Research ArticleINNATE LYMPHOID CELLS

Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans

See allHide authors and affiliations

Science Immunology  07 Jun 2019:
Vol. 4, Issue 36, eaav7638
DOI: 10.1126/sciimmunol.aav7638
  • Fig. 1 The number of ILC2s rapidly increases in the peribronchial and perivascular region after rIL-33 treatment.

    IL-13–eGFP mice were treated with three doses of rIL-33 (1 μg per dose), Alt (10 μg), or PBS (25 μl) over 1 week and culled 24 hours after the final dose. The frequency of ILC2s (GFP+CD45+LinCD3NKp46CD127+CD90.2+KLRG1+CD25varIL-13+IL-5+) in the (A) airways (BAL fluid), (B) lung, and (C) lung-draining lymph nodes (lung dLN; mediastinal). Live viable PCLSs of 200-μm thickness were obtained and stained for CD31 (magenta; the lung structure and blood vessels), CD4 (cyan; T cells, orange arrow), EpCAM (red; to visualize bronchial epithelium), and GFP (ILC2s, white arrow). Images of 1024 μm by 1024 μm field of view (FOV) were taken under a 20× objective using an inverted confocal microscope. (D) Images showing ILC2 (GFP+CD4) CD4+ T cells (CD4+GFP) location in PBS-, rIL-33–, and Alt-treated mice. Scale bars, 150 μm. (E) Number of ILC2s (GFP+CD4) in lung sections per FOV taken under a 10× objective. (F) Schematic illustration of the lung depicting the anatomical location in the lung where PCLSs were prepared. Representative images show two regions of the lung slice from a rIL-33–treated mouse showing distribution of ILC2s and CD4+ T cells. Scale bars, 50 μm. n = 4 mice per group [mock (PBS)], n = 6 mice per group (Alt or rIL-33 treatment). Data are representative of four experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 2 rIL-33 stimulation induces ILC2 motility around blood vessels and airways.

    IL-13–eGFP mice were treated with three doses of rIL-33 (1 μg per dose) over 1 week and culled 24 hours after the final dose. Live viable PCLSs of 200-μm thickness were obtained and stained for CD31 (magenta; the lung structure and blood vessels), CD4 (cyan; T cells, orange arrow), EpCAM (red; to visualize bronchial epithelium), and GFP (ILC2s, white arrow), and time-lapse video was taken (1024 μm by 1024 μm FOV, 45-min duration under a 20× objective using an inverted confocal microscope). (A) Static image depicting the location of ILC2s and CD4+ T cells. Scale bar, 100 μm. (B) Zoomed in section of the blood vessel in (A). Scale bar, 20 μm. (C) High-power images of boxed cells in (B) showing differences in pattern of cell movement (oscillatory versus amoeboid movement). ILC2s and CD4+ T cells dynamics were tracked and plotted as (D) individual tracks or (E) tracks commencing from centroid and overlaid. (F) Track speed, (G) track length, and (H) track displacement were quantified. Representative images shown in (A) to (C) are from rIL-33–treated mice, where n = 6 mice per treatment (three slices per mouse were imaged). For (F) to (H) in box and whiskers plots, each dot represents an individual cell. Data are representative of four experiments, where n = 6 mice per treatment. **P < 0.01 and ****P < 0.0001.

  • Fig. 3 rIL-33 stimulation induces ILC2 motility around blood vessels and airways.

    IL-13–eGFP mice were treated with three doses of rIL-33 (1 μg per dose) or Alt (10 μg) over 1 week. Live PCLSs were obtained, ILC2 dynamics were compared between the two, and the differences were plotted as (A) individual tracks and (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length, and (E) track displacement. Intravital microscopy (IVM) was performed in live IL-13–eGFP mice after rIL-33 treatment (one 512 μm by 512 μm FOV in a 1-hour-duration video). (F) Static images of different frames captured during the course of the video depicting amoeboid shape changes of ILC2s at separate time points. Scale bars, 20 μm. n ≥ 4 mice per group. Data are representative from four experiments. ****P < 0.0001. Quantifications from (A) to (E) are representative of four experiments, where n = 6 mice per treatment (three slices per mouse were imaged). For (F), intravital microscopy images are representative of six individual IL-33–treated mice.

  • Fig. 4 ILC2s use distinct chemotactic pathways to home to inflammatory sites in the lung.

    IL-13–eGFP mice were treated with three doses of rIL-33 (1 μg per dose) or PBS (25 μl) over 1 week and culled 24 hours after the final dose. (A) The percentage of murine ILC2s (CD45+LinNKp46CD3) expressing CCR1, CCR4, and CCR8. CCL8 levels in murine (B) BAL and (C) lung. (D) Location of CCL8 expression and ILC2s and (E) quantified CCL8 deposits in PCLSs stained for CD31 (magenta; the lung structure and blood vessels), CCL8 (cyan; yellow arrow), EpCAM (red; to visualize bronchial epithelium), and GFP (ILC2s, white arrow), images of 1024 μm by 1024 μm FOV. Scale bars, 150 μm. Human ILC2 lines were generated, and migration to varying concentrations of (F) PGD2 and (G) CCL8 was determined. (H) Peak migratory responses of a human ILC2 cell line to IL-25, TGF-β, rIL-33, CCL8, and PGD2. IL-13–eGFP mice treated with rIL-33 were also treated with 5 μg of purified anti-mouse CCR8 Ab intraperitoneally, rCCL8 intranasally, or an isotype control, and PCLSs were obtained and stained. (I) Localization of ILC2s in live PCLSs. (J) Number of ILC2s per FOV under 10× objective. Time-lapse imaging of 45 min duration was performed, and ILC2 (K) track from centroid, (L) track length, (M) track speed, and (N) track displacement were quantified. In box and whiskers graphs, each data point represents an individual cell. BALB/c mice treated with rIL-33 were further treated with rCCL8, αCCR8, or isotype (Iso) control Ab. (O) Percentage of IL-13+IL-5+ ILC2s (CD45+linNKp46CD3GATA-3+). (P) Representation histogram of MFI of IL-13 and IL-5 and quantification of MFI for (Q) IL-13 and (R) IL-5 from GATA+ ILC2s. For (A) to (E), n ≥ 4 mice per group. Data are representative of four experiments. For (F) to (H), n = 3 individual donors. Data are representative of three experiments. For (I) to (R), n = 5 mice per group. Data are representative from two experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001.

  • Fig. 5 ECM proteins, collagen-IV, and fibronectin promote increased ILC2 motility.

    Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin, collagen-I, collagen-IV, or serum-free coating (control) for 24 hours. Cell movement was imaged via the JuLI imaging system and plotted as (A) individual tracks, (B) track speed dot plots, and (C) track speed spider plots. IL-13–eGFP mice were treated with three doses of rIL-33 (1 μg per dose) or PBS (25 μl) over 1 week and culled 24 hours after the final-dose PCLS was obtained. (D) SHG imaging of PCLSs revealing collagen fibers, representative maximum intensity projections. Scale bars, 50 μm. (E) GLCM analysis of SHG imaging. a.u., arbitrary units. (F) Images of fibronectin expression and localization. PCLS stained for CD31 (magenta; the lung structure and blood vessels), fibronectin (cyan; yellow arrow), EpCAM (red; to visualize bronchial epithelium), and GFP (ILC2s, white arrow), and images of 1024 μm by 1024 μm FOV were taken. Scale bars, 150 μm. For (A) to (C), n = 3 donors (in triplicate). Data are representative of three experiments. For (D) to (F), n = 6 (in triplicate). ****P < 0.0001.

  • Fig. 6 Collagen-I enhances ILC2 actin cytoskeletal remodeling and polarity.

    Human ILC2 lines were seeded on tissue culture plates coated with either 10% FBS, fibronectin (FBN), collagen-I, collagen-IV, or serum-free coating (control) and imaged after 12 hours. (A) Bright-field images depicting change in shape. (B) Actin remodeling after staining with phalloidin (green) and DAPI (cyan) and imaging using Airyscan detection (maximum intensity projections). Scale bars, 5 μm. (C) Cell area. (D) Cell perimeter. n = 3 donors. Data are representative of two experiments. ***P < 0.001.

  • Fig. 7 Blocking collagen fibrillogenesis in vivo increases ILC2 dynamics in the inflamed lung.

    IL-13–eGFP mice treated with rIL-33 were further treated with BAPN along with controls were culled 24 hours after the final dose. ILC2 dynamics from live PCLSs were plotted as either (A) individual tracks or (B) tracks commencing from centroid and overlaid. Differences in tracks between treatments were quantified as (C) track speed, (D) track length, and (E) track displacement. Total ILC2s in (F) lungs, (G) BAL, and (H) blood were enumerated. For (A) to (E), n ≥ 4 mice per group. Data are representative from four experiments. For (F) to (H), n = 6 mice per group. Data are representative from two experiments. ***P < 0.001 and ****P < 0.0001.

  • Fig. 8 Blocking collagen fibrillogenesis reduces eosinophil accumulation in the inflamed lung.

    BALB/c mice were treated with rIL-33 or PBS with or without BAPN. (A) Airway responsiveness to methacholine. BL, baseline; veh, vehicle. Lung (B) alveolar macrophages (AM), (C) dendritic cells (DC), (D) neutrophils, and (E) eosinophils were enumerated by flow cytometry. (F) CCL24 protein in lung tissue. (G) Congo red-stained eosinophils (blue arrow) in lung histological samples. Scale bar, 50 μm. (H) Quantification of eosinophil from histological samples. For (A) to (E), n ≥ 6 mice per group. Data are representative from two experiments. For (G) and (H), n ≥ 4 mice per group. Data are representative of two experiments. *P < 0.05 and **P < 0.01. ns, not significant.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/36/eaav7638/DC1

    Fig. S1. Gating strategy for identification of ILC2 populations, related to Fig. 1 (A to C).

    Fig. S2. Quantification of ILC2s in different tissues, related to Fig. 1 (A to C).

    Fig. S3. Gating strategy for identification of ILC2 populations based on GATA-3 expression, related to Fig. 1 (A to C).

    Fig. S4. Distribution of ILC2s and CD4+ T cells in rIL-33–treated mice lungs, related to Fig. 1 (D and F).

    Fig. S5. Chemokine receptor expression on ILCs, related to Fig. 4 (A and B).

    Fig. S6. Phenotype and IL-13 production by human ILC2 lines, related to Figs. 4 and 5.

    Fig. S7. ILC2 motility on ECM proteins, collagen-III, and proteoglycans, versican and tenascin-C, related to Fig. 5 (A to C).

    Fig. S8. BAPN fails to affect ILC2 cytokine production and eosinophil movement, related to Fig. 8G.

    Table S1. Antibodies used for imaging of PCLS.

    Table S2. Antibodies used for flow cytometry.

    Table S3. Antibodies used for sorting human ILC2.

    Movie S1. Time-lapse imaging of rIL-33–treated mouse lung PCLSs showing ILC2 movement and location, related to Fig. 2A.

    Movie S2. Time-lapse imaging of rIL-33–treated mouse lung PCLSs depicting differences in movement of independent ILC2s, related to Fig. 2B.

    Movie S3. Time-lapse imaging of rIL-33–treated mouse lung PCLSs depicting high-power videos differences in movement of independent ILC2s, related to Fig. 2C.

    Movie S4. Intravital imaging of rIL-33–treated mouse lung depicting amoeboid ILC2 movement, related to Fig. 2H.

    Movie S5. Influence of ILC2 survival and cytokines on ILC2 motility, related to Fig. 5 (A to C).

    Movie S6. Time-lapse imaging of lung PCLSs from rIL-33–treated mouse treated with anti-CCR8 Ab or isotype Ab, related to Fig. 4I.

    Movie S7. Collagen-I–induced ILC2 shape change and elongation of cell body, related to Fig. 5A.

    Movie S8. Time-lapse imaging of a lung PCLSs assessing the effect of BAPN on rIL-33–treated mouse lung eosinophil movement, related to Fig. 8G.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Gating strategy for identification of ILC2 populations, related to Fig. 1 (A to C).
    • Fig. S2. Quantification of ILC2s in different tissues, related to Fig. 1 (A to C).
    • Fig. S3. Gating strategy for identification of ILC2 populations based on GATA-3 expression, related to Fig. 1 (A to C).
    • Fig. S4. Distribution of ILC2s and CD4+ T cells in rIL-33–treated mice lungs, related to Fig. 1 (D and F).
    • Fig. S5. Chemokine receptor expression on ILCs, related to Fig. 4 (A and B).
    • Fig. S6. Phenotype and IL-13 production by human ILC2 lines, related to Figs. 4 and 5.
    • Fig. S7. ILC2 motility on ECM proteins, collagen-III, and proteoglycans, versican and tenascin-C, related to Fig. 5 (A to C).
    • Fig. S8. BAPN fails to affect ILC2 cytokine production and eosinophil movement, related to Fig. 8G.
    • Table S1. Antibodies used for imaging of PCLS.
    • Table S2. Antibodies used for flow cytometry.
    • Table S3. Antibodies used for sorting human ILC2.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Time-lapse imaging of rIL-33–treated mouse lung PCLSs showing ILC2 movement and location, related to Fig. 2A.
    • Movie S2 (.mp4 format). Time-lapse imaging of rIL-33–treated mouse lung PCLSs depicting differences in movement of independent ILC2s, related to Fig. 2B.
    • Movie S3 (.mp4 format). Time-lapse imaging of rIL-33–treated mouse lung PCLSs depicting high-power videos differences in movement of independent ILC2s, related to Fig. 2C.
    • Movie S4 (.mp4 format). Intravital imaging of rIL-33–treated mouse lung depicting amoeboid ILC2 movement, related to Fig. 2H.
    • Movie S5 (.mp4 format). Influence of ILC2 survival and cytokines on ILC2 motility, related to Fig. 5 (A to C).
    • Movie S6 (.mp4 format). Time-lapse imaging of lung PCLSs from rIL-33–treated mouse treated with anti-CCR8 Ab or isotype Ab, related to Fig. 4I.
    • Movie S7 (.mp4 format). Collagen-I–induced ILC2 shape change and elongation of cell body, related to Fig. 5A.
    • Movie S8 (.mp4 format). Time-lapse imaging of a lung PCLSs assessing the effect of BAPN on rIL-33–treated mouse lung eosinophil movement, related to Fig. 8G.

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article