Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia
Fig 5
MLKL is required for PFT induced necroptosis.
Western blot for pMLKL in MH-S whole cell lysates following their challenge with A) S. marcescens (Sma), ΔshlA, S. pneumoniae (Spn) and Δply and B) S. aureus (Sau), L. monocytogenes (Lmo) and UPEC. C) Immunofluorescence for MLKL (Green) and lipid membrane (Red) in uninfected and Sma infected MH-S cells. Nuclei were stained with DAPI (blue). D) LDH release assay of infected THP-1 macrophages following mock or pretreatment with the MLKL inhibitor necrosulfonamide (100μM). MOIs were: Sma = 1,Spn = 100, Sau = 10, UPEC = 100. E) LDH release assay of THP-1 macrophages following challenge with recombinant pneumolysin (rPly), with and without pretreatment with necrosulfonamide (NSA, 100μM, black bars). LDH release assay of F) Sma infected or G) rPly challenged THP-1 macrophages transfected with siRNA targeting MLKL. As control scrambled (Scram) siRNA was used. H) Alveolar macrophage numbers in BALF of MLKL KO mice 18h after intratracheal infection with S. marcescens. Student t-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, during LDH assays each with 8 biological replicates.