Research ArticleINFLAMMATION

The cysteinyl leukotriene 3 receptor regulates expansion of IL-25–producing airway brush cells leading to type 2 inflammation

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Science Immunology  05 Oct 2018:
Vol. 3, Issue 28, eaat9453
DOI: 10.1126/sciimmunol.aat9453
  • Fig. 1 BrCs, chemosensory cholinergic cells, express IL-25 and the CysLT generating machinery.

    Tracheas of naïve WT (A), ChAT(BAC)-eGFP (B), and Il25F25/F25 (C) mice were stained for DCLK1, Gα-gustducin, ChAT-eGFP, IL-25–RFP, and Hoechst 33342 (blue). Scale bars, 20 μm. (D) Principal components analysis of BrCs (EpCAM+CD45eGFP+), EpCAM+ EpCs (EpCAM+CD45eGFP), and resident hematopoietic cells (CD45+) analyzed by RNA-seq using the top 500 most variable transcripts. Numbers in parentheses indicate percent variance described by each principal component. (E to H) EpCAM+ EpC and BrC-enriched transcripts were determined using criteria of fourfold or greater expression in one relative to the other with a P adjusted value of <0.05. (E) Volcano plot representing the fold change (log2 fold change) and FDR (−log10 P adjusted value) of differentially expressed genes in EpCAM+ EpCs compared with BrCs. Genes highlighted in purple are fourfold differentially expressed. Genes highlighted in red are 32-fold differentially expressed. (F) Transcripts for genes encoding taste receptors, taste receptor signaling machinery, acetyl choline synthesis machinery, and enzymes involved in lipid mediator generation showing specific enrichment in BrCs compared with EpCAM+ EpCs. (G) Selected genes differentially expressed in BrCs compared with EpCAM+ EpCs and CD45+ cells. y axis indicates average of normalized TPM. (H) Transcription factors identified in the RIKEN transcription factor database that are enriched in BrCs compared with EpCAM+ EpCs.

  • Fig. 2 Common aeroallergens induce BrC expansion in a CysLT-dependent fashion.

    (A to E) WT, Stat6−/−, and Ltc4s−/− mice were given a single dose of intranasal Alternaria, Df, or PBS, and tracheal BrCs were assessed 3 days after the challenge. (A to C) DCLK1 (red) immunofluorescence in the tracheal epithelium from WT, Stat6−/−, and Ltc4s−/− after PBS or Alternaria challenges. Circles and the rectangle indicate clusters of DCLK1+ cells. Confocal maximum projection images of full-thickness z-stacks of the tracheal epithelium in tracheal whole mounts. Scale bar, 50 μm. Right panel represents magnified doublets and triplets from Alternaria-challenged WT mice. Scale bars, 20 μm. (D) Immunohistochemical quantitation of BrCs in the trachea of indicated genotypes after challenge with PBS or a 30-μg dose of Alternaria. (E) Quantitation of BrCs in the trachea after challenge with a single 100-μg dose of Df or PBS. Data in (A) to (C) are representative of at least three experiments, and data in (D) and (E) are means ± SEM from three or more experiments; each circle represents a separate mouse. *P < 0.05, **P < 0.01.

  • Fig. 3 CysLT3R is a BrC-associated receptor and regulates aeroallergen-induced BrC expansion.

    (A to G) WT and Oxgr1−/− mice were given a single dose of intranasal Alternaria, Df, or PBS and were assessed 3 days after the challenge. (A) Transcripts of the three CysLT receptors and indicated BrC-specific genes were assessed in the trachea of PBS- or Alternaria-challenged WT mice on day 3 by quantitative PCR. Correlations between CysLTRs and BrC genes were performed using linear regression (R2). (B) ISH for Oxgr1 in cross sections of trachea from naïve WT and Oxgr1−/− mice. Positive red staining in WT mice is indicated by arrows. (C) X-gal reactivity in cross sections of trachea from naïve WT and Oxgr1–/− mice. E. coli β-galactosidase activity in Oxgr1−/− mice produces blue precipitates (arrows) absent in WT mice. (D) Colocalization (arrows) of X-gal reactivity and DCLK1 in cross sections of tracheas from Oxgr1−/− mice that carry the targeted insertion of lacZ, encoding E. coli β-galactosidase. Scale bars, 25 μm (B to D). (E) DCLK1 immunofluorescence in whole mounts of trachea from WT and Oxgr1−/− mice after PBS or Alternaria challenges. Circles indicate clusters of DCLK1+ cells. Confocal maximum projection images of full-thickness z-stacks of the tracheal epithelium in tracheal whole mounts. Scale bar, 50 μm. (F and G) Quantitation of BrCs in the trachea of WT and Oxgr1−/− mice challenged with Alternaria (F) or Df (G). Data in (F) and (G) are means ± SEM from at least three experiments; each circle represents a separate mouse. *P < 0.05, ** P < 0.01.

  • Fig. 4 CysLT3R regulates BrC expansion through an IL-25–dependent but STAT6-independent pathway.

    (A to E) WT, Oxgr1−/−, Stat6−/−, and Il25F25/F25 mice were given intranasal LTE4 or PBS daily for 4 days, and DCLK1+ and/or IL-25–RFP+ cells were evaluated in the trachea 24 hours after the last challenge. (A) DCLK1+ cells in the tracheal epithelium of WT mice after PBS or LTE4 challenges; three-dimensional confocal microscopy presenting apical (top) and sagittal (bottom) views of the EpCs derived from full-thickness z-stack images of the tracheal epithelium. Circles indicate doublets of DCLK1+ cells. Scale bar, 50 μm. (B) Quantitation of DCLK1+ cells in WT, Oxgr1−/−, and Stat6−/− mice after LTE4 challenges. (C and D) DCLK1+ and IL-25–RFP+ cells in tracheas of Il25F25/F25 mice with and without challenges with LTE4 and quantitation of cells positive for IL-25–RFP. Scale bars, 25 μm. (E) WT and Oxgr1−/− mice were given intranasal LTE4 or PBS challenges as above and injected intraperitoneally with 100 μg of IL-25 antibody (35B) or rat IgG1k on days 0 and 2, and DCLK1+ cells were evaluated in the trachea 24 hours after the last challenge. Results are means ± SEM pooled from two to three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 CysLT3R regulates IL-25–dependent type 2 lung inflammation.

    (A) WT and Oxgr1−/− mice were given four daily doses of 0 or 0.25 nmol intranasal LTE4 and analyzed 24 hours after the last inhalation. Flow cytometry analysis of dispersed lung cells showing CD45+ cells, eosinophils (CD45+SiglecF+CD11b+CD11cSSChi), ILC2s (CD45+linThy1.2+CD44+ICOS+), and CD4+ T cells (CD45+TCRβ+CD4+). (B) WT and Oxgr1−/− mice were given daily LTE4 as above and treated with 100 μg of IL-25 antibody or rat IgG1k on days 0 and 2 intraperitoneally. Lung CD45+ cells, eosinophils, KLRG1+ ILC2s, and CD4+ T cells 24 hours after the last LTE4 inhalation. (C) WT and Oxgr1−/− mice were given a single dose of Alternaria (30 μg) or PBS intranasally on day 0. The total number of lung inflammatory cells (CD45+), eosinophils, ILC2s, and CD4 T cells was enumerated 3 days later. (D) Alternaria-treated mice were given IL-25 antibody intraperitoneally on days 0 and 2. The total number of lung inflammatory cells (CD45+), eosinophils, KLRG1+ ILC2s, and CD4 T cells on day 3. Data are means ± SEM from three experiments; each circle represents a separate mouse. *P < 0.05, **P < 0.01, ***P < 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/28/eaat9453/DC1

    Fig. S1. Histological and flow cytometrical characterization of BrCs.

    Fig. S2. Colocalization of X-gal and DCLK1 in whole tracheal mounts.

    Fig. S3. Flow cytometry data and gating strategy for eosinophils, ILC2s, CD4 T cells, macrophages, and DCs.

    Fig. S4. LTE4-elicited airway inflammation is STAT6-dependent.

    Fig. S5. IL-25–dependent BrC expansion and lung inflammation schema.

    Table S1. Reagents.

    Table S2. Raw data (Excel file).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Histological and flow cytometrical characterization of BrCs.
    • Fig. S2. Colocalization of X-gal and DCLK1 in whole tracheal mounts.
    • Fig. S3. Flow cytometry data and gating strategy for eosinophils, ILC2s, CD4 T cells, macrophages, and DCs.
    • Fig. S4. LTE4-elicited airway inflammation is STAT6-dependent.
    • Fig. S5. IL-25–dependent BrC expansion and lung inflammation schema.
    • Table S1. Reagents.

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    Other Supplementary Material for this manuscript includes the following:

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