Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28-Deficient Mice Have Reduced IgA Antibody-Secreting Cells and an Altered Microbiota in the Colon.

doi: 10.4049/jimmunol.1700037

Figure Lengend Snippet: FIGURE 1. Generation of CCL28-deficient mice. CCL28-deficient mice (CCL28-knockout/EGFP–knock-in mice) were generated on the C57BL/6 background by replacing exon 1 of the CCL28 gene with a gene cassette encoding EGFP and Neor. (A) Targeting strategy: the murine genomic CCL28 locus, the targeting vector construct, and the targeted allele with a cassette encoding EGFP and Neor are shown. (B) Genomic PCR analysis. PCR was performed for CCL28 using genomic DNA prepared from the tails of WT mice, heterozygous CCL28-deficient mice (CCL28+/2), and homozygous CCL28-deficient mice (CCL282/2). Representative results from two separate experiments are shown. (C) RT-PCR analysis. CCL28 expression was determined by RT-PCR using cDNA samples derived from the indicated tissues. GAPDH was used as an internal control. Represen- tative results from two separate experiments are shown.

Article Snippet: In brief, microbial cells in midlogarithmic growth phase were resuspended at 105 cells per milliliter in 1 mM potassium phosphate buffer (PPB; a mixture of 1 mM K2HPO4 and 1 mM KH2PO4; pH 7.2), mixed with an equal volume of 1 mM PPB, with or without recombinant mouse CCL28 (R&D Systems) in 2-fold serial dilutions, and incubated in a U-bottom 96-well microtest plate at room temperature for 2 h with brief mixing every 15 min. After appropriate dilutions with 1 mM PPB, cells were plated on agar plates and grown at 37 ̊C overnight.

Techniques: Knock-Out, Knock-In, Generated, Plasmid Preparation, Construct, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28-Deficient Mice Have Reduced IgA Antibody-Secreting Cells and an Altered Microbiota in the Colon.

doi: 10.4049/jimmunol.1700037

Figure Lengend Snippet: FIGURE 2. Immunofluorescence staining of CCL28 and IgA in the colon and sublingual gland. (A) Immunofluorescence staining of CCL28. Tissue sections of the colon and sublingual gland from WT and CCL28- deficient (CCL28-KO) mice were stained for CCL28 (green) and DAPI (blue). Scale bars, 100 mm. Representative results from three separate experiments are shown. (B) Double immunofluorescence staining of CCL28 and CD31. Tissue sections of the colon (Figure legend continues)

Article Snippet: In brief, microbial cells in midlogarithmic growth phase were resuspended at 105 cells per milliliter in 1 mM potassium phosphate buffer (PPB; a mixture of 1 mM K2HPO4 and 1 mM KH2PO4; pH 7.2), mixed with an equal volume of 1 mM PPB, with or without recombinant mouse CCL28 (R&D Systems) in 2-fold serial dilutions, and incubated in a U-bottom 96-well microtest plate at room temperature for 2 h with brief mixing every 15 min. After appropriate dilutions with 1 mM PPB, cells were plated on agar plates and grown at 37 ̊C overnight.

Techniques: Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28-Deficient Mice Have Reduced IgA Antibody-Secreting Cells and an Altered Microbiota in the Colon.

doi: 10.4049/jimmunol.1700037

Figure Lengend Snippet: FIGURE 7. Severe DSS-induced colitis of CCL28-deficient mice. Co- litis was induced in WT and CCL28-deficient (CCL28-KO) mice by ad- ministration of 2% DSS in the drinking water for 7 d, followed by regular drinking water. (A) Body weight. Mice were weighed daily. (B) Macroscopic images of the colon on day 7. (C) The average length of the colon on day 7 (left panel). The length ratio of DSS(+)/DSS(2) is also shown (right panel). The data are shown as mean 6 SE of six mice. (Figure legend continues)

Article Snippet: In brief, microbial cells in midlogarithmic growth phase were resuspended at 105 cells per milliliter in 1 mM potassium phosphate buffer (PPB; a mixture of 1 mM K2HPO4 and 1 mM KH2PO4; pH 7.2), mixed with an equal volume of 1 mM PPB, with or without recombinant mouse CCL28 (R&D Systems) in 2-fold serial dilutions, and incubated in a U-bottom 96-well microtest plate at room temperature for 2 h with brief mixing every 15 min. After appropriate dilutions with 1 mM PPB, cells were plated on agar plates and grown at 37 ̊C overnight.

Techniques:

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: 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