An NLRP3 inflammasome-triggered cytokine storm contributes to Streptococcal toxic shock-like syndrome (STSLS)
Fig 2
NLRP3 was mainly responsible for inflammasome activation in response to S. suis infection.
(A) 293T cells were transfected with plasmids expressing Myc-tagged ASC, Flag-tagged pro-caspase-1, and Flag-tagged pro-IL-1β and a plasmid co-expressing GFP with NLRP3, NLRP1, NLRC4, or AIM2, followed by infection with S. suis strain SC-19 or stimulation with poly (dA:dT). Then, the cell supernatants were collected for western blotting with antibodies against casp1 and IL-1β and for the determination of IL-1β with a commercial ELISA kit (two-tailed, unpaired t-tests, n = 5). (B) The THP-1 nlrp3 knockout cell line (THP-1-nlrp3-/-) and its control cell line (THP-1-nlrp3+/+) were primed with LPS, followed by infection with S. suis strains or by stimulation with ouabain. The cellular proteins were subjected to western blot analysis for the expression of actin, NLRP3, casp1 and IL-1β, and the supernatants of cell cultures were collected for detection of casp1 and IL-1β via western blot assay, and the 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. In addition, the IL-1β and LDH concentrations in the supernatants were also determined (two-tailed, unpaired t-tests, n = 5). (C) THP-1 cells were primed with LPS, followed by infection with an S. suis strain or treatment with ATP in the presence of the specific P2X7 antagonist KN-62, the ROS scavenger N-acetyl-L-cysteine (NAC), the phagocytosis inhibitor cytochalasin B, the lysosomal inhibitor bafilomycin A, or the caspase-1 inhibitor (casp1 inh) Ac-YVAD-CHO. IL-1β in the cell culture supernatants with different treatments was detected using a commercial ELISA kit to reflect inflammasome activation (two-tailed, unpaired t-tests, n = 5). (D) THP-1 cells were primed with LPS for 4 h and then inoculated in K+-rich media or Na+-rich media, followed by infection with S. suis. IL-1β in the supernatants of cell cultures with different treatments was detected using a commercial ELISA kit to reflect inflammasome activation (two-tailed, unpaired t-tests, n = 5). Error bars represented the mean ± standard deviations.