Francisella induced microparticulate caspase-1/gasdermin-D activation is regulated by NLRP3 independent of Pyrin

Abstract

Although the study of pathogen sensing by host defense systems continues to uncover a role for inflammasome components specific to particular pathogens, gaps remain in our knowledge. After internalization, Francisella escapes from the phagosome in mononuclear cells and is thought to be detected by intracellular pathogen-response-receptors pyrin and Aim2 in human and murine models, respectively. However, it remains controversial as to the role of pyrin in detecting Francisella. Our current work aims to study the contribution of inflammasome sensor, Pyrin in regulating microparticulate caspase-1/GSDM-D activation by Francisella. Our findings suggest that NLRP3 is central to the activation/release of active caspase-1/GSDM-D encapsulated in microparticles (MP) by Francisella. We also provide evidence that this regulation is independent of pyrin, implicated in sensing cytosolic Francisella in NLRP3-/- conditions where endogenous Pyrin is present. Absence of NLRP3 completely abrogated Francisella mediated MP caspase-1/GSDM-D activation and release both before and after internalization of the pathogen. However, deletion of pyrin not only enhanced both LPS and Francisella mediated MP active caspase-1/GSDM-D release, but pyrin overexpression resulted in a reduction of inflammasome activation and release; suggesting an inhibitory role of pyrin in LPS and Francisella mediated MP responses. This NLRP3 dependence and inhibitory effect of pyrin correlated with cytokine release as well. These observations also correlated with MPs ability to induce cell death; as LPS and Francisella-induced MPs from pyrin-deficient cells were more potent than wild-type monocytes whereas, NLRP3-/- MPs failed to induce cell death. Taken together, we report that NLPR3 not only mediates Francisella induced cytokine responses, but is also critical for cytokine-independent microparticle-induced inflammasome activation and endothelial cell injury independent of pyrin.

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.