Fig 1.
Microparticle caspase-1 and GSDM-D release by Francisella regulated by NLRP3 and not Pyrin.
A) Microparticles (MP) fractions were isolated from CAS9, CAS9/NLRP3-KO and CAS9/PYRIN-KO cells stimulated with LPS (1μg/ml) for 2h or left untreated, Francisella (25MOI) for 2h, 4h or left untreated. Microparticulate p20 caspase-1 and p30 GSDM-D release was abrogated in NLRP3 KO by both LPS and Francisella, but not in PYRIN-KO when compared to CAS9 cells. * # CAS9 vs CAS9/NLRP3 KO; $ & CAS9/NLRP3 KO vs CAS9/PYRIN KO. B) Cell lysates were analyzed and confirmed for NLRP3 and Pyrin, as well as effects on pro-caspase-1 and pro-GSDM-D. C) Cells were treated with LPS or Francisella overnight (17h) and analyzed for the presence of active p20 caspase-1 and cleaved GSDM-D. Overnight experiments were performed to allow internalization of Francisella and thereby induce downstream signaling mediated by internalized Francisella. * # CAS9/PYRIN KO vs CAS9/NLRP3 KO. Immunoblot data is representative of n = 4 experiments.
Fig 2.
Francisella mediated inflammasome responses and cell injury is enhanced in absence of pyrin and reduced upon overexpression of Pyrin in PYRIN-KO/KI cells.
A) CAS9, CAS9/PYRIN-KO and CAS9/PYRIN-KO/KI cells were stimulated with LPS (1μg/ml) for 2h or left untreated. Microparticulate p20 caspase-1 and p30 GSDM-D levels were analyzed. p20 active caspase-1 levels in MP- *CAS9 vs CAS9/PYRIN-KO/KI; # CAS9/PYRIN-KO/KI vs CAS9/PYRIN KO. B) CAS9, CAS9/NLRP3-KO, CAS9/PYRIN-KO and CAS9/PYRIN-KO/KI cells were stimulated with LPS (1μg/ml) or Francisella for 2h for IL-18; and 17h for IL-1β. Released cytokines were measured in the supernatants from each condition respectively. * # CAS9 vs CAS9/NLRP3 KO; $ & CAS9/PYRIN-KO/KI vs CAS9/PYRIN KO C) MPs from CAS9, CAS9/PYRIN-KO, CAS9/NLRP3-KO and CAS9/PYRIN-KO/KI stimulated with LPS (1μg/ml) for 2h or left untreated were subjected to human pulmonary microvascular endothelial cells (HPVEC); co-cultured overnight and analyzed for endothelial cell death by Trypan Blue (cell death assay) and MTS (cell viability assay). Data is representative of n = 4 experiments. * CAS9 vs CAS9/NLRP3 KO; # CAS9/PYRIN-KO/KI vs CAS9/PYRIN KO.
Fig 3.
Microparticulate active caspase-1 and GSDM-D release by LPS and Francisella is negatively regulated by Pyrin overexpression.
A) Cell lysates (CE) and microparticles (MP) fractions were isolated from THP-1 EGFP, THP1-EGFP overexpressed Pyrin cells stimulated with LPS (1μg/ml) for 2h or left untreated and analyzed for the presence of active p20 caspase-1 and p30 GSDM-D. Pyrin-EGFP overexpression was confirmed at 112kD which was not detected in the vector control cells. Endogenous pyrin at 86kD was detected in both cell types. Overexpressing Pyrin significantly inhibited caspase-1 activation (p20) and GSDM-D cleavage (p30) in microparticles. Densitometry scans of active caspase-1 levels from n = 4 experiments. * p20 active caspase-1 levels in MP THP1-EGFP vs THP1-EGFP Pyrin. # p30 active GSDM-D levels in MP THP1-EGFP vs THP1-EGFP Pyrin. B) Similar experiments were performed also with overnight (17h) stimulation with LPS and Francisella. THP-1 EGFP, THP1-EGFP overexpressed Pyrin cells were stimulated with LPS (1μg/ml), Francisella (25MOI) for 17h or left untreated and analyzed for the presence of active p20 caspase-1 and p30 GSDM-D. C) CAS9, THP-1 EGFP, THP1-EGFP overexpressed Pyrin were stimulated with LPS (1μg/ml) or Francisella for 4h, overnight (17h) or left alone. IL-1β was measured from supernatants. Release of IL-1β was significantly diminished with overexpression of Pyrin. Data is representative of n = 4 experiments.THP1-EGFP vs THP1-EGFP Pyrin * IL-1β levels at 4 and 17h upon Francisella stimulation; # IL-1β by LPS stimulation for 4 and 17h.