Figure 6. RIPK3-activated necroptosis and apoptosis pathways are both required for protection against IAV in vivo.

(A) Survival analysis of age- and sex-matched ripk3^+/+ and ripk3^−/− mice infected with PR8 (4000 EID₅₀/mouse by i.n.). (B) Survival analysis of age- and sex-matched mlkl^+/+, mlkl^−/−, and mlkl^−/−fadd^−/− double knock-out mice with PR8 (4000 EID₅₀/mouse by i.n.). Data are pooled from two independent experiments. (C) Virus titers were determined on infected lungs 3 or 9 d.p.i. by plaque assay (n=3–5). (D) Paraffin embedded sections of ripk3^+/+ and ripk3^−/− mouse lungs 6 d.p.i. were stained with anti-IAV antibodies, and slides were scanned and virus spread was quantified using Aperio ImageScope. Representative images of anti-IAV stained 11 ripk3^+/+ (top) and 10 ripk3^−/− (bottom) lungs are shown to the left, and quantification of virus spread from lungs of individual mice are shown to the right. (E) Representative images of H&E-stained 11 ripk3^+/+ and 10 ripk3^−/− lungs 6 d.p.i at two magnifications reveal severe edema and alveolar damage in ripk3^−/− pulmonary tissue (left). Quantification of alveolar fibrin deposition from lungs of individual mice is shown to the right. The extent and severity of pulmonary damage was determined from blinded sections examined by a pathologist and scored for the distribution of alveolar fibrin deposition on a severity scale of 1–5. These scores were converted to a semi-quantitative scale as described previously (Matute-Bello et al., 2011). (F) Representative images of CD3⁺ staining in 11 ripk3^+/+ and 10 ripk3^−/− lungs 6 d.p.i. Quantification of CD3⁺ staining from lungs of individual mice are shown to the right. (G) Quantification of frequencies of IAV-specific (DbPA224) CD8⁺ T cells and percentage of IAV PA224 peptide-stimulated poly-functional IFN-γ⁺TNF-α⁺CD8⁺ T cells in BAL fluid from ripk3^+/+ and ripk3^−/− animals (n=3–5) at 9 d.p.i. The low pathogenic HKx31 reassortant of PR8 was used in these experiments to avoid 9 d.p.i. lethality effects seen with PR8. Error bars represent mean +/− S.D. * p <0.05; *** p <0.0005. See also Fig. S6.