H3N1 infection causes AHR and inflammation. (a) Change in lung resistance (RL) in 8-week-old BALB/c mice (n = 7–9 per group; anesthetized, tracheotomized, intubated and mechanically ventilated) challenged with methacholine nebulized into the airways 5 d after infection with H3N1 or allantoic fluid (mock infection control (mock)). *P < 0.01 and **P < 0.001, compared with mock infection (two-way analysis of variance (ANOVA)). (b) Macrophages (MΦ), neutrophils (Neu), eosinophils (Eos) and lymphocytes (Lym) in BAL fluid 5 d after treatment as in a. ND, not detectable. *P < 0.001, compared with mock infection (Student’s two-tailed t-test). (c) Lung sections obtained from mock- or H3N1-infected BALB/c or C57BL/6 mice, stained with hematoxylin and eosin. Scale bars, 200 μm. (d) Change in lung resistance in 8-week-old C57BL/6 mice (n = 4 per group), treated and assessed as in a. *P ≤ 0.05 and **P ≤ 0.001. (e) Cells in BAL fluid 5 d after treatment as in d. *P < 0.001 (Student’s two-tailed t-test). (f,g) AHR in 8-week-old wild-type (WT) or Rag2−/− mice (n = 6–7 per group) 5 d after infection with H3N1 or allantoic fluid, assessed as change in lung resistance (f) or cells in BAL fluid (g), as in a,b. *P ≤ 0.01 and **P ≤ 0.001 (two-way ANOVA (f) or Student’s two-tailed t-test (g)). Data are representative of three independent experiments (mean and s.e.m.).

ST2-deficient mice fail to develop H3N1-induced AHR. (a,b) AHR in 8-week-old ST2-deficient (Il1rl1^−/−) mice and their heterozygous (Il1rl1^+/−) littermates (n = 4–6 per group) 5 d after infection with H3N1 or allantoic fluid, assessed (as in Fig. 1a,b) as change in lung resistance (a) and cells in BAL fluid (b). *P < 0.05, **P < 0.01 and ***P < 0.001 (two-way ANOVA (a) or Student’s two-tailed t-test (b)). (c,d) Change in lung resistance in 8-week-old wild-type mice (c) and Rag2^−/− mice (d; n = 3–4 per group) treated with two injections of mAb to ST2 (0.5 mg) or control immunoglobulin G (IgG), on days −1 and day +4, and infected with H3N1 or allantoic fluid on day 0, assessed (as in Fig. 1a) on day +5. *P < 0.001, compared with H3N1 plus IgG (two-way ANOVA). (e) Change in lung resistance in 8-week-old ST2-deficient mice and their heterozygous littermates (n = 3 per group) sensitized with saline (control) or OVA-alum on day 0, then challenged with OVA on days 7–9 and assessed (as in Fig. 1a) on day 10. *P < 0.05 and **P < 0.001, compared with saline (two-way ANOVA). Data are representative of three independent experiments (mean and s.e.m.).

H3N1-induced AHR requires TLR7 and MyD88. (a) Change in lung resistance in 8-week-old wild-type and Tlr7^−/− mice (n = 5–6 per group) 5 d after infection with H3N1 or allantoic fluid (assessed as in Fig. 1a). (b) Cells in BAL fluid 5 d after treatment as in a. (c) Change in lung resistance in 8-week-old wild-type and Myd88^−/− mice (n = 4–6 per group) treated and assessed as in a. (d) Cells in the BAL fluid 5 d after treatment as in c. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with H3N1 (two-way ANOVA (a,c) or Student’s two-tailed t-test (b,d)). Data are representative of two independent experiments (mean and s.e.m.).

H3N1-induced production of IL-33 in alveolar macrophages. (a) Enzyme-linked immunosorbent assay of IL-33 in lungs of BALB/c mice (n = 3 per group) obtained on days 0, 1, 4 and 7 after infection with H3N1 or mock infection and homogenized in 1 ml PBS. *P < 0.05 and **P < 0.001, compared with mock infection (Student’s two-tailed t-test). (b) Flow cytometry sorting (top) of CD45⁺ interstitial macrophages (IM; F4/80⁺CD11c⁻), alveolar macrophages (AM; F4/80⁺CD11c⁺) and DCs (F4/80⁻CD11c⁺) from the lungs of BALB/c mice on day 1 after infection with H3N1 or mock infection, followed by staining with mAb to IL-33 or isotype-matched control antibody (below). Numbers adjacent to cell designation (top) or in quadrants (below) indicate percent cells in each. (c) Absolute number of IL-33⁺ interstitial macrophages, alveolar macrophages and DCs in the mice in b. *P < 0.001, compared with mock infection (Student’s two-tailed t-test). (d) IL-33 expression by CD45⁻ nonhematopoietic lung cells from the mice in b. (e) Quantitative RT-PCR analysis of IL-33 mRNA in alveolar macrophages, bone marrow–derived DCs (BMDC) or the MLE mouse lung epithelial cell line infected for 24 h in vitro with H3N1 at a multiplicity of infection of 5 (top), or in alveolar macrophages infected for 24 h in vitro with H3N1 at various multiplicities of infection (bottom). *P < 0.05 and **P < 0.001, compared with mock infection (Student’s two-tailed t-test). (f) Enzyme-linked immunosorbent assay of IL-33 in supernatants of alveolar macrophages or bone marrow–derived DCs infected for 24 or 96 h in vitro with H3N1 (multiplicity of infection, 5; 5 × 10⁵ cells per well, in 24 well plates) or given mock infection. *P < 0.001, compared with mock infection (Student’s two-tailed t-test). Data are representative of three independent experiments (a,e,f; mean and s.e.m.) or three experiments (b–d; mean and s.e.m. in c).

H3N1 infection results in a greater abundance of natural helper cells in the lungs. (a) Gating strategy for lung Lin⁻ST2⁺ cells among CD45⁺ cells. Numbers adjacent to outlined areas indicate percent cells in each gate. SSC, side scatter; FSC, forward scatter. (b,c) Expression of Sca-1 and c-Kit (b) and of c-Kit, CD90.2 (Thy-1.2), CD25, CD1d and major histocompatibility complex class II (MHCII; c) by lung cells from 8-week-old BALB/c mice 5 d after infection with H3N1 or mock infection, assessed after gating on the Lin⁻ST2⁺ subset. Numbers adjacent to outlined areas (b) indicate percent cells in each. FMO (c), fluorescence minus one (control). (d) Flow cytometry analysis of the expression of c-Kit and Sca-1 (bottom) by lung cells from 8-week-old BALB/c mice (n = 3 per group) 1, 3, 6, 9 or 15 d after infection as in b,c, assessed after gating on the Lin⁻ST2⁺ subset among CD45⁺ cells (top). Numbers adjacent to outlined areas (top) or in quadrants (bottom) indicate percent cells in each. (e,f) Frequency (e) and absolute number (f) of CD45⁺Lin⁻ST2⁺ c-Kit⁺Sca-1⁺ lung cells in d. *P < 0.05 and **P < 0.001, compared with day 0 (Student’s two-tailed t-test). (g) Flow cytometry analysis of the expression of c-Kit and Sca-1 (middle) and of CD90.2 (Thy-1.2) and Sca-1 (bottom) by CD45⁺ lung cells or BAL fluid cells from 8-week-old Rag2^−/− mice (n = 3 per group) 5 d after infection as in b,c, assessed after gating on the Lin⁻ST2⁺ subset (top). Data are representative of or three experiments (mean and s.e.m. in e,f).

IL-13 and natural helper cells cause AHR. (a,b) Change in lung resistance (a) and cells in BAL fluid (b) in 8-week-old wild-type and Il13^−/− mice after infection with H3N1 or mock infection (n = 5–6 per group; assessed as in Fig. 1a,b). *P ≤ 0.05 and **P < 0.001 (two-way ANOVA (a) or Student’s two-tailed t-test (b)). (c) Quantitative RT-PCR analysis of IL-13 mRNA (top; relative to expression at day 0) and enzyme-linked immunosorbent assay of IL-13 protein (bottom) in homogenates of H3N1-infected BALB/c lungs (n = 3 per group). *P < 0.001, compared with day 0 (Student’s two-tailed t-test). (d) Flow cytometry analysis of intracellular IL-13 and Sca-1 in CD45⁺Lin⁻ST2⁺c-kit⁺Sca-1⁺ lung cells left unstimulated (US) or stimulated for 5 h with the phorbol DC ester PMA plus ionomycin (PMA + iono), assessed after gating (as in top row) on Lin⁺ST2⁺ cells (middle row) or Lin⁻ST2⁺ cells (bottom row). (e) Intracellular IL-13 and Sca-1 in lung cells obtained from wild-type or Rag2^−/− mice IL-13 on day 5 d after infection with H3N1 or mock infection and stimulated for 5 h with PMA plus ionomycin (bottom), assessed after gating on Lin⁻ST2⁺c-Kit⁺ cells (top). (f) Absolute number of Lin⁻ST2⁺c-Kit⁺Sca-1⁺ cells (left) or Lin⁻ST2⁺c-Kit⁺Sca-1⁺IL-13⁺ cells (right) in the lungs of mice in e. *P ≤ 0.001 (Student’s two-tailed t-test). (g) Flow cytometry (top) of lung interstitial macrophages (F4/80⁺CD11c⁻), alveolar macrophages (F4/80⁺CD11c⁺) and DCs (F4/80⁻CD11c⁺) among lung leukocytes (CD45⁺) from the mice in e; below, intracellular IL-13 expression in single cells. Data are representative of three experiments (a–d; mean and s.e.m. in c) or three independent experiments (e–g; mean and s.e.m. in f).

Natural helper cells are essential for H3N1-induced AHR in Rag2^−/− mice. (a,b) Change in lung resistance (a) and cells in BAL fluid (b) in 8-week-old Rag2^−/− mice (n = 4 per group) left undepleted (mock and H3N1) or depleted of CD90.2⁺ (Thy-1.2⁺) cells by three injections of mAb to CD90.2 (30-H12; 0.5 mg per mouse) on days −3, 0 and +3 (H3N1 + mAb to CD90.2), and infected with H3N1 or mock infected on day 0, analyzed 5 d after infection (as in Fig. 1a,b). *P < 0.05 and **P < 0.001, compared with H3N1 infection (two-way ANOVA (a) or Student’s two-tailed t-test (b)). (c,d) Change in lung resistance (c) and cells in BAL fluid (d) in Il13^−/− recipients (n = 4 per group) given purified natural helper cells (Lin⁻ST2⁺ subsets) from Il13^+/+ (Rag2^−/−) donors or Il13^−/− donors treated intranasally with IL-33 (1 μg) 5 d before adoptive transfer of cells (1× 10⁵ cells/mouse) by intratracheal injection, followed by mock infection or infection of recipients with H3N1 and analysis 5 d after infection (as in Fig. 1a,b). *P ≤ 0.001 (two-way ANOVA (c) or Student’s two-tailed t-test (d)). Data are representative of two (a,b) or three (c,d) independent experiments (mean and s.e.m.).