Fig. 5

Caspase-8 deficiency in macrophages alters the response to Toll-like receptor activation and macrophage polarization in vitro. a Casp8 ^fl/fl (control), Cre ^LysM Casp8 ^fl/fl, RIPK3 ^−/−, and RIPK3 ^−/− Cre ^LysM Casp8 ^fl/fl BMDMs were stimulated with LPS (10 ng/ml), imiquimod (5 μg/ml), and CpG (5 μg/ml) with or without Nec-1 (30 μM) for 3, 6, and 12 h and evaluated for transcript and supernatant levels of IL-6. Data are represented as mean ± SD of biological triplicates, and experiments were repeated twice. b and c Control, control + Z-IETD-FMK (20 μM), and Cre ^LysM Casp8 ^fl/fl BMDMs were cultured with M1-polarizing conditions (primed overnight with IFN-γ 100 ng/ml and stimulated for 3 h with LPS 10 ng/ml) and M2-polarizing conditions (stimulated for 24 h with IL-4 40 ng/ml) with or without Nec-1. Principal component analysis of gene expression was performed in BMDMs under (b) M1- and M2-polarizing conditions and (c) M1- and M2-polarizing conditions + Nec-1. Data are represented as biological triplicates. FMK fluoromethylketone, IFN interferon, IL interleukin, LPS lipopolysaccharide, Nec-1 necrostatin-1, PCA principal component analysis, Z-IETD Z-Ile-Glu-(O-ME)-Thr-Asp-(O-Me)