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.

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.

Tetraspanin CD82 regulates S1PR1-mediated hematopoietic stem and progenitor cell mobilization.

Hematopoietic stem and progenitor cell (HSPC) mobilization into the blood occurs under normal physiological conditions and is stimulated in the clinic to enable the isolation of HSPCs for transplantation therapies. In the present study, we identify the tetraspanin CD82 as a novel regulator of HSPC mobilization. Using a global CD82 knockout (CD82KO) mouse, we measure enhanced HSPC mobilization after granulocyte-colony stimulating factor (G-CSF) or AMD3100 treatment, which we find is promoted by increased surface expression of the sphingosine 1-phosphate receptor 1 (S1PR1) on CD82KO HSPCs. Additionally, we identify a disruption in S1PR1 internalization in CD82-deficient HSPCs, suggesting that CD82 plays a critical role in S1PR1 surface regulation. Finally, combining AMD3100 and anti-CD82 treatments, we detect enhanced mobilization of mouse HSPCs and human CD34+ cells in animal models. Together, these data provide evidence that CD82 is an important regulator of HSPC mobilization and suggests exploiting the CD82 scaffold as a therapeutic target to enhance stem cell isolation.

High-mobility group box 1 is a driver of inflammation throughout pregnancy.

A proinflammatory response driven by high-mobility group box 1 (HMGB1) is important for the success of both the early stages of pregnancy and parturition initiation. However, the tight regulation of HMGB1 within these two stages is critical, as increased HMGB1 can manifest into pregnancy-related pathologies. Although during the early stages of pregnancy HMGB1 is critical for the development and implantation of the embryo, and uterine decidualization, high levels within the uterine cavity have been linked to pregnancy failure. In addition, chronic inflammation, resultant from increased HMGB1 within the maternal circulation and gestational tissues, also increases the risk for preterm labor, preterm birth, or infant mortality. Due to the link between HMGB1 and several pregnancy pathologies, the possibility of leveraging HMGB1 as a biomarker has been assessed. However, data are limited that demonstrate how known HMGB1 inhibitors could reduce inflammation within pregnancy. Thus, further research is warranted to improve our understanding of the potential of HMGB1 as a therapeutic target to reduce inflammation within pregnancy. This review aims to describe what is understood about the role of HMGB1 that drives inflammation throughout pregnancy and highlight its potential as a biomarker and therapeutic target within this context.

Tetraspanin Scaffold Proteins Function as Key Regulators of Hematopoietic Stem Cells.

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the development, maintenance, and regeneration of all the blood forming cells in the body, and as such, are critical for a number of patient therapies. For successful HSPC transplantation, stem cells must traffic through the blood and home to the bone marrow (BM) microenvironment or "niche," which is composed of soluble factors, matrix proteins, and supportive cells. HSPC adhesion to, and signaling with, cellular and extracellular components of the niche provide instructional cues to balance stem cell self-renewal and differentiation. In this review, we will explore the regulation of these stem cell properties with a focus on the tetraspanin family of membrane proteins. Tetraspanins are molecular scaffolds that uniquely function to distribute proteins into highly organized microdomains comprising adhesion, signaling, and adaptor proteins. As such, tetraspanins contribute to many aspects of cell physiology as mediators of cell adhesion, trafficking, and signaling. We will summarize the many reports that identify tetraspanins as markers of specific HSPC populations. Moreover, we will discuss the various studies establishing the functional importance of tetraspanins in the regulation of essential HSPC processes including quiescence, migration, and niche adhesion. When taken together, studies outlined in this review suggest that several tetraspanins may serve as potential targets to modulate HSPC interactions with the BM niche, ultimately impacting future HSPC therapies.

The Role of Danger Associated Molecular Patterns in Human Fetal Membrane Weakening.

The idea that cellular stress (including that precipitated by stretch), plays a significant role in the mechanisms initiating parturition, has gained considerable traction over the last decade. One key consequence of this cellular stress is the increased production of Danger Associated Molecular Patterns (DAMPs). This diverse family of molecules are known to initiate inflammation through their interaction with Pattern Recognition Receptors (PRRs) including, Toll-like receptors (TLRs). TLRs are the key innate immune system surveillance receptors that detect Pathogen Associated Molecular Patterns (PAMPs) during bacterial and viral infection. This is also seen during Chorioamnionitis. The activation of TLR commonly results in the activation of the pro-inflammatory transcription factor Nuclear Factor Kappa-B (NF-kB) and the downstream production of pro-inflammatory cytokines. It is thought that in the human fetal membranes both DAMPs and PAMPs are able, perhaps via their interaction with PRRs and the induction of their downstream inflammatory cascades, to lead to both tissue remodeling and weakening. Due to the high incidence of infection-driven Pre-Term Birth (PTB), including those that have preterm Premature Rupture of the Membranes (pPROM), the role of TLR in fetal membranes with Chorioamnionitis has been the subject of considerable study. Most of the work in this field has focused on the effect of PAMPs on whole pieces of fetal membrane and the resultant inflammatory cascade. This is important to understand, in order to develop novel prevention, detection, and therapeutic approaches, which aim to reduce the high number of mothers suffering from infection driven PTB, including those with pPROM. Studying the role of sterile inflammation driven by these endogenous ligands (DAMPs) activating PRRs system in the mesenchymal and epithelial cells in the amnion is important. These cells are key for the maintenance of the integrity and strength of the human fetal membranes. This review aims to (1) summarize the knowledge to date pertinent to the role of DAMPs and PRRs in fetal membrane weakening and (2) discuss the clinical potential brought by a better understanding of these pathways by pathway manipulation strategies.

The tetraspanin CD82 regulates bone marrow homing and engraftment of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cell (HSPC) transplantation represents a treatment option for patients with malignant and nonmalignant hematological diseases. Initial steps in transplantation involve the bone marrow homing and engraftment of peripheral blood-injected HSPCs. In recent work, we identified the tetraspanin CD82 as a potential regulator of HSPC homing to the bone marrow, although its mechanism remains unclear. In the present study, using a CD82 knockout (CD82KO) mouse model, we determined that CD82 modulates HSPC bone marrow maintenance, homing, and engraftment. Bone marrow characterization identified a significant decrease in the number of long-term hematopoietic stem cells in the CD82KO mice, which we linked to cell cycle activation and reduced stem cell quiescence. Additionally, we demonstrate that CD82 deficiency disrupts bone marrow homing and engraftment, with in vitro analysis identifying further defects in migration and cell spreading. Moreover, we find that the CD82KO HSPC homing defect is due at least in part to the hyperactivation of Rac1, as Rac1 inhibition rescues homing capacity. Together, these data provide evidence that CD82 is an important regulator of HSPC bone marrow maintenance, homing, and engraftment and suggest exploiting the CD82 scaffold as a therapeutic target for improved efficacy of stem cell transplants.