The Placental NLRP3 Inflammasome and Its Downstream Targets, Caspase-1 and Interleukin-6, Are Increased in Human Fetal Growth Restriction: Implications for Aberrant Inflammation-Induced Trophoblast Dysfunction

Abstract

Fetal growth restriction (FGR) is commonly associated with placental insufficiency and inflammation. Nonetheless, the role played by inflammasomes in the pathogenesis of FGR is poorly understood. We hypothesised that placental inflammasomes are differentially expressed and contribute to the aberrant trophoblast function. Inflammasome gene expression profiles were characterised by real-time PCR on human placental tissues collected from third trimester FGR and gestation-matched control pregnancies (n = 25/group). The functional significance of a candidate inflammasome was then investigated using lipopolysaccharide (LPS)-induced models of inflammation in human trophoblast organoids, BeWo cells in vitro, and a murine model of FGR in vivo. Placental mRNA expression of NLRP3, caspases 1, 3, and 8, and interleukin 6 increased (>2-fold), while that of the anti-inflammatory cytokine, IL-10, decreased (<2-fold) in FGR compared with control pregnancies. LPS treatment increased NLRP3 and caspase-1 expression (>2-fold) in trophoblast organoids and BeWo cell cultures in vitro, and in the spongiotrophoblast and labyrinth in the murine model of FGR. However, the LPS-induced rise in NLRP3 was attenuated by its siRNA-induced down-regulation in BeWo cell cultures, which correlated with reduced activity of the apoptotic markers, caspase-3 and 8, compared to the control siRNA-treated cells. Our findings support the role of the NLRP3 inflammasome in the inflammation-induced aberrant trophoblast function, which may contribute to FGR.

Keywords: NLRP3; apoptosis; caspase-1; cytokines; fetal growth restriction; inflammasomes; placental function.

Figure 1

Real-time PCR validation of DAMPs: independent validation of DAMPs-involved mRNA gene expression relative to 18S rRNA. Statistical significance was determined using a Mann–Whitney U test and indicated by *** (p < 0.001).

Figure 2

Real-time PCR validation of pro- and anti-inflammatory cytokines: independent validation of mRNA expression of the pro- and anti-inflammatory cytokines relative to 18S rRNA. Statistical significance was determined using a Mann–Whitney U test and indicated by *** (p < 0.001).

Figure 3

NLRP3 mRNA expression in pre-term and term groups. Data from the pre-term group are shown in blue; from the term group in red. A Mann–Whitney U test was performed to determine statistical differences between the groups. Statistical significance is denoted by *** p < 0.001 vs. corresponding group indicated. Non-statistical significance is denoted by ns.

Figure 4

Placental NLRP3 protein expression in control and FGR pregnancies. (a) A representative immunoblot shows immunoreactive NLRP3 protein at 110 kDa in placentae obtained from FGR and gestation-matched control pregnancies (n = 5 in each group). GAPDH (37 kDa) was used as a loading control. (b). Semi-quantitation of immunoreactive NLRP3 protein normalised to GAPDH demonstrates a significant increase in NLRP3 protein in the placentae from FGR-affected pregnancies compared with gestation-matched control pregnancies. Statistical difference in placental NLRP3 protein content between FGR and control is denoted by * p < 0.05 (t-test).

Figure 5

Spatial distribution of NLRP3 in first and third trimester placental tissues. NLRP3 protein localization in first trimester placental tissues (7 weeks of gestation) shows the presence of immunoreactive NLRP3 protein in the villous cytotrophoblasts (VCT), syncytiotrophoblast (STB), and in some stromal cells (STR). Image represents a 100× magnification. Representative images of term control and term FGR placental tissues for NLRP3 immunostaining in STB, STR, and in endothelial cells (EC) surrounding the fetal capillaries in both control and FGR placentae. Image represents a 200× magnification. Rabbit IgG was used as a negative control. Scale bar represents 100 μm.

Figure 6

Expression of Ki67 and NLRP3 in human trophoblast organoids obtained from first trimester pregnancies (6–12 weeks of gestation). (a) Ki67, a marker for proliferating cells in the VCT, was detected in red in both untreated (UT) and in LPS-treated trophoblast organoids. NLRP3 protein localisation in the VCT and STB is shown in red in both UT and LPS-treated trophoblast organoids. Nuclear staining was performed using DAPI. (b) shows concentrations of NLRP3 and caspase-1 in the human placental organoids cultured over 72 h following treatment with or without LPS. Protein concentration of NLRP3 and caspase-1 significantly increased in the conditioned media obtained from LPS treated placental organoids, compared with untreated organoid cultures (n = 3). Statistical difference in NLRP3 and caspase-1 protein between untreated and LPS treated organoid cultures is denoted by ** p < 0.005 (t-test).

Figure 7

Effect of LPS on BeWo cell function in vitro. Confluent cultures treated in the presence of 1 ng/mL LPS show significant increases in (a) NLRP3, (b) caspase-1, (c) IL-6, (i) IL-1β, (h) IL-18, (g) CGB, and (e) caspase-3 and (f) Caspase-8 mRNA, and a significantly decreased (d) IL-10 mRNA relative 18S rRNA (a–i). Significant differences are denoted by * p < 0.05; ** p < 0.005.

Figure 8

Effect of LPS on protein expression of NLRP3, caspase-1, pro- and anti-inflammatory cytokines, and trophoblast differentiation marker; and activity of apoptosis markers in BeWo cells. LPS treatment increases protein concentrations of (a) NLRP3, (b) caspase-1, (c) IL-6, (i) IL-1β, (h) IL-18, and (g) βhCG, and increases activity of (f) caspase-3 and (e) 8; but decreases (d) IL-10 protein expression when compared to untreated cells. Significant differences are denoted by * p < 0.05; **p < 0.005.

Figure 9

Effect of NLRP3 siRNA inactivation and LPS treatment on mRNA expression of NLRP3, caspase-1, pro- and anti-inflammatory cytokines, trophoblast differentiation, and apoptosis markers in BeWo cells. siNLRP3 treatment significantly decreases LPS-induced BeWo cell expression of (a) NLRP3, (b) caspase-1, (c) IL-6, (i) IL-1β, (h) IL-18, (g) CGB, and (e) caspase-3 and (f) Caspase-8 mRNA, but increases (d) IL-10 mRNA relative to 18S rRNA, compared to siCONT treated cells. Significant differences are denoted by * p < 0.05.

Figure 10

Effect of NLRP3 siRNA inactivation and LPS on protein expression of NLRP3, caspase-1, pro- and anti-inflammatory cytokines, trophoblast differentiation marker, and activity of apoptosis markers in BeWo cells. Protein concentrations of (a) NLRP3, (b) caspase-1, (c) IL-6, (i) IL-1β, (h) IL-18, (g) βhCG, and (e) caspase-3 and (f) caspase- 8 activities significantly decrease in siNLRP3 treated cells compared to siCONT treated cells. In contrast, (d) IL-10 protein concentrations significantly increase in siNLRP3 + LPS treated BeWo cells, compared to siCONT treated cells. Significant differences are denoted by * p < 0.05; ** p < 0.005.

Figure 11

Placental NLRP3 and caspase-1 protein in murine pregnancy in vivo. Placental NLRP3 and caspase-1 expression levels associated with inflammation are investigated using immunohistochemistry in the placentae obtained from LPS-induced inflammation in a murine model of pregnancy. As shown, NLRP3 localisation is observed in the spongiotrophoblast (Sp) and labyrinth (Lab) in the placentae from both control and LPS-treated mice. Scale bar denotes 100 μm.