Staphylococcus aureus Leukocidin A/B (LukAB) Kills Human Monocytes via Host NLRP3 and ASC when Extracellular, but Not Intracellular
Fig 5
LukAB activates the NLRP3 inflammasome leading to cell death and cytokine secretion.
(A) THP1 cells were transduced with shRNA against NLRP3, ASC or a non-targeting control. Cell lysates were analyzed by immunoblot to confirm knock down of NLRP3 and ASC. (B) Surface CD11b levels were evaluated by flow cytometry. (C) THP1 shRNA cells were incubated in the presence of FLICA-1 then intoxicated with LukAB (50 ng/mL) for 1 hour then analyzed by flow cytometry. (D) THP1 shRNA cells were primed with 500 ng/mL LTA for 3 hours followed by intoxication with LukAB (50ng/mL) for one hour. Culture supernatants were collected and analyzed for secretion of the indicated cytokines. (E) THP1 shRNA cells were intoxicated with LukAB (50 ng/mL) for 1 hour and culture supernatants were collected for analysis of LDH release. (F) THP1 shRNA cells were incubated with propidium iodide then intoxicated with LukAB (50 ng/mL) for 1 hour then analyzed by flow cytometry. (G and H) THP1 cells were incubated in media supplemented with an additional 25 mM NaCl or KCl. Cells were incubated with FLICA-1 (G) or propidium iodide (H) then intoxicated with LukAB (50 ng/mL) for 1 hour and analyzed by flow cytometry. Bars represent the mean ± standard error of the mean for at least two independent experiments, each performed in triplicate. Asterisks indicate significance at a p-value of ≤ 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.