Placental thromboinflammation impairs embryonic survival by reducing placental thrombomodulin expression

Gestational vascular diseases, such as preeclampsia (PE), affect ∼5% to 7% of pregnancies and are a major cause of maternal, fetal, and neonatal morbidity and mortality.¹  Gestational vascular diseases are closely associated with altered activation of the hemostatic system, but the underlying mechanisms remain poorly defined.²  Maternal and embryonic factors coordinately control aspects of placental hemostasis, including platelet activation.^3-6  Placental thrombomodulin (TM) deficiency hinders normal placental development (eg, reduces trophoblast proliferation and increases trophoblast death) and results in embryonic lethality.^5,7,8  Maternal platelet deficiency partially rescues TM-null embryos, establishing a crucial role of maternal platelet activation in the TM-dependent developmental phenotype.⁹  Intriguingly, excess platelet activation, for example, resulting from procoagulant extracellular vesicles (EVs), causes trophoblast inflammasome activation and results in a PE-like phenotype in mice.¹⁰  Decreased TM expression has been shown in PE patients and is possibly caused by an angiogenic imbalance.¹¹  Accordingly, we initially speculated that loss of trophoblast TM expression unleashes platelet-dependent placental inflammasome activation, thus promoting embryonic lethality. However, our results imply a converse and unidirectional relationship of placental inflammasome activation and placental TM expression: inflammasome activation reduces placental TM expression and hence placental development and function.

Wild-type C57BL/6 mice were obtained from The Jackson Laboratory. TM-deficient and CMV^Cre mice have been published previously.^8,12  TM^hi-LoxP/LoxP mice were newly generated (supplemental Methods and supplemental Figure 7, available on the Blood Web site). Pregnant mice were injected with EVs as described earlier.¹⁰  Anakinra or soluble thrombomodulin (sTM; solulin) were injected intraperitoneally 30 minutes prior to each EV injection.^10,13  All animal experiments were conducted following standards and procedures approved by the local Animal Care and Use Committee (Landesverwaltungsamt, Halle, Germany).

Human placenta samples from PE pregnancies and from normotensive control pregnancies (supplemental Table 1) were collected at the Universitätsfrauenklinik-Erlangen (Erlangen, Germany) in accordance with the guidelines and with the approval of the local Ethics Committee after obtaining informed patient consent.¹⁰ 

EVs were prepared using previously described methods (see the supplemental Methods for details).¹⁰ 

Platelet-rich plasma (PRP) was prepared as previously described.¹⁰  Differentiated mouse trophoblast stem (mTS) cells were treated with interleukin 1β (IL-1β) or EV (final procoagulant activity, 7.5 nM thrombin equivalent) and PRP and, in some experiments, with anakinra or solulin along with EV and PRP for 24 hours in differentiation medium. Human trophoblast-like cells (JEG-3) were treated with EV (final procoagulant activity, 7.5 nM thrombin equivalent) and PRP for 24 hours.

Immunoblotting was conducted as previously described (see the supplemental Methods for details).¹⁰ 

Trophoblast proliferation was studied using a bromodeoxyuridine proliferation assay (in vitro) or Ki-67 immunohistochemistry (ex vivo).⁸ 

The data are summarized as the means ± standard error of the mean. Statistical analyses performed are delineated in the figure legends. Post hoc comparisons of analysis-of-variance (ANOVA) results were corrected with the Tukey method. The Kolmogorov-Smirnov test or D’Agostino-Pearson normality test was used to determine whether the distribution of the data were consistent with a Gaussian distribution. Prism 5 (www.graphpad.com) software was used for statistical analyses. Statistical significance was accepted at values of P < .05.

To determine whether the lethality of TM-null embryos results from excess inflammasome activation, we treated pregnant TM^+/− female mice mated with TM^+/− male mice with the IL-1R antagonist anakinra (20 mg/kg). Although this dose efficiently prevented inflammasome-induced embryonic death,¹⁰  it did not improve the survival of TM-null embryos at day 10.5 postcoitus (Figure 1A). Similarly, a higher anakinra dose (40 mg/kg) or genetically superimposed Nlrp3 deficiency did not impact the lethality of TM-null embryos (Figure 1A). Hence, the lethality of TM-deficient embryos is independent of the NLRP3 inflammasome.

We next hypothesized that trophoblast inflammasome activation and associated IL-1β release reduce trophoblast TM expression and that reduced trophoblast TM expression contributes to embryonic demise associated with placental inflammasome activation. Indeed, exposure of differentiated mTS cells to IL-1β reduced TM expression (messenger RNA and protein) and promoted TM shedding (Figure 1B-C; supplemental Figure 1). Furthermore, injection of endothelial cell–derived EVs into pregnant C57BL/6 mice at days 10.5 and 11.5 postcoitus, which causes placental inflammasome activation,¹⁰  reduced trophoblast TM expression (Figure 1D-E; supplemental Figure 2). Similarly, exposure of mTS cells or human placenta-derived JEG-3 (choriocarcinoma) cells to increasing numbers of EVs in the presence of platelets reduced TM expression in vitro (Figure 1F-G; supplemental Figure 1). Importantly, anakinra restored placental TM expression in EV-injected pregnant C57BL/6 mice, showing that inflammasome activation impairs placental TM expression (Figure 1D-E).

To scrutinize the translational relevance, we analyzed human placenta samples from PE patients and healthy controls. TM expression was reduced in trophoblast cells of PE placentae compared with control placentae whereas a reduction of TM expression was not detectable in embryonic endothelial cells (Figure 1H; supplemental Figure 2). Furthermore, TM expression correlated negatively with IL-1β expression and correlated positively with platelet counts in pregnant women without or with PE (Figure 1I-J).

Hence, although embryonic lethality of TM-deficient embryos is not a consequence of NLRP3 inflammasome activation, we speculated that the reverse is true: inflammasome activation, as observed in PE, reduces placental TM expression, which impairs placental development (eg, reduced trophoblast proliferation) and embryonic outcome.⁸  Indeed, TM expression in human PE placentae correlated positively with trophoblast proliferation (Figure 1K-L), another consequence of placental TM deficiency.⁸  In pregnant mice or in mTS cells, EVs reduced trophoblast proliferation (supplemental Figure 3), which was restored upon anakinra treatment (Figure 1M-O). Therefore, inflammasome-associated loss of TM promotes trophoblast growth arrest, reflecting the findings in TM-null placentae.⁸ 

To scrutinize the functional relevance of reduced TM expression to the placental and embryonic defects associated with placental inflammasome activation, we aimed to restore TM levels. First, we used solulin, a modified version of sTM with increased stability,¹⁴  which markedly reduced EV-induced platelet activation (supplemental Figure 4). sTM prevented EV-induced and platelet-mediated inflammasome activation, as indicated by NLRP3 expression and IL-1β cleavage in mTS cells (supplemental Figure 5). Furthermore, upon solulin treatment, placental TM expression was maintained and EV-induced fetal death, intrauterine growth restriction, placental inflammasome activation, and reduced trophoblast proliferation were prevented (Figure 2A-E; supplemental Figure 6a-c). In vitro, solulin restored cell proliferation (supplemental Figure 6d). Aspirin treatment of EV-injected pregnant mice likewise maintained placental TM expression, indicating that platelet inhibition is sufficient to sustain TM expression (Figure 2F).

To determine whether restoring TM expression in embryonic placentae is sufficient to rescue EV-induced and inflammasome-mediated embryonic demise, we genetically restored TM expression in the placenta. We generated mice with conditional (Cre-dependent) TM expression (TM^hi-LoxP/LoxP mice; supplemental Figure 7). Male TM^hi-LoxP/LoxP mice were mated with female mice homozygous for X-linked Cre recombinase (CMV^Cre mice; homozygous mice were obtained by inbreeding and genotyping the progeny). This strategy resulted in Cre-mediated, constitutive TM expression only within embryonic tissues, including trophoblast cells (supplemental Figure 7). These pregnant female mice were injected with EVs at days 10.5 and 11.5 postcoitus. Genetically maintained placental TM expression protected against EV-induced fetal death, intrauterine growth restriction, and placental inflammasome activation, and restored trophoblast proliferation (Figure 2G-L). Taken together, these findings show that restoration of placental TM levels in pregnant mice, either upon systemically inhibiting platelet activation (using solulin or aspirin) or locally within the placenta, prevents EV-induced placental inflammasome activation, placental dysfunction, and embryonic demise.

These results establish a unidirectional relation of inflammasome activation and reduced trophoblast TM expression, which contributes to the placental defects and embryonic demise observed in PE (Figure 2M). The impaired survival and growth of embryos with partially reduced TM expression in the placenta secondary to EV-induced maternal PE is in agreement with the impaired survival and development of embryos with genetically superimposed partial loss of TM function and additional maternal risk factors.¹⁵  Restoring TM levels may be a new and, as solulin cannot cross the placental barrier, safe therapy for placental defects associated with excess platelet activation and placental inflammasome activation, as observed in PE.^10,16-18  However, our data do not support the converse hypothesis that the placental defects observed in TM-null embryos are mechanistically linked to NLRP3 inflammasome activation. Whether platelet-dependent placental inflammasome activation may be a contributing factor to pregnancy complications in less severe cases of thrombophilia, such as those observed in pregnancies of homozygous factor V Leiden carriers,¹⁵  remains to be established.

The authors thank Kathrin Deneser, Julia Judin, René Rudat, and Rumiya Makarova for excellent technical support. The authors also thank Satish Ranjan for support in some animal experiments.

This work was supported by Deutsche Forschungsgemeinschaft grants KO-5736/1-1 (S.K.), 361210922/GRK2408 (B.I.), IS-67/4-3 (B.I.), and IS-67/16-1 (B.I.).

Contribution: S.K. designed, performed, and interpreted the results of the in vivo, in vitro, and ex vivo experiments and prepared the manuscript; K.K.S., P.M., F.L., D.G., A.G., R.R., and A.E. supported the mouse experiments and ex vivo analysis; H.H. and M.R. provided human placenta samples; and B.I. designed and interpreted the experimental work and prepared the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Shrey Kohli, Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, Leipzig University, Liebigstr 27A, 04103 Leipzig, Germany; e-mail: shrey.kohli@medizin.uni-leipzig.de; and Berend Isermann, Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, Leipzig University, Liebigstr 27A, 04103 Leipzig, Germany; e-mail: berend.isermann@medizin.uni-leipzig.de.