Groups of 10 wildtype, cryopyrin^−/− (CRYO^−/−) and casapse-1 (CASP1^−/−) mice were infected i.n. with 8×10³ EID₅₀ (= 1LD₅₀) of PR8 and survival was monitored daily for 14d. Differences in group survival were analyzed with Cox proportional hazards test and p<0.05 was considered statistically significant.

Groups (n=4) of B6 (WT), cryopyrin^−/− (CRYO^−/−) and ipaf^−/− (IPAF^−/−) mice were infected i.n with 4×10³ EID₅₀ of PR8 influenza virus and sampled 3d later to determine levels of IL-1β (A), IL-18 (B), TNF-α (C), IL-6 (D), IFN-γ (E), IL-12p40 (F), KC (G), and MIP-2 (H), in BAL fluid. Results are mean ± S.D. and are representative of three independent experiments. Data were analyzed with Student’s t-test (*, p<0.05).

Groups of 4 B6 (WT) and caspase-1^−/− (CASP1^−/−) mice were infected i.n with 4×10³ EID₅₀ of PR8 influenza virus and levels of IL-1β (A), IL-18 (B), TNF-α (C), IL-6 (D), IFN-γ (E), IL-12p40 (F), KC (G), and MIP-2 (H) were measured in BAL fluid 3d later. Results are mean ± S.D.. Data were analyzed with Student’s t-test (*, p<0.05) and are representative of three independent experiments.

(A) Dendritic cells from WT, CRYO^−/− and CASP1^−/− mice were transfected with 2 μg purified influenza A viral RNA for 24 h and cell-free supernatants were analyzed by ELISA for production of IL-1β. Data are representative of two independent experiments. Groups of 4 C57BL/6 WT, CRYO^−/− and CASP1^−/− mice were infected i.n. with 2×10³ EID₅₀ of PR8 influenza virus and cells recovered by BAL on d3 (B) and d6 (C) were characterized by flow cytometry (see Experimental Procedures). Results are mean ± S.D. Data were analyzed with ANOVA (*, p<0.05) and are representative of three independent experiments.

Groups of 4 WT, CRYO ^−/− and CASP1^−/− mice were infected i.n. with 4×10³ EID₅₀ of PR8 influenza. Seven days following infection, the frequency of antibody secreting cells in the draining mediastinal lymph node was determined by ELISPOT for the indicated isotype (A–C). Antigen-specific serum IgG was measured by ELISA on d11 following infection (D). Also on d11, antigen-specific CD8⁺ T cell numbers were enumerated from the BAL by tetramer staining (E). Virus titers in lungs were determined on d3 (F) and d6 (G) by standard MDCK plaque assay. Data were analyzed with ANOVA (*, p<0.05) and are representative of three independent experiments.

Groups of 4 WT and cryopyrin^−/− mice were infected i.n. with 8×10³ EID₅₀ of PR8 influenza virus. Lung samples were collected on d3 and processed for routine H&E staining of 4μ paraffin sections. (A), top panels, ×200, WT airway epithelium is intact with only small foci of necrosis (arrow) and peribronchiolar inflammation; * shows unobstructed airway lumen. Cryopyrin^−/−, the small bronchi and bronchioles show diffuse necrosis with loss of the airway epithelium and obstruction of the lumen by debris (*). (B), bottom panels ×400, Intact wildtype epithelium with limited foci of necrosis (arrow), and diffuse necrosis of CRYO^−/− bronchiolar epithelium (arrows). Histologic evaluation was performed by an experienced veterinary pathologist (KLB). Groups of 4 wildtype and cryopyrin^−/− mice were infected i.n. with 8×10³ EID₅₀ of PR8 influenza virus. Lung samples were collected on d11 and 4μm paraffin sections processed for routine H&E (C,D) or Masson’s Trichrome staining (E). (C) ×200, the WT mice show only focal and minimal collagen deposition in the alveoli and occasionally in the terminal airways (arrows). This collagen deposition is exuberant in the mutant groups and often occludes alveoli and terminal airways (arrows). (D) ×400, arrows indicate areas of collagen deposition. (E) ×200, bottom row Masson’s Trichrome stain confirms collagen deposition. In the WT groups the insult was sufficiently severe to cause basement membrane damage, evidenced by a thin layer of collagen lining the alveolar spaces. In the mutant lungs, deposition of collagen is abundant and forms concentric layers that occlude alveoli and terminal airways.