Research ArticleALLERGY

Inception of early-life allergen–induced airway hyperresponsiveness is reliant on IL-13+CD4+ T cells

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Science Immunology  07 Sep 2018:
Vol. 3, Issue 27, eaan4128
DOI: 10.1126/sciimmunol.aan4128
  • Fig. 1 T cells and ILCs are equally important sources of IL-13 in neonatal mice exposed to rIL-33.

    (A) BALB/c mice ages 3 days and 6 to 8 weeks were exposed to intermittent intranasal rIL-33 (50 μg/kg) or PBS for 2 weeks (red arrows). Harvest was carried out 24 hours after the final dose. Numbers of CD3+CD4+IL-13+ T cells compared with ILCs (LinnegCD45+CD90+IL-13+) in lungs of (B) adult and (C) neonatal BALB/c mice. (D) Pulmonary eosinophils (SiglecF+CD11clow/neg) enumerated by flow cytometry. Airway resistance to methacholine (Mch) in (E) neonatal and (F) adult mice exposed to intranasal rIL-33. (G) IL-13 gene expression in the lung. (H) Levels of IL-13 in the lungs. (I) IL-5 gene expression in the lung. Expression of (J) Muc5ac and (K) Muc5b. n = 6 to 8 rIL-33, n = 4 to 5 PBS. Data are representative of at least two experiments. *P < 0.05, **P < 0.01.

  • Fig. 2 In response to allergen, T cells and ILCs are equally important sources of IL-13.

    Airway responsiveness to methacholine in (A) neonatal and (B) adult mice. (C) Pulmonary eosinophils (SiglecF+CD11clow/neg). Numbers of LinnegCD45+CD90+IL-13+ILCs and CD3+CD4+IL-13+ T cells in lungs of (D) adult and (E) neonatal mice. (F) Levels of IL-13 in the lung of mice exposed to HDM. IL-13+ ILCs and T cells in lungs of (G) adult and (H) neonatal mice exposed to ALT. (I) Levels of IL-13 in the lung of mice exposed to A. alternata (ALT). n = 6 to 8 allergen-exposed, n = 4 to 6 PBS. Data are representative of two experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 IL-13 is a feature of allergen-activated neonatal T cells.

    CD4+ T cells from HDM and A. alternata (ALT)–treated neonatal and adult mice were classified as either IL-13+ or IL-13neg and analyzed for surface markers. Cells were defined as either (A and B) memory-like (CD44+CD62Llow), (C and D) epithelial associated (CD103+), or (E and F) activated (CD69+/ICOS+). The proportion of neonatal and adult CD4+ T cells from (G and H) PBS control, (I and J) HDM-treated, or (K and L) ALT-exposed mice expressing cytokine were also enumerated. n = 7 to 9 allergen-exposed, n = 5 PBS. Data are representative of two experiments. Human cord blood was analyzed for (M) total CD4+ T cells and total ILC2 (LinnegCD161+CD127+CRTH2+CD56neg) and (N) IL-13+ CD4+ T cells and IL-13+ ILC2. (O) Neonatal T cells expressed TCR-β but lacked expression of CD1d dimer. Values are expressed as a percentage of live CD45+ lymphoid cells. n = 20. **P < 0.01, ***P < 0.001.

  • Fig. 4 IL-33 is not critical for initiation of allergic airways disease phenotype.

    Three-day-old WT BALB/c and Il33−/−mice were exposed to intermittent doses of HDM or PBS. (A) Airway responsiveness to methacholine in neonatal WT and Il33−/− mice. (B) Pulmonary eosinophils (SiglecF+CD11clow/neg). Numbers of pulmonary (C) LinnegCD45+IL-13+ILCs and (D) CD3+CD4+IL-13+ T cells. Levels of (E) IL-33, (F) IL-5, and (G) IL-13 in the lung. n = 7 to 12 HDM, n = 6 to 8 PBS. Data are representative of two experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 T cell–derived IL-13 is essential for the inception of AHR.

    Three-day-old WT BALB/c and Cd4-cre Il-4Il13fl/fl (CD4Cre) mice were exposed to intermittent doses of HDM, A. alternata (ALT), or PBS for 2 weeks. (A) Representative flow cytometry plots of cytokine expressing (IL-5 and/or IL-13) CD3+CD4+ T cells. Airway responsiveness to methacholine in neonatal mice exposed to (B) HDM or (C) ALT. Lung eosinophils enumerated by flow cytometry in mice exposed to (D) HDM or (E) ALT. LinnegCD45+CD90+IL-13+ILCs in mice exposed to (F) HDM or (G) ALT. IL-5+ T cells to in mice exposed to (H) HDM or (I) ALT. IL-13 levels in mice exposed (J) HDM or (K) ALT. Muc5ac gene expression in mice exposed to (L) HDM or (M) ALT. n = 6 to 8 allergen-exposed, n = 4 to 6 PBS. Data are representative of at least two experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 Protection from AHR in neonatal mice can be achieved by reduction of IL-13+ T cells.

    (A) BALB/c mice aged 3 days were exposed to intermittent intranasal A. lwoffii F78 (1.3 × 108 cfu for the first 2 weeks and then 2 × 108 cfu) or PBS (black arrows), followed by HDM (10 μg for the first 2 weeks and then 15 μg) or PBS for 3 weeks (red arrows). Analysis was carried out 24 hours after the final dose. (B) AHR to methacholine after 3 weeks of A. lwoffii F78 and HDM coexposure. (C) Numbers of eosinophils (SiglecF+CD11clow/neg) in the lung. (D) IL-33 and (E) IL-13 levels from lung homogenate. (F) Numbers of ILCs (LinnegCD45+IL-13+) and (G) CD3+CD4+IL-13+ T cells. (H) IL-5 levels in the lung tissue. (I) DC populations in the lung enumerated by flow cytometry. cDC (CD11b+CD11chigh), pDC (CD11cintLy6c+CD64neg), MoDC (CD11c+Ly6c+CD64+). n = 8 for HDM and bacteria exposed groups, n = 5 for PBS. *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative of three experiments.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/27/eaan4128/DC1

    Fig. S1. Defining IL-13+ CD4+ T cells and ILCs in neonatal mice.

    Fig. S2. A. alternata induces AHR in neonatal mice.

    Fig. S3. Allergen exposure in neonatal SCID mice does not result in AHR.

    Table S1. Antibodies used for flow cytometry.

    Table S2. Raw data sets.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Defining IL-13+ CD4+ T cells and ILCs in neonatal mice.
    • Fig. S2. A. alternata induces AHR in neonatal mice.
    • Fig. S3. Allergen exposure in neonatal SCID mice does not result in AHR.
    • Table S1. Antibodies used for flow cytometry.
    • Legend for table S2

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    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Raw data sets.

    Files in this Data Supplement:

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