Toll-like receptor 4 signalling in inflammation pathways to term and preterm delivery

Abstract

A pro-inflammatory signalling cascade initiates the events that bring about parturition, including uterine contractions, cervical ripening and the rupture of fetal membranes. One mechanism by which this inflammatory process can be initiated is via toll-like receptor (TLR)- mediated activation, elicited by danger associated molecular patterns (DAMPs) or other endogenous TLR4 regulators, such as platelet activating factor, surfactant protein A (SP-A), high mobility group box 1 (HMGB1), heat shock protein 70 (HSP70), uric acid and oxysterols which are released from gestational tissues after necrotic cell death as a result of tissue stretch, injury and remodelling in late gestation. It is postulated that preterm delivery results from a pathological process that involves an early increase in DAMPs and TLR4 regulators, premature activation of TLRs and the inflammatory pathways associated with on-time labour, leading to preterm delivery. This premature release of DAMPs and TLR4 regulators can be triggered by non-infectious events, leading to sterile inflammation and preterm delivery in the absence of infection. Many of the DAMPs and TLR4 regulators produced by the gestational tissues in late pregnancy and labour are known to trigger TLR4 signalling, raising the possibility that TLR4 acts as a point of convergence in the mechanistic pathways linking infection-driven and sterile preterm labour, and term labour, in mice and potentially humans. Prior to this study, the role of TLR4 and its association with DAMPs and TLR4 regulators in the timing of labour had not been evaluated in normal term parturition or sterile inflammation-driven preterm delivery. We hypothesised that TLR4 activation by DAMPs and TLR4 regulators acts upstream of the gene expression and leukocyte recruitment steps that mediate the physiological and sterile inflammation in normal term delivery and preterm delivery respectively. In this thesis we describe experiments in mouse models, particularly mice with null mutation in the Tlr4 gene, which demonstrate that TLR4 plays a critical role in the timing of labour. TLR4-deficient mice were found to have reduced expression of pro-inflammatory genes Il1b, Il6, Il12b, Tnf and Il17a, which was mirrored by immune cell populations with reduced percentages and numbers of inflammatory leukocytes in the gestational tissues, including placental neutrophils and uterine dendritic cells. The compromised pro-inflammatory environment likely impacted upon the downstream uterine activation genes Ptgfr, Oxtr and Gja1 which were expressed at lower levels in TLR4-deficient mice compared to wildtype controls, in the gestational tissues in late gestation. This led to an increased length of pregnancy and elevated pup mortality in the TLR4-deficient mice. We then attempted to induce preterm birth using the endogenous TLR4 regulator carbamyl PAF (cPAF), which was administered intraperitoneally into pregnant mice. cPAF administration induced pro-inflammatory cytokine expression in the gestational tissues, leading to preterm delivery in wildtype mice and decreased fetal viability. TLR4-deficient females were resistant to cPAF induced preterm birth. Furthermore, cPAF-induced preterm delivery was blocked by administering the TLR4 antagonist (+)-naltrexone to mice, which prevented initiation of the pro-inflammatory cytokine cascade, strongly indicating that cPAF-mediated induction of inflammation within the gestational tissues and the resulting preterm delivery are TLR4 dependent. In summary, this thesis provides new evidence about the association between cPAF and TLR4 in inducing downstream inflammatory pathways, including cytokine expression and leukocyte recruitment that trigger uterine activation genes in both preterm and term delivery. This includes a crucial role of TLR4 as a key mediator of sterile inflammation in preterm and term delivery. The study provides fundamental insights on the inflammatory pathways underpinning the parturition cascade, and may inform future clinical studies to investigate prevention or delay of spontaneous preterm delivery in humans.