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. 2020 Jul 9;26(9):712–726. doi: 10.1093/molehr/gaaa054

The alarmin interleukin-1α causes preterm birth through the NLRP3 inflammasome

K Motomura g1,g2, R Romero g1,g3,g4,g5,g6,g7, V Garcia-Flores g1,g2, Y Leng g1,g2, Y Xu g1,g2, J Galaz g1,g2, R Slutsky g1, D Levenson g1,g2, N Gomez-Lopez g1,g2,g8,✉
PMCID: PMC7473789  PMID: 32647859

Abstract

Sterile intra-amniotic inflammation is a clinical condition frequently observed in women with preterm labor and birth, the leading cause of neonatal morbidity and mortality worldwide. Growing evidence suggests that alarmins found in amniotic fluid, such as interleukin (IL)-1α, are central initiators of sterile intra-amniotic inflammation. However, the causal link between elevated intra-amniotic concentrations of IL-1α and preterm birth has yet to be established. Herein, using an animal model of ultrasound-guided intra-amniotic injection of IL-1α, we show that elevated concentrations of IL-1α cause preterm birth and neonatal mortality. Additionally, using immunoblotting techniques and a specific immunoassay, we report that the intra-amniotic administration of IL-1α induces activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in the fetal membranes, but not in the decidua, as evidenced by a concomitant increase in the protein levels of NLRP3, active caspase-1, and IL-1β. Lastly, using Nlrp3−/− mice, we demonstrate that the deficiency of this inflammasome sensor molecule reduces the rates of preterm birth and neonatal mortality caused by the intra-amniotic injection of IL-1α. Collectively, these results demonstrate a causal link between elevated IL-1α concentrations in the amniotic cavity and preterm birth as well as adverse neonatal outcomes, a pathological process that is mediated by the NLRP3 inflammasome. These findings shed light on the mechanisms underlying sterile intra-amniotic inflammation and provide further evidence that this clinical condition can potentially be treated by targeting the NLRP3 inflammasome.

Keywords: amniotic cavity, chorioamnionitis, caspase-1, decidua, fetal membranes, interleukin-1β, prematurity, sterile intra-amniotic inflammation

Introduction

Preterm birth, the leading cause of perinatal morbidity and mortality worldwide (Blencowe et al., 2012; Liu et al., 2015; Rizzolo et al., 2020), is a pregnancy complication that is often preceded by spontaneous preterm labor (Romero et al., 1994; Muglia and Katz, 2010). While preterm labor has many known etiologies (Romero et al., 2014a; Strauss et al., 2018; Brosens et al., 2019), intra-amniotic inflammation is the best-characterized and has been studied the most extensively (Romero et al., 1988, 2007; Hirsch et al., 1995; Baggia et al., 1996; Gomez et al., 1997; Hirsch et al., 1999; Hirsch and Muhle, 2002; Sadowsky et al., 2003; Park et al., 2005; Hirsch et al., 2006; Mendelson, 2009; Adams Waldorf et al., 2011; Senthamaraikannan et al., 2016; Gomez-Lopez et al., 2019b). Intra-amniotic inflammation is often associated with microbial invasion of the amniotic cavity (i.e. intra-amniotic infection) (Romero and Mazor, 1988; Romero et al., 1989c, 2001; Gravett et al., 2004; Gomez-Lopez et al., 2018a; Pacora et al., 2019; Tang et al., 2019b), which can be treated with antibiotics (Lee et al., 2016; Oh et al., 2019; Yoon et al., 2019). However, we have recently shown, using both cultivation and molecular microbiology techniques, that a subset of women with preterm labor and birth present intra-amniotic inflammation without detectable microorganisms (Romero et al., 2014b,c). This clinical condition is defined as sterile intra-amniotic inflammation (Romero et al., 2014c) and is thought to be initiated by endogenous danger signals derived from damaged and necrotic cells, termed damage-associated molecular patterns or alarmins (Matzinger, 1998; Oppenheim and Yang, 2005; Lotze et al., 2007). While less common, sterile intra-amniotic inflammation has also been observed in women with other pregnancy complications such as an asymptomatic short cervix (Romero et al., 2015c), preterm prelabor rupture of membranes (Romero et al., 2015b), and clinical chorioamnionitis at term (Romero et al., 2015d; Gomez-Lopez et al., 2019d). Moreover, sterile intra-amniotic inflammation has been shown to have deleterious effects on offspring and is observed more frequently than microbial-associated intra-amniotic inflammation in women with preterm labor and intact membranes (Romero et al., 2014c); therefore, elucidation of the mechanisms that lead to this clinical condition is crucial.

Alarmins, namely interleukin (IL)-1α (Romero et al., 1992, 2015a), high-mobility group box-1 (HMGB1) (Romero et al., 2011, 2012), S100 calcium-binding protein B (S100B) (Friel et al., 2007) and heat shock protein 70 (HSP70) (Chaiworapongsa et al., 2008), are thought to mediate sterile intra-amniotic inflammation as they are elevated in amniotic fluid of women with inflammation-associated preterm labor and birth. Specifically, IL-1α is the top-ranked protein derived from a network connectivity analysis of inflammatory-related proteins from women with preterm labor and sterile intra-amniotic inflammation, indicating that this cytokine plays a central role in the inflammatory milieu in premature parturition (Romero et al., 2015a). IL-1α is constitutively produced in almost all cell types in its active form (Kong et al., 2006; Dinarello, 2009; Bersudsky et al., 2014) and functions as a transcription regulator (Buryskova et al., 2004; Werman et al., 2004; Zamostna et al., 2012). However, IL-1α is a dual-function cytokine, meaning it is localized intracellularly under homeostatic conditions but in the setting of inflammation can serve to respond to cellular damage and act as an alarmin (Chen et al., 2007; Eigenbrod et al., 2008; Cohen et al., 2010; Rider et al., 2011). Indeed, the systemic administration of the alarmin IL-1α induces preterm birth in mice (Romero et al., 1991; Romero and Tartakovsky, 1992), and its intra-amniotic administration results in fetal damage in sheep (Emerson et al., 1997; Berry et al., 2011; Kallapur et al., 2011; Wolfs et al., 2011). However, the causal link between increased intra-amniotic concentrations of IL-1α and preterm birth has yet to be established.

The mechanisms whereby alarmins induce sterile intra-amniotic inflammation and preterm birth involve the activation of the NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome (Gotsch et al., 2008; Gomez-Lopez et al., 2017, 2018b, 2019a,c). This inflammasome includes: the NLRP3 sensor molecule; the adaptor protein ASC (apoptosis‐associated speck‐like protein containing a C-terminal caspase recruitment domain); and pro-caspase-1 (Martinon et al., 2002; Franchi et al., 2009; Guo et al., 2015). Upon activation, the NLRP3 inflammasome releases active caspase-1 (Sutterwala et al., 2006, 2014), which promotes the maturation of IL-1β and IL-18 into their bioactive forms (Black et al., 1989; Kostura et al., 1989; Cerretti et al., 1992; Thornberry et al., 1992; Ghayur et al., 1997; Gu et al., 1997; Dinarello, 1998; Fantuzzi and Dinarello, 1999; Sansonetti et al., 2000). Herein, we hypothesized that NLRP3 inflammasome activation is implicated in the mechanisms whereby IL-1α induces adverse pregnancy outcomes.

In the current study, we investigated whether the ultrasound-guided intra-amniotic administration of IL-1α induces preterm birth and adverse neonatal outcomes in mice. Additionally, we evaluated whether the NLRP3 inflammasome is activated by IL-1α and, finally, whether Nlrp3 deficiency could ameliorate the IL-1α-induced adverse pregnancy outcomes.

Materials and methods

Ethical approval

All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Wayne State University under Protocol No. 18-03-0584.

Mice

C57BL/6 (wild-type or Nlrp3+/+) mice (JAX stock #000664) and B6.129S6-Nlrp3tm1Bhk/J (Nlrp3−/−) mutant mice (JAX stock #021302) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and bred in the animal care facility at C.S. Mott Center for Human Growth and Development at Wayne State University (Detroit, MI, USA). All mice were kept under a circadian cycle (light:dark = 12:12 h). Females, 8–12 weeks old, were bred with males of proven fertility. Female mice were checked daily between 8:00 a.m. and 9:00 a.m. for the appearance of a vaginal plug, which indicated 0.5 days post coitum (dpc). Females were then housed separately from the males, their weights were monitored daily, and a weight gain of 2 g or more by 12.5 dpc confirmed pregnancy. All animals were randomly assigned to experimental or control groups prior to the following experiments.

Ultrasound-guided intra-amniotic administration of IL-1α

Pregnant wild-type mice were anesthetized on 16.5 dpc by inhalation of 2–3% isoflurane (Aerrane, Baxter Healthcare Corporation, Deerfield, IL, USA) and 1–2 l/min of oxygen in an induction chamber. Anesthesia was maintained with a mixture of 1.75–2% isoflurane and 2.0 l/min of oxygen during the ultrasound procedure, which was performed using the Vevo® 2100 Imaging System (VisualSonics Inc., Toronto, Ontario, Canada). Mice were positioned on a heating pad and stabilized with adhesive tape. Fur removal from the abdomen was accomplished by applying Nair depilatory cream (Church & Dwight Co., Inc., Ewing, NJ, USA) to that area. Body temperature was maintained at 37 ± 1°C and detected using a rectal probe (VisualSonics). Respiratory and heart rates were monitored by electrodes embedded in the heating pad. An ultrasound probe was anchored and mobilized with a mechanical holder, and the transducer was slowly moved toward the Aquasonic CLEAR ultrasound gel (Parker Laboratories, Inc., Fairfield, NJ, USA) applied on the abdomen. Ultrasound-guided intra-amniotic injection of IL-1α (Cat #200-LA/CF, R&D Systems, Inc., Minneapolis, MN, USA) at concentrations of 0.15 ng, 0.5 ng, 1 ng, 5 ng, 10 ng, 100 ng, 200 ng, 250 ng, or 500 ng per 25 µl of sterile 1× PBS (Fisher Scientific Bioreagents, Fair Lawn, NJ, USA) was performed in each amniotic sac using a 30-gauge needle (BD PrecisionGlide Needle, Becton Dickinson, Franklin Lakes, NJ, USA). The syringe was stabilized with a mechanical holder (VisualSonics). Three of the mice injected with 100 ng/25 µl underwent dystocia, defined as disturbed progression of labor (duration of labor ≥ 6 h), and were excluded from the study group. Control wild-type dams were injected with 25 µl of PBS. The number of animals injected per group is shown in each figure legend. Following the ultrasound, mice were placed under a heat lamp until they regained full motor function, which occurred 5–10 min after heating. Two separate groups of wild-type mice were intra-amniotically injected with 100 ng of IL-1α: the first was performed to determine the dosage of IL-1α required to induce adverse perinatal outcomes (as described above), and the second was performed in parallel with Nlrp3−/− mice to elucidate the role of the NLRP3 inflammasome in IL-1α-induced preterm birth.

Video monitoring of pregnancy outcomes

Immediately after intra-amniotic injection of IL-1α or PBS, dams were monitored until delivery using a video camera and infrared light (Sony Corporation, Tokyo, Japan). Gestational length was defined as the time elapsed from the detection of the vaginal plug (0.5 dpc) through the delivery of the first pup. Preterm birth was defined as delivery occurring before 18.5 dpc. The rate of preterm birth was represented as the percentage of females delivering preterm among the total number of mice. The rate of neonatal mortality was determined for each litter (pups delivered from the same dam) and defined as the proportion of delivered pups found dead among the total litter size. The average of the rates of neonatal mortality per litter was then calculated and plotted for each experimental group. Representative photographs of fetuses (17.5 dpc) from dams injected with IL-1α or PBS were also taken.

Tissue sampling from dams intra-amniotically injected with IL-1α

Pregnant wild-type mice were intra-amniotically injected with 100 ng of IL-1α (n = 8) or PBS (control: n = 8), as described above. Mice were euthanized on 17.5 dpc (16 h post-injection), and animal dissection was performed to obtain the decidua (decidua basalis) and fetal membranes, as previously described (Arenas-Hernandez et al., 2015; Faro et al., 2019; Gomez-Lopez et al., 2019c). Tissues were snap-frozen in liquid nitrogen and stored at −80°C until analysis.

Immunoblotting for inflammasome-related proteins

Tissue lysates of the fetal membranes and decidua (n = 6, each) were prepared by mechanically homogenizing the snap-frozen fetal membranes and decidua in 1× PBS (Thermo Fisher Scientific, Inc., Rockford, IL, USA) containing a complete protease inhibitor cocktail (Roche Applied Sciences, Mannheim, Germany). Lysates were centrifuged at 15 700×g for 5 min at 4°C and the supernatants were stored at −80°C until use. Lysates and culture supernatants of murine bone marrow-derived macrophages that were stimulated in vitro to induce inflammasome activation were utilized as positive controls for the expression of NLRP3, pro-caspase-1, caspase-1 p20, and caspase-1 p35, as previously described (Faro et al., 2019; Gomez-Lopez et al., 2019c). Prior to immunoblotting, total protein concentration was determined using the Pierce BCA Protein Assay Kit (Cat#23225; Pierce Biotechnology, Thermo Fisher Scientific). Fetal membranes and decidual tissue lysates (50 μg per well), cell lysates (10 μg per well), and concentrated cell supernatants (20 μl) were subjected to electrophoresis in 4–12% sodium dodecyl sulphate-polyacrylamide gels (Cat#NP0336BOX, Invitrogen, Thermo Fisher Scientific). All samples and positive controls were run in the same gels. Separated proteins were then transferred onto nitrocellulose membranes (Cat#1620145, Bio-Rad, Hercules, CA, USA). Next, the nitrocellulose membranes were submerged in blocking solution (StartingBlock T20 Blocking Buffer, Thermo Fisher Scientific) for 30 min at room temperature and then probed overnight at 4°C with the following mouse antibodies: mouse anti-NLRP3 (Cat#AG-20B-0014-C100, 1 μg/ml, Adipogen Life Sciences, San Diego, CA, USA) and rat anti-caspase-1 (Cat#14-9832-82, 5 μg/ml, Invitrogen). Finally, the nitrocellulose membranes were stripped with Restore PLUS Western Blot Stripping Buffer (Pierce Biotechnology, Thermo Fisher Scientific) for 15 min, washed with 1× PBS, blocked, and re-probed for 1 h at room temperature with a mouse anti-β-actin monoclonal antibody (Cat#A5441, Sigma-Aldrich). Chemiluminescent signals were detected with the ChemiGlow West Substrate Kit (ProteinSimple, San Jose, CA, USA) and images were acquired using the ChemiDoc Imaging System (Bio-Rad). Quantification was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA). Briefly, each individual protein band on the blot image was automatically quantified by the software. The target protein expression in each individual sample was then normalized using the internal control, β-actin, in the same sample.

The positive controls contain high amounts of pro-caspase-1, caspase-1 p35 and caspase-1 p20; thus, the total protein amounts loaded onto the gels of the positive controls were reduced compared to those of samples. Consequently, a longer exposure time was required to detect the target protein bands in the positive controls. Therefore, although the samples and positive controls were run in the same immunoblot, the images are shown separately in Fig. 4.

Figure 4.

Figure 4.

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Intra-amniotic injection of IL-1α increases the protein quantities of active caspase-1 in the murine fetal membranes prior to preterm birth. Pregnant C57BL/6 dams were intra-amniotically injected with interleukin (IL)-1α (100 ng/25 µl) or saline (1× PBS) in each amniotic sac under ultrasound guidance on 16.5 days post coitum (dpc). The fetal membranes and decidua were collected 16 h after intra-amniotic injection. Immunoblotting of pro-caspase 1, caspase-1 p20 and caspase-1 p35 in the fetal membranes (A–D) and decidua (E–H) prior to IL-1α-induced preterm birth (n = 6 dams each). Supernatants and lysates from mouse bone marrow derived-macrophages (BMDM) stimulated with lipopolysaccharide and nigericin were used as positive controls in (A) and (E). Although the positive controls and samples were run in the same immunoblot, the images are shown separately, as the exposure times of the samples and the positive controls were different (see the Methods section). Data are shown as box and whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum and maximum ranges. The P-values were determined by a Mann–Whitney U-test. Whole immunoblot images are shown in Supplementary Figs S1, S2, S5 and S6.

ELISA determination of IL-1β concentrations in the fetal membranes and decidua

Tissue lysates of the fetal membranes and decidua (n = 8, each) were prepared by mechanical homogenization of the snap-frozen samples of these tissues in Cell Lysis Buffer 2 (Cat#895347; R&D Systems). Lysates were centrifuged at 15 700×g for 5 min at 4°C and the supernatants were stored at −80°C until use. Prior to the ELISA, total protein concentration was determined using the Pierce BCA Protein Assay Kit. Concentrations of IL-1β in the tissue lysates were determined using a sensitive and specific ELISA assay kit (Cat#MLB00C; R&D Systems). This ELISA kit was validated in our laboratory prior to the execution of this study. The concentrations of IL-1β were obtained by interpolation from the standard curve. The sensitivity of the assay was 2.31 pg/ml. The inter- and intra-assay coefficients of variation were less than 10%. The concentrations of IL-1β were normalized with the total protein amounts of the tissue lysate.

Statistical analysis

Data analysis was performed using GraphPad Prism version 8.0.1 for Windows (GraphPad Software, San Diego, CA, USA). Differences in the rate of preterm birth between groups were analyzed using a Fisher’s exact test. Kaplan–Meier survival curves were used to plot and compare the gestational length data. A log-rank test was used to evaluate differences between the survival curves. For the neonatal mortality among wild-type dams intra-amniotically injected with IL-1α (100 ng/25 µl or 200–500 ng/25 µl) or PBS, the statistical significance of group comparisons was assessed using a Kruskal–Wallis test, followed by a Dunn’s test to determine differences between PBS- and IL-1α-injected groups. For protein expression and protein concentration, the statistical significance of between-group comparisons was assessed using a Mann–Whitney U-test. For the statistical evaluation of the neonatal mortality among Nlrp3−/− and Nlrp3+/+ dams injected with IL-1α or PBS, two-way ANOVA tests, followed by a Tukey’s test to determine differences between each groups, were used. A P-value <0.05 was regarded as statistically significant for all tests.

Results

Intra-amniotic administration of IL-1α induces preterm birth

Pathophysiological concentrations of IL-1α in amniotic fluid of women with intra-amniotic inflammation/infection range from 157 pg/ml to 10 ng/ml (Romero et al., 1992). Accordingly, we intra-amniotically injected concentrations of IL-1α ranging from 0.15 ng to 10 ng in wild-type dams (n = 8); yet, no preterm birth was observed (data not shown). However, we observed increased neonatal mortality among dams treated with 1 ng and 10 ng of IL-1α compared to controls (control: 11.3%, 1 ng IL-1α: 44.4%, 10 ng IL-1α: 33.3%). An earlier study showed that the systemic administration of 1 µg of IL-1α induced preterm birth in mice (Romero et al., 1991); therefore, we reasoned that by increasing the intra-amniotic dose of this alarmin, preterm birth could be induced. First, we chose to test whether the intra-amniotic injection of 100 ng of IL-1α (10× more than 10 ng) could induce preterm birth (Fig. 1A). At this concentration, preterm birth was induced in 60% (3/5) of the wild-type dams (Fig. 1B). To determine the dose of IL-1α that would induce preterm birth in all dams, we next injected 500 ng of IL-1α and observed a 100% rate of preterm birth. The intra-amniotic injection of lower IL-1α concentrations (250 ng and 200 ng) resulted in the same outcome. Accordingly, we combined dams injected within this range (200–500 ng of IL-1α) into the same experimental group, which had a 100% (5/5) rate of preterm birth (Fig. 1B). Gestational lengths of these dams are also shown in Fig. 1C. These data indicate that increased concentrations of the alarmin IL-1α in the amniotic cavity can induce preterm birth.

Figure 1.

Figure 1.

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Intra-amniotic injection of IL-1α induces preterm birth in mice. (A) Experimental design for the intra-amniotic administration of the alarmin interleukin (IL)-1α. Pregnant C57BL/6 dams were intra-amniotically injected with IL-1α (100 ng/25 µl (n = 5) or 200–500 ng/25 µl (n = 5 total)) or saline (1× PBS) (n = 4) in each amniotic sac under ultrasound guidance on 16.5 days post coitum (dpc) and monitored until delivery. (B) Preterm birth rates of dams intra-amniotically injected with IL-1α (100 ng/25 µl or 200–500 ng/25 µl) or PBS. (C) Kaplan–Meier survival curves showing the gestational lengths of dams intra-amniotically injected with IL-1α (100 ng/25 µl or 200–500 ng/25 µl) or PBS.

Intra-amniotic administration of IL-1α induces neonatal mortality

A large proportion (79.5%) of the neonates born to wild-type dams injected with 100 ng of IL-1α, as well as all of those (100%) born to wild-type dams injected with 200–500 ng, failed to thrive (Fig. 2A). The neonatal mortality in dams injected with 200–500 ng was significantly higher than in the control group (Fig. 2A). In addition to evaluating neonatal mortality, we assessed whether the intra-amniotic injection of IL-1α would alter fetal growth parameters. Fetuses from the control group (injected with PBS) appeared well developed and had no visible abnormalities at 17.5 dpc (Fig. 2B). In contrast, most of the fetuses born to dams intra-amniotically injected with 100 ng of IL-1α appeared smaller than those from controls at 17.5 dpc, and some also appeared to be abnormally red or inflamed, implying the existence of systemic fetal inflammation (Fig. 2C). Lastly, all of the fetuses born to dams injected with 250 ng of IL-1α were visibly smaller and extremely red at delivery (17.5 dpc), suggesting severe inflammation (Fig. 2D). These results show that increased concentrations of the alarmin IL-1α in the amniotic cavity can induce adverse neonatal outcomes.

Figure 2.

Figure 2.

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Intra-amniotic injection of IL-1α induces neonatal mortality in mice. Pregnant C57BL/6 dams were intra-amniotically injected with interleukin (IL)-1α (100 ng/25 µl (n = 5) or 200–500 ng/25 µl (n = 5 total)) or saline (1× PBS) (n = 4) in each amniotic sac under ultrasound guidance on 16.5 days post coitum (dpc). Neonatal mortality and fetal morphology were recorded. (A) Mortality rates of neonates from dams intra-amniotically injected with IL-1α (100 ng/25 µl or 200–500 ng/25 µl) or PBS. The bar graph shows the average rate of neonatal mortality per litter. The P-values were determined by a Kruskal–Wallis test, followed by a Dunn’s test to determine differences between PBS control and IL-1α-administered groups. (B–D) The size of fetuses from dams intra-amniotically injected with IL-1α (100 ng/25 µl or 250 ng/25 µl) or PBS on 17.5 dpc. Ruler numbers indicate centimeters.

Intra-amniotic administration of IL-1α increases the concentration of NLRP3 in the fetal membranes and decidua prior to preterm birth

Our previous studies indicated that intra-amniotic inflammation induced by alarmins is mediated through the NLRP3 inflammasome (Gomez-Lopez et al., 2016, 2017, 2018b, 2019c; Plazyo et al., 2016). Specifically, we found that alarmins such as HMGB1 and S100B induce the activation of the NLRP3 inflammasome in the fetal membranes, leading to the cleavage of caspase-1 and the subsequent release of the mature form of IL-1β (Plazyo et al., 2016; Gomez-Lopez et al., 2019c). Therefore, we next investigated whether IL-1α activates this inflammasome prior to preterm birth. Consistent with other alarmins (Plazyo et al., 2016; Gomez-Lopez et al., 2019c), the protein expression of NLRP3, the sensor molecule of the NLRP3 inflammasome, was greater in the fetal membranes (Fig. 3A and B) and decidua (Fig. 3C and D) of dams injected with IL-1α than of those injected with PBS. This finding confirms that IL-1α induces priming of the NLRP3 inflammasome in both the fetal membranes and decidua prior to preterm birth.

Figure 3.

Figure 3.

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Intra-amniotic injection of IL-1α increases the concentration of NLRP3 in the murine fetal membranes and decidua prior to preterm birth. Pregnant C57BL/6 dams were intra-amniotically injected with interleukin (IL)-1α (100 ng/25 µl) or saline (1× PBS) in each amniotic sac under ultrasound guidance on 16.5 days post coitum (dpc). The fetal membranes and decidua were collected 16 h after intra-amniotic injection. Immunoblotting of NOD-like receptor family, pyrin domain containing 3 (NLRP3) in the fetal membranes (A, B) and decidua (C, D) prior to IL-1α-induced preterm birth (n = 6 dams each). Supernatants and lysates from mouse bone marrow derived-macrophages (BMDM) stimulated with lipopolysaccharide and nigericin were used as positive controls in (A) and (C). Data are shown as box and whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum and maximum ranges. The P-values were determined by a Mann–Whitney U-test. ACTB, β-actin; L, BMDM lysate; S, BMDM supernatant. Whole immunoblot images are shown in Supplementary Figs S1–S4.

Intra-amniotic administration of IL-1α increases active capase-1 in the fetal membranes prior to preterm birth

Following priming, the NLRP3 inflammasome induces the cleavage of pro-caspase-1, which produces the active forms of caspase-1 (p10 and p20) (Martinon et al., 2002; Franchi et al., 2009; Latz et al., 2013; Sutterwala et al., 2014; Guo et al., 2015). Therefore, we next investigated whether IL-1α induced caspase-1 cleavage in the fetal membranes and decidua prior to preterm birth. Pro-caspase-1 was significantly increased in the fetal membranes (Fig. 4A and B) from dams intra-amniotically injected with IL-1α compared to those from dams injected with PBS. Moreover, caspase-1 p20 was significantly increased in the fetal membranes of dams intra-amniotically injected with IL-1α compared to those injected with PBS (Fig. 4A and C). Caspase-1 p35 tended to be greater in the fetal membranes of dams intra-amniotically injected with IL-1α compared to those injected with PBS; yet this did not reach statistical significance (Fig. 4A and D). In the decidua, pro-caspase-1 was significantly increased in dams intra-amniotically injected with IL-1α compared to those from dams injected with PBS (Fig. 4E and F), while caspase-1 p20 was unaffected (Fig. 4E and G). Yet, caspase-1 p35 was significantly elevated in the decidua of dams intra-amniotically injected with IL-1α (Fig. 4E and H). These results indicate that, in the fetal membranes, IL-1α activates the NLRP3 inflammasome leading to the cleavage of caspase-1 into its active form.

Intra-amniotic administration of IL-1α increases the concentrations of IL-1β in the fetal membranes and decidua prior to preterm birth

Following the NLRP3 inflammasome activation, active caspase-1 drives the release of mature IL-1β into the extracellular space (Black et al., 1989; Cerretti et al., 1992; Thornberry et al., 1992; Dinarello, 1998; Sansonetti et al., 2000). Thus, we next investigated whether IL-1α causes upregulation of IL-1β in the fetal membranes and decidua prior to preterm birth. Consistent with the upregulation of NLRP3 and caspase-1 activation, the concentration of IL-1β was significantly increased in the fetal membranes from dams intra-amniotically injected with IL-1α compared to those from dams injected with PBS (Fig. 5A). The concentrations of IL-1β were also significantly increased in the decidua from dams intra-amniotically injected with IL-1α compared to those from dams injected with PBS (Fig. 5B). These data support our hypothesis that IL-1α causes NLRP3 inflammasome activation, which leads to the release of IL-1β by the fetal membranes.

Figure 5.

Figure 5.

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Intra-amniotic injection of IL-1α causes increased concentrations of IL-1β in the murine fetal membranes and decidua prior to preterm birth. Pregnant C57BL/6 dams were intra-amniotically injected with interleukin (IL)-1α (100 ng/25 µl) or saline (1× PBS) in each amniotic sac under ultrasound guidance on 16.5 days post coitum (dpc). The fetal membranes and decidua were collected 16 h after intra-amniotic injection. Concentrations of IL-1β in the lysates of the fetal membranes (A) and decidua (B) prior to IL-1α-induced preterm birth were determined by ELISA. The IL-1β concentrations were normalized with the total protein amounts of the tissue lysates (n = 8 dams each). Data are shown as box and whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum and maximum ranges. The P-values were determined by a Mann–Whitney U-test.

Intra-amniotic administration of IL-1α induces preterm birth predominately through the NLRP3 inflammasome

Based on the aforementioned experiments, we have shown that IL-1α induces NLRP3 inflammasome activation in the fetal membranes, leading to the processing of IL-1β. Thus, to demonstrate the causal relationship between the NLRP3 inflammasome and alarmin-induced preterm birth, we determined whether mice deficient for Nlrp3 showed greater resistance to IL-1α-induced adverse pregnancy and neonatal outcomes. The rate of preterm birth in Nlrp3−/− dams intra-amniotically injected with IL-1α was significantly lower (3/13, 23.1%) than that in Nlrp3+/+ dams (6/8, 75.0%) (Fig. 6A). Similarly, the gestational lengths of Nlrp3−/− dams intra-amniotically injected with IL-1α were significantly longer than those of Nlrp3+/+ dams (Fig. 6B). Moreover, neonatal mortality was significantly lower in the Nlrp3−/− dams intra-amniotically injected with IL-1α (25.8%) compared to that of Nlrp3+/+ dams (78.1%) (Fig. 6C). Nlrp3+/+ and Nlrp3−/− dams intra-amniotically injected with PBS delivered at term and had low rates of neonatal mortality (Fig. 6A–C). These results demonstrate that the intra-amniotic administration of IL-1α induces preterm birth and neonatal mortality predominately through the NLRP3 inflammasome.

Figure 6.

Figure 6.

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IL-1α-induced preterm birth and neonatal mortality are reduced in Nlrp3−/− mice. Pregnant Nlrp3−/− and Nlrp3+/+ dams were intra-amniotically injected with interleukin (IL)-1α (100 ng/25 µl) (Nlrp3−/− dams, n = 13; Nlrp3+/+ dams, n = 8) or saline (1× PBS) (Nlrp3−/− dams, n = 5; Nlrp3+/+ dams, n = 4) in each amniotic sac under ultrasound guidance on 16.5 days post coitum (dpc) and monitored until delivery. (A) Preterm birth rates of Nlrp3−/− and Nlrp3+/+ dams injected with IL-1α or PBS. The P-values were determined by a Fisher’s exact test. (B) Kaplan–Meier survival curves showing the gestational lengths of Nlrp3−/− and Nlrp3+/+ dams intra-amniotically injected with IL-1α (100 ng/25 µl) or PBS. The P-values were determined by a log-lank test (a, P = 0.03; b, P = 0.01). (C) Mortality rates of neonates from Nlrp3−/− and Nlrp3+/+ dams intra-amniotically injected with IL-1α or PBS. The bar graph shows the average rate of neonatal mortality per litter. The P-values were determined by two-way ANOVA, followed by a Tukey’s test to determine differences between each group.

Discussion

In the current study, we report that the ultrasound-guided intra-amniotic administration of IL-1α induced high rates of preterm birth and neonatal mortality. The intra-amniotic injection of IL-1α led to increased concentrations of NLRP3 and IL-1β in the fetal membranes and decidua prior to preterm birth. In addition, the intra-amniotic injection of IL-1α resulted in augmented concentrations of active caspase-1 in the fetal membranes, but not in the decidua, indicating activation of the NLRP3 inflammasome in the amniotic cavity. Lastly, Nlrp3 deficiency mitigated IL-1α-induced adverse pregnancy and neonatal outcomes.

IL-1α is regarded as one of the most important cytokines in the initiation of sterile inflammation (Rock et al., 2010; Di Paolo and Shayakhmetov, 2016). Interestingly, during apoptosis, any intracellular IL-1α is sequestered and consumed by scavenging macrophages (Cohen et al., 2010; Berda-Haddad et al., 2011; Di Paolo and Shayakhmetov, 2016), whereas in the context of tissue damage or necrosis, IL-1α is released into the microenvironment (Cohen et al., 2010, 2015). Extracellular IL-1α recruits innate immune cells such as macrophages (Cohen et al., 2010) and neutrophils (Eigenbrod et al., 2008; Cohen et al., 2010), which may then release their own inflammatory mediators, resulting in an inflammatory feedback loop (Di Paolo and Shayakhmetov, 2016; Voronov et al., 2018). Indeed, high levels of IL-1α are associated with various autoimmune and inflammatory conditions, such as rheumatoid arthritis (Chu et al., 1992; Alam et al., 2017), psoriasis (Dinarello et al., 2012; Bou-Dargham et al., 2017), and reperfusion injuries (Dinarello et al., 2012), all of which are characterized by a sterile inflammatory response. In the context of pregnancy, IL-1α was the first alarmin to be implicated in the mechanisms leading to preterm and term parturition (Romero et al., 1989a, 1991, 1992; Romero and Tartakovsky, 1992), followed by demonstrations that other alarmins, such as HMGB1 (Gomez-Lopez et al., 2016) and S100B (Gomez-Lopez et al., 2019c), can cause preterm birth and adverse neonatal outcomes. Herein, we provided a causal link between increased intra-amniotic concentrations of IL-1α and adverse perinatal outcomes. This finding further supports our principal hypothesis that alarmins can induce sterile intra-amniotic inflammation leading to preterm labor and birth.

The concentration of IL-1α required to induce significant rates of preterm birth in mice is higher than the pathophysiological concentration observed in the amniotic fluid of women with intra-amniotic inflammation (Romero et al., 1992). This phenomenon was also observed with the intra-amniotic injection of the alarmin S100B (Gomez-Lopez et al., 2019c) and can be explained by several factors that include the inability of a single alarmin to recapitulate the interconnected alarmin network observed in a clinical setting (Romero et al., 2015a) and the fundamental mouse-human difference (Nemzek et al., 2008).

Herein, we showed that the intra-amniotic injection of IL-1α induced a significant rate of neonatal mortality. Moreover, neonates born to IL-1α-injected dams exhibited a red and inflamed appearance, suggesting that these offspring underwent fetal inflammatory response syndrome (Gomez et al., 1998; Romero et al., 1998; Gotsch et al., 2007). This fetal inflammatory process is characterized by elevated concentrations of pro-inflammatory mediators in the fetal/neonatal circulation (Gomez et al., 1998; Romero et al., 1998; Madsen-Bouterse et al., 2010; Chaiworapongsa et al., 2011) and leukocyte infiltration in the umbilical cord (Yoon et al., 2000; Kim et al., 2001, 2015; Pacora et al., 2002), which can lead to adverse short- and long-term neonatal outcomes (Yoon et al., 1999; Gotsch et al., 2007; Musilova et al., 2018; Tang et al., 2019a). Such a fetal inflammatory response has been well described in the context of microbial-associated intra-amniotic inflammation and can also occur in the context of sterile intra-amniotic inflammation (Romero et al., 2014c, 2015b). This concept is supported by in vivo studies demonstrating that intra-amniotic administration of IL-1α results in fetal injury in an ovine model (Emerson et al., 1997; Berry et al., 2011; Kallapur et al., 2011; Wolfs et al., 2011), and that the intra-amniotic administration of other alarmins in mice (e.g. HMGB1, S100B and HSP70) can induce intra-amniotic inflammation and neonatal mortality (Gomez-Lopez et al., 2016, 2019c; Schwenkel et al., 2020). Together with our current findings, these data provide a link between sterile intra-amniotic inflammation and adverse neonatal outcomes.

Previous studies have demonstrated a role for the NLRP3 inflammasome in the mechanisms leading to preterm labor and birth in the setting of intra-amniotic inflammation (Gomez-Lopez et al., 2017, 2018b, , 2019c; Faro et al., 2019). Our prior study found that women who underwent spontaneous preterm labor with sterile intra-amniotic inflammation had increased amniotic fluid concentrations of extracellular ASC (Gomez-Lopez et al., 2018b), the adaptor protein of the inflammasome that is released upon activation (Baroja-Mazo et al., 2014; Franklin et al., 2014). We subsequently found that the alarmin S100B induced preterm birth and neonatal mortality in mice by activating the NLRP3 inflammasome in the fetal membranes, and treatment with the NLRP3 inhibitor MCC950 prevented these adverse perinatal outcomes (Gomez-Lopez et al., 2019c). In addition, we reported in vitro and in vivo evidence indicating that the prototypical alarmin HMGB1 induces preterm birth by activating the NLRP3 inflammasome in the chorioamniotic membranes (Gomez-Lopez et al., 2016; Plazyo et al., 2016). These findings, in addition to the alarmin enrichment observed in the amniotic cavity of women with intra-amniotic inflammation (Romero et al., 1992, 2015a), led us to propose that the mechanisms whereby alarmins induce preterm birth involve the activation of the NLRP3 inflammasome (Gomez-Lopez et al., 2019a). In line with this hypothesis, in the current study, we found that the intra-amniotic administration of IL-1α induces the activation of the NLRP3 inflammasome in the fetal membranes, as evidenced by the concomitant increase of NLRP3, active caspase-1, and IL-1β. Moreover, a reduced rate of preterm birth and neonatal mortality in response to IL-1α injection was observed in Nlrp3−/− mice compared to Nlrp3+/+ mice. This significant mitigation of IL-1α-induced consequences in the setting of Nlrp3 deficiency further supports our understanding of NLRP3 inflammasome activation in the amniotic cavity leading to preterm parturition, and demonstrates that the mechanisms whereby IL-1α induces preterm birth are primarily mediated through the NLRP3 inflammasome.

In contrast to our findings in the fetal membranes, we show herein that the intra-amniotic injection of IL-1α induces increased protein expression of NLRP3 and IL-1β, but not the active form of caspase-1, in the decidual tissues. This is in line with previous reports showing that, in the decidua, caspase-1 is not cleaved into its active forms in response to the intra-amniotic injection of lipopolysaccharide (Faro et al., 2019). Yet, several studies have indicated that the decidua is a source of IL-1β (Romero et al., 1989d; Liang et al., 1996; Ammala et al., 1997; Ibrahim et al., 2016), suggesting that inflammasome-independent mechanisms are involved in the processing of this cytokine at the maternal-fetal interface. Examples of such mechanisms include cathepsin G (Hazuda et al., 1990), cathepsin C (Kono et al., 2012), collagenase (Hazuda et al., 1990), mast cell chymase (Mizutani et al., 1991), matrix metalloproteinases 2, 3 and 9 (Schonbeck et al., 1998), neutrophil elastase (Hazuda et al., 1990) and neutrophil proteinase 3 (Coeshott et al., 1999), among others. Thus, it is tempting to hypothesize that such inflammasome-independent mechanisms may be involved in the activation of IL-1β in the decidual tissues of mice intra-amniotically injected with IL-1α.

It is worth mentioning that a small proportion of Nlrp3−/− mice delivered preterm upon intra-amniotic injection with IL-1α. This suggests that, while IL-1α may activate the NLRP3 inflammasome, this alarmin may also trigger inflammasome-independent mechanisms leading to preterm birth. IL-1α and IL-1β were first described as human pyrogens (Dinarello et al., 1974), and although IL-1α and IL-1β have different amino acid sequences (Di Paolo and Shayakhmetov, 2016), they stimulate the same cell surface receptor IL-1 receptor (IL-1R) I (Di Paolo and Shayakhmetov, 2016), eliciting biological responses similar to multipotent pro-inflammatory cytokines (Weber et al., 2010). The binding of IL-1α to IL-1RI leads to the activation of the nuclear factor kappa B pathway, subsequently resulting in the release of additional pro-inflammatory cytokines (Weber et al., 2010). IL-1α is released into the affected environment where it stimulates the production of chemokines to initiate the infiltration of additional inflammatory cells, resulting in a self-perpetuating inflammatory loop (Di Paolo and Shayakhmetov, 2016; Voronov et al., 2018). Therefore, besides the pathway of NLRP3 inflammasome activation shown in the current study, IL-1α may directly stimulate IL-1RI, inducing multiple inflammatory processes in the amniotic cavity. In line with this concept, previous studies reported that IL-1α directly stimulates human amnion cells, thereby inducing the production of prostaglandin E2 (Romero et al., 1989b; Bry and Hallman, 1991), which has both physiological and pathological roles during pregnancy (Romero et al., 1987; Challis et al., 2002; Guo et al., 2019). Thus, in addition to the inhibition of the NLRP3 inflammasome, which has proven to be effective for preventing alarmin-induced preterm birth and adverse neonatal outcomes (Gomez-Lopez et al., 2019c), targeting of the IL-1RI signaling pathway may also represent a possible therapeutic option for treating sterile intra-amniotic inflammation. Indeed, a previous study reported that the natural IL-1R antagonist prevents endotoxin-induced preterm birth (Romero and Tartakovsky, 1992). In addition, recent studies investigated the prevention of preterm birth by modification of IL-1R signaling using a non-competitive IL-1R inhibitor in mice (Nadeau-Vallee et al., 2015, 2017). However, future studies are required to investigate the effectiveness of blocking IL-1R in the prevention of preterm birth in larger animals as well as any potential harmful side effects this treatment may have during pregnancy.

A limitation of our study is that we could not weigh every pup at the time of delivery, as they were born at different times throughout the day and night. This prevented the consistent evaluation of pup and placenta weights at the same time point (i.e. immediately after delivery), which would allow for valid comparisons between groups. Yet, we report the rates of neonatal mortality in IL-1α-injected mice, which is the most impactful quantitative measurement of the adverse outcomes resulting from the intra-amniotic administration of this alarmin.

Conclusion

The data presented herein show that the intra-amniotic administration of the alarmin IL-1α induces preterm birth and adverse neonatal outcomes in mice by initiating intra-amniotic inflammatory responses. Specifically, IL-1α activates the NLRP3 inflammasome in the fetal membranes, as evidenced by increased expression of inflammasome components and downstream mediators. Moreover, Nlrp3 deficiency mitigated IL-1α-induced preterm birth and neonatal mortality. Collectively, these results demonstrate a causal link between elevated IL-1α concentrations in the amniotic cavity and preterm birth as well as adverse neonatal outcomes, a pathological process that is mediated by the NLRP3 inflammasome. These findings also shed light on the mechanisms underlying sterile intra-amniotic inflammation, and provide further evidence that this clinical condition may potentially be treated by targeting the NLRP3 inflammasome.

Data availability statement

The data underlying this article are available in the article.

Supplementary data

Supplementary data are available at Molecular Human Reproduction online.

Authors’ roles

N.G.-L. and R.R. conceived and designed the study. K.M., V.G.-F., Y.L. and Y.X. performed experiments and analyzed data. N.G.-L., K.M., J.G., R.S. and D.L. analyzed data and drafted the manuscript. All authors approved the final version of the manuscript.

Funding

This research was supported, in part, by the Perinatology Research Branch (PRB), Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health and Human Services (NICHD/NIH/DHHS), and, in part, with federal funds from the NICHD/NIH/DHHS under Contract No.HHSN275201300006C. This research was also supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health. R.R. contributed to this work as part of his official duties as an employee of the United States government.

Conflict of interest

The authors declare no potential conflicts of interest.

Supplementary Material

gaaa054_Supplementary_Data
Click here for additional data file. (366.3KB, pdf)

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