NLRP3 inflammasome function and pyroptotic cell death in human placental Hofbauer cells

Abstract

Alterations in the number and protein/gene expression of Hofbauer cells (HBCs) may play a role in microbial-driven/cytokine-mediated placental inflammation, and in subsequent pregnancy complications such as villitis, histologic chorioamnionitis, and the fetal inflammatory response syndrome. Pyroptosis is an inflammatory form of cell death mediated by the inflammasome, a multi-protein complex which drives the processing and secretion of interleukin 1 beta (IL-1β). Pyroptosis can be triggered by bacterial lipopolysaccharide (LPS) and adenosine triphosphate (ATP) in non-placental macrophages through activation of the NLRP3 inflammasome. However, the role of inflammasome activation and pyroptosis in HBC pathophysiology remains unclear. HBCs isolated from human term placentas were treated with or without LPS or ATP, alone or in combination. Treatment of HBCs with both LPS and ATP induced the rapid secretion of high levels of IL-1β and at the same time, cell death associated with nuclear condensation and cellular swelling. HBC treatment with both LPS and ATP induced caspase-1 activation, gasdermin D (GSDMD) cleavage, which mediates pyroptosis, and IL-1β processing. Caspase-1 activation, GSDMD cleavage, IL-1β processing, and IL-1β secretion were all significantly reduced following NLRP3 knockdown; inhibition of caspase-1; and inhibition of P2X7, the receptor that mediates K+ efflux. Together, our data indicate that LPS and ATP treatment stimulated NLRP3 inflammasome activation and pyroptosis in HBCs leading to the rapid release of IL-1β. Since the localization of HBCs confers a unique ability to influence microbial-associated placental and fetal inflammation, these studies suggest a key role for the inflammasome and pyroptosis in mediating HBC driven inflammation.

Keywords: Infection; Inflammasome; Macrophage; Placenta; Pyroptosis.

Conflict of interest statement

Declaration of Competing Interest The authors declare that there are no conflict of interest.

Copyright © 2020 Elsevier B.V. All rights reserved.

Figures

Figure 1.. Combination LPS and ATP induce a rapid secretion of IL-1β by HBCs.

HBCs were treated with media alone (Ctrl), LPS alone, ATP alone, or combination LPS and ATP (LPS+ATP) for 20–120 min. Levels of secreted IL-1β, quantitated in four independent experiments, were determined by ELISA. Results are presented as Mean ± SEM. *p<0.05: LPS+ATP vs LPS, ATP, and Ctrl; +p<0.05 LPS+ATP vs ATP, and Ctrl.

Figure 2.. LPS and ATP treatment of HBCs promotes pyroptosis.

HBCs were treated with or without LPS or ATP, alone or in combination. After 40 min cells were stained with H33342 (Blue) and SYTOX® Orange (Pink). (A) Left hand panels show bright field (black and white) images of HBCs at 40x magnification; right hand panel shows the enlarged bright field image of HBCs treated with LPS+ATP. (B) Fluorescent microscopy of HBCs at 10x magnification. Results are representative of five independent experiments.

Figure 3.. Modulation of the NLRP3 inflammasome and GSDMD in HBCs by LPS and ATP treatment.

HBCs were treated with media alone (Ctrl) or with combination LPS and ATP (LPS+ATP) either alone or with inhibitors to caspase-1 (WEHD or Vx-765) or P2X7 (KN-62). (A) After 40 min HBC protein was analyzed by Western blot (n=4–5) for expression of NLRP3; pro- and active-GSDMD; pro- and active-caspase-1; and pro- and active-IL-1β. Blots are from a representative experiment. Charts show protein expression levels as determined by densitometry after normalization to HSP90. (B) After 40 min HBC supernatants were analyzed by ELISA for IL-1β secretion (n=5). Data are either presented as Mean ± SEM for bar charts or are presented as medians and percentiles; the lines inside the box indicate the median, the ends of the box describe the lower and upper quartiles, and the whiskers define the smallest and largest values. *p<0.05 relative to LPS+ATP.

Figure 4.. The NLRP3 inflammasome mediates pyroptotic IL-1β secretion by HBCs treated with LPS and ATP.

HBCs were treated with media alone (Ctrl), with LPS alone or with combination LPS and ATP (LPS+ATP) either alone or scrambled or NLRP3 siRNA. (A) After 40 min HBC protein was analyzed by Western blot for expression of NLRP3; pro- and active-GSDMD; pro- and active-caspase-1; and pro- and active-IL-1β. Blots are from a representative experiment. Charts show protein expression levels as determined by densitometry after normalization to HSP90 (n=5). (B) After 40 min HBC supernatants were analyzed by ELISA for IL-1β secretion (n=5). Data are either presented as Mean ± SEM for bar charts or are presented as medians and percentiles; the lines inside the box indicate the median, the ends of the box describe the lower and upper quartiles, and the whiskers define the smallest and largest values. *p<0.05 relative to scrambled siRNA control unless otherwise specified.