Stretch Causes cffDNA and HMGB1-Mediated Inflammation and Cellular Stress in Human Fetal Membranes.

Danger-associated molecular patterns (DAMPs) are elevated within the amniotic cavity, and their increases correlate with advancing gestational age, chorioamnionitis, and labor. Although the specific triggers for their release in utero remain unclear, it is thought that they may contribute to the initiation of parturition by influencing cellular stress mechanisms that make the fetal membranes (FMs) more susceptible to rupture. DAMPs induce inflammation in many different tissue types. Indeed, they precipitate the subsequent release of several proinflammatory cytokines that are known to be key for the weakening of FMs. Previously, we have shown that in vitro stretch of human amnion epithelial cells (hAECs) induces a cellular stress response that increases high-mobility group box-1 (HMGB1) secretion. We have also shown that cell-free fetal DNA (cffDNA) induces a cytokine response in FM explants that is fetal sex-specific. Therefore, the aim of this work was to further investigate the link between stretch and the DAMPs HMGB1 and cffDNA in the FM. These data show that stretch increases the level of cffDNA released from hAECs. It also confirms the importance of the sex of the fetus by demonstrating that female cffDNA induced more cellular stress than male fetuses. Our data treating hAECs and human amnion mesenchymal cells with HMGB1 show that it has a differential effect on the ability of the cells of the amnion to upregulate the proinflammatory cytokines and propagate a proinflammatory signal through the FM that may weaken it. Finally, our data show that sulforaphane (SFN), a potent activator of Nrf2, is able to mitigate the proinflammatory effects of stretch by decreasing the levels of HMGB1 release and ROS generation after stretch and modulating the increase of key cytokines after cell stress. HMGB1 and cffDNA are two of the few DAMPs that are known to induce cytokine release and matrix metalloproteinase (MMP) activation in the FMs; thus, these data support the general thesis that they can function as potential central players in the normal mechanisms of FM weakening during the normal distension of this tissue at the end of a normal pregnancy.

Validation of a mouse 3D gastruloid-based embryotoxicity assay in reference to the ICH S5(R3) guideline chemical exposure list.

There is growing interest in establishing alternative methods in place of conventional animal tests to assess the developmental and reproductive toxicity (DART) of chemicals. Gastruloids are 3D aggregates of pluripotent stem cells that spontaneously exhibit axial elongation morphogenesis similar to gastrulation. They have been explored as in vitro embryogenesis models for developmental and toxicological studies. Here, a mouse gastruloid-based assay was validated for DART assessment in accordance with the ICH S5(R3) guideline, which provides the plasma concentration data of various reference drugs in rodents, specifically Cmax and AUC for NOAEL and LOAEL. First, adverse effect concentrations of the reference drugs and their known metabolites on gastruloid development were determined based on morphological impact, namely reduced growth or aberrant elongation. Then, the NOAEL to LOAEL concentration range obtained from the gastruloid assay was compared with that in rodents to examine similarities in sensitivity between the in vitro and in vivo assays for each chemical. For 18 out of the 24 reference drugs that have both NOAEL and LOAEL information in rodents, the sensitivity of the gastruloid assay was comparable to the in vivo assay within an 8-fold concentration margin. For 7 out of the 8 additional reference drugs that have only NOAEL or LOAEL information in rodents, the gastruloid assay was in line with the in vivo data. Altogether, these results support the effectiveness of the gastruloid assay, which may be exploited as a non-animal alternative method for DART assessment.

Fetal DNA Causes Sex-Specific Inflammation From Human Fetal Membranes.

Inflammation is central to the mechanisms of parturition, but the lack of understanding of how it is controlled in normal parturition hampers our ability to understand how it may diverge resulting in preterm birth. Cell-free fetal DNA is found in the amniotic fluid, and it is thought to be able to activate inflammation as a danger-associated molecular pattern. Although its levels increases with gestational age, its effect has not been studied on the human fetal membranes. Thus, the aim of this study was to determine if the fetal DNA can trigger inflammation in the human fetal membranes and, thus, potentially contribute to the inflammatory load. Isolated human amniotic epithelial cells and fetal membrane explants were treated apically with fetal DNA causing the translocation of NF-KB into the nucleus of cells and throughout the cells of the explant layers with time. Fetal membrane explants were treated apically with either small or larger fragments of fetal DNA. IL-6, TNFα, and GM-CSF secretion was measured by ELISA, and pro-MMP2 and pro-MMP9 activity was measured by zymography from apical and basal media. Increased apical IL-6 secretion and basal pro-MMP2 activity was seen with small fragments of fetal DNA. When the data were disaggregated based on fetal sex, males had significant increases in IL-6 secretion and basal increased activity in pro-MMP2 and 9, whereas females had significantly increased basal secretion of TNFα. This was caused by the smaller fragments of fetal DNA, whereas the larger fragments did not cause any significant increases. Male fetal DNA had significantly lower percentages of methylation than females. Thus, when the cytokine and pro-MMP activity data were correlated with methylation percentage, IL-6 secretion significantly correlated negatively, whereas GM-CSF secretion positively correlated. These data support the role of fetal DNA as an inflammatory stimulus in the FM, as measured by increased NF-κB translocation, cytokine secretion, and increased pro-MMP activity. However, the data also suggested that the responses are different from FM tissues of male and female fetuses, and both the fragment size and methylation status of the fetal DNA can influence the magnitude and type of molecule secreted.

RAGE against the Machine: Can Increasing Our Understanding of RAGE Help Us to Battle SARS-CoV-2 Infection in Pregnancy?

The receptor of advanced glycation end products (RAGE) is a receptor that is thought to be a key driver of inflammation in pregnancy, SARS-CoV-2, and also in the comorbidities that are known to aggravate these afflictions. In addition to this, vulnerable populations are particularly susceptible to the negative health outcomes when these afflictions are experienced in concert. RAGE binds a number of ligands produced by tissue damage and cellular stress, and its activation triggers the proinflammatory transcription factor Nuclear Factor Kappa B (NF-κB), with the subsequent generation of key proinflammatory cytokines. While this is important for fetal membrane weakening, RAGE is also activated at the end of pregnancy in the uterus, placenta, and cervix. The comorbidities of hypertension, cardiovascular disease, diabetes, and obesity are known to lead to poor pregnancy outcomes, and particularly in populations such as Native Hawaiians and Pacific Islanders. They have also been linked to RAGE activation when individuals are infected with SARS-CoV-2. Therefore, we propose that increasing our understanding of this receptor system will help us to understand how these various afflictions converge, how forms of RAGE could be used as a biomarker, and if its manipulation could be used to develop future therapeutic targets to help those at risk.