recombinant mouse ccl28  (R&D Systems)

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: 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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.78206

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: 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: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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:

Article Snippet: Peptide, recombinant protein , Recombinant Mouse CCL28 Protein , R&D Systems , Cat#533-VI , Chemotaxis: 50 nM; neutrophil stimulation: 50 nM.

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: 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: Gene therapy

Article Title: Plasmids encoding the mucosal chemokines CCL27 and CCL28 are effective adjuvants in eliciting antigen-specific immunity in vivo

doi: 10.1038/gt.2009.112

Figure Lengend Snippet: Expression of bioactive chemokines from plasmid-encoded chemokine. (a) Reverse transcriptase (RT)-PCR analysis of lymphocytes from the spleen (Spl), mesenteric lymph node (MLN) or Peyer’s patch (PP) from wild-type or CCR10 knockout BALB/c mice. (b) Schematic representation of chemokine expression plasmids. All genes were cloned into the multiple cloning region of the expression vector pVAX (kanamycin resistance) using the restriction enzyme sites, EcoR1 and Xho1. (c) Expression of pCCL27 and pCCL28 was confirmed by a T7-coupled transcription/translation reticulocyte lysate system (TNT). The gel shows size markers (designated M, lane 1), pVAX background control (V, lane 2), 10.9 kDa pCCL27 protein (lane 3) and 12.6 kDa pCCL28 protein (lane 4). (d and e) Expression of bioactive chemokine protein translated from the plasmid forms of CCL27 (d) and CCL28 (e). ELISA was carried out using supernatants from pCCL27- or pCCL28-transfected RD cells (pg ml−1). For each chemokine, vector background control is included (gray bar) vs pCCL27 or pCCL28 (black bar). Data are shown as pg ml−1 of chemokine protein±s.d. of triplicate wells. (f) Migration index of splenocytes, MLN, PP/cryopatch cells and lung cells in response to secreted CCL27- or CCL28-containing supernatants from transfected RD cells.

Article Snippet: After 48 h, cell supernatants were harvested and analyzed for the presence of murine CCL27 or CCL28 protein by commercial ELISA kits (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Plasmid Preparation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Clone Assay, Enzyme-linked Immunosorbent Assay, Transfection, Migration

Journal: Gene therapy

Article Title: Plasmids encoding the mucosal chemokines CCL27 and CCL28 are effective adjuvants in eliciting antigen-specific immunity in vivo

doi: 10.1038/gt.2009.112

Figure Lengend Snippet: CCR10 ligands induce long-lived IgG, IgA and neutralizing antibody and protect mice against influenza A/PR8/34 lethal mucosal challenge. (a) Influenza-specific long-lived IgG and IgA titers were determined at time of challenge. Individual serum samples were harvested from mice from each immunization group (n=10 mice per group) and (ng ml−1) levels of pPR8HA-specific IgG (gray bar) and IgA (black bar) were determined by ELISA using a recombinant standard of known concentration. [**P<0.01, for comparisons between chemokine plasmid adjuvanted groups (n=10 mice per experimental group vs n=10 pPR8HA alone]. (b) Coimmunization with CCL27 and CCL28 chemokines elicits anti-influenza A/PR8/34 neutralizing antibodies. Serial fourfold dilutions of individual serum samples from immunized mouse groups were incubated with influenza A/PR8/34 (100 TCID50 virus per well), and results from the chicken erythrocyte sedimentation assay are shown as the serum dilution at which 2000TCID50/ml of influenza A/PR8/34 can be neutralized. Data represent individual mouse sera (n=10) in six replicates per dilution for each immunized mouse group. Representative wells of neutralized virus (influenza A/PR8/34 neutralized wells, pool of erythrocytes at bottom of well) and non-neutralized virus (influenza A/PR8/34 positive in well, agglutination or fuzzy well is present) are shown for each immunization group in panel b. (c and d) Systemic coimmunization with mucosal chemokines protects mice from influenza A/PR8/34 intranasal challenge. Average percent weight loss (morbidity) and death (survival) in groups of mice immunized and challenged. Vector-immunized mice (diamonds) lost weight rapidly (c) and died (n=10) by day 9 post infection (d). Mice that were immunized with pPR8HA (triangles) alone lost an average 18% weight (c) by day 10, whereas 80% died (d) by day 10 post challenge. Mice immunized with CCR10 ligands, pCCL27 and pCCL28, were protected from weight loss (squares and circles, respectively, c) and were protected from mortality (d) in the days after challenge. Using log-rank analysis, pVAX vs pR8HA (P=0.0005), pVAX vs CCL27 (P<0.0001) and pVAX vs CCL28 (P<0.0001); pR8HA vs pR8HA/CCL27 (P=0.0003) and pR8HA vs pR8HA/CCL28 (P=0.0003).

Article Snippet: After 48 h, cell supernatants were harvested and analyzed for the presence of murine CCL27 or CCL28 protein by commercial ELISA kits (R&D Systems, Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Plasmid Preparation, Incubation, Virus, Sedimentation, Agglutination, Infection

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: In gradient experiments, CCL20 and CCL28 recombinant proteins (R&D Systems) were loaded in the bottom chamber with a CCL20/CCL28 ratio varying from 0:100% to 100:100% of their maximum chemotactic activity (10 and 200 ng/mL, respectively).

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: In gradient experiments, CCL20 and CCL28 recombinant proteins (R&D Systems) were loaded in the bottom chamber with a CCL20/CCL28 ratio varying from 0:100% to 100:100% of their maximum chemotactic activity (10 and 200 ng/mL, respectively).

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