Impact of influenza A infection on subsequent house dust mite-induced inflammation in early life. (a) Eight-day-old mice were infected with influenza virus (Flu) or given PBS alone. Seven days later, PBS- and Flu-infected groups are exposed either to 3 weeks of HDM or Sal and killed 72 h after the last HDM exposure. (b) Cellular profile in BAL fluid showing the number of total cells, macrophages (Macs), mononuclear cells (MNC), neutrophils (Neutro), eosinophils (Eos), and the percentage of eosinophils. (c) Lung histopathology was evaluated by H&E to assess the degree of total lung inflammation and eosinophilia. All images taken at × 50 and × 400 total magnification as indicated. (d) Serum Ig levels measured by ELISA showing HDM-specific IgE, IgG1, and IgG2a and Flu-specific IgG1 and IgG2a. For all data, n=8–12 mice per group. One of two representative experiments is shown. Data represent mean±s.e.m. P<0.001 compared with *Sal, ‡Flu, and §HDM, respectively, except Flu-IgG1, P<0.01 compared with ‡Flu. ELISA, enzyme-linked immunosorbent assay; H&E, hematoxylin and eosin; HDM, house dust mite; PBS, phosphate-buffered saline.