Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia
Fig 4
RIP3 is required for PFT induced necroptosis.
A) LDH release assay of THP-1 cells transfected with siRNAs targeting RIP3, caspase-1 (Casp-1), caspase-8 (Casp-8) and a scramble control, infected with (A) Sma or challenged with B) recombinant pneumolysin (rPly). C) LDH release assay of MH-S macrophages infected with Sma at an MOI of 0.1 mock or following pretreatment with RIP3 inhibitor GSK’872. D) LDH release assay of BMDM from WT-C57BL/6, Caspase 1/11 KO, Caspase 3 KO and RIP3 KO mice, infected with Sma at an MOI of 0.5 or 0.1. E) LDH release of BMDM from wildtype mice, Caspase 1/11 (Casp1/11) KO, RIP3 KO, and BMDM from wildtype mice pretreated with necrostatin-5 (100μM) following their infection with S. aureus (MOI 10), L. monocytogenes (MOI 10) and S. pneumoniae (MOI 100). F) LDH release assay of BMDM from wild type C57BL/6 or RIP3 KO mice following their challenge with recombinant pneumolysin (rPly). G) LDH release assay of MH-S macrophages challenged with rPly with and without pretreatment with GSK’872 (10 μM). H) Alveolar macrophage numbers in BALF of RIP3 KO mice 18h after intratracheal infection with S. marcescens. Mann-Whitney tests were applied for two-group comparisons, for multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05. Data are representative of ≥3 separate experiments, each with 8 biological replicates.