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Neutrophil cytoplasts induce TH17 differentiation and skew inflammation toward neutrophilia in severe asthma

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Science Immunology  03 Aug 2018:
Vol. 3, Issue 26, eaao4747
DOI: 10.1126/sciimmunol.aao4747
  • Fig. 1 Neutrophilic inflammation in a murine model of HDM and LPS.

    (A) Schematic diagram showing the allergen sensitization and challenge protocol. After sensitization with HDM/Veh or HDM/LPS (protocol day 0 to 2) followed by HDM challenge (intranasally, protocol day 7 to 14), the inflammatory response was assessed 24 hours later on protocol day 15 (n = 8 mice). (B) BALF total cell count (C) and leukocyte differential (% total leukocytes) were determined. Eos, eosinophils; Macs, macrophages; PMNs, polymorphonuclear neutrophils. (D and E) MLNs were collected on protocol day 7, and dissociated cells were restimulated with PMA and ionomycin. (D) Representative flow cytometry plot and (E) MLN total cell count of CD4+ T cells expressing IL-17, IFN-γ, IL-5, and IL-13 (n = 10 mice). **P < 0.01 and ***P < 0.001 by Student’s t test.

  • Fig. 2 LPS promotes NETosis during allergen sensitization.

    (A) BALFs and MLNs were harvested on protocol day 3 after allergen sensitization with HDM/Veh or HDM/LPS. (B) BALF total cell count and (C) leukocyte differential count on protocol day 3 from HDM/Veh- or HDM/LPS-sensitized mice. Data are representative of two independent experiments with n > 3. (D) PicoGreen assay showing NET-associated DNA present in BALF (n = 4 mice). (E) Western blot showing citH3 in BALFs (top) and Ponceau S stain as loading control (bottom). (F) Representative flow cytometry plot showing the presence of DNA-positive neutrophils (PMNs) and DNA-negative cytoplasts in BALFs from HDM/Veh (left) and HDM/LPS (right) mice (n = 8). SSC, side scatter. (G) MLN total cell count for CD45+CD11b+Ly6g+ cells and (H) DNA-positive PMN and DNA-negative cytoplast count (n = 4 mice). *P <0.05 and **P < 0.01 by two-tailed unpaired Student’s t test, where indicated, or Mann-Whitney U test.

  • Fig. 3 DNase instillation altered levels of NETs but not of neutrophilia.

    Mice were subjected to DNase (intranasally, 6 hours after sensitization with HDM/LPS) or PBS control, and tissues were harvested at the end of the sensitization period (day 3). (A) PicoGreen assays showing the amount of DNA in BALFs (n = 5 mice). (B) Western blot showing citH3 (top) and Ponceau S stain as a loading control (bottom; n = 5 mice). (C to E) To assess inflammatory responses, BAL was performed at the end of the allergen (HDM) challenge on protocol day 15. (C) BALF total cell count and (D and E) leukocyte differential (% total leukocytes and cell count) were enumerated (n = 3 mice). *P < 0.05 and **P < 0.01 by Student’s t test.

  • Fig. 4 Enucleated cytoplasts formed after NETosis are intact and retain functional responses.

    Neutrophils (PMNs) and neutrophil cytoplasts were flow-sorted from HDM/LPS-treated mouse lungs (protocol day 3). (A and B) The morphology and size of the sorted PMNs and cytoplasts were determined by phase-contrast microscopy. ****P < 0.0001 by two-tailed Student’s t test. (C to E) Chemotaxis to LTB4 was assessed using a microfluidic device with sorted cells (see Materials and Methods). (C) Fluorescent microscopic image showing one chemotaxis unit in the microfluidic device. (D) PMNs or (E) cytoplasts were loaded into the microfluidic chamber with an LTB4 (100 nm) gradient and time lapse, and phase-contrast microscopic imaging was performed for 6 hours (representative images). (F) Measurements of the number of cells that entered the migration microchannels per unit at various conditions. ****P < 0.0001 using one-way ANOVA. (G) Measurements of chemotaxis velocity of PMNs in the migration channels and chemokinesis velocity of cytoplasts in the cell loading channel. ****P < 0.0001 using one-way ANOVA. (H) Individual trajectories of cytoplast chemokinesis in the cell loading channel. (I to K) Phagocytosis by PMNs and cytoplasts was determined using pHrodo-coupled E. coli particles. (I) Absence of nuclei in cytoplasts confirmed by DAPI staining. (J) Phagocytosis of pHrodo-coupled E. coli particles leading to fluorescent color change in PMNs and cytoplasts. (K) Phagocytosis index (% total) was determined. This experiment was performed three times. Values represent the mean, and error bars represent the SEM. (L) The killing capacity of sorted neutrophils and cytoplasts toward S. pneumoniae (serotype 1) was determined at different time points indicated. This experiment was performed two times. Values represent the mean between duplicate controls, and error bars represent the SEM used in a representative experiment.

  • Fig. 5 Deficiency in PAD4 results in decreased cytoplasts, neutrophils, and IL-17.

    WT and PAD4−/− mice were sensitized with HDM/LPS, and BALFs were collected on day 3. (A) BALF total cell count and (B) leukocyte differential were enumerated (n = 5 mice). (C) NETosis was monitored by BALF DNA levels on day 3 (n = 5 mice) and (D) Western blot for citH3 (top). Ponceau S stain was used as a loading control (bottom). (E) Representative flow cytometry plot showing DNA-positive PMNs and DNA-negative cytoplasts in BALFs collected on protocol day 3 after HDM/LPS sensitization from WT and PAD4−/− mice. (F) Percent cytoplasts were enumerated by flow cytometry criteria. (G to K) In addition to the postsensitization period, the resulting antigen-driven lung inflammation in WT and PAD4−/− mice was determined on protocol day 15 after HDM/LPS sensitization, followed by HDM challenge. (G) BALF total cell count and (H) BALF differential count were measured. Lung histology between WT and PAD4−/− mice was evaluated by (I) hematoxylin and eosin staining. (J) PAS staining. (K) Resistance of the respiratory system (Rrs) was measured in anesthetized mice that were mechanically ventilated in the presence of ascending doses of inhaled methacholine (MCh). (L) IL-17 and IFN-γ. (M) IL-13 and IL-5. Values represent the mean, and error bars represent SEM [for (M), error bars represent SD]. *P < 0.05 and **P < 0.01 using a Student’s t test.

  • Fig. 6 Lung cytoplasts interact with DCs to induce antigen-specific T lymphocytes.

    (A) Schematic diagram showing the antigen-specific T cell activation protocol. Briefly, DCs were harvested from the lungs of HDM/Veh- or HDM/LPS-sensitized mice and were incubated overnight with cytoplasts or neutrophils (PMNs) at the indicated cell ratios (top of plots) of DC/cytoplasts (1:2 and 1:0.5) and DC/PMNs (1:10). The HDM/Veh and HDM/LPS DCs were then cocultured with naïve CD4+ T cells from DO11.10 mice in the presence of ovalbumin (OVA) peptide, and T cells were restimulated and stained for intracellular cytokines (see Materials and Methods). (B) Representative flow cytometry plots showing T cells expressing the indicated cytokines. Numbers outside the box represent percentage of CD4+ T cells. Bar graphs (right) show the absolute cell count for the number of cells expressing the indicated cytokine. Data are representative of two experiments.

  • Fig. 7 Select cytoplast surface proteins are distinct from intact neutrophils.

    BALF from HDM/LPS-sensitized mice were stained with different antibodies to detect the expression of these markers on neutrophils and cytoplasts. The expression of the markers is shown in the flow cytometry plots along with the percentage of positivity for each cell population. This experiment was performed twice.

  • Fig. 8 Lung NETosis in SA correlates with BALF IL-17.

    (A) PicoGreen assays of BALF DNA in biospecimens from HDs and patients with NSA and SA. (B) BALF DNA levels were further stratified into low neutrophil (PMN) (<5%) and high PMN (>5%). (C) Representative Western blot showing citH3 in BALFs from HD, NSA, and SA with low or high PMN count (top) and Ponceau S stain as a loading control (bottom). (D) Correlation between BALF DNA and PMN count in SA. (E) Correlation between BALF IL-17 levels and number of PMNs. (F) Correlation between BALF IL-17 levels and number of BALF cytoplasts. Pearson correlation r values and significance are noted for each correlation, and regression lines are shown.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/26/eaao4747/DC1

    Fig. S1. A mouse model of neutrophilic airway inflammation is associated with the neutrophil recruitment and TH17 activation during sensitization.

    Fig. S2. Gating strategy to identify neutrophils and cytoplasts.

    Fig. S3. Anti–IL-17 administration during the sensitization phase blunts neutrophilia without affecting cytoplast generation.

    Fig. S4. Protease-free DNase does not affect neutrophilia.

    Fig. S5. Citrullinated histone identified on neutrophils from WT mice.

    Fig. S6. Cytoplast promotes T cell responses via contact-dependent mechanism.

    Fig. S7. Gating strategies for detection of cytoplasts in human BALF from severe asthmatics.

    Table S1. Patient characteristics.

    Table S2. Neutrophil and DNA high asthma patient symptoms, lung function, and comorbidities.

    Movie S1. Murine lung neutrophils were flow-sorted after HDM/LPS treatment (3-day exposure), and chemotaxis to LTB4 was monitored in a microfluidic chamber.

    Movie S2. Murine lung cytoplasts were flow-sorted after HDM/LPS treatment (3-day exposure), and chemotaxis to LTB4 was monitored in a microfluidic chamber.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. A mouse model of neutrophilic airway inflammation is associated with the neutrophil recruitment and TH17 activation during sensitization.
    • Fig. S2. Gating strategy to identify neutrophils and cytoplasts.
    • Fig. S3. Anti–IL-17 administration during the sensitization phase blunts neutrophilia without affecting cytoplast generation.
    • Fig. S4. Protease-free DNase does not affect neutrophilia.
    • Fig. S5. Citrullinated histone identified on neutrophils from WT mice.
    • Fig. S6. Cytoplast promotes T cell responses via contact-dependent mechanism.
    • Fig. S7. Gating strategies for detection of cytoplasts in human BALF from severe asthmatics.
    • Table S1. Patient characteristics.
    • Table S2. Neutrophil and DNA high asthma patient symptoms, lung function, and comorbidities.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Murine lung neutrophils were flow-sorted after HDM/LPS treatment (3-day exposure), and chemotaxis to LTB4 was monitored in a microfluidic chamber.
    • Movie S2 (.avi format). Murine lung cytoplasts were flow-sorted after HDM/LPS treatment (3-day exposure), and chemotaxis to LTB4 was monitored in a microfluidic chamber.
    • Raw data (Microsoft Excel format)

    Files in this Data Supplement:

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