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. Author manuscript; available in PMC: 2023 Feb 1.
Published in final edited form as: J Reprod Immunol. 2021 Dec 2;149:103457. doi: 10.1016/j.jri.2021.103457

Human fetal membrane IL-1β production in response to bacterial components is mediated by uric-acid induced NLRP3 inflammasome activation

Alex S Miller 1, Tiffany N Hidalgo 1, Vikki M Abrahams 1,*
PMCID: PMC8792319  NIHMSID: NIHMS1762014  PMID: 34875574

Abstract

Inflammatory interleukin-1β (IL-1β) is an important mediator of preterm birth. IL-1β secretion is mediated by the inflammasome that processes pro-IL-1β into its active form. However the mechanisms involved at the level of the fetal membrane (FM) are not fully understood. This study sought to determine the FM compartment involved in IL-1β production in response to bacterial components and to evaluate the mechanism of inflammasome activation. Since IL-18 is also mediated by the inflammasome and IL-8 is a chemoattractant that contributes to neutrophil recruitment in chorioamnionitis, we also evaluated the production of these factors. A human explant system was used to evaluate the response of the chorion, amnion, and intact FMs to the bacterial components lipopolysaccharide (LPS), peptidoglycan (PGN), or muramyl dipeptide (MDP). The chorion was the major source of IL-1β and IL-8 production in response to LPS, PGN, and MDP. LPS, PGN, and MDP induced FM IL-1β and IL-18 secretion in a non-pyroptotic manner through activation of the NLRP3 inflammasome with contributions from ATP release through Pannexin-1, and ROS signaling. Since LPS, PGN, and MDP are not known to activate NLRP3 directly, the role of uric acid as a potential mediator was assessed. FMs produced elevated uric acid in response to LPS, PGN and MDP. FM IL-1β secretion was inhibited by allopurinol, which blocks uric acid production, for LPS and PGN, and to a lesser degree, MDP. These findings shed light on the mechanisms by which fetal membrane inflammation and subsequent preterm birth may arise.

Keywords: Bacteria, Infection, Inflammasome, Inflammation, Fetal Membrane, Pyroptosis

1. Introduction

Preterm birth complicates ~10% of pregnancies in the United States, a rate that has remained static over the past decade (March of Dimes, 2020). At least half of these deliveries occur secondary to preterm premature rupture of membranes (PPROM), clinical chorioamnionitis, or preterm labor associated with inflammation and/or infection (Norwitz and Caughey 2011). Studies have identified interleukin-1β (IL-1β), a pro-inflammatory cytokine, as an important mediator of PPROM and spontaneous preterm birth. In vitro, IL-1β stimulates the expression of uterine activation proteins which contribute to the onset of labor (Zaragoza et al. 2006), induces oxytocin secretion in uterine smooth muscle cells (Friebe-Hoffmann et al. 2001), and promotes local progesterone metabolism in human cervical fibroblasts (Roberson et al. 2012). In humans, elevated IL-1β serum concentrations are independently associated with an elevated risk of preterm birth (Gargano et al. 2008), while in animal models, administration of IL-1 and inhibition of IL-1 induce and prevent preterm birth, respectively (Romero et al. 1991, Romero et al. 1992, Sadowsky et al. 2006).

IL-1β production is classically a two-step process: induction of pro-IL-1β, typically following activation of a Toll like receptor (TLR) or Nod protein, followed by processing of the pro-form into active secreted IL-1β by the inflammasome. Inflammasomes are protein complexes that classically includes a Nod-like receptor (NLR), the adaptor molecule apoptosis-associated speck-like protein containing a CARD domain (ASC), and caspase-1 (Jin and Flavell 2010, Netea et al. 2010). This second step requires an additional activation signal that triggers the sensor component of the inflammasome, leading to inflammasome assembly and caspase-1-dependent cleavage of pro-IL-1β (Jin and Flavell 2010, Netea et al. 2010). Additional signals that can potentiate inflammasome activation, include ATP-associated potassium efflux acting through the P2X7 receptor and pannexin-1, or by ROS production (Jin and Flavell 2010, Hung et al. 2013). IL-1β secretion then occurs either in a cell conserving manner or via pyroptosis, an inflammatory form of cell death mediated by the inflammasome (Christgen et al. 2020). During pyroptosis, in addition to processing pro-IL-1β, active caspase-1 also cleaves gasdermin D (GSDMD) resulting in membrane pore formation and cell death with resultant cytokine release (Liu et al. 2016). Production of the inflammatory factor, IL-18 is also mediated through a similar mechanism whereby pro-IL-18 is processed into active IL-18 by the inflammasome (Christgen et al. 2020).

We previously reported that normal human fetal membranes (FM) collected at term without labor secrete IL-1β in response to a range of bacterial TLR and Nod protein ligands (Hoang et al. 2014). However, the mechanism of inflammasome activation in human FM tissue leading to IL-1β production following exposure to bacterial components is incompletely understood. Therefore, this study sought to determine the FM compartment involved in IL-1β production in response to bacterial components that specifically activate distinct TLRs and Nod proteins, and to evaluate the mechanism involved with a focus on the NLRP3 inflammasome. Since IL-18 is also mediated by the inflammasome (Christgen et al. 2020), and IL-8 is a chemoattractant that contributes to neutrophil recruitment in chorioamnionitis (Cherouny et al. 1993), we also evaluated FM production of these factors in response to bacterial components.

Herein we report that the chorion is the major FM sensor of bacterial components and source of FM inflammatory factors. We also report that FM IL-1β and IL-18 production following exposure to the bacterial components lipopolysaccharide (LPS), peptidoglycan (PGN), and muramyl dipeptide (MDP) which activate TLR4, TLR2, and Nod2, respectively, occurs in a non-pyroptotic fashion involving the NLRP3 inflammasome with contributions from ATP via pannexin-1 and ROS. Finally, we report that inhibition of uric acid production by the xanthine oxidase inhibitor, allopurinol, reduces IL-1β, but not IL-18, production in response to bacterial components.

2. Materials and Methods

2.1. Study approvals

Human tissue collection was approved by Yale University’s Human Research Protection Program. All samples were collected through the Yale University Reproductive Sciences (YURS) Biobank following patient consent.

2.2. Preparation of human FM explants

Human FMs were collected from a total of 23 planned uncomplicated cesarean deliveries at term (37–41 weeks of gestation) in the absence of labor or known infection or inflammation. After washing the FMs with sterile PBS supplemented with penicillin (100U/mL) and streptomycin (100μg/mL) (Life Technologies, Grand Island, NY), adherent blood clots were removed and full thickness FM explants in which the chorion and amnion were intact were obtained using a 6mm biopsy punch. In instances where the chorion or amnion were treated separately, the chorion and amnion were separated by gently stretching apart the layers prior to obtaining biopsies of the isolated chorion and amnion layers prior to collecting a 6mm biopsy punch. The FM intact explants, or separated chorion and amnion explants, were placed in 0.4μm pore cell culture inserts (BD Falcon, Franklin Lakes, NJ) with 500μL of Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT). These were placed in a 24-well plate containing 500μL of DMEM/10% FBS. The next day, the medium was removed and replaced with serum-free Opti-MeM media (Life Technologies) prior to treatment.

2.3. Treatment of human FM explants

Intact FM, chorion, and amnion explants were either untreated (media control) or treated with lipopolysaccharide isolated from Escherichia coli 0111:B4 at 100ng/mL (LPS; Sigma-Aldrich, St. Louis, MO); peptidoglycan isolated from Staphylococcus Aureus at 10μg/mL (PGN; InvivoGen, San Diego, CA); flagellin at 1μg/mL (InvivoGen); L18-MDP, D-glutamyl-meso-diaminopimelic acid at 100μg/mL (iE-DAP; InvivoGen); or a synthetic derivative of muramyl dipeptide at 10μg/mL (MDP; InvivoGen). Doses were based on previous studies (Hoang et al. 2014, Cross et al. 2017, Potter et al. 2020). For some experiments, FMs were pre-treated for 1 hour with either the NLRP3 inhibitor 3,4-methylenedioxy-β-nitrostyrene (MCC950; 10μM; Cayman Chemical, Ann Arbor, MI); the pannexin-1 inhibitor, carbenoxolone (10μM; Sigma-Aldrich); the caspase-1 inhibitor, Z-WEHD-FMK (1μM; R&D Systems, Minneapolis, MN); the reactive oxygen species (ROS) inhibitor, diphenyleneiodonium (DPI; 10μM; Sigma-Aldrich); or the xanthine oxidase inhibitor, allopurinol (400 μM; Selleck Chemicals, Houston, TX). After 24 hours, cell-free culture supernatants were collected and FM tissues snap-frozen, and both then stored at −80°C.

2.4. FM secretome analysis

Analysis of FM culture supernatants was performed by ELISA for the following inflammatory factors: IL-1β, IL-8, and IL-18 (R&D Systems). Measurement of uric acid release was performed using a colorimetric uric acid assay kit (Sigma-Aldrich). Lactate dehydrogenase release as a marker of cell death was measured on FM culture supernatants using CyQuant LDH cytotoxicity assay (Thermo Fisher Scientific, Waltham, MA).

2.5. Western blot analysis

Total protein lysates from FM explants were collected by tissue homogenization using a beadbug microtube homogenizer with microtubes pre-filled with high impact zirconium beads (Benchmark Scientific; Sayreville, NJ). Western blot analysis was performed as previously described (Tong et al. 2021). Membranes were probed with either the anti-human total GSDMD mouse monoclonal antibody (mAb) (64-Y) (1:1000, sc81868; Santa Cruz Biotechnology, Inc, Dallas, TX) or the anti-human cleaved GSDMD (Asp275) rabbit mAb (1:500, E7H9G; Cell Signaling Technology, Danvers, MA). β-Actin was used as a loading control (1:5000, Sigma-Aldrich). Chemiluminescence was detected and images collected using an Amersham Imager 680 (General Electric, Boston, MA), and semiquantitative densitometry was performed using Image Studio Lite (LI-COR, Lincoln, NE).

2.6. Statistical analysis

All FM experiments were performed at least 3 times. Data is reported as mean +/− SEM. Statistical significance was set at p<0.05. Analysis was performed using Prism software (Graphpad, Inc., La Jolla, CA) For normally distributed data, significance was determined using either ANOVA with Dunnett’s multiple comparison test or a paired t-test. For data not normally distributed, significance was determined using either the Friedman test or the Kruskal-Wallis test with Dunn’s multiple comparisons test or a paired Wilcoxon matched-pairs signed rank test.

3. Results

3.1. FM production of inflammatory factors in response to bacterial components is generated predominantly by the chorion.

First, the inflammatory response of FMs to the bacterial components LPS, PGN, flagellin, iE-DAP and MDP which active TLR4, TLR2, TLR5, Nod1, and Nod2, respectively was analyzed. We focused on inflammatory IL-1β and IL-18 since IL-1β is associated with tissue injury (Dinarello 1997), and preterm birth (Christiaens et al. 2008, Kemp et al., Adams Waldorf et al. 2011), and both are mediated by the inflammasome. Since chorioamnionitis is characterized by a neutrophil infiltrate, we also evaluated the neutrophil chemoattractant IL-8 (Kobayashi 2008). Bacterial LPS, PGN, and MDP significantly increased intact human FM explant secretion of IL-1β by 18.3±7.0-fold; 84.4±41.0-fold; and 29.3±14.2-fold, respectively when compared to the untreated control (Figure 1A; i). Similarly, FM IL-8 secretion significantly increased 3.7±0.6-fold; 3.6±0.6-fold; and 2.7±0.6-fold in response to LPS, PGN, and MDP, respectively, when compared to the untreated control (Figure 1A; ii). Intact FM IL-18 secretion significantly increased 8.4±1.6-fold in response to LPS, and 15.0±6.6-fold in response to PGN when compared to the untreated control. While not significant, intact FM IL-18 secretion in response to MDP increased 15.1±8.4-fold (Figure 1A; iii). Flagellin only significantly increased FM IL-8 secretion by 2.1±0.4-fold, while iE-DAP had no significant effect on FM secretion of IL-1β, IL-8 or IL-18 (Figure 1A).

Figure 1. FM production of inflammatory factors in response to bacterial components is generated predominantly by the chorion.

Figure 1.

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(A) Intact human FM explants or (B) FM explants separated into the chorion and amnion compartments from 8–10 patients were untreated or treated with either LPS; PGN; Flagellin; iE-DAP; or MDP. After 24hr, cell-free culture supernatants were collected and measured for i) IL-1β (n=8–10); ii) IL-8 (n=8); and iii) IL-18 (n=8–10). *p<0.05 vs the untreated control unless indicated otherwise.

To determine which FM compartment was responsible for generating the inflammatory cytokine response after exposure to theses bacterial components, isolated chorion and amnion tissues were treated separately, and the supernatants evaluated. IL-1β secretion was significantly increased in supernatants from isolated chorion in response to LPS, PGN, and MDP when compared to the untreated control, and the chorion secreted significantly more IL-1β under all bacterial component conditions when compared to the amnion. There was no significant change in levels of IL-1β secreted by the amnion in response to any of the bacterial components when compared to the untreated control (Figure 1B; i). IL-8 secretion was significantly increased in supernatants from the isolated chorion in response to LPS, PGN, and MDP. The amnion also secreted significantly more IL-8 in response to LPS and PGN when compared to the untreated control. However, again, the chorion secreted significantly more IL-8 under all conditions when compared to the amnion (Figure 1B; ii). While there was no significant difference in the levels of secreted IL-18 by the separated chorion or amnion under all conditions, with the exception of the control, the chorion appeared to be the major source in the presence of the bacterial components LPS, PGN, iE-DAP and MDP (Figure 1B; iii).

3.2. FM secretion of IL-1β and IL-18 in response to bacterial components is mediated by the NLRP3 inflammasome through a non-pyroptotic mechanism.

Having demonstrated that bacterial LPS, PGN, and MDP increased intact FM secretion of IL-1β and IL-18, we next evaluated the mechanism of inflammasome activation involved by systematically using inhibitors to NLRP3 (MCC950), Pannexin-1 (Carbenoxolone), Caspase-1 (Z-WEHD-FMK), and ROS (DPI). As shown in Figure 2, LPS, PGN and MDP significantly increased intact FM secretion of (A) IL-1β and (B) IL-18 when compared to the untreated control under media conditions. NLRP3 inhibition significantly inhibited LPS, PGN, and MDP-induced FM IL-1β secretion by 82.1±4.6%, 77.0±7.8%, and 95.0±2.1%, respectively (Figure 2A; i). Pannexin-1 inhibition significantly inhibited LPS, PGN, and MDP-induced FM IL-1β secretion by 83.3±2.5%, 80.1±7.9%, and 98.0±1.4%, respectively (Figure 2A; ii). Caspase-1 inhibition, significantly inhibited FM IL-1β production following stimulation by LPS by 26.9±4.1% and by MDP by 68.6±20.7%, but had no significant effect on IL-1β secretion in response to PGN (Figure 2A; iii). ROS inhibition, significantly reduced LPS, PGN, and MDP-induced FM IL-1β secretion by 86.5±3.6%; 67.3±14.8% and 98.2±1.6%, respectively (Figure 2A; iv).

Figure 2. FM secretion of IL-1β and IL-18 in response to bacterial components is mediated by the NLRP3 inflammsome with contrubtions from ATP via Pannexin-1 and ROS signaling.

Figure 2.

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A) Intact human FM explants from 4–7 patients were untreated or treated with either LPS; PGN; or MDP in the presence of either media; i) the NLRP3 inhibitor, MCC950; ii) carbenoxolone which inhibits pannexin-1; iii) the caspase-1 inhibitor; Z-WEHD-FMK; or iv) the ROS inhibitor, DPI. After 24hr cell-free culture supernatants were collected and measured for (A) IL-1β (n=4–7) or (B) IL-18 (n=4–6). *p<0.05 vs the untreated control under each condition (media or inhibitor) unless indicated otherwise.

IL-18 secretion was similarly evaluated. Inhibition of NLRP3 significantly inhibited LPS, PGN, and MDP-induced FM IL-18 secretion by 80.1±9.3%, 89.5±6.4%, and 94.4±5.6%, respectively (Figure 2B; i). Pannexin-1 inhibition, significantly reduced FM IL-18 secretion following exposure to LPS, PGN and MDP by 83.3±7.8%, 96.5±3.5%, and 100.0±0.0%, respectively (Figure 2B; ii). Caspase-1 inhibition, significantly inhibited FM IL-1β production following stimulation by LPS by 93.8±4.6% and by MDP by 96.8±2.1%, but had no significant effect on IL-1β secretion in response to PGN (Figure 2B; iii). ROS inhibition significantly reduced LPS, PGN, and MDP-induced IL-18 secretion by 94.4±5.1%; 76.5±19.2% and 99.9±0.1%, respectively (Figure 2B; iv).

Evaluation for pyroptosis was determined by measuring FM release of LDH and FM expression of cleaved GSDMD. As shown in Figure 3A, compared to the untreated control there was no significant change in the levels of LDH release from FM explants following treatment with either LPS, PGN, and MDP. Similarly, cleaved and full length GSDMD were detectable in all samples tested, and densitometry of cleaved GSDMD/total GSDMD was not significantly different across all treatment groups (Figure 3B). There was also no significant differences in the absolute levels of cleaved or full length GSDMD after normalization to β-actin between the treatment groups (data not shown).

Figure 3. FM secretion of IL-1β and IL-18 in response to bacterial components is mediated through a non-pyroptotic mechanism.

Figure 3.

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Intact human FM explants from 3–8 patients were untreated or treated with either LPS; PGN; or MDP. (A) After 24hr, cell-free culture supernatants were collected and measured for LDH release (n=8). (B) After 24hr, FM explants were homogenized for protein and expression of cleaved and total GSDMD evaluated by Western blot. Image shows one representative blot while the bar chart shows quantification of protein expression as determined by densitometry (n=3).

3.3. Endogenous uric acid mediates FM NLRP3 inflammasome activation and IL-1β production.

Having demonstrated that FM IL-1β and IL-18 secretion is mediated by the NLRP3 inflammasome in response to LPS, PGN and MDP, we next sought to determine what could be providing the secondary signal for NLRP3 since these bacterial components are not known to be able to activate this NLR (Koizumi et al. 2011). Based on prior studies demonstrating the role of endogenous uric acid in the direct activation of the NLRP3 inflammasome in the placental trophoblast (Mulla et al. 2011, Mulla et al. 2013), supernatants were analyzed to determine whether treatment of FMs with LPS, PGN, or MDP increased uric acid production. FM uric acid levels were significantly increased following treatment with LPS, PGN, and MDP by 3.1±0.4-fold, 6.9±1.3-fold, and 4.7±1.3-fold, respectively when compared to the untreated control (Figure 4A). Given this finding, we then assessed the FM IL-1β and IL-18 response following pre-treatment with allopurinol, a xanthine oxidase inhibitor that reduces uric acid production (Negi et al. 2020). When administered prior to bacterial component exposure, allopurinol significantly reduced FM IL-1β production in response to LPS by 63.9±7.6% and to PGN by 59.1±14.8% (Figure 4B; i). While MDP-induced IL-1β was not significantly reduced by allopurinol, it was downregulated by 29.6±15.7% (Figure 4B; i). In contrast, allopurinol had no effect on FM secretion of IL-18 in response to LPS, PGN or MDP (Figure 4B; ii).

Figure 4. Endogenous uric acid mediates FM inflammasome activation and IL-1β but not IL-18 secretion.

Figure 4.

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(A) Intact human FM explants from 7 patients were untreated or treated with either LPS; PGN; or MDP. After 24hr, cell-free culture supernatants were collected and measured for uric acid. *p<0.05 vs the untreated control. (B) Intact human FM explants from 6 patients were untreated or treated with either LPS; PGN; or MDP in the presence of either media or allopurinol. After 24hr, cell-free culture supernatants were collected and measured for i) IL-1β or ii) IL-18. *p<0.05 vs the untreated control under either media or allopurinol unless indicated otherwise.

4. DISCUSSION

Chorioamnionitis, PPROM and preterm birth are a major contributors to neonatal mortality and morbidity. When associated with infection, bacterial colonization is frequently polymicrobial, with common implicated pathogens including Ureaplasma urealyticum, Mycoplasma hominis, Gardnerella vaginalis, bacteroides, aerobes including Group B streptococcus (GBS), and gram-negative rods including Escherichia coli (Tita and Andrews 2010). In our study, LPS, PGN, and MDP, bacterial components that activate TLR4, TLR2 and Nod2 respectively, upregulated FM production of the inflammatory factors IL-1β, IL-8, and IL-18. LPS and PGN are common cell wall components of all Gram-negative and Gram-positive bacteria, respectively, while MDP is generated by all Gram-negative and Gram-positive bacteria. Thus, our findings using these bacterial components are clinically relevant (Goldenberg et al. 2002, Goncalves et al. 2002, Lamont 2003, DiGiulio et al. 2008, Hecht et al. 2008, DiGiulio 2012). IL-1β is an inflammatory cytokine that has been linked to preterm birth and intraamniotic infection (Hillier et al. 1993). IL-8, a neutrophil chemoattractant, has similarly been associated with development of histologic chorioamnionitis and subsequent preterm birth (Cherouny et al. 1993). IL-18, like IL-1β, relies on the inflammasome for processing. Unlike IL-1β, however, literature regarding the association between IL-18 and preterm birth is mixed (Pacora et al. 2000, Menon et al. 2001, Zhu et al. 2021Gomez-Lopez, 2017 #4373).

In our current study, the relative cytokine/chemokine contribution of the chorion exceeded that of the amnion, consistent with other in vitro studies (Zaga-Clavellina et al. 2014, Boldenow et al. 2015), and with clinical observations where IL-1β was preferentially localized to the chorion from women with chorioamnionitis and preterm birth (Gomez-Lopez et al. 2017a, Gomez-Lopez et al. 2017b). This indicates that the chorion is a major sensor of bacterial components and the main source of the inflammatory mediators IL-1β and IL-8. Some studies have isolated amniotic epithelial cells to examine responses to bacteria and bacterial components (Motedayyen et al. 2018, Motedayyen et al. 2019); and one study that screened TLR function in these cells found only flagellin, that stimulates TLR5, and Malp2, that stimulates TLR2/6, elicited a low response (Gillaux et al. 2011). Indeed, in our system the separated amnion was able to respond to TLR2 and TLR4 activation to promote elevated IL-8 secretion, albeit at low levels. This indicates that the amnion is not devoid of innate immune sensing, but is not as capable as the chorion. One explanation for this may be as simple as the cellular density, since the amnion is an epithelial layer, while the chorion is a thicker compartment consisting of mainly cytotrophoblast and syncytiotrophoblast cells (Lim 2017). There may also be differences in the levels and signaling capacities of TLRs and NLRs between the chorion and amnion. Finally, that the chorion would be the primary responder to bacterial insults suggests a physiological role for this compartment in preventing the movement of pathogens from the maternal to the fetal side.

Following our characterization of this inflammatory response to bacterial components, we then assessed the mechanism of FM IL-1β and IL-18 production since both are mediated by the inflammasome. There are number of inflammasomes that have been demonstrated via PCR or immunohistochemistry in the chorioamniotic membranes including the NLRP1, NLRP3, NLRC4, AIM2, and Pyrin inflammasomes (Hoang et al. 2014, Bryant et al. 2017, Gomez-Lopez et al. 2017b, Romero et al. 2018, Zhu et al. 2021). However, NLRP3 is the only inflammasome that has been noted to be primed and activated in human FMs following spontaneous labor occurring both preterm and at term, with and without histologic chorioamnionitis (Gomez-Lopez et al. 2017a, Gomez-Lopez et al. 2017b, Romero et al. 2018).

Using inhibitors to systematically assess the mechanism of FM IL-1β and IL-18 production following exposure to LPS, PGN, and MDP, there was common involvement of NLRP3 with contributions from ATP release through Pannexin-1, and ROS signaling following exposure to each bacterial component. Caspase-1 dependent FM IL-1β and IL-18 production was found in response to LPS and MDP, but not after exposure to PGN. Canonical NLRP3 inflammasome activation results in caspase-1 cleavage of pro-IL-1β and pro-IL-18 into their active forms. However, caspase-4 and caspase-5, which are homologous with mouse caspase-11, have also been associated with noncanonical inflammasome activation resulting in caspase-1 independent IL-1β production (Downs et al. 2020). Thus, PGN may trigger a non-canonical inflammasome pathway in human FMs for both IL-1β and IL-18 production. Indeed, caspase-4 and caspase-5 have been identified in placental tissue, although only caspase-4 has been reported to be upregulated in the setting of parturition both preterm and at term, with and without histologic chorioamnionitis (Gomez-Lopez et al. 2017a, Gomez-Lopez et al. 2017b, Romero et al. 2018). Alternatively, Monteleone et al. previously reported two separate mechanisms of IL-1β secretion in macrophages following exposure to PGN: a fast, caspase-1 and GSDMD-dependent secretion; and a second, slow, caspase-1 and GSDMD-independent secretion (Monteleone et al. 2018). Further study is needed to assess the non-caspase-1 dependent pathways of FM IL-1β and IL-18 production following exposure to bacterial PGN.

Regardless of whether caspase-1 was involved or not, we saw no clear evidence of pyroptosis following FM exposure to bacterial components based on LDH or cleaved GSDMD levels. Notably, cleaved and full length GSDMD were detectable in all samples with no significant differences between treated and untreated explants either in absolute levels of cleaved or full length GSDMD normalized to β-actin, or the relative ratio of cleaved to full length GSDMD. In contrast, though GSDMD was present in amniotic fluid at term regardless of labor status, Gomez-Lopez et al., previously reported that in vivo concentrations of GSDMD, measured by ELISA, were elevated in amniotic fluid and chorioamniotic membranes from women who underwent spontaneous labor at term or spontaneous preterm birth in the setting of intra-amniotic infection or inflammation (Gomez-Lopez et al. 2019b, Gomez-Lopez et al. 2021). However, the presence of GSDMD may not necessarily imply a pyroptotic pathway; full length GSDMD has been previously implicated in non-pyroptotic IL-1β secretion via the release of small extracellular vesicles intestinal epithelial cells (Bulek et al. 2020). Thus, alternate pathways of FM GSDMD upregulation and non-pyroptotic IL-1β and IL-18 secretion should be explored.

Since LPS, PGN, and MDP are not known to be able to activate NLRP3 directly (Jin and Flavell 2010), we questioned what could be providing the second signal for inflammasome activation following stimulation by these TLR and Nod protein agonists. One signal that can directly active NLRP3 is the damage associated molecular pattern (DAMP), uric acid (Martinon et al. 2006, Mulla et al. 2011). Endogenous uric acid-mediated activation of the NLRP3 inflammasome has been previously demonstrated in a trophoblast model in the setting of either antiphospholipid antibodies (Mulla et al. 2013) or excess glucose (Han et al. 2015). Though first trimester uric acid levels are not associated with risk of preterm birth (Laughon et al. 2011), third trimester serum uric acid levels have been associated with elevated risk of preterm labor in women with preeclampsia (Ryu et al. 2019). Further study demonstrated that use of allopurinol, a xanthine oxidase inhibitor that decreases both uric acid and ROS production, inhibited IL-1β production in an excess glucose exposure trophoblast model (Negi et al. 2020). In our study, FM explants produced elevated uric acid levels in response to the bacterial components LPS, PGN, and MDP. Furthermore, allopurinol reduced FM IL-1β secretion following stimulation by LPS and PGN, indicating this may be the mechanism by which these bacterial components can induce FM NLRP3 inflammasome activation and subsequent IL-1β secretion. This is suspected to be mediated by uric acid suppression instead of ROS, given our finding that allopurinol did not inhibit FM IL-18 production in response to LPS, PGN and MDP, while the ROS inhibitor, DPI did. Although allopurinol did not significantly inhibit MDP-induced IL-1β there was a small reduction, and MDP did significantly upregulate FM uric acid. Together this indicates that MDP may utilize both uric acid and an alternative mechanism to activate NLRP3. Indeed, Nod2 which senses MDP, may form a complex with NLRP1 leading to caspase-1 activation; and a similar complex has been demonstrated with NLRP3 (Hsu et al. 2008). Alternatively, Singh et al., reported that Nod2-induced inflammasome activation occurs through a WNT/β-CATENIN-dependent pathway in macrophages (Singh et al. 2015).

IL-18 and IL-1β are both cytokines within the IL-1 family, synthesized by precursor proteins, and processed through the inflammasome; however key differences may explain their noncongruent responses in the presence of allopurinol. Pro-IL-18 is constitutively expressed under steady state, while pro-IL-1β is not and requires an inflammatory stimulus mediated through a TLR or Nod protein (Zhu and Kanneganti 2017). Additionally pro-IL-18 is mediated through mitogen activated protein kinase 38 (MAPK p38), while pro-IL-1β is mediated through NFκB, not seen with IL-18 (Lee et al. 2004). In addition to activating the NLRP3 inflammasome, uric acid can also prime TLRs (specifically TLR2 and TLR4) (Crisan et al. 2016). Therefore allopurinol’s putative prevention of uric acid-mediated TLR priming may explain the differential dependency of uric acid for FM IL-18 and IL-1β production as well as providing an alternate explanation for the lack of significant suppression with MDP, a Nod2 agonist. Thus, an additional activation signal for NLRP3 would be required for the FM IL-18 response that we observe in our system. Gomez-Lopez et al., demonstrated that following ultrasound-guided intraamniotic administration, the alarmin S100B induced the activation of the FM NLRP3 inflammasome, triggering IL-1β secretion and increasing the rate of preterm labor and birth in mice (Gomez-Lopez et al. 2019a). NLRP3 activation by S100 proteins have also been reported in other cellular systems (Simard et al. 2013, Kim et al. 2018, Shi et al. 2019).

In summary, IL-1β and IL-18 production in human FMs following exposure to the bacterial components LPS, PGN, and MDP occured in a non-pyroptotic fashion involving the NLRP3 inflammasome with contributions ATP, via pannexin-1, and ROS signaling. Inhibition of uric acid production by the xanthine oxidase inhibitor, allopurinol, reduced IL-1β, but not IL-18, production in response to bacterial components. Since IL-1β is an important mediator of tissue injury, these findings shed new light on the mechanisms by which bacterial associated fetal membrane inflammation and subsequent preterm birth may arise.

Highlights.

  • The chorion is the source of major fetal membrane (FM) inflammatory factors

  • Bacterial components induced FM IL-1β and IL-18 via the NLRP3 inflammasome

  • FM IL-1β and IL-18 secretion occurred in a non-pyroptotic manner

  • Endogenous uric acid contributed to FM inflammasome activation and IL-1β secretion

Acknowledgements

Vikki M Abrahams, PhD holds a 2021 Next Gen Pregnancy Research Grant from the Burroughs Wellcome Fund. The authors would like to thank the Yale University Reproductive Sciences Biobank and the staff of Labor and Delivery, Yale-New Haven Hospital for their help with fetal membrane sample collection.

Funding

This study was supported in part by a grant from the NIAID, NIH (R01AI121183, to VMA) and by a Next Gen Pregnancy Research Grant from the Burroughs Wellcome Fund grant (NPG125, to VMA).

Footnotes

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Declaration of competing interest

The authors declare that there are no conflict of interest.

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