Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia
Fig 2
RIP1 and not caspases are required for S. marcescens induced macrophage cell death.
A) LDH release assay of MH-S macrophages infected with a high and low MOI of S. marcescens (Sma) following mock or pretreatment with the general caspase inhibitor Z-VAD-FMK (GI), caspase-1 inhibitor Z-WEHD-FMK (CI) caspase-3 inhibitor Z-DEVD-FMK (C3), caspase-8 inhibitor Z-YVAD-FMK (C8), and caspase-9 inhibitor Z-LEHD-FMK (C9), all at a 10 μM concentration. Percent positive cells for B) caspase-1 and C) caspase-3/7 activity 2h after Sma infection following FLICA staining and measured by FACs analyses. For controls, cells with nigericin induced pyroptosis (Ni +LPS; lipopolysaccharide at 10 ng/mL for 4h then Ni at 10 μM for 6 h) and cycloheximide (CHX; 2,000 μg/ml) induced apoptosis were measured, respectively. D) LDH release assay of MH-S macrophages infected with Sma at an MOI of 1 following their mock or pretreatment with necrostatin-1s, -1, -5, or -7 at a concentration of 100 μM. E) LDH release assay of MH-S macrophages infected with Sma after pretreatment with necrostatin-5 at increasing concentrations. F) LDH release assay of MH-S macrophages treated with CHX after pretreatment with necrostatin-5 (Nec 5; 100 μM) or ZVAD (10 μM). For multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001. Data are representative of ≥3 separate experiments, each with 8 biological replicates.