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Neutrophils restrain allergic airway inflammation by limiting ILC2 function and monocyte–dendritic cell antigen presentation

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Science Immunology  08 Nov 2019:
Vol. 4, Issue 41, eaax7006
DOI: 10.1126/sciimmunol.aax7006
  • Fig. 1 Neutrophil-depleted mice display augmented type 2 inflammation after 3 weeks of HDM exposure.

    (A) Balb/c mice were administered HDM or PBS intranasally three times per week for up to 3 weeks, and at 24 hours before each HDM/PBS administration, mice were intraperitoneally dosed with either 100 μg of neutrophil-depleting antibody, 1A8, or isotype control antibody, 2A3. At 24-hour, 1-week, and 3-week time points (in each instance 24 hours after the final HDM/PBS exposure), lung tissue and BALF were collected (Ŧ). Total numbers of neutrophils in the lungs (B) and airways (C) were determined by flow cytometry. (D) Total cell numbers in the lung were assessed after 3 weeks of HDM exposure by trypan blue exclusion. (E) Representative H&E-stained lung sections from mice exposed to PBS or HDM for 3 weeks and treated with 2A3 or 1A8. (F) The number of eosinophils in the lung was quantified by flow cytometry at 3 weeks. The number of CD4+ T cells expressing T1ST2 (G) or IL-13 (H) in the lung was assessed by flow cytometry after 3 weeks of PBS/HDM exposure. (I) After 3 weeks of HDM/PBS exposure, concentrations of HDM-specific IgE and IgG1 in the serum were determined by ELISA. Figures present combined data from two independent experiments with four to six mice per group in each experiment. Results depicted as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 using Mann-Whitney statistical test.

  • Fig. 2 Augmented TH2 cytokine production by ILC2s in neutrophil-depleted mice administered HDM for 1 week.

    (A) Balb/c mice were intranasally administered HDM or PBS three times per week for 1 week, and at 24 hours before each HDM treatment, mice were intraperitoneally dosed with either 100 μg of neutrophil-depleting antibody, 1A8, or isotype control antibody, 2A3. At 24 hours after the final HDM administration, BALF was collected (Ŧ). (B) Concentrations of IL-4, IL-5, and IL-13 protein were assessed using BALF by ELISA. In some experiments, CD4+ T cells and ILC2s (pooled from three mice per data point) were isolated from the airways by FACS, at 24 hours after the final HDM exposure, for subsequent mRNA gene expression analysis. (C) Relative expression of Il-4, Il-5, Il-13, and Gata3 in T cells and ILC2s derived from BAL of HDM-treated mice, as determined by qPCR. At the same time point, the number of IL-13+ ILC2s and IL-5+ ILC2s (D) and geometric expression of IL-13 and IL-5 in ILC2s (E) in the BAL were assessed by flow cytometry. Figures present combined data from two independent experiments with four to six mice per group in each experiment (B, D, and E) or from one experiment whereby each data point represents cells pooled from three independent mice (C). Results depicted as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 using Mann-Whitney statistical test.

  • Fig. 3 HDM-exposed neutrophil-depleted mice exhibit augmented monocytosis, DC numbers, and antigen presentation.

    Balb/c mice were intranasally administered HDM or PBS three times per week for 1 week. At 24 hours before each HDM/PBS administration, mice were intraperitoneally treated with either 100 μg of neutrophil-depleting antibody, 1A8, or isotype control antibody, 2A3. At 24 hours after the final HDM/PBS exposure, lung tissue was collected. (A) Lung monocyte subsets identified as Ly6Clow and Ly6Chigh were enumerated by flow cytometry. (B) Numbers of lung moDCs were also determined by flow cytometry. The number of moDCs (C) and CD86+ moDCs (D) in the MLNs was enumerated by flow cytometry. In some experiments, the final dose of HDM was admixed with 100 μg of Alexa Fluor 488–labeled OVA (E), and MLNs were collected (Ŧ) at 24 hours after the final HDM/PBS exposure. (F) The number of OVA+ moDCs within the MLNs was enumerated by flow cytometry. Figures present data combined from two independent experiments with four to six mice per group for each experiment (A to D) or from one experiment with five mice per group (F). Results depicted as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 using Mann-Whitney statistical test.

  • Fig. 4 Neutrophil-depleted mice display increased numbers of monocyte and DC progenitors within their bone marrow owing to a dysregulated IL-23–IL-17–G-CSF axis.

    Balb/c mice were intranasally administered HDM or PBS three times per week for 1 week. At 24 hours before each HDM/PBS administration, mice were intraperitoneally treated with either 100 μg of neutrophil-depleting antibody, 1A8, or isotype control antibody, 2A3. At 24 hours after the final HDM/PBS exposure, BALF, lung tissue, blood, and bone marrow were collected. (A) The number of Ly6Clow and Ly6Chigh monocytes in the blood was quantified by flow cytometry. (B) The percentage of MDPs and CDPs within the bone marrow was assessed by flow cytometry. (C) The concentration of G-CSF in the serum, lung homogenate, and BALF was determined by ELISA. Expression of Csf3 (G-CSF gene; D), Il17 (E), and Il23 (F) was assessed in whole lung by qPCR. (G) Schematic depicting the negative feedback pathway by which tissue neutrophils limit granulopoiesis by modulation of the IL-23–IL-17–G-CSF axis. Figures present data from two independent experiments with four to six mice per group in each experiment. Results depicted as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 using Mann-Whitney statistical test.

  • Fig. 5 Neutralization of G-CSF abrogates the augmented monocytes, bone marrow progenitors, and TH2 cytokines observed in neutrophil-depleted HDM-exposed mice.

    (A) Balb/c mice were intranasally administered HDM three times per week for 1 week. At 24 hours before each HDM administration, mice were treated with either 100 μg of neutrophil-depleting antibody, 1A8, or isotype control antibody, 2A3. To neutralize G-CSF, mice were also intraperitoneally administered 100 μg of anti–G-CSF or isotype control, 2A3, at 24 hours before each HDM dose. At 24 hours after the final HDM administration, BAL, lung tissue, and bone marrow were collected (Ŧ). The number of lung and airway neutrophils (B) and lung Ly6Clow and Ly6Chigh monocytes (C) was determined by flow cytometry. (D) The percentage of MDPs and CDPs within the bone marrow was assessed by flow cytometry. (E) The concentrations of BALF IL-4, IL-5, and IL-13 were assessed by ELISA. (F) Geometric mean of IL-13 by lung ILC2s was assessed by flow cytometry. Figures present data combined from two independent experiments with four to six mice per group in each experiment. Results depicted as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 using Mann-Whitney statistical test.

  • Fig. 6 Coadministration of recombinant G-CSF augments numbers of HDM-induced neutrophils, monocytes, and bone marrow progenitors and ILC2 cytokine responses.

    (A) Balb/c mice were intranasally administered PBS or HDM three times per week for 1 week. Mice were also intranasally administered 100 ng of recombinant G-CSF (in 50 μl of PBS) and intraperitoneally administered 2 μg of recombinant G-CSF (in 200 μl of PBS), or respective PBS controls, daily throughout the study. At 24 hours after the final HDM administration, BAL, lung tissue, and bone marrow were collected (Ŧ). The number of lung and airway neutrophils (B) and lung Ly6Clow and Ly6Chigh monocytes (C) was determined by flow cytometry. (D) The percentage of MDPs and CDPs within the bone marrow was assessed by flow cytometry. (E) The concentrations of BALF IL-4, IL-5, and IL-13 were assessed by ELISA. (F) Geometric mean of IL-13 by BAL ILC2s was assessed by flow cytometry. Figures present data from one experiment with four to six mice per group in each experiment. Results depicted as means ± SEM. *P < 0.05 and **P < 0.01 using Mann-Whitney statistical test.

  • Fig. 7 G-CSF augments TH2 cytokine production from IL-33–expanded airway Csf3r-expressing ILC2s.

    Balb/c mice were intranasally administered HDM three times per week for 1 week. At 24 hours after the final HDM administration, BAL was collected. (A) Relative expression of mRNA Csf3r in T cells and ILC2s isolated by FACS from the BAL of HDM-exposed mice, as determined by qPCR. (B) Balb/c mice were intranasally administered 1 μg of recombinant IL-33 three times per week for 1 week. At 24 hours after the final IL-33 administration, BAL was collected. (C) Relative expression of mRNA Csf3r in T cells and ILC2s isolated by FACS from the BAL of IL-33–exposed mice, as determined by qPCR. In some experiments, 100 ng of recombinant G-CSF was co-administered with IL-33 (B), and CD4+ T cells and ILC2s were isolated from the airways by FACS, at 24 hours after the final IL-33 administration, for subsequent mRNA gene expression analysis. (D) Relative expression of IL-4 and IL-13 in T cells and ILC2s derived from BAL, as determined by qPCR. (E) From the same experiments, BALF concentrations of IL-5 and IL-13 were determined by ELISA. Figures present data from one experiment with four to six mice per group in each experiment. Results depicted as means ± SEM. *P < 0.05 and **P < 0.01 using Mann-Whitney statistical test.

  • Fig. 8 G-CSF directly augments TH2 cytokine production from human ILC2s.

    (A) Human ILC2s were isolated from peripheral blood and expanded in bulk culture with IL-7 (5 ng/ml) and IL-33 (15 ng/ml) before the addition of medium or medium containing recombinant G-CSF (100 ng/ml) for 72 hours. (B) Relative expression of CSF3R was assessed by qPCR after 72 hours. (C) The levels of IL-5 and IL-13 in the ILC2 supernatant were assessed by a multiplex cytokine assay and expressed as a donor-specific fold change after G-CSF treatment. (D) Expression of IL5, IL13, and GATA3 was assessed by qPCR after 72 hours and presented as a donor-specific fold change after G-CSF treatment. Results depicted as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 using a paired t test.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/41/eaax7006/DC1

    Fig. S1. Intranasal administration of HDM induces neutrophilic inflammation in the lungs and airways of mice.

    Fig. S2. Intranasal administration of HDM induces an increase in neutrophil proteases in the lungs and airways of mice.

    Fig. S3. Intraperitoneal administration of anti-Ly6G antibody, 1A8, systemically depletes neutrophils in HDM-treated mice.

    Fig. S4. Intraperitoneal administration of anti-Ly6G antibody, 1A8, specifically depletes neutrophils.

    Fig. S5. Neutrophil-depleted mice display augmented type 2 inflammation, epithelial remodeling, and airway resistance after 3 weeks of HDM exposure.

    Fig. S6. Neutrophil-depleted mice display comparable airway hyperresponsiveness after 3 weeks of HDM exposure.

    Fig. S7. Augmented TH2 cytokine levels in neutrophil-depleted mice administered HDM for 1 week.

    Fig. S8. ILC2s are the predominant source of IL-13 after 1 week of HDM administration.

    Fig. S9. Gating strategy for isolation of ILC2s and CD4+ T cells by FACS.

    Fig. S10. Augmented IL-5 and IL-13 levels in lungs of neutrophil-depleted mice administered HDM for 1 week are primarily derived from ILC2s.

    Fig. S11. ILC2s from neutrophil-depleted mice administered HDM for 1 week produce more IL-5 and IL-13 on a per-cell basis.

    Fig. S12. HDM-exposed neutrophil-depleted mice exhibit augmented DC numbers and antigen presentation.

    Fig. S13. Lung concentrations of classical monocyte chemokines and flow cytometry gating strategy to identify MDPs and CDPs.

    Fig. S14. G-CSF is elevated in the serum of neutrophil-depleted mice administered HDM.

    Fig. S15. Neutrophil-depleted mice administered HDM produce elevated levels of IL-17 and IL-23.

    Fig. S16. Neutralization of IL-17 reverts the effects of neutrophil depletion in HDM-administered mice.

    Fig. S17. G-CSF directly augments TH2 cytokine production from mouse ILC2s.

    Fig. S18. Neutrophil depletion alters the IL-23–IL-17–G-CSF regulatory feedback pathway to exacerbate TH2 cytokine production and allergen sensitization.

    Table S1. Raw data file.

    Table S2. List of antibodies used for flow cytometry staining.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Intranasal administration of HDM induces neutrophilic inflammation in the lungs and airways of mice.
    • Fig. S2. Intranasal administration of HDM induces an increase in neutrophil proteases in the lungs and airways of mice.
    • Fig. S3. Intraperitoneal administration of anti-Ly6G antibody, 1A8, systemically depletes neutrophils in HDM-treated mice.
    • Fig. S4. Intraperitoneal administration of anti-Ly6G antibody, 1A8, specifically depletes neutrophils.
    • Fig. S5. Neutrophil-depleted mice display augmented type 2 inflammation, epithelial remodeling, and airway resistance after 3 weeks of HDM exposure.
    • Fig. S6. Neutrophil-depleted mice display comparable airway hyperresponsiveness after 3 weeks of HDM exposure.
    • Fig. S7. Augmented TH2 cytokine levels in neutrophil-depleted mice administered HDM for 1 week.
    • Fig. S8. ILC2s are the predominant source of IL-13 after 1 week of HDM administration.
    • Fig. S9. Gating strategy for isolation of ILC2s and CD4+ T cells by FACS.
    • Fig. S10. Augmented IL-5 and IL-13 levels in lungs of neutrophil-depleted mice administered HDM for 1 week are primarily derived from ILC2s.
    • Fig. S11. ILC2s from neutrophil-depleted mice administered HDM for 1 week produce more IL-5 and IL-13 on a per-cell basis.
    • Fig. S12. HDM-exposed neutrophil-depleted mice exhibit augmented DC numbers and antigen presentation.
    • Fig. S13. Lung concentrations of classical monocyte chemokines and flow cytometry gating strategy to identify MDPs and CDPs.
    • Fig. S14. G-CSF is elevated in the serum of neutrophil-depleted mice administered HDM.
    • Fig. S15. Neutrophil-depleted mice administered HDM produce elevated levels of IL-17 and IL-23.
    • Fig. S16. Neutralization of IL-17 reverts the effects of neutrophil depletion in HDM-administered mice.
    • Fig. S17. G-CSF directly augments TH2 cytokine production from mouse ILC2s.
    • Fig. S18. Neutrophil depletion alters the IL-23–IL-17–G-CSF regulatory feedback pathway to exacerbate TH2 cytokine production and allergen sensitization.
    • Legend for table S1
    • Table S2. List of antibodies used for flow cytometry staining.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data file.

    Files in this Data Supplement:

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