concentrated uterine flushings and 0.5 lg of recombinant human CCL28 protein (rCCL28; R&D Systems) as a control were loaded into and electrophoresed on 12% SDSPAGE gels followed by electrotransfer onto nitrocellulose membranes.

Techniques: Expressing, Quantitative RT-PCR, Control, In Situ Hybridization, Labeling, Negative Control, Western Blot, Positive Control

Journal: Biology of reproduction

Article Title: Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

doi: 10.1095/biolreprod.116.141903

Figure Lengend Snippet: FIG. 4. Expression of CCR3 and CCR10 in conceptuses and expression of CCR10 in chorioallantoic membranes. A) RT-PCR analyses of CCL28, CCR3, and CCR10 mRNAs in the uterine endometrium and conceptuses at Days 12 and 15 of pregnancy. Expression of CCL28 and CCR10, but not CCR3, mRNAs was detectable in conceptuses on Days 12 and 15 of pregnancy. RPL7 was used as a positive control. RTase þ/, with (þ) or without () reverse transcriptase; M, molecular marker; D12 Endo, endometrium from Day 12 of pregnancy; D15 Endo, endometrium from Day 15 of pregnancy; D12 Con, Day 12 conceptus; D15 Con, Day 15 conceptus. B) Immunohistochemical analysis of the CCR10 protein in a Day 12 conceptus. CCR10 protein was detected in conceptus trophec- toderm derived from Day 12 of pregnancy. D12P, Day 12 of pregnancy; Tr, trophectoderm; En, endoderm. Bar¼ 50 lm. C) Real-time RT-PCR analysis of the expression of CCR10 mRNA in porcine chorioallantoic tissues during pregnancy. Analysis of the expression of CCR10 mRNA in chorioallantoic tissue samples on Days 30, 60, 90, and 114 of pregnancy revealed that the expression of CCR10 mRNA increased during late pregnancy (linear effect of day, P , 0.01). The data are reported as expression relative to that detected on Day 30 of pregnancy after normalization of the transcript amount to the endogenous RPL7 control, and are presented as least-squares means with SEM.

Article Snippet: Fifty-five micrograms of protein from concentrated uterine flushings and 0.5 lg of recombinant human CCL28 protein (rCCL28; R&D Systems) as a control were loaded into and electrophoresed on 12% SDSPAGE gels followed by electrotransfer onto nitrocellulose membranes.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Reverse Transcription, Marker, Immunohistochemical staining, Derivative Assay, Quantitative RT-PCR, Control

Journal: Biology of reproduction

Article Title: Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

doi: 10.1095/biolreprod.116.141903

Figure Lengend Snippet: FIG. 5. Effect of CCL28 on pTr cell proliferation and migration. A) RT-PCR analysis of CCR3 and CCR10 mRNAs in the pTr cell line. The pTr cell line and PBMC as a control expressed CCR10 mRNA, but pTr cells did not express CCR3 mRNA. RPL7 was used as a positive control for RT-PCR. RTase þ/, with (þ) or without () reverse transcriptase; M, molecular marker; D12 Endo, endometrium from Day 12 of pregnancy. B) Effect of rCCL28 on pTr cell proliferation. After serum starvation for 24 h, pTr cells were treated with 0, 0.1, 1, 10, or 100 ng/ml rCCL28 at 378C for 48 h in triplicate. C) Effect of rCCL28 on pTr cell migration. Serum-starved pTr cells were seeded on 8-lm-pore Transwell inserts and treated with different doses (0, 10, or 100 ng/ml) of rCCL28 or 1 lg/ml of rSPP1 as a positive control in triplicate for 12 h. Unmigrated cells on the upper side of the inserts were removed, and cells that had migrated were counted systematically in five nonoverlapping locations. Each independent experiment for cell proliferation and migration was replicated three times. The asterisks denote statistically significant differences (*P , 0.05; **P , 0.01).

Article Snippet: Fifty-five micrograms of protein from concentrated uterine flushings and 0.5 lg of recombinant human CCL28 protein (rCCL28; R&D Systems) as a control were loaded into and electrophoresed on 12% SDSPAGE gels followed by electrotransfer onto nitrocellulose membranes.

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Control, Positive Control, Reverse Transcription, Marker

Journal: Aging (Albany NY)

Article Title: CCL28 promotes locomotor recovery after spinal cord injury via recruiting regulatory T cells

doi: 10.18632/aging.102239

Figure Lengend Snippet: CCL28 is upregulated in the spinal cord after SCI. ( A , D ) qRT-PCR analysis of CCL28 mRNA level (A), ELISA analysis of CCL28 protein concentration ( B ), and Western blotting analysis of CCL28 protein expression ( C ) and band intensity analysis of CCL28 ( D ) in the spinal cord at different time points after sham or SCI surgery (n=5). GAPDH was used as a reference or loading control. Data are mean ± SD. Data were compared with sham group and statistical analysis was performed using Student’s t -test. **, P<0.01; *, P<0.05; NS, not significant.

Article Snippet: Neutralizing antibodies against mouse IL-1β (AF401), TNF-α (AF410), CCL28 (MAB533), CCR10 (MAB2815), CCR3 (MAB1551), CD25 (AF2438) and IgG isotype control (43414), and recombinant mouse CCL28 (533-VI) were purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Protein Concentration, Western Blot, Expressing, Control

Journal: Aging (Albany NY)

Article Title: CCL28 promotes locomotor recovery after spinal cord injury via recruiting regulatory T cells

doi: 10.18632/aging.102239

Figure Lengend Snippet: IL-1β and TNF-α upregulate CCL28 through activating NF-κB after SCI. ( A , B ) Mice were pre-injected with control antibody (Ctrl Ab) or neutralizing antibodies against IL-1β (anti-IL-1β) and/or TNF-α (anti-TNF-α) into the intraspinal cord for 12 hrs, and then subjected to sham or SCI surgery. After another 12 hrs, the spinal cord samples were analyzed by qRT-PCR and Western blotting for detecting CCL28 mRNA level ( A ) and protein level ( B ) (n=5). ( C , D ) Mice were pre-injected with equal volume of vehicle or 60 mg/kg ML120B into the intraspinal cord for 12 hrs, and then subjected to sham or SCI surgery. After another 12 hrs, the CCL28 mRNA level ( C ) and protein level ( D ) in the spinal cord was analyzed as in ( A , B ) (n=5). GAPDH was used as a reference or loading control. Data are mean ± SD. The statistical analysis was performed using Student’s t -test. **, P<0.01; NS, not significant.

Article Snippet: Neutralizing antibodies against mouse IL-1β (AF401), TNF-α (AF410), CCL28 (MAB533), CCR10 (MAB2815), CCR3 (MAB1551), CD25 (AF2438) and IgG isotype control (43414), and recombinant mouse CCL28 (533-VI) were purchased from R&D Systems.

Techniques: Injection, Control, Quantitative RT-PCR, Western Blot

Journal: Aging (Albany NY)

Article Title: CCL28 promotes locomotor recovery after spinal cord injury via recruiting regulatory T cells

doi: 10.18632/aging.102239

Figure Lengend Snippet: Spinal cord recruits Treg cells through CCL28-CCR10 axis after SCI. ( A ) Mouse peripheral blood mononuclear cells (PBMCs) were seeded in the upper chambers and pretreated with control antibody (Ctrl Ab), neutralizing antibodies against CCL28, CCR10 or CCR3 for 1 hr. The percentage of CD4 + CD25 + FOXP3 + Treg cells among the CD4 + cells recruited to the lower chambers with medium containing mouse recombinant CCL28 (rMCCL28) or 1% mouse control serum was analyzed by flow cytometry (n=6 replicates in each group). ( B ) Mice were pre-injected with Ctrl Ab or neutralizing antibodies against CCL28 (anti-CCL28), CCR10 (anti-CCR10) or CCR3 (anti-CCR3) into the intraspinal cord for 12 hrs, and then subjected to sham or SCI surgery. After another 12 hrs, the percentage of CD4 + CD25 + FOXP3 + Treg cells in the spinal cord was determined by flow cytometry analysis (n=5). ( C ) Mice were pre-injected with Ctrl Ab, anti-CCL28 or anti-CCR10 and rMCCL28 or 1% mouse control serum as indicated into the intraspinal cord for 12 hrs, and then subjected to sham or SCI surgery. After another 12 hrs, the percentage of CD4 + CD25 + FOXP3 + Treg cells in the spinal cord were determined (n=5). Data are mean ± SD. The statistical analysis was performed using Student’s t -test. **, P<0.01; NS, not significant.

Article Snippet: Neutralizing antibodies against mouse IL-1β (AF401), TNF-α (AF410), CCL28 (MAB533), CCR10 (MAB2815), CCR3 (MAB1551), CD25 (AF2438) and IgG isotype control (43414), and recombinant mouse CCL28 (533-VI) were purchased from R&D Systems.

Techniques: Control, Recombinant, Flow Cytometry, Injection

Journal: Aging (Albany NY)

Article Title: CCL28 promotes locomotor recovery after spinal cord injury via recruiting regulatory T cells

doi: 10.18632/aging.102239

Figure Lengend Snippet: CCL28 promotes locomotor recovery after SCI through recruiting Treg cells. ( A , B ) Mice were pre-injected with Ctrl Ab or anti-CCL28 into the intraspinal cord for 12 hrs, and then subjected to sham or SCI surgery. The injection of Ctrl Ab or anti-CCL28 was repeated at 14 days after injury. The locomotion recovery was monitored using the BMS open-field test to determine locomotor capabilities ( A ) and the percentage of Treg cells at 28 days after injury was assessed by FACS analysis (n=8). Data are mean ± SEM. Data were analyzed by repeated measures analysis of variance (ANOVA). ** P<0.01 represents the comparison between SCI + Ctrl Ab group and SCI + anti-CCL28 group. ( C ) Mice were pre-injected with Ctrl Ab, anti-CCR10, anti-CD25, rMCCL28 or 1% mouse control serum as indicated into the intraspinal cord for 12 hrs, and then subjected to sham or SCI surgery. The injection of Ctrl Ab, anti-CCR10, anti-CD25, rMCCL28 or 1% mouse control serum was repeated at 14 days after injury. The locomotion recovery was monitored (n=8). Data are mean ± SEM. Data were analyzed by repeated measures analysis of variance (ANOVA). ** P<0.01 represents the comparison between SCI + Ctrl serum group and SCI + rMCCL28. # P<0.01 represents the comparison between SCI + rMCCL28 + Ctrl Ab group and SCI + rMCCL28 + anti-CCR10 group. & P<0.01 represents the comparison between SCI + rMCCL28 + Ctrl Ab group and SCI + rMCCL28 + anti-CD25. ( D ) The percentage of CD4 + CD25 + FOXP3 + Treg cells in the spinal cord from SCI + rMCCL28 + Ctrl Ab group and SCI + rMCCL28 + anti-CD25 group was determined (n=8). Data are mean ± SD. The statistical analysis was performed using Student’s t -test. **, P<0.01.

Article Snippet: Neutralizing antibodies against mouse IL-1β (AF401), TNF-α (AF410), CCL28 (MAB533), CCR10 (MAB2815), CCR3 (MAB1551), CD25 (AF2438) and IgG isotype control (43414), and recombinant mouse CCL28 (533-VI) were purchased from R&D Systems.

Techniques: Injection, Comparison, Control

Journal: Aging (Albany NY)

Article Title: CCL28 promotes locomotor recovery after spinal cord injury via recruiting regulatory T cells

doi: 10.18632/aging.102239

Figure Lengend Snippet: Treg cells mediate immune suppression in the spinal cord after SCI. ( A ) Mice were treated as in . ELISA analysis of cytokine concentration in the spinal cord at 7 days after sham or SCI surgery (n=5). ( B , C ) Mice were treated as in . ( B ) The cytokine concentration in the spinal cord at 7 days after sham or SCI surgery were determined as in ( A ) (n=5). ( C ) The proliferation rate of effector T cells was determined by [ 3 H]-thymidine incorporation analysis (n=5). c.p.m., counts per minute of incorporated [ 3 H]-thymidine. Data are mean ± SD. The statistical analysis was performed using Student’s t -test. **, P<0.01; *, P<0.05. ( D ) A brief schematic model of this study. After SCI, inflammatory cells infiltrate into the spinal cord and secrete cytokines, including IL-1β and TNF-α, which promptly induces the production of CCL28 via NF-κB activation. Responding to increased CCL28 in the focal sites, CCR10-expressing Treg cells are recruited and then exert their immune suppressive activities, restricting the inflammation to a controllable extent along with the time consumed. Owing to the activity of Treg cells recruited by CCL28, the local levels of IL-1β and TNF-α are decreased, thereby in turn relieving the stimulative effect on CCL28 upregulation, through this negative feed-back loop, CCL28 functions to suppress inflammation, reduce secondary damage and promotes locomotor recovery after SCI.

Article Snippet: Neutralizing antibodies against mouse IL-1β (AF401), TNF-α (AF410), CCL28 (MAB533), CCR10 (MAB2815), CCR3 (MAB1551), CD25 (AF2438) and IgG isotype control (43414), and recombinant mouse CCL28 (533-VI) were purchased from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Activation Assay, Expressing, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Invasion of Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β (mean ± SEM, n = 3). *P < 0.05 vs. cells without CCL28 and TGF-β; #P < 0.05, ##P < 0.005 vs. TGF-β–only–treated cells by 1-way ANOVA with multiple-comparisons test. (B) Invasion of Ca9.22 and YD10B OSCC cells with CCL28 and/or TGF-β into the CAMs of fertilized eggs (mean ± SEM, n = 3). Representative images of CAM. Scale bars: 100 μm. Cells invaded into the mesoderm layer of CAMs are quantified by the mean fluorescence. *P < 0.05, **P < 0.01 vs. cells without CCL28 and TGF-β; #P < 0.05, ##P < 0.001 vs. TGF-β–only–treated cells by 1-way ANOVA with multiple-comparisons test. (C) Expression levels and cellular localization of E-cadherin and β-catenin in Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β. Representative immunofluorescence images. Scale bars: 100 μm. (D) Expression levels of E-cadherin, β-catenin, and EMT-regulating transcription factors in Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β. (E) Cytosolic and nuclear β-catenin levels in Ca9.22 and YD10B OSCC cells treated with CCL28 and/or TGF-β. (D and E) Representative Western blot images.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Fluorescence, Expressing, Immunofluorescence, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Invasion of CCL28-knockdown OSCC cells. (B) Invasion of CCR3-knockdown OSCC cells. (C) Invasion of CCR10-knockdown OSCC cells. (A–C) OSCC cells were transduced with lentiviral particles with control shRNAs or 3 different shRNAs targeting CCL28, CCR10, or CCR3. Knockdown of CCL28, CCR10, or CCR3 in transduced cells was confirmed by Western blotting (top panels). Cell invasion is quantified as the number of invaded cells per field (mean ± SEM, n = 3). *P < 0.05, **P < 0.005 vs. control shRNA–transfected cells without CCL28; #P < 0.05, ##P < 0.01 vs. CCL28-, CCR3-, or CCR10-specific shRNA–transfected cells without CCL28 by 1-way ANOVA with multiple-comparisons test. (D) Invasion of CCL28- or CCR10-knockdown OSCC cells labeled with CFDA-SE and then suspended in a DMEM/Matrigel (4:1) mixture on the CAMs of fertilized eggs (mean ± SEM, n = 3). Representative images of CAM. Scale bars: 100 μm. Cells invaded into the mesoderm layer are quantified by the mean fluorescence. *P < 0.05 versus control shRNA–transfected cells without CCL28; #P < 0.01 vs. CCL28- or CCR10-knockdown cells without CCL28 by 1-way ANOVA with multiple-comparisons test. (E) CCL28, CCR3, or CCR10 mRNA levels in normal and HNSCC tissues. The data were obtained from the TCGA database. Box plots show the median and interquartile range. *P < 0.0001 vs. normal tissue by 2-tailed Student’s t test. (F) Kaplan-Meier survival curves for HNSCC patients with high or low expression of CCL28, CCR3, or CCR10 mRNA by the log-rank test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Knockdown, Transduction, Control, Western Blot, shRNA, Transfection, Labeling, Fluorescence, Expressing

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Representative pathway reporter array (n = 2) for wild-type and CCR10-knockdown (KD) OSCC cells in the absence or presence of CCL28 (20 ng/mL). Reporter gene activities in CCL28-treated cells were normalized by those in untreated cells and represented as fold changes. (B) Correlations between CCL28 mRNA expression and RARβ mRNA expression in patients with HNSCC by Pearson’s correlation analysis. Scatter plots represent normalized RSEM values for each gene. (C) RARβ and RARβ2 expression in response to CCL28 treatment (20 pg/mL) in Ca9.22, YD10B, HSC2, or HSC3 OSCC cells. (D) RARβ and RARβ2 expression in CCL28-overexpressing or CCL28-knockdown Ca9.22 or YD10B OSCC cells. (E) RARβ expression in response to CCL28 treatment (20 pg/mL) in CCR3- or CCR10-downregulated Ca9.22 or YD10B OSCC cells. (C–E) Representative Western blot images. (F) Invasion of OSCC cells treated with the RARβ-selective antagonist LE135 or the inverse pan-RAR agonist BMS493 in the presence of CCL28 (20 pg/mL) (mean ± SEM, n = 3). *P < 0.05 and **P < 0.005 versus CCL28-untreated cells; #P < 0.05 and ##P < 0.01 versus CCL28-only-treated cells by 1-way ANOVA with multiple-comparisons test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) RARβ and RARβ2 expression levels in OSCC cells treated with CCL28 (20 pg/mL) and/or the selective RARα antagonist ER50891 (ER). (B) Invasion of OSCC cells treated with CCL28 (20 pg/mL) and/or the selective RARα antagonist ER50891 (ER) (mean ± SEM, n = 3). *P < 0.001 versus CCL28-untreated control cells; #P < 0.005 and ##P < 0.001 versus CCL28-only-treated cells by 1-way ANOVA with multiple-comparisons test. (C) Interaction between RARα and HDACs or DNMT in OSCC cells treated with CCL28 (20 pg/mL). Immune complexes were obtained using a Pierce Co-IP kit. (A and C) Representative Western blot images. (D) Acetylated histone H3 levels and HDAC1 interaction at the RARB promoter region of OSCC cells treated with CCL28 (20 pg/mL). Histone modification (H3K9ac) and HDAC1 binding were analyzed by ChIP-qPCR. Data are presented as the percentage of the total chromatin input (% input), and graphs are representative.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Expressing, Control, Co-Immunoprecipitation Assay, Western Blot, Modification, Binding Assay, ChIP-qPCR

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) RANKL and OPG levels secreted by CCL28-treated OSCC cells into the culture media, and the RANKL/OPG ratio (mean ± SEM, n = 3). *P < 0.05 vs. CCL28-untreated cells by 2-tailed Student’s t test. (B) RANKL levels secreted by OSCC cells treated with the selective RARα antagonist ER50891 or the RARβ antagonist LE135 in the presence of CCL28 (mean ± SEM, n = 3). *P < 0.05 versus CCL28-untreated cells; #P < 0.05 versus CCL28-only-treated cells by 1-way ANOVA with multiple comparisons test. (C) RANKL and OPG levels secreted by CCL28-treated osteoblasts into the culture media, and the RANKL/OPG ratio (mean ± SEM, n = 3). *P < 0.05 and **P < 0.01 versus CCL28-untreated cells by 1-way ANOVA with multiple comparisons test. (D) Secreted levels of RANKL and OPG by CCL28-treated osteoblasts in the presence of conditioned media (CM) from OSCC cell lines, and the RANKL/OPG ratio (mean ± SEM, n = 3). #P < 0.05 and ##P < 0.01 versus control cells without CM; *P < 0.05 versus CM-only-treated cells by 1-way ANOVA with multiple-comparisons test. (E) Osteoclast formation in CCL28-treated BMMs in the presence of RANKL (mean ± SEM, n = 3). Representative images at ×100 original magnification. *P < 0.05 versus RANKL-only-treated cells by 1-way ANOVA with multiple comparisons test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Control

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: CCL28 was intraperitoneally administered to mice subcutaneously injected with Ca9.22 OSCC cells in the calvaria (n = 5 for control and n = 10 for experimental groups). (A) Tumor size (mean ± SEM). #P < 0.001 versus vehicle-treated mice by 1-way ANOVA with multiple comparisons test. (B) Representative CT 3D images of calvarial osteolytic lesions. (C) Bone morphometric parameters BV/TV and BS/TV (mean ± SEM). (D) Serum levels of bone turnover markers (mean ± SEM). (E) Representative images of H&E and TRAP staining in calvarial tissue sections. Scale bars: 100 μm. (F) Oc.S/BS determined from TRAP staining as the percentage of bone surface in contact with osteoclasts (mean ± SEM). (C, D, and F) #P < 0.05, ##P < 0.01, and ###P < 0.005 versus control mice; *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple comparisons test. (G) Ki67, CD31, and RAR expression levels in calvarial tumor tissues of OSCC-injected mice. Left panel: Representative images of immunohistochemically stained tumor tissues. Scale bars: 100 μm. Graph shows quantified data. *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple-comparisons test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Injection, Control, Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: CCL28 was intraperitoneally administered to mice injected with YD10B OSCC cells into the bone marrow of the right tibia (n = 5 for control and n = 7 for experimental groups). (A) Representative CT 3D images of osteolytic lesions in the tibia. (B) Bone morphometric parameters (mean ± SEM). (C) Serum levels of bone turnover markers (mean ± SEM). (D) Representative images of H&E and TRAP staining in tibial tissue sections. Scale bars: 100 μm. (E) Tumor area determined from H&E staining as the percentage of the total tumor area per tissue area. (F) Oc.S/BS determined from TRAP staining as the percentage of bone surface in contact with osteoclasts (mean ± SEM). (B, C, E, and F) #P < 0.05, ##P < 0.01, and ###P < 0.005 versus control mice; *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple comparisons test. (G) Ki67, CD31, and RARβ expression levels in tibial tumor tissues of OSCC-injected mice. Left panel: Representative images of immunohistochemically stained tumor tissues. Scale bars: 100 μm. Right panel: Ki67-positive cells, CD31-positive vessels, and RARβ-positive cells were counted in tumor tissues. *P < 0.05 and **P < 0.01 versus OSCC cell–injected mice by 1-way ANOVA with multiple comparisons test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Injection, Control, Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: CCL28-induced RAR β expression inhibits oral squamous cell carcinoma bone invasion

doi: 10.1172/JCI125336

Figure Lengend Snippet: (A) Representative images of IHC staining of CCL28, CCR3, CCR10, and RARβ in normal oral mucosa and OSCC tissues. Scale bars: 100 μm. Magnified images of the boxed area are shown in the insets. Scale bars: 20 μm. (B) Frequency of histoscores in normal oral mucosa and OSCC tissues. (C) Kaplan-Meier survival curve of patients with OSCC stratified based on CCL28, CCR3, CCR10, or RARβ expression by the log-rank test.

Article Snippet: CCL28, RANKL, or OPG levels in cell culture media were measured with commercially available kits for CCL28 (BioLegend), RANKL (EIAab), or OPG (Boster) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Expressing

Journal: Mucosal immunology

Article Title: Th22 cells are efficiently recruited in the gut by CCL28 as an alternative to CCL20 but do not compensate for the loss of Th17 cells in treated HIV-1-infected individuals.

doi: 10.1038/s41385-020-0286-6

Figure Lengend Snippet: Fig. 2 Expression of CCL28 by enterocytes in the duodenal mucosa. CCL28 production in the duodenal mucosa of treated HIV- 1-infected (n = 10) and uninfected individuals (n = 10). Chemokine expression was quantified per surface unit of epithelium using NIS- element (Nikon). CCL28 (red) was stained by immunohistochemistry on an ApoTome (Zeiss, original magnification ×63). Cell nuclei were counterstained (DAPI, blue). Representative treated HIV-1-infected and uninfected individuals are shown. Groups were compared with the Wilcoxon’s rank-sum test. Median bars are shown.

Article Snippet: Measure of the proliferation and apoptosis rates of Th17 and Th22 cells Th17 and Th22 cells were cultured overnight in presence of CCL20 and/or CCL28 recombinant proteins (R&D Systems) and stained with Annexin V-FITC and Propidium Iodide (556547, BD Biosciences) to measure apoptosis, and with anti-Ki67-FITC (556026, BD Biosciences) to measure proliferation, by flow cytometry.

Techniques: Expressing, Infection, Staining, Immunohistochemistry

Journal: Mucosal immunology

Article Title: Th22 cells are efficiently recruited in the gut by CCL28 as an alternative to CCL20 but do not compensate for the loss of Th17 cells in treated HIV-1-infected individuals.

doi: 10.1038/s41385-020-0286-6

Figure Lengend Snippet: Fig. 3 Impact of the interactions between IL-17A/Th17 cells and IL-22/Th22 cells on CCL20 and CCL28 expression by enterocytes. Effect of IL-17A (white bars) and IL-22 (gray bars) on a CCL20 and b CCL28 mRNA expression by enterocytes. Monolayers of differentiated human primary enterocytes on transwell inserts were stimulated by 0.5, 5, and 50 ng/mL of cytokine. CCL20 and CCL28 mRNA was quantified in the enterocytes by qRT-PCR. CCL20 and CCL28 expression following cytokine stimulation was normalized relatively to the expression in unstimulated epithelial cells (set to 1) and expressed as fold change (log2 scale). Presented data were obtained from at least eight independent experiments performed with different donors. Cuzick’s test for trend was used to compare chemokine expression across the increasing concentrations of cytokines (the corresponding P value is shown on the line above the IL17-A and IL-22 bars); paired Wilcoxon’s test was used to compare chemokine expression upon stimulation vs. unstimulated condition (intradonor pairing); P value is shown above each bar; *P < 0.05; **P < 0.01. Means and SEM are shown. c Effect of Th22:IEC coculture on CCL20 and CCL28 mRNA expression by enterocytes. Enterocytes were cocultured with FACS-sorted Th22 or Th17 cells, added in the bottom chamber for 15 h. CCL20 and CCL28 mRNA were quantified in the enterocytes by qRT-PCR. CCL20 and CCL28 expression in Th22:IEC coculture was normalized relatively to the expression in Th17:IEC coculture (set to 1) and expressed as fold change (log2 scale). Enterocytes production of d CCL20 and e CCL28 proteins in Th22:IEC and Th17:IEC cocultures. CCL20 and CCL28 were quantified in the bottom chamber by ELISA. Presented data were obtained from ten independent experiments performed with different donors. Paired Wilcoxon’s test was used to compare CCL20 and CCL28 in Th22:IEC vs. Th17:IEC cocultures (intradonor pairing); *, P < 0.05. IEC, intestine epithelial cells. Means and SEM are shown.

Article Snippet: Measure of the proliferation and apoptosis rates of Th17 and Th22 cells Th17 and Th22 cells were cultured overnight in presence of CCL20 and/or CCL28 recombinant proteins (R&D Systems) and stained with Annexin V-FITC and Propidium Iodide (556547, BD Biosciences) to measure apoptosis, and with anti-Ki67-FITC (556026, BD Biosciences) to measure proliferation, by flow cytometry.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: CCL28 expression is upregulated after anti-angiogenesis therapy by hypoxia-sensitive transcription factor CEBPB in lung adenocarcinoma

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Tumor-derived CCL28 recruits pericytes to promote vascular normalization in the tumor microenvironment

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Tumor-derived CCL28 promotes the expression of angiopoietin-1 via CCR3 in pericytes

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Retinoic acid signaling is activated by CCL28 in pericytes through CCR3 A , Volcano plot of changes in metabolic pathways after CCL28 stimulation. B Volcano plot of the enrichment of gene expression after CCL28 stimulation. C Diagram of the metabolic conversion process in the retinoic acid metabolic signaling pathway. D and E Expression of RDH13 and DHRS11 detected by qPCR and western blot with or without exogenous supplement of CCL28. F Correlation of expression of CCL28 with RDH13 in lung adenocarcinoma. G The protein level of DHRS11 and RDH13 stimulated with or without CCL28 and CCR3 neutralizing antibody in pericytes (left) and gray value was calculated(right). H Knockdown efficiency of RDH13 was confirmed by qPCR. I and J Relative expression of RXRα and ANGPT1 after knockdown of RDH13 with or without stimulation of CCL28. K Representative immunofluorescence images of PAN-CK, NG2, CCL28 with DHRS11 or RDH13 or Angiopoietin-1 on biopsy tissues from lung cancer patients (left panel). Scale bar = 100 μm. The correlation between the expression of CCL28 and the levels of DHRS11, RDH13, and angiopoietin-1 (right panel). Data with error bars are shown as mean ± SEM. Each symbol represents data from a replicate. Each panel is a representative experiment of at least three independent biological replicates. *, **, *** represent p < 0.05, p < 0.01 and p < 0.001, respectively. Abbreviation: MFI, Mean fluorescence intensity

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Expressing, Western Blot, Knockdown, Immunofluorescence, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Both CCL28 and retinoic acid could promote vascular normalization in vivo

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: In Vivo

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: CCL28 is involved in bevacizumab-mediated vascular normalization

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: A schematic diagram of tumor microenvironment modulation effects of CCL28

Article Snippet: Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A–B ) cDNA was isolated from a panel of human pancreatic cancer cell lines, Panc1, MiaPaCa2, Capan2, Capan1, HPAFII, and Hs766t, and screened by RT-PCR for expression of CCR (A), CXCR, CX 3 CR, or XCR (B) family of receptors. C–D ) The same panel of human cell lines was probed for expression of the known ligands for CCR10 (C) and CXCR6 (D). E ) RT-PCR analysis of patient-derived pancreatic cells (MCW PDAC cell lines) as well as a human pancreatic epithelial nestin-expressing (HPNE) confirmed CXCR6-CXCL16 and CCR10-CCL28 transcript expression. F ) Pancreatic tumor cells derived from the KPC murine model exhibited varying levels of the ligands and receptors. GAPDH and actin were analyzed as loading controls. Regions of the MBL and NRAMP genes were assessed as genomic DNA controls. Positive control was RNA from PBMCs.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Positive Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A ) Normal pancreatic tissue was sectioned and processed for histopathologic analysis for CCL28, CCR10 and CK19. The arrowhead denotes the epithelial cell lining. B) Tissue from pancreatic tumor was sectioned and processed for immunohistochemical staining with antibodies specific for CCL28, CCR10, CK19, α-SMA as well as H&E, Masson’s trichrome (3C) or Movat’s pentachrome (5C). ‘T’ denotes the tumor cells and ‘S’ denotes the stromal compartments of the tissue section as defined by CK19, α-SMA and trichrome staining. C–D ) Representative stained tissue was scored by an investigator blinded to the tissue or antibody source for staining intensity. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Tissue staining shown in A and B representative of tissues sections from 14 normal, 12 PanIN, and 12 PDAC patients, based on clinical diagnosis and examination by a board-certified pathologist.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunohistochemical staining, Staining, Biomarker Discovery

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A) Sections of pancreatitis tissue were processed for immunostaining of CCR10 and CCL28, CXCR6, and CXCL16. B–C) . Scoring for the number of ductal (CK19+) or stromal (SMA+) regions for stain of CXCL16, CXCR6, CCL28 or CCR10. Gray line is the mean staining intensity of normal pancreatic tissue shown in and included as a reference. D ) Parallel tissue sections were processed for immunohistochemistry for CXCL12, CXCR7, CXCR4 and CK19. E ) Quantitative staining intensity of CXCL12, CXCR7 and CXCR4 in CK19+ cells. *** = P ≤ 0.001, **** = P ≤ 0.0001. Tissue staining shown in A and D are representative of tissue specimens from 5–9 individual patients clinically and pathologically diagnosed with pancreatitis.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunostaining, Staining, Immunohistochemistry

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A ) Sandwich ELISA revealed that the panel of tissue culture pancreatic cancer cells secrete CCL28 when stimulated with 50 ng/mL IFNγ. Levels of CCL28 were undetectable in HPSCs treated with IFNγ. B ) Flow cytometric analysis of HPSCs revealed the cell surface expression of CCR10 (top panel). The mean fluorescence intensity (MFI) was determined for cells stained with the isotype control primary antibody (light gray) and anti-CCR10 antibody (dark gray). C ) CCL28 stimulates chemotaxis. HPSCs were plated in the top well of a transwell insert in serum free media. The bottom well contained media (serum free; SF or full growth; FG). Recombinant CCL28 [30 nM] was added to the bottom or top well as indicated. After 4h, inserts were swabbed, stained with DAPI and enumerated. D ) CCL28-mediated HPSC migration is dose dependent. HPSCs were plated in the top well of a transwell insert. The bottom well contained either full growth media (+; positive control) or serum-free media that either lacked stimulant (-; negative control), or increasing concentrations of recombinant CCL28. E ) HPSC migration is mitigated by an anti-CCL28 neutralizing antibody. The transwell migration setup as above was modified to include the following conditions: no stimulation (NS; negative control), 10 ng/mL TGF-β (positive control) or 30 nM recombinant CCL28. Neutralizing antibody to CCL28 was added to the bottom well for 30 minutes prior to incubation with the cells. F ) CCL28-mediated directional migration of HPSCs is through a G-coupled protein receptor pathway. HPSCs were pretreated with pertussis toxin (PTX) or vehicle, prior to CCL28 stimulation. G) Transcript expression of CCR10 in HPSC cells. RNA was isolated and used as a template for RT-PCR analysis using CCR10 or GAPDH as a loading control. H) CCL28 stimulates chemotactic migration of patient-derived HSPC2 cells. HPSC2 cells were added to a transwell insert and placed in a well containing either serum free medium in the absence (NS) or presence of CCL28 (CCL28) or full growth medium (FG) as a positive control (FG). * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Sandwich ELISA, Expressing, Fluorescence, Staining, Control, Chemotaxis Assay, Recombinant, Migration, Positive Control, Negative Control, Modification, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Three independent HPSC lines were treated with 30 nM CCL28, left untreated (NS), treated with 10 ng/mL TGF-β (C+), treated with SB-431542 [25 µM], a TGF-β1R inhibitor (C-) or treated with vehicle controls (V). A) Representative immunofluorescence images show digital intensity “heat maps” of detected αSMA, corresponding to intensity color bar scale. Scale bars represent 50 µm. B) Semi-quantitative imaging analysis of the mean integrated intensity (Integ Int) of αSMA detection normalized for each HPSC line (HPSC, 2, 3) using the vehicle (V) set as 1 arbitrary unit. HPSC: #, P = 0.0179. ***, P ≤ 0.0002. HPSC2: #, P = 0.0144; ****, P <0.0001; HPSC3, *****, P < 0.0001. C) Representative images of HPSC-derived ECMs produced under experimental conditions as in A and analyzed using Orientation-J plugin of Image-J software. Color tones were normalized using hue values for common mode angle (cyan/green boarded color) visualization as represented on the orientation bar. D) Box and whisker plot of mean percent of fibers distributed within 15° angles from the mode corresponding to the indicated experimental conditions. HPSC: #, P = 0.0304; *, P = 0.028; HPSC2: *, P = 0.0117; HPSC3: C + versus Vs, *, P = 0.0101. No substantial ECM production was obtained from HPSC3 under TGF-β inhibition and was designated as not available (NA).

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunofluorescence, Imaging, Derivative Assay, Produced, Software, Whisker Assay, Inhibition

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Schematic representation of the experimental plan ( A ). Briefly, PDAC epithelial cells were seeded to the bottom well of a transwell dish (1) then stimulated with 50 ng/mL IFNγ to elicit chemokine secretion. HPSCs were plated onto the top chamber of the transwell insert (2) while CCL28 neutralizing antibody was added to the bottom chamber. The insert with HPSCs was added (3) and incubated for 4h prior to (4) fixing, DAPI-staining and cell counting. Stimulation of Panc1 ( B–C ) and MiaPaCa2 ( D–E ) cells by IFNγ resulted in directional migration of HPSCs that was inhibited by the neutralizing CCL28 antibody. HPSC migrate towards PDAC tumor cells and not non-transformed epithelial HPNE cells ( F–G ). Representative images ( C, E ) of 5 independent biological replicates are presented. * = P ≤ 0.05. ** = P ≤ 0.01.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Incubation, Staining, Cell Counting, Migration, Transformation Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Normal pancreatic duct epithelial cells and quiescent stromal fibroblasts almost no ligand CCL28 (purple circles) and basal levels of the receptor CCR10 (left panel). In inflammation, such as in pancreatitis, CCL28 expression is increased by cytokines such as IFNγ, which also participate in the activation of stellate cells into cancer-associated fibroblasts. CCL28 directs the migration of stellate cells toward the epithelium (middle panel). CCL28 produced by transformed ductal cells direct the sustained migration of activated stellate cells into the remodeling tumor microenvironment (right panel).

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Expressing, Activation Assay, Migration, Produced, Transformation Assay