Utility of small molecule TLR4 antagonist (+)-naltrexone in suppression of preterm birth induced in mice by platelet activating factor

Introduction: Microbial and sterile triggers of inflammation can activate Toll-like receptors (TLR) to initiate synthesis of pro-inflammatory cytokines that in turn drive the parturition cascade leading to preterm delivery (PTD). Platelet activating factor (PAF) has been identified as a key endogenous sterile inflammatory trigger that is elevated in the amniotic fluid in both normal and PTD in human. Intrauterine administration of PAF in mice results in PTD. We investigated the role of TLR4 in PAF-induced PTD. Materials and Methods: BALB/c females were administered PAF or vehicle i.p on gestational day (gd) 16.5, followed by treatment with the small molecule TLR4 antagonist, (+)-Naltrexone, or vehicle control. Mice were observed for PTD, and in the absence of PTD mice were killed on gd 18.5 and implantation sites, fetal viability and fetal and placental weights were determined. Results: PAF administration lead to PTD in 64% (9/14 mice), with reduced numbers of viable fetuses compared to vehicle controls. (+)- Naltrexone administration to mice given PAF was able to prevent PAF-induced PTD and viable fetuses were comparable in number to control groups. Secondly, wildtype and Tlr4-/- BALB/c female mice were mated to males of the same genotype, and were challenged with PAF or vehicle on gd 16.5. Tlr4-/- females had 17% (2/12 mice) of PTD, with the number of viable fetuses similar to wildtype females when administered with PAF, suggesting that PAF may activate TLR4- independent pathways to drive fetal inflammatory injury. Conclusion: PAF is a clear mediator of inflammation-driven PTD, through TLR4-dependent and TLR4- independent pathways

Chorioamnionitis disrupts erythropoietin and melatonin homeostasis through the placental-fetal-brain axis during critical developmental periods

Introduction: Novel therapeutics are emerging to mitigate damage from perinatal brain injury (PBI). Few newborns with PBI suffer from a singular etiology. Most experience cumulative insults from prenatal inflammation, genetic and epigenetic vulnerability, toxins (opioids, other drug exposures, environmental exposure), hypoxia-ischemia, and postnatal stressors such as sepsis and seizures. Accordingly, tailoring of emerging therapeutic regimens with endogenous repair or neuro-immunomodulatory agents for individuals requires a more precise understanding of ligand, receptor-, and non-receptor-mediated regulation of essential developmental hormones. Given the recent clinical focus on neurorepair for PBI, we hypothesized that there would be injury-induced changes in erythropoietin (EPO), erythropoietin receptor (EPOR), melatonin receptor (MLTR), NAD-dependent deacetylase sirtuin-1 (SIRT1) signaling, and hypoxia inducible factors (HIF1α, HIF2α). Specifically, we predicted that EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α alterations after chorioamnionitis (CHORIO) would reflect relative changes observed in human preterm infants. Similarly, we expected unique developmental regulation after injury that would reveal potential clues to mechanisms and timing of inflammatory and oxidative injury after CHORIO that could inform future therapeutic development to treat PBI.Methods: To induce CHORIO, a laparotomy was performed on embryonic day 18 (E18) in rats with transient uterine artery occlusion plus intra-amniotic injection of lipopolysaccharide (LPS). Placentae and fetal brains were collected at 24 h. Brains were also collected on postnatal day 2 (P2), P7, and P21. EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α levels were quantified using a clinical electrochemiluminescent biomarker platform, qPCR, and/or RNAscope. MLT levels were quantified with liquid chromatography mass spectrometry.Results: Examination of EPO, EPOR, and MLTR1 at 24 h showed that while placental levels of EPO and MLTR1 mRNA were decreased acutely after CHORIO, cerebral levels of EPO, EPOR and MLTR1 mRNA were increased compared to control. Notably, CHORIO brains at P2 were SIRT1 mRNA deficient with increased HIF1α and HIF2α despite normalized levels of EPO, EPOR and MLTR1, and in the presence of elevated serum EPO levels. Uniquely, brain levels of EPO, EPOR and MLTR1 shifted at P7 and P21, with prominent CHORIO-induced changes in mRNA expression. Reductions at P21 were concomitant with increased serum EPO levels in CHORIO rats compared to controls and variable MLT levels.Discussion: These data reveal that commensurate with robust inflammation through the maternal placental-fetal axis, CHORIO impacts EPO, MLT, SIRT1, and HIF signal transduction defined by dynamic changes in EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α mRNA, and EPO protein. Notably, ligand-receptor mismatch, tissue compartment differential regulation, and non-receptor-mediated signaling highlight the importance, complexity and nuance of neural and immune cell development and provide essential clues to mechanisms of injury in PBI. As the placenta, immune cells, and neural cells share many common, developmentally regulated signal transduction pathways, further studies are needed to clarify the perinatal dynamics of EPO and MLT signaling and to capitalize on therapies that target endogenous neurorepair mechanisms

Toll-like Receptor-4: A New Target for Preterm Labour Pharmacotherapies?

Inflammatory activation, a major driver of preterm birth and subsequent neonatal morbidity, is an attractive pharmacological target for new preterm birth therapeutics. Inflammation elicited by intraamniotic infection is causally associated with preterm birth, particularly in infants delivered ≤34 weeks' gestation. However, sterile triggers of PTB, including placental ischaemic injury, uterine distention, cervical disease, or imbalance in the immune response, also act through inflammatory mediators released in response to tissue damage. Toll-like Receptors (TLRs) are critical upstream gate-keepers controlling the inflammatory activation that precedes preterm delivery, as well as in normal term labour. In particular, TLR4 is implicated for its capacity to sense and integrate a range of disparate infectious and sterile pro-inflammatory triggers, and so acts as a point-ofconvergence through which a range of infectious and sterile agents can activate and accelerate the parturition cascade. Recent studies point to the TLR4 signalling complex as a tractable target for the inhibition of fetal, placental & intraamniotic inflammatory cytokine production. Moreover, studies on mice show that novel small molecule antagonists of TLR4 signalling are highly effective in preventing preterm birth induced by bacterial mimetic LPS, heat-killed E. coli or the TLR4-dependent pro-inflammatory lipid, Platelet Activating Factor (PAF). In this review, we discuss the role of TLR4 in regulating the timing of birth and the potential utility of TLR4 antagonists as novel therapeutics for preterm delivery

Toll-like receptor-4 antagonist (D)-naltrexone protects against carbamyl- platelet activating factor (cPAF)-induced preterm labor in mice

Spontaneous preterm labor is frequently caused by an inflammatory response in the gestational tissues elicited by either infectious or sterile agents. In sterile preterm labor, the key regulators of inflammation are not identified, but platelet-activating factor (PAF) is implicated as a potential rate-limiting effector agent. Since Toll-like receptor (TLR)-4 can amplify PAF signaling, we evaluated whether TLR4 contributes to inflammation and fetal loss in a mouse model of PAF-induced sterile preterm labor, and whether a small-molecule TLR4 inhibitor, (þ)-naltrexone, can mitigate adverse PAF-induced effects. The administration of carbamyl (c)-PAF caused preterm labor and fetal loss in wild-type mice but not in TLR4-deficient mice. Treatment with (þ)-naltrexone prevented preterm delivery and alleviated fetal demise in utero elicited after cPAF administered by i.p. or intrauterine routes. Pups born after cPAF and (þ)-naltrexone treatment exhibited comparable rates of postnatal survival and growth to carrier-treated controls. (þ)-Naltrexone suppressed the cPAF-induced expression of inflammatory cytokine genes Il1b, Il6, and Il10 in the decidua; Il6, Il12b, and Il10 in the myometrium; and Il1b and Il6 in the placenta. These data demonstrate that the TLR4 antagonist (þ)-naltrexone inhibits the inflammatory cascade induced by cPAF, preventing preterm birth and perinatal death. The inhibition of TLR4 signaling warrants further investigation as a candidate strategy for fetal protection and delay of preterm birth elicited by sterile stimuli

Expression of GBS virulence genes in high vaginal swabs of symptomatic pregnant women at Hospital Tengku Ampuan Afzan, Kuantan

During pregnancy, group B streptococcus (GBS) colonization is known as one of the risk factors for preterm birth and consequently neonatal infections. Previous in-vitro experiments using human cells and in vivo animal models have portrayed the important roles of these virulence factors including hemolytic pigment (CylE), hyaluronidase (HylB), serine-rich protein (Srr) and bacterial surface adhesion of GBS (BsaB) in mediating GBS colonization and intrauterine ascending infection, leading to preterm delivery. The aim of this study is to investigate the association between mRNA expression of women and preterm delivery. GBS isolates were obtained from high vaginal swabs of pregnant women(n=40) with gestational age less than 37 weeks and symptoms including preterm labour, preterm premature rupture of membrane (pPROM), vaginal discharge and vaginal bleeding. Socio-demographic details, obstetric history and delivery outcomes of these women were also enquired. RNA was extracted from these GBS isolates and RT-qPCR was performed to determine the relative mRNA expression of GBS virulence genes including CylE,HylB, Srr and BsaB. Socio-demographic details and obstetric history were not found to be associated with the delivery outcomes of these women. Women with preterm labour and pPROM who delivered prematurely were demonstrated with higher expression of HylB and Cyl Egenes, in comparison to women with term delivery. The expression of Srr and BsaB genes were both similar between symptomatic pregnant women who had term and preterm delivery. Theseresultssuggestthatfollowingvaginalcolonization,bothCylEandHylBgenespossiblycontributetointrauterineascendinginfectionandinflammation,leading to preterm delivery in humans.Thus, hemolytic pigment and hyaluronidase may be targeted for exploratory and pre-clinical stages of vaccine development, which is a good alternative to intrapartum antibiotic prophylaxis in order to prevent neonatal GBS infections

Expression of GBS virulence genes in high vaginal swabs of symptomatic pregnant women at Hospital Tengku Ampuan Afzan, Kuantan

During pregnancy, group B streptococcus (GBS) colonization is known as one of the risk factors for preterm birth and consequently neonatal infections. Previous in-vitro experiments using human cells and in vivo animal models have portrayed the important roles of these virulence factors including hemolytic pigment (CylE), hyaluronidase (HylB), serine-rich protein (Srr) and bacterial surface adhesion of GBS (BsaB) in mediating GBS colonization and intrauterine ascending infection, leading to preterm delivery. The aim of this study is to investigate the association between mRNA expression of women and preterm delivery. GBS isolates were obtained from high vaginal swabs of pregnant women(n=40) with gestational age less than 37 weeks and symptoms including preterm labour, preterm premature rupture of membrane (pPROM), vaginal discharge and vaginal bleeding. Socio-demographic details, obstetric history and delivery outcomes of these women were also enquired. RNA was extracted from these GBS isolates and RT-qPCR was performed to determine the relative mRNA expression of GBS virulence genes including CylE,HylB, Srr and BsaB. Socio-demographic details and obstetric history were not found to be associated with the delivery outcomes of these women. Women with preterm labour and pPROM who delivered prematurely were demonstrated with higher expression of HylB and Cyl Egenes, in comparison to women with term delivery. The expression of Srr and BsaB genes were both similar between symptomatic pregnant women who had term and preterm delivery. Theseresultssuggestthatfollowingvaginalcolonization,bothCylEandHylBgenespossiblycontributetointrauterineascendinginfectionandinflammation,leading to preterm delivery in humans.Thus, hemolytic pigment and hyaluronidase may be targeted for exploratory and pre-clinical stages of vaccine development, which is a good alternative to intrapartum antibiotic prophylaxis in order to prevent neonatal GBS infections