The alarmin S100A12 causes sterile inflammation of the human chorioamniotic membranes as well as preterm birth and neonatal mortality in mice†

Abstract

Sterile inflammation is triggered by danger signals, or alarmins, released upon cellular stress or necrosis. Sterile inflammation occurring in the amniotic cavity (i.e. sterile intra-amniotic inflammation) is frequently observed in women with spontaneous preterm labor resulting in preterm birth, the leading cause of neonatal morbidity and mortality worldwide; this condition is associated with increased amniotic fluid concentrations of alarmins. However, the mechanisms whereby alarmins induce sterile intra-amniotic inflammation are still under investigation. Herein, we investigated the mechanisms whereby the alarmin S100A12 induces inflammation of the human chorioamniotic membranes in vitro and used a mouse model to establish a causal link between this alarmin and adverse perinatal outcomes. We report that S100A12 initiates sterile inflammation in the chorioamniotic membranes by upregulating the expression of inflammatory mediators such as pro-inflammatory cytokines and pattern recognition receptors. Importantly, S100A12 induced the priming and activation of inflammasomes, resulting in caspase-1 cleavage and the subsequent release of mature IL-1β by the chorioamniotic membranes. This alarmin also caused the activation of the chorioamniotic membranes by promoting MMP-2 activity and collagen degradation. Lastly, the ultrasound-guided intra-amniotic injection of S100A12 at specific concentrations observed in the majority of women with sterile intra-amniotic inflammation induced preterm birth (rates: 17% at 200 ng/sac; 25% at 300 ng/sac; 25% at 400 ng/sac) and neonatal mortality (rates: 22% at 200 ng/sac; 44% at 300 ng/sac; 31% at 400 ng/sac), thus demonstrating a causal link between this alarmin and adverse perinatal outcomes. Collectively, our findings shed light on the inflammatory responses driven by alarmins in the chorioamniotic membranes, providing insight into the immune mechanisms leading to preterm birth in women with sterile intra-amniotic inflammation.

Keywords: Amniotic cavity; NLRP3; calgranulin C; caspase-1; chorioamnionitis; decidua; fetal membranes; funisitis; inflammasome; interleukin-1β; matrix metalloproteinase-2; pregnancy.

Figure 1

Incubation with S100A12 upregulates the expression of inflammatory and pattern recognition receptor genes in the human chorioamniotic membranes. (A) Experimental design. The chorioamniotic membranes from women who delivered at term without labor were incubated with S100A12 (black dots) or PBS (control, white dots) for 24 h (n = 5–8 per group). Messenger RNA abundance of NFKB1 (B), TNF (C), IL6 (D), IL1A (E), IL1B (F), IL18 (G), RAGE (H), TLR2 (I), and TLR4 (J). Relative gene expression is presented as –ΔCT values. The p-values were determined by either paired student’s t-tests (NFKB1, IL6, RAGE, and TLR2) or Wilcoxon signed-rank tests (TNF), based on their normality of distribution.

Figure 2

Incubation with S100A12 increases the concentrations of inflammatory cytokines released by the human chorioamniotic membranes. The chorioamniotic membranes from women who delivered at term without labor were incubated with S100A12 (black dots) or PBS (control, white dots) for 24 h (n = 12 per group). The concentrations of IL-6 (A) and IL-8 (B) in culture supernatants were evaluated by specific ELISA kits. The P-values were determined by paired student’s t-tests.

Figure 3

Incubation with S100A12 induces the transcriptional priming of inflammasomes in the human chorioamniotic membranes. The chorioamniotic membranes from women who delivered at term without labor were incubated with S100A12 (black dots) or PBS (control, white dots) for 24 h (n = 5–8 per group). Messenger RNA abundance of NLRP1 (A), NLRP3 (B), NLRC4 (C), AIM2 (D), NOD1 (E), and NOD2 (F). Relative gene expression is presented as –ΔCT values. The P-values were determined by paired student’s t-tests.

Figure 4

Incubation with S100A12 increases the protein expression of inflammasome sensor molecules in the human chorioamniotic membranes. The chorioamniotic membranes from women who delivered at term without labor were incubated with S100A12 (black dots) or PBS (control, white dots) for 24 h (n = 10–14 per group). The concentrations of NLRP1 (A), NLRP3 (B), NLRC4 (C), AIM2 (D), NOD1 (C), and NOD2 (F) in the tissue lysates of human chorioamniotic membrane samples were evaluated by specific ELISA kits. The P-values were determined by Wilcoxon signed-rank tests.

Figure 5

Incubation with S100A12 can activate caspase-1 and promote the maturation of IL-1β in the human chorioamniotic membranes. The chorioamniotic membranes from women who delivered at term without labor were incubated with S100A12 (black dots) or PBS (control, white dots) for 24 h (n = 6–15 per group). (A) The mRNA expression of CASP1. Relative gene expression is presented as a –ΔCT value. The P-value was determined by paired student’s t-test. (B) The concentrations of caspase-1 in the tissue lysates of human chorioamniotic membrane samples were evaluated by a specific ELISA kit. The P-values were determined by Wilcoxon signed-rank tests. (C and D) Immunoblotting of pro-caspase-1, caspase-1 p20, caspase-1 p10, and GAPDH in the human chorioamniotic membranes. The P-values were determined by Wilcoxon signed-rank tests. (E) The concentrations of mature IL-1β in culture supernatants as evaluated by a specific ELISA kit. The P-values were determined by Wilcoxon signed-rank tests.

Figure 6

Incubation with S100A12 can trigger the activation of human chorioamniotic membranes. The chorioamniotic membranes from women who delivered at term without labor were incubated with S100A12 (black dots) or PBS (control, white dots) for 24 h (n = 6 per group). Zymography showing the activation of (A) MMP-2 and (B) MMP-9 in human chorioamniotic membranes. (C) Trichrome staining of human chorioamniotic membranes incubated with S100A12 or PBS (control). Magnification = ×20, scale bar = 100 μm. The P-value was determined by a Wilcoxon signed-rank test.

Figure 7

Intra-amniotic injection of S100A12 induces preterm birth and adverse neonatal outcomes in mice. (A) Amniotic fluid concentrations of S100A12 from women with preterm labor without intra-amniotic inflammation (blue dots) and preterm labor with sterile intra-amniotic inflammation (red dots). Lines indicate the median of each group. The median and maximum values of S100A12 concentrations in women with preterm labor and sterile intra-amniotic inflammation are shown. The P-value was determined by a Mann–Whitney U-test. (B) Experimental design for the intra-amniotic administration of S100A12. Pregnant C57BL/6 mice were intra-amniotically injected with S100A12 (100 ng/25 μL, n = 5; 200 ng/25 μL, n = 6; 300 ng/25 μL, n = 4; 400 ng/25 μL, n = 8; 850 ng/25 μL, n = 9) or PBS (control, n = 6) in each amniotic sac under ultrasound guidance on 16.5 days post coitum (dpc). Pregnancy and neonatal outcomes were monitored until delivery. (C) Gestational length and rate of preterm birth of dams intra-amniotically injected with S100A12 or PBS. (D) Mortality of neonates born to dams intra-amniotically injected with S100A12 or PBS. The P-value was determined by a Mann–Whitney U-test.