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

Highlights

• LPS and ATP induce rapid secretion of high levels of IL-1β by Hofbauer cells (HBC).

• HBC IL-1β triggered by LPS and ATP is mediated by the NLRP3 inflammasome.

• The NLRP3 inflammasome also triggers HBC pyroptotic cell death.

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.

Introduction

Hofbauer cells (HBCs) are large, pleiomorphic, highly vacuolated placental macrophages, thought to be of fetal origin, and located beneath the syncytium and adjacent to fetal capillaries (Castellucci et al., 1980), a site critical for the protection of the fetus against microbes migrating from the mother. Recent studies have indicated that alterations in the number and protein/gene expression of HBCs may play a role in microbial-driven/cytokine-mediated inflammation in placenta, and in subsequent pregnancy complications such as villitis, histologic chorioamnionitis (HCA), and the fetal inflammatory response syndrome (FIRS) (Tang et al., 2011a; Reyes and Golos, 2018).

Based on their surface expression markers, HBCs can be classified as anti-inflammatory M2 macrophages (Joerink et al., 2011; Tang et al., 2011b), supporting their role in angiogenesis and development (Anteby et al., 2005; Ingman et al., 2010). However, while expressing M2 markers, HBCs can also produce robust inflammatory cytokine responses towards bacterial and viral triggers (Young et al., 2015; Hendrix et al., 2020; Schliefsteiner et al., 2020), indicating that HBCs actively sense and respond to infections (Tang 2011 103). Yet, little is known about the mechanisms involved.

One important inflammatory factor that is associated with pregnancy complications including preterm labor, chorioamnionitis, and FIRS is interleukin 1 beta (IL-1β) (Menon et al., 2010; Nadeau-Vallee et al., 2016; Xiong and Wintermark, 2020). Experimental models have demonstrated a role for placental-derived IL-1β in mediating placental inflammation and injury, and poor fetal/neonatal outcomes (Girard et al., 2010; Adams Waldorf et al., 2011; Leitner et al., 2014; Bergeron et al., 2016; Brien et al., 2017; Presicce et al., 2018). Furthermore, macrophages may play a contributing role (Girard et al., 2010). Indeed, we previously reported that HBCs infected with a live herpes virus (MHV-68) secreted IL-1β, which in turn induced a pro-neutrophilic response in endothelial cells (Hendrix et al., 2020).

Since IL-1β is such a potent inflammatory cytokine that if not properly controlled causes tissue injury, its production and secretion is regulated by a two-step process culminating in activation of the inflammasome, a multi-protein complex which, classically though caspase-1, drives the processing and secretion of mature IL-1β (Franchi et al., 2009). The first step is a priming signal whereby microbial activation of a pattern recognition receptor, such as a Toll-like receptor (TLR), upregulates expression of pro-IL-1β and the Nod-like receptor (NLR) that will form the inflammasome. The second step is a triggering signal whereby activation of the NLR leads to inflammasome assembly and activation (Franchi et al., 2009). In non-placental macrophages, the NLPR3 inflammasome has been well described. Bacterial lipopolysaccharide (LPS) can prime macrophages to increase NLRP3 and pro-IL-1β expression, while adenosine triphosphate (ATP) activates the NLRP3 inflammasome (Mariathasan et al., 2006; Bauernfeind et al., 2009; Netea et al., 2009). In parallel, caspase-1 activation of gasdermin D (GSDMD) leads to pyroptosis, a form of rapid cell death that contributes to IL-1β release (He et al., 2015; Shi et al., 2015). Since the mechanism by which IL-1β is produced by HBC remains unclear, the aim of this study was to explore the regulation of NLRP3 inflammasome function and pyroptotic cell death in HBCs pathophysiology.