Inhibition of the NLRP3 inflammasome can prevent sterile intra-amniotic inflammation, preterm labor/birth, and adverse neonatal outcomes†

Abstract

Sterile intra-amniotic inflammation is commonly observed in patients with spontaneous preterm labor, a syndrome that commonly precedes preterm birth, the leading cause of perinatal morbidity and mortality worldwide. However, the mechanisms leading to sterile intra-amniotic inflammation are poorly understood and no treatment exists for this clinical condition. Herein, we investigated whether the alarmin S100B could induce sterile intra-amniotic inflammation by activating the NLRP3 inflammasome, and whether the inhibition of this pathway could prevent preterm labor/birth and adverse neonatal outcomes. We found that the ultrasound-guided intra-amniotic administration of S100B induced a 50% rate of preterm labor/birth and a high rate of neonatal mortality (59.7%) without altering the fetal and placental weights. Using a multiplex cytokine array and immunoblotting, we reported that S100B caused a proinflammatory response in the amniotic cavity and induced the activation of the NLRP3 inflammasome in the fetal membranes, indicated by the upregulation of the NLRP3 protein and increased release of active caspase-1 and mature IL-1β. Inhibition of the NLRP3 inflammasome via the specific inhibitor MCC950 prevented preterm labor/birth by 35.7% and reduced neonatal mortality by 26.7%. Yet, inhibition of the NLRP3 inflammasome at term did not drastically obstruct the physiological process of parturition. In conclusion, the data presented herein indicate that the alarmin S100B can induce sterile intra-amniotic inflammation, preterm labor/birth, and adverse neonatal outcomes by activating the NLRP3 inflammasome, which can be prevented by inhibiting such a pathway. These findings provide evidence that sterile intra-amniotic inflammation could be treated by targeting the NLRP3 inflammasome.

Keywords: S100B; acute chorioamnionitis; alarmins; amniotic fluid; caspase-1; cytokines; damage-associated molecular patterns; danger signals; funisitis; inhibitor; interleukin-1β; mice.

Figure 1.

Intra-amniotic administration of the alarmin S100B. (A) Pregnant C57BL/6 dams were intra-amniotically injected on 16.5 days post coitum (dpc) with 60 ng/25 μL of S100B or saline (1X phosphate-buffered saline; PBS). (B) Intra-amniotic injections were performed using a high-frequency ultrasound system. After injection, mice were monitored by camera until delivery and the (C) rate of preterm labor/birth, (D) gestational length, and (E) rate of neonatal mortality were recorded. Black lines represent medians. n = 8–10 dams per group.

Figure 2.

Fetal and placental growth parameters after intra-amniotic administration of the alarmin S100B. (A) Sizes of fetuses and placentas from dams intra-amniotically injected with S100B. Ruler lines indicate centimeters. The (B) placental and (C) fetal weights were recorded. Gray lines represent means ± standard deviation. n = 4–16 litters per group.

Figure 3.

Amniotic fluid cytokines in dams intra-amniotically injected with the alarmin S100B. Amniotic fluid concentrations of IL-1β, IFN-γ, IL-12p70, IL-6, IL-10, IL-2, IL-4, and IL-5 are shown. Lines represent medians. n = 6–10 dams per group.

Figure 4.

Protein quantities of the NLRP3 inflammasome components in the fetal membranes from dams intra-amniotically injected with the alarmin S100B. Images showing immunoblotting of (A) NLRP3 and mature IL-1β or (B) pro-caspase-1, caspase-1 p35, and caspase-1 p20 in fetal membranes lysates. Bone marrow-derived macrophages served as positive controls for NLRP3, pro-caspase-1, and caspase-1 p35, and culture supernatants from the same cells were positive controls for active IL-1β and caspase-1 p20. Lines represent medians. n = 6 dams per group.

Figure 5.

Inhibition of the NLRP3 inflammasome via MCC950 in dams intra-amniotically injected with S100B. (A) Pregnant C57BL/6 dams were intraperitoneally injected on 16.5 days post coitum(dpc) with 50 mg/kg of the NLRP3 inhibitor MCC950 followed by intra-amniotic administration of S100B (60 ng/25 μL) or saline (1X phosphate-buffered saline; PBS). After injection, mice were monitored by camera until delivery and the (B) rate of preterm labor/birth, (C) gestational length, and (D) rate of neonatal mortality were recorded. *P < 0.004. n = 6–16 dams per group.

Figure 6.

Administration of MCC950 at term gestation. (A) Pregnant C57BL/6 dams were intraperitoneally injected from 18.5 days post coitum (dpc) until delivery with 50 mg/kg of the NLRP3 inhibitor MCC950. After injection, mice were monitored by camera until delivery and the (B) gestational length was recorded. n = 10 dams per group.

Figure 7.

Conceptual framework. The alarmin S100B can initiate an intra-amniotic inflammatory response characterized by the activation of the NLRP3 inflammasome (complex including the NLRP3 sensor molecule, the ASC adaptor protein, and pro-caspase-1). This pathway leads to the processing and release of caspase-1 and the subsequent maturation of IL-1β which, in turn, can lead to preterm labor and birth. Inhibiting the NLRP3 inflammasome (e.g. MCC950) represents a novel strategy to prevent sterile intra-amniotic inflammation, preterm labor and birth, and adverse neonatal outcomes.