An NLRP3 inflammasome-triggered cytokine storm contributes to Streptococcal toxic shock-like syndrome (STSLS)
Fig 5
The membrane perforation activity of SLY was mainly responsible for inflammasome activation by S. suis.
THP-1 cells were differentiated into macrophage-like cells by treatment with 50 nM PMA overnight and then primed with LPS for 4 h, followed by infection with S. suis strains or by stimulation with ouabain or recombinant SLY (rSLY) for 2 h. (A) The THP-1 cells were primed with LPS and then treated with SC-19, heat-killed SC-19 or ouabain. The cellular proteins were subjected to western blot analysis to assess actin, casp1, IL-1β, and GSDMD expression, and the supernatants of the cell cultures were collected for detection of casp1, IL-1β, and GSDMD by western blot assay. Symbols of “black triangle” and “asterisk” indicate the corresponding specific and non-specific protein band. (B) The THP-1 cells were primed with LPS and then treated with ouabain, SC-19 or its isogenic mutants dlta (Δdlta), cpsEF (ΔcpsEF) or sly (Δsly) or the mutant strain msly (P353L). The cellular proteins were subjected to western blot analysis to assess actin, casp1, IL-1β, and GSDMD expression, and the supernatants of cell cultures were collected for detection of casp1, IL-1β, and GSDMD by western blot assay. Symbols of “black triangle” and “asterisk” indicate the corresponding specific and non-specific protein band. (C) Densitometric analysis of mature IL-1β secretion was calculated based on the western blot signal from mature IL-1β in the supernatant / signal from cellular actin, and the concentrations of IL-1β, TNF-α, and LDH in the supernatants of THP-1 cells treated with heat-killed or live SC-19, various mutants or ouabain were also detected (two-tailed, unpaired t-tests, n = 5). (D) THP-1 cells were primed with LPS and then treated with different concentrations of purified recombinant SLY (rSLY). The cellular proteins were subjected to western blot analysis to assess actin, casp1, IL-1β, and GSDMD expression, and the supernatants of cell cultures were collected for detection of casp1, IL-1β, and GSDMD by western blot assay. Symbols of “black triangle” and “asterisk” indicate the corresponding specific and non-specific protein band. (E) Densitometric analysis of mature IL-1β secretion was calculated based on the western blot signal from mature IL-1β in the supernatant / signal from cellular actin, and the concentrations of IL-1β in the supernatants of THP-1 cells treated with different concentrations of rSLY were detected (two-tailed, unpaired t-tests, n = 5). (F) THP-1 cells were primed with LPS and then treated with ouabain or SC-19 in the presence of different concentrations of soluble cholesterol. The cellular proteins were subjected to western blot analysis to assess actin, casp1, IL-1β, and GSDMD expression, and the supernatants of cell cultures were collected for detection of casp1 and IL-1β by western blot assay. (G) Detection of IL-1β in the supernatants of THP-1 cell cultures treated with ouabain or SC-19 in the presence of different concentrations of soluble cholesterol (two-tailed, unpaired t-tests, n = 5). “NC” indicates that the cells were not stimulated with LPS, while “CON” indicates that the cells were primed with LPS but not treated with another stimulator. Error bars represented the mean ± standard deviations.