Figure 1.

High mortality and macrophage-related overactivation of inflammatory responses in infected mice. Mice (n = 6 or 10, per group) were infected with freshly prepared influenza virus A/FM/1/47 (H1N1) strain. Mice (n = 10, per group) and monitored daily for survival and body weight. For lung histopathology, the mice (n = 6, per group) were euthanized with 10% phenobarbital to harvest the whole lungs on day 4 post-infection. (A) Percentage survival. Data below individual survival curves represent the number of survivors/total number of mice. (B) Bodyweight. Mice were weighed every day. (C) Lung histopathology score. Data were calculated by evaluating the histopathological severity of the analyzed sections of each lung (six separate random fields per tissue section); the scores were assigned as: normal = 0; minor = 1; mild= 2; intermediate = 3; and severe = 4. (D) Lung/body index was calculated as follows: whole lung weight (mg)/body weight. (E, F) The right lobes of the mice were homogenized to quantify the total protein (E) and inflammatory cytokine TNF-α (F). (G-H) Flow cytometric analysis of the macrophage number and phenotype in BALF; macrophages (CD11b and F4/80 positive cells), M1 polarized macrophages (CD11b, F4/80 and CD86 triple-positive cells), and M2 polarized macrophages (CD11b, F4/80 and CD206 triple-positive cells). Mean ± SD from at least three independent experiments are shown. **p < 0.01. (I) H&E staining of the lung tissues. Scale bar: 40 μm. (J) Immunofluorescence histochemical staining of the lung tissues. Scale bar: 50 μm.