ID: 2228

• Title:
  Nanosecond Pulsed Electric Fields Activate the NLRP3 Inflammasome in Innate Immune Cells in a Dose-Dependent Manner.
  
  Authors:
  

  Chittams-Miles, Alexandra E.- 1
  Mazzarda, Flavia- 2
  Pittaluga, Julia- 3
  Vernier, P. Thomas- 1, 
  Muratori, Claudia- 1,4
  
  1. Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.  
  2. University of Montpellier, Institut de Génétique Humaine, CNRS, Montpellier, France
  3. LifeNet Health, Virginia Beach, VA
  4. Department of Electrical and Computer Engineering, Old Dominion University, Norfolk, VA, USA
  

  
  
  Abstract:
  Summary: Multiple studies have shown that pulsed electric fields (PEF) ablation technologies, especially nanosecond duration PEF (nsPEF), have immunomodulatory effects. However, the effects of these pulses on immune cells have not been systematically investigated. Our results show that nsPEF activate the innate immune platform known as the NLRP3 inflammasome, a critical regulator of inflammation expressed in innate immune cells which initiates the caspase-1-mediated release of proinflammatory cytokines such as IL-1� and IL-18. We present evidence that nsPEF cause inflammasome activation.
  
  
  
  Introduction: The NLRP3 inflammasome is a multiprotein complex that leads to a form of programmed cell death called pyroptosis that is responsible for the secretion of highly active proinflammatory cytokines like interleukin-1β (IL-1β) and IL-18. This form of cell death occurs as a result of exposure to pathogen- or damage- associated molecular patterns (PAMPs or DAMPs). The inflammasome also plays an important role in T-cell stimulation, particularly in antitumor responses. Stimuli that are known to cause inflammasome activation include ion flux, formation of reactive oxygen species (ROS), and cell volume changes. nsPEF permeabilize the plasma membrane and membranes of intracellular structures such as the mitochondria and the endoplasmic reticulum (ER). Cell swelling, ER stress, ion flux, and ROS production are well documented effects of nsPEF permeabilization. In this study, we are investigating whether the damage created by nsPEF is sufficient to cause NLRP3 inflammasome activation. Understanding the mechanisms of innate immune stimulatory effects of PEF is essential to determining new approaches in tumor immunotherapy.
  
  
  
  Material and Methods: In this study, we used the J774A.1 mouse macrophage cell line and bone-marrow-derived macrophages (BMDMs) from wild-type or NLRP3 knockout mice. For all the experiments, the cells were primed with lipopolysaccharide (LPS, 1 μg/mL) or Pam3Cys (300 ng/mL) for 4 hours to stimulate the upregulation of NLRP3 inflammasome precursors pro-IL-1β and NLRP3. 5 mM ATP was used as positive control for inflammasome activation. Cells were exposed to nsPEF in suspension in 1 mm electroporation cuvettes. Macrophages were treated with increasing numbers of 200 ns pulses (25, 50, or 100 pulses, 9 kV/cm, 10 Hz). IL-1β release was measured by ELISA or western blot at 1 h post treatment. Caspase-1 activation was measured at 30 min by western blot analysis.
  
  
  
  Results: nsPEF ability to activate the inflammasome was tested using both low and high 200 ns pulse doses. Low-dose treatments caused approximately 20% cell death (25 p J774A.1 and 50 p BMDM) while high-dose treatments caused 50% cell death (50 p J774A.1 and 100 p BMDM). In both J774A.1 and BMDM, we observed IL-1β release and caspase-1 cleavage after a high-dose treatment. To confirm NLRP3 inflammasome activation, we used the specific blocker MCC950. In cells treated with MCC950, we saw a reduction in nsPEF-induced cell death and a reduction of IL-1β release. This suggests that the NLRP3 inflammasome is indeed activated in response to nsPEF.
  
  
  
  Conclusion: Our experiments demonstrate a dose-dependent activation of the NLRP3 inflammasome by 200 ns PEF. IL-1β release from BMDM from NLRP3 knockout mice and possible non-canonical activation of the NLRP3 inflammasome will also be discussed. Understanding the mechanism of NLRP3 inflammasome activation in response to nsPEF will be critical for developing applications of nsPEF in cancer therapy.
  
  
  
  Keywords:
  NLRP3 Inflammasome, nsPEF, Immune Stimulation
  
  Refs:
  
  
  Topic 1:
  1. Biological responses (molecular, subcellular, cellular and intercellular)
  
  Topic 2:
  12. Biomedical applications