DOI: 10.5061/dryad.59zw3r2j6
title: Data from: CCL28 modulates neutrophil responses during infection with mucosal pathogens

description: The chemokine CCL28 is highly expressed in mucosal tissues, but its role
 during infection is not well understood. Here we show that CCL28 promotes
 neutrophil accumulation in the gut of mice infected with Salmonella and in
 the lung of mice infected with Acinetobacter. Neutrophils isolated from
 the infected mucosa expressed the CCL28 receptors CCR3 and, to a lesser
 extent, CCR10, on their surface. The functional consequences of CCL28
 deficiency varied between the two infections: Ccl28-/- mice were highly
 susceptible to Salmonella gut infection but highly resistant to otherwise
 lethal Acinetobacter lung infection. In vitro, unstimulated neutrophils
 harbored pre-formed intracellular CCR3 that was rapidly mobilized to the
 cell surface following phagocytosis or inflammatory stimuli. Moreover,
 CCL28 stimulation enhanced neutrophil antimicrobial activity, production
 of reactive oxygen species, and formation of extracellular traps, all
 processes largely dependent on CCR3. Consistent with the different
 outcomes in the two infection models, neutrophil stimulation with CCL28
 boosted the killing of Salmonella but not Acinetobacter. CCL28 thus plays
 a critical role in the immune response to mucosal pathogens by increasing
 neutrophil accumulation and activation, which can enhance pathogen
 clearance but also exacerbate disease depending on the mucosal site and
 the infectious agent.
description: See manuscript materials and methods for details
description: # CCL28 Manuscript Source Data File
 [https://doi.org/10.5061/dryad.59zw3r2j6](https://doi.org/10.5061/dryad.59zw3r2j6) ## Description of the data and file structure These are source data files for the manuscript: CCL28 modulates neutrophil responses during infection with mucosal pathogens. eLife (2024). Walker, Perez-Lopez, et al. Data is contained in files in accordance to the respective manuscript figure names. Each file contains data from separate panels in each figure on separate tabs. The materials and methods and the figure legends contain all information needed for analysis of each data file. Further description of individual files and tabs: ### Files and variables #### File: elife\_CCL28\_Figure1\_SourceData.xlsx **Description**: File contains data from mice gavaged with streptomycin 24h prior to oral infection with approximately 10^9 CFU S. enterica serovar Typhimurium (STm). A) At 4 days post-infection (dpi), CCL28 in feces was quantified by ELISA. Data comprise two independent experiments. B) STm CFU in the fecal content collected 1-3 dpi, and in the cecal content 3 dpi from wild-type (WT) and Ccl28-/- (CCL28-KO) littermate mice. C) STm CFU recovered from the Peyer’s patches, mesenteric lymph nodes, spleen, bone marrow, and blood at 3 dpi. Data comprise eight independent experiments. E) Frequency of neutrophils in the live CD45+ cells obtained from the gut mucosa of WT or CCL28-KO mice. Naive mouse data shown comprise four independent experiments; 2 dpi data comprise four independent experiments; 3 dpi data comprise eight independent experiments. F) Relative expression levels (qPCR) of Cxcl1, Tnfa, Ifng, Csf3, Il1b, and Il17a in the cecal tissue of WT or CCL28-KO mice 3 days post-STm infection, relative to uninfected control mice. Data comprise four independent experiments. G) Sum histopathological analysis scores of the cecum collected from STm-infected WT or CCL28-KO mice, 3 dpi. H) Cecal histopathology scores showing the individual analyzed parameter scores of each mouse. #### File: 3dpi-STm\_WT\_Cecum\_H\_E\_100x.png **Description**: Source image for Figure 1I of representative WT cecal sections stained with H&E, 200X magnification. #### File: 3\_dpi-STm\_CCL28-KO\_Cecum\_H\_E\_100x.png **Description**: Source image for Figure 1I of representative CCL28-KO cecal sections stained with H&E, 200X magnification. #### **File: elife\_CCL28\_Figure1-Supp1\_SourceData.xlsx** **Description**: File contains data from mice gavaged with streptomycin 24h prior to oral infection with approximately 109 CFU S. enterica serovar Typhimurium (STm). Data comprise 4 independent experiments. A) STm CFU in the fecal content collected at 1 and 2 dpi, and in the cecal content 2 dpi from WT and CCL28-KO littermate mice. B) STm CFU recovered from the Peyer’s patches, mesenteric lymph nodes, spleen, bone marrow, and blood from WT and CCL28-KO littermate mice at 2 dpi. #### File: elife\_CCL28\_Figure1-Supp2\_SourceData.xlsx **Description**: File contains data from mice infected by intraperitoneal injection with 103 STm or sterile PBS. A) CCL28 in serum at 4 dpi quantified by ELISA from mock-infected (UI) and STm-infected (WT) mice. Data comprise two independent experiments. B) STm CFU in the spleen, liver, and blood of WT and CCL28-KO mice 4 dpi Ccl28-/- mice. Data shown comprise two independent experiments C-D) In vitro antimicrobial activity of CCL28 against STm wild-type, STm phoQ, E. coli K12, and A. baumannii. C) CFU of STm WT, STm phoQ, Acinetobacter, and E. coli K12, enumerated after 2h incubation with recombinant murine CCL28 at the indicated concentrations. D) CFU of STm WT remaining after incubation with 50 nM recombinant murine CCL28 or 25 nM CCL11, enumerated at 75 min and 150 min. #### File: elife\_CCL28\_Figure1-Supp4\_SourceData.xlsx **Description**: File contains data from flow cytometry quantification of live, CD45+ CD11b- immune cells recovered from WT and CCL28 mouse gut, blood, and bone marrow, before (Naïve) and during STm infection (2 dpi and 3 dpi). Naive samples comprise data from 5 independent experiments; 2 dpi samples comprise data from 4 independent experiments; 3 dpi samples comprise data from 6 independent experiments. A) Percent abundance of B cells (CD11b- CD3- CD19+). B) Percent abundance of CD8+ T cells (CD11b- CD19- CD3+ CD8+ CD4-). C) Percent abundance of CD4+ T cells (CD11b- CD19- CD3+ CD4+ CD8-). #### File: elife\_CCL28\_Figure1-Supp5\_SourceData.xlsx **Description**: File contains data from flow cytometry quantification of live, CD45+ CD11b- immune cells recovered from WT and CCL28 mouse gut, blood, and bone marrow, before (Naïve) and during STm infection (2 dpi and 3 dpi). Naive samples comprise data from 5 independent experiments; 2 dpi samples comprise data from 4 independent experiments; 3 dpi samples comprise data from 6 independent experiments. A) Percent abundance of Neutrophils (CD11b+ Ly6G+) in the blood and bone marrow. B) Percentage of eosinophils (CD11b+ Ly6G- SiglecF+ side scatter-high) expressing surface CCR3. C) Percent abundance of eosinophils (CD11b+ Ly6G- SiglecF+ side scatter-high). D) Percent abundance of macrophage-like cells (CD11b+ Ly6G- SiglecF- F4/80+ CD11c-). E) Percent abundance of conventional dendritic cell-like cells (CD11b+ Ly6G- SiglecF- CD11c+ F4/80-). #### **File: elife\_CCL28\_Figure1-Supp6\_SourceData.xlsx** **Description**: File contains data from ELISAs on fecal and cecal supernatant from WT and CCL28-KO mice gavaged with streptomycin 24h prior to oral infection with approximately 10^9 CFU S. enterica serovar Typhimurium (STm) and sacrificed 2 or 3 dpi. A) Levels of myeloperoxidase (MPO) B) Levels of neutrophil elastase. C) Levels of S100A9. #### File: elife\_CCL28\_Figure2\_SourceData.xlsx **Description**: File contains data from WT and CCL28-KO mice intratracheally infected with approximately 10^8 CFU Acinetobacter baumannii (Ab). A) Mortality over the 10 days following Ab infection. Data comprise two independent experiments. B-H) WT and CCL28-KO mice were intratracheally infected with Ab and sacrificed 1 day post-infection (dpi). Data comprise three independent experiments. B) Ab CFU quantified from bronchoalveolar lavage (BAL) fluid 1 dpi. C) Ab CFU quantified from lung tissue 1 dpi. D) Ab CFU quantified from blood 1 dpi. F) Frequency of neutrophils in the live CD45+ population obtained from the BAL, lung, blood, and bone marrow 1 dpi, determined by flow cytometry. G) The number of total live host cells per mL of BAL from naive mice and Ab-infected WT and CCL28-KO mice 1 dpi. H) Percent abundance of different leukocyte populations as a proportion of the live CD45+ cell population in the BAL. I) Representative immunofluorescence image of lungs from WT and Ccl28-/- mice, uninfected or infected with Ab, stained for the neutrophil marker Ly6G (magenta). DAPI (blue) was used to label nuclei. J) Quantification of Ly6G+ cells per high-power field (HPF) from immunofluorescence images of lungs from WT and CCL28-KO mice 1 dpi. K) Histopathological analysis of Ab-infected mouse lungs 1 dpi. L) Relative expression levels (qPCR) of Cxcl1, Tnfa, Ifng, Csf3, Il1b, and Il17a in the Ab-infected mouse lungs 1 dpi. Data comprise three independent experiments. #### File: 1dpi-Ab\_Ccl28-KO\_Lung\_Ly6G-IF\_400x.png **Description**: Source image for Figure 2I, representative lung immunofluorescence section from Ab-infected CCL28-KO lung stained with Ly6G-AlexaFluor555 and DAPI, 400X magnification.  #### File: 1dpi-Ab\_WT\_Lung\_Ly6G-IF\_400x.png **Description**: Source image for Figure 2I, representative lung immunofluorescence section from Ab-infected WT lung stained with Ly6G-AlexaFluor555 and DAPI, 400X magnification. #### **File: Uninfected\_Ccl28-KO\_Lung\_Ly6G-IF\_400x.png** **Description**: Source image for Figure 2I, representative lung immunofluorescence section from uninfected CCL28-KO lung stained with Ly6G-AlexaFluor555 and DAPI, 400X magnification. #### File: Uninfected\_WT\_Lung\_Ly6G-IF\_400x.png **Description**: Source image for Figure 2I, representative lung immunofluorescence section from uninfected WT lung stained with Ly6G-AlexaFluor555 and DAPI, 400X magnification. #### File: elife\_CCL28\_Figure2-Supp1\_SourceData.xlsx **Description**: File contains data from flow cytometry quantification of live, CD45+ CD11b- immune cells recovered from WT and CCL28 mouse BAL fluid, lung tissue, blood, and bone marrow, before (Naive) and during Ab infection (1 dpi). Samples not collected from mice are indicated. Naive samples comprise data from 6 independent experiments; 1 dpi samples comprise data from 3 independent experiments. A) Percent abundance of Neutrophils (CD11b+ Ly6G+). B) Percent abundance of eosinophils (CD11b+ Ly6G- SiglecF+ side scatter-high). C) Percent abundance of macrophage-like cells (CD11b+ Ly6G- SiglecF- F4/80+ CD11c-). D) Percent abundance of conventional dendritic cell-like cells (CD11b+ Ly6G- SiglecF- CD11c+ F4/80-). #### File: elife\_CCL28\_Figure2-Supp2\_SourceData.xlsx **Description**: File contains data from flow cytometry quantification of live, CD45+ CD11b- immune cells recovered from WT and CCL28 mouse BAL fluid, lung tissue, blood, and bone marrow, before (Naive) and during Ab infection (1 dpi). Samples not collected from mice are indicated. Naive samples comprise data from 6 independent experiments; 1 dpi samples comprise data from 3 independent experiments. A) Percent abundance of B cells (CD11b- CD3- CD19+). B) Percent abundance of CD8+ T cells (CD11b- CD19- CD3+ CD8+ CD4-). C) Percent abundance of CD4+ T cells (CD11b- CD19- CD3+ CD4+ CD8-). #### File: elife\_CCL28\_Figure2-Supp3\_SourceData.xlsx **Description**: File contains data from ELISAs on BAL supernatant from naive WT and Ab-infected WT and CCL28-KO mice, 1 dpi. A) Levels of myeloperoxidase (MPO). B) Levels of neutrophil elastase. C) Levels of S100A9. #### File: elife\_CCL28\_Figure3\_SourceData.xlsx **Description**: File contains data from analysis of CCR3 and CCR10 expression by mouse neutrophils from tissues during infection and upon stimulation with proinflammatory stimuli and phagocytosis. A) Percent of neutrophils staining CCR3+ isolated from the gut tissues, blood, and bone marrow of WT mice, 3 days after STm infection. B) Percent of neutrophils staining CCR10+ isolated from the gut tissues, blood, and bone marrow of WT mice, 3 days after STm infection. C) Percent of neutrophils staining CCR3+ isolated from the BAL, blood, and bone marrow of WT mice, 1 day after Ab infection. D) Percent of neutrophils staining CCR10+ isolated from the BAL, blood, and bone marrow of WT mice, 1 day after Ab infection. E) Percent of bone marrow neutrophils staining CCR3+, unstimulated and following stimulation with the cytokines IFNg + TNFa + GM-CSF, fMLP, PMA, or LPS. F) Percent of bone marrow neutrophils staining CCR10+, unstimulated and following stimulation with the cytokines IFNg + TNFa + GM-CSF, fMLP, PMA, or LPS. G) Percent of bone marrow neutrophils staining CCR3+, unstimulated and following stimulation with the cytokines IFNg + TNFa + GM-CSF, beads, or cytokines and beads combined. H) Percent of bone marrow neutrophils staining CCR10+, unstimulated and following stimulation with the cytokines IFNg + TNFa + GM-CSF, beads, or cytokines and beads combined. I) Percent of enriched bone marrow neutrophils staining CCR3+, unstimulated or following infection with STm at a multiplicity of infection of 10 for 1h, and treated with either cytochalasin D or vehicle. J) Percent of enriched bone marrow neutrophils staining CCR10+, unstimulated or following infection with STm at a multiplicity of infection of 10 for 1h, and treated with either cytochalasin D or vehicle. #### File: elife\_CCL28\_Figure3-Supp1\_SourceData.xlsx **Description**: Files contains data from analysis of CCR3 and CCR10 on neutrophils isolated from the STm-infected gut and Ab-infected lung mucosa in infected WT and CCL28-KO mice. A) Percent of gut neutrophils from STm-infected WT mice expressing CCR3 alone, CCR10 alone, and both CCR3 and CCR10 on their surface, 3 dpi. Samples comprise data from 6 independent experiments. B) Percentage of CCR3+ and CCR10+ neutrophils obtained from the gut, blood, and bone marrow of WT and CCL28-KO mice infected with STm for 3 days. Samples comprise data from 6 independent experiments. C) Percent of BAL-associated neutrophils from Ab-infected WT mice expressing CCR3 alone, CCR10 alone, and both CCR3 and CCR10 on their surface. Samples comprise data from 2 independent experiments. D) Percentage of CCR3+ and CCR10+ neutrophils obtained from the BAL, lung, blood, and bone marrow of WT and CCL28-KO mice infected with Ab for 3 days. Samples comprise data from 2 independent experiments. #### File: elife\_CCL28\_Figure4\_SourceData.xlsx **Description**: Files contain data from analysis of surface and intracellular CCR3 expression of BM-derived neutrophils following ex vivo and in vitro infection with STm or Ab. A) Percentage of surface CCR3+ neutrophils following STm infection (MOI=10) at indicated time points. B) Percentage of intracellular CCR3+ neutrophils following STm infection (MOI=10) at indicated time points. C) Percentage of surface CCR3+ neutrophils following Ab infection (MOI=10) at indicated time points. D) Percentage of intracellular CCR3+ neutrophils following Ab infection (MOI=10) at indicated time points. E) Percentage of surface and intracellular CCR3+ neutrophils isolated from the gut, blood, and BM of wildtype mice infected with STm for 3 days. F) Percentage of surface and intracellular CCR3+ neutrophils isolated from the BAL, blood, and BM of wildtype mice infected with Ab for 1 day. #### File: elife\_CCL28\_Figure4-Supp1\_SourceData.xlsx **Description**: Files contain data from analysis of surface and intracellular CCR10 expression of BM-derived neutrophils following ex vivo and in vitro infection with STm or Ab. A) Percentage of surface CCR10+ neutrophils following STm infection (MOI=10) at indicated time points. B) Percentage of intracellular CCR10+ neutrophils following STm infection (MOI=10) at indicated time points. C) Percentage of surface CCR10+ neutrophils following Ab infection (MOI=10) at indicated time points. D) Percentage of intracellular CCR10+ neutrophils following Ab infection (MOI=10) at indicated time points. E) Percentage of surface and intracellular CCR10+ neutrophils isolated from the gut, blood, and BM of wildtype mice infected with STm for 3 days. F) Percentage of surface and intracellular CCR10+ neutrophils isolated from the BAL, blood, and BM of wildtype mice infected with Ab for 1 day. #### File: elife\_CCL28\_Figure5\_SourceData.xlsx **Description**: Files contain data from experiments analyzing CCL28 capacity to stimulate neutrophil antimicrobial activity. A) Chemotaxis index of WT murine bone marrow neutrophils stimulated with IFNg + TNFa + GM-CSF for 4h before adding 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), were placed in separate lower compartments, and the plate was incubated for 2h. Cells that migrated to the lower compartment were enumerated by flow cytometry, and 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) Percentage of STm surviving following 2.5h co-culture with bone marrow neutrophils with or without CCL28 or CCL11 added. Data for each infection comprise three independent experiments. C) Percentage of Ab surviving following 4.5h co-culture with bone marrow neutrophils with or without CCL28 or CCL11 added. Data for each infection comprise three independent experiments. D) Percentage of STm surviving following 2.5h co-culture with bone marrow neutrophils with or without CCL28 or CCL11 added, in the presence or absence of the CCR3 antagonist SB328437. Data comprise three independent experiments. E) Percentage ROS+ cells (as measured by H2DCFDA conversion to fluorescent DCF) detected by flow cytometry of bone marrow neutrophils, uninfected or infected with STm, with or without CCL28 for 4h. F) Percentage ROS+ cells following stimulation with CCL28 and infection with STm (as indicated) in the presence of an anti-CCR3 antibody or isotype control antibody. G) Percentage ROS+ cells following stimulation with CCL28 and infection with STm (as indicated) in the presence of an anti-CCR10 antibody or isotype control antibody. I) Percentage of human peripheral blood neutrophils forming NETs based on observed morphology, following stimulation with platelets and CCL28, and co-incubated with the CCR3 antagonist SB328437, the CCR10 antagonist BI-6901, or both, as indicated. #### File: elife\_CCL28\_Figure5-Supp1\_SourceData.xlsx **Description**: Files contain data from analysis of Helix+ MPO+ neutrophils of human neutrophil samples activated with platelets and CCL28, with or without CCR3 or CCR10 antagonists. B) Percentage of Helix+ MPO+ neutrophils in the indicated treatment groups. #### File: No\_chemokine\_DAPI.tiff **Description**: Source image for Figure 5H, representative fluorescent image of human neutrophils stimulated with platelets + no chemokine then stained with DAPI and Helix to observe NET morphology, imaged under the DAPI filter. #### File: No\_Chemokine\_Helix.tiff **Description**: Source image for Figure 5H, representative fluorescent image of human neutrophils stimulated with platelets + no chemokine then stained with DAPI and Helix to observe NET morphology, imaged under the FITC filter. #### File: CCL28\_DAPI.tiff **Description**: Source image for Figure 5H, representative fluorescent image of human neutrophils stimulated with platelets + CCL28 then stained with DAPI and Helix to observe NET morphology, imaged under the DAPI filter. #### File: CCL28\_Helix.tiff **Description**: Source image for Figure 5H, representative fluorescent image of human neutrophils stimulated with platelets + CCL28 then stained with DAPI and Helix to observe NET morphology, imaged under the FITC filter. #### File: CCL28\_SB\_DAPI.tiff **Description**: Source image for Figure 5H, representative fluorescent image of human neutrophils stimulated with platelets + CCL28 + SB328437 then stained with DAPI and Helix to observe NET morphology, imaged under the DAPI filter. #### File: CCL28\_SB\_Helix.tiff **Description**: Source image for Figure 5H, representative fluorescent image of human neutrophils stimulated with platelets + CCL28 + SB328437 then stained with DAPI and Helix to observe NET morphology, imaged under the FITC filter. #### File: CCL28\_BI\_DAPI.tiff **Description**: Source image for Figure 5H, representative fluorescent image of human neutrophils stimulated with platelets + CCL28 + BI-6901 then stained with DAPI and Helix to observe NET morphology, imaged under the DAPI filter. #### File: CCL28\_BI\_Helix.tiff **Description**: Source image for Figure 5H, representative fluorescent image of human neutrophils stimulated with platelets + CCL28 + BI-6901 then stained with DAPI and Helix to observe NET morphology, imaged under the FITC filter. #### File: CCL28\_SB\_BI\_DAPI.tiff **Description**: Source image for Figure 5H, representative fluorescent image of human neutrophils stimulated with platelets + CCL28 + SB328437 + BI-6901 then stained with DAPI and Helix to observe NET morphology, imaged under the DAPI filter. #### File: CCL28\_SB\_BI\_Helix.tiff **Description**: Source image for Figure 5H, representative fluorescent image of human neutrophils stimulated with platelets + CCL28 + SB328437 + BI-6901 then stained with DAPI and Helix to observe NET morphology, imaged under the FITC filter. Please contact [[manuelar@health.ucsd.edu](mailto:manuelar@health.ucsd.edu)] for additional information.