Fig 1.
Inflammasome activation contributed to STSLS.
Mice were infected with S. suis epidemic strain SC-19, which causes STSLS in humans, mice, and pigs, and then treated with the caspase-1 inhibitor (casp1 inh) Ac-YVAD-CHO or PBS as a control at 1 h post-infection. Infection of mice with the strain P1/7, which induces only sporadic cases of meningitis and sepsis in pigs, was used as a control. (A) Cytokine levels in peritoneal lavage fluids at 6 h post-infection were determined using ELISA kits (two-tailed, unpaired t-tests, n = 5). (B) The bacterial load in the blood was determined to evaluate the effect of caspase-1 (casp1) signaling on S. suis clearance (two-tailed, unpaired t-tests, n = 5). (C) Blood levels of AST, ALT, LDH, and CK at 6 h post-infection (two-tailed, unpaired t-tests, n = 5) (D) H&E staining of infected tissue sections from mice at 6 h post-infection with S. suis epidemic strain SC-19 with or without casp1 inh treatment. Congestion in the lung and spleen is indicated by a “red arrow”, infiltration of inflammatory cells in the lung is indicated by a “hollow arrow”, vacuolated degeneration in the liver is indicated by a “black arrow”, and necrosis in the liver is indicated by a “yellow arrow”. (E) Survival of mice infected with S. suis epidemic strain SC-19 with or without casp1 inh treatment (log-rank test, n = 10). (F) Clinical signs of mice infected with S. suis epidemic strain SC-19 with or without casp1 inh treatment were monitored and scored (two-way RM ANOVA, n = 10). Error bars represented the mean ± standard deviations.
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.
Fig 3.
Evaluation of the role of NLRP3 inflammasome activation in STSLS with MCC950.
Mice were infected with S. suis epidemic strain SC-19 and then treated with MCC950 (NLRP3 inhibitor) or control to evaluate the role of NLRP3 in STSLS. (A) Cytokine levels in peritoneal lavage fluids at 6 h post-infection were determined using ELISA kits (two-tailed, unpaired t-tests, n = 5). (B) Blood levels of AST, ALT, LDH and CK at 6 h post-infection (two-tailed, unpaired t-tests, n = 5). (C) H&E staining of infected tissue sections from mice at 6 h post-infection with S. suis epidemic strain SC-19 with or without MCC950 treatment. Congestion in the lung and spleen is indicated by a “red arrow”, infiltration of inflammatory cells in the lung is indicated by a “hollow arrow”, vacuolated degeneration in the liver is indicated by a “black arrow”, and necrosis in the liver is indicated by a “yellow arrow”. (D) Clinical symptom scores of mice infected with S. suis epidemic strain SC-19 and treated with or without the NLRP3 inhibitor MCC950 (two-way RM ANOVA, n = 10). (E) Survival of mice infected with S. suis epidemic strain SC-19 and treated with or without the NLRP3 inhibitor MCC950 (log-rank test, n = 10). (F) The bacterial load in the blood at 6 h post-infection was determined to evaluate the role of NLRP3 in S. suis clearance (two-tailed, unpaired t-tests, n = 5). Error bars represented the mean ± standard deviations.
Fig 4.
nlrp3 was required for STSLS caused by S. suis.
The nlrp3-deficient mice (nlrp3-/-) and its wild-type mice (nlrp3+/+) were infected with S. suis epidemic strain SC-19. (A) Cytokine levels in peritoneal lavage fluids at 6 h post-infection were determined using ELISA kits (two-tailed, unpaired t-tests, n = 4). (B) The bacterial load in the blood at 6 h post-infection was determined (two-tailed, unpaired t-tests, n = 4). (C) Blood levels of AST, ALT, LDH and CK at 6 h post-infection (two-tailed, unpaired t-tests, n = 4). (D) H&E staining of infected tissue sections from nlrp3-/- or nlrp3+/+ mice at 6 h post-infection with S. suis epidemic strain SC-19. Congestion in the lung and spleen is indicated by a “red arrow”, infiltration of inflammatory cells in the lung is indicated by a “hollow arrow”, vacuolated degeneration in the liver is indicated by a “black arrow”, and necrosis in the liver is indicated by a “yellow arrow”. (E) Clinical symptom scores of mice infected with S. suis epidemic strain SC-19 (two-way RM ANOVA, n = 6). (F) Survival of mice infected with S. suis epidemic strain SC-19 (log-rank test, n = 6). Error bars represented the mean ± standard deviations.
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.
Fig 6.
The high membrane perforation activity of S. suis was required for STSLS.
Mice were infected with S. suis epidemic strain SC-19 or the mutant [msly (P353L)] containing the point substitution P353L, which lacks hemolytic activity. (A) Cytokine levels in peritoneal lavage fluids at 3.5 or 6 h post-infection were detected using ELISA kits (two-tailed, unpaired t-tests, n = 5). (B) Blood values of AST, ALT, LDH and CK at 6 h post-infection (two-tailed, unpaired t-tests, n = 5). (C) The bacterial load in the blood at 3.5 or 6 h post-infection was detected (two-tailed, unpaired t-tests, n = 5). (D) H&E staining of infected tissue sections from mice at 6 h post-infection with S. suis epidemic strain SC-19 or mutant [msly (P353L)]. Congestion in the lung and spleen is indicated by a “red arrow”, infiltration of inflammatory cells in the lung is indicated by a “hollow arrow”, vacuolated degeneration in the liver is indicated by a “black arrow”, and necrosis in the liver is indicated by a “yellow arrow”. (E) Clinical signs of mice infected with S. suis were monitored and scored (two-way RM ANOVA, n = 10). (F) Survival of mice infected with S. suis strains (log-rank test, n = 10). Error bars represented the mean ± standard deviations.
Fig 7.
Schematic representation of the mechanism underlying STSLS.
The present study indicated an important mechanism by which the epidemic S. suis strain causes STSLS. First, infection with S. suis can activate the transcription of genes involved in the inflammasome through pattern-recognition receptors, such as Toll-like receptor (TLR). Then, the high SLY expression level allows the strain to exert high levels of membrane perforation activity, which can further result in several events, including cytosolic K+ efflux, an essential event for NLRP3 inflammasome activation. Subsequently, the high level of inflammasome activation results in GSDMD, pro-IL-1β and pro-IL-18 cleavage, and the GSDMD cleavage leads to pyroptosis and facilitates secretion of mature IL-1β and IL-18, which may further induce the production of downstream cytokines, such as IFN-γ and IL-17A, causing a cytokine storm and multiple organ dysfunction, the main characteristics of STSLS.