Fig. 6: Caspase-8 mediates experimental cerebral malaria (ECM) in PbA-infected mice.

C57BL/6, Casp1/11^−/−, Gsdmd^−/−, Ripk3^−/− (control), Ripk3^−/−/Casp8^−/−, Ripk3^−/−/Casp8/1/11^−/−, and Ripk3^−/−/Casp8^−/−/Gsdmd^−/− mice were used in different experiments as indicated. CD11b⁺ cells were purified from splenocytes harvested on days 0 and 5 post infection. Cells were lysed in RIPA buffer to prepare the lysates, and splenocytes were spin down 400 × g for 5 min to prepare supernatants. Western blots were revealed with (a) anti-caspase-1 and anti-caspase-11 or (b) anti-caspase-8 antibodies. c Splenocytes from uninfected controls and PbA-infected mice were stimulated in vitro with 1 μg/ml of LPS, and 48 h later the levels of IL-1β (top panel, ***P < 0.0001) and TNFα (bottom panel, *P = 0.0104 and **P = 0.0056) measured in culture supernatants by ELISA. For IL-1β: C57BL/6 (control n = 4, +LPS n = 3; infected n = 6), Ripk3^−/−/Casp8^−/− n = 4, Ripk3^−/−/Casp8/1/11^−/− n = 6, and Ripk3^−/− n = 3. For TNFαː C57BL/6 (control n = 4, infected n = 6), Ripk3^−/−/Casp8^−/− n = 4, Ripk3^−/−/Casp8/1/11^−/− (n = 6, + LPS n = 5), and Ripk3^−/− n = 3. d Survival of PbA-infected mice was followed by 21 days, and in (e), each of the classic CM symptoms (ruffled fur, abnormal posture, unbalancing, limb paralysis, convulsion, coma, and death) was given a score (0, 2, 4, 8, or 10). f After 6 days of infection, mice were injected i.v. with 0.2 mL of 1% Evans blue (120 mg kg⁻¹  d⁻¹) at 6 days post infection of vehicle-treated mice. One hour later, mice were killed, and brain color was assessed. Naive mice were also injected with Evans blue and used as unstained control. In a, b, blots are representative of two different experiments. In c, mean ± s.e.m of two to three different experiments are shown; statistical analysis by one-way ANOVA. The survival curve was analyzed by log-rank test.