The transcription factor Nrf2 is decreased after spontaneous term labour in human fetal membranes where it exerts anti-inflammatory properties

Highlights

• Nrf2 expression in fetal membranes is lower after spontaneous term labour and delivery.

• Nrf2 expression is lower in fetal membranes with preterm histologic chorioamnionitis.

• Nrf2 siRNA increased cytokine-induced pro-inflammatory cytokines from primary amnion cells.

• Nrf2 siRNA augmented in vitro models of infection-induced cytokines from primary amnion cells.

Abstract

Introduction

Inflammation plays a central role in the terminal processes of human labour and delivery, including the rupture of fetal membranes. Recent studies show a role for the transcription factor Nrf2 (NF-E2-related factor 2) in regulating inflammation. The aims of this study were to determine the effect of human spontaneous term and preterm labour on Nrf2 expression in fetal membranes; and Nrf2 siRNA knockdown on pro-inflammatory cytokines.

Methods

Fetal membranes, from term and preterm, were obtained from non-labouring and labouring women. Primary amnion cells were used to determine the effect of Nrf2 siRNA knockdown on pro-inflammatory cytokines in the presence of inflammation or infection.

Results

Nrf2 mRNA expression and nuclear protein expression were significantly decreased after spontaneous term labour and delivery. There was, however, no effect of spontaneous preterm labour and delivery on Nrf2 mRNA expression and nuclear protein expression. On the other hand, Nrf2 gene expression was significantly lower in fetal membranes obtained from women at preterm with histologic chorioamnionitis compared to fetal membranes obtained from women at preterm without histologic chorioamnionitis. Nrf2 silencing by siRNA in primary amnion cells was associated with a significant increase in IL-6 and IL-8 mRNA expression and release induced by IL-1β, TNF-α, flagellin and poly(I:C).

Discussion

Nrf2 has an anti-inflammatory effect in human fetal membranes. It is decreased with term labour and preterm chorioamnionitis; and Nrf2 silencing increases inflammation- and infection-induced pro-inflammatory cytokines. Further studies are required to determine if agents that can increase Nrf2 expression may be a potential therapeutic strategy in the treatment and management of infection-induced preterm labour.

Introduction

Preterm birth is the major cause of perinatal morbidity and mortality accounting for up to 85% of all early infant deaths [1], [2]. Of infants surviving preterm birth, 20–25% will have at least one major disability including long-term neurocognitive deficits, pulmonary dysfunction and ophthalmological disorders [3], [4]. Compared to infants delivered at term, there is an increased cost of health care for the preterm infant during the first 10 years of life [5]; for the severely disabled survivors of preterm birth, there are substantial societal costs beyond childhood [6].

A major cause of preterm birth is the premature rupture of fetal membranes (PROM). Rupture of membranes precedes the initiation of uterine contractions in at least 10% of term and nearly 40% of preterm births [7]. The physiology of rupture of membranes is yet to be fully elucidated; however apoptosis and degradation of the extracellular matrix (ECM) are key features [8]. These processes are activated by pro-inflammatory cytokines, which are increased with labour onset as well as ascending infection; leucocyte infiltration can further amplify this inflammatory response, leading to the production of proteolytic enzymes in the fetal membranes, predisposing them to rupture [9], [10]. Important in the recognition of pathogenic microorganisms to trigger the inflammatory response in these uterine tissues are Toll-like receptors (TLRs) [11]. By elucidating the physiological mechanisms that are involved in fetal membrane rupture, as well as pathological activation at preterm, can we begin to develop clinically useful interventions that could improve neonatal outcomes.

Recently, the transcription factor Nrf2 (NF-E2-related factor 2), has been shown to play a central role in the regulation of inflammation. Under basal conditions, Nrf2 is sequestered inactive in the cytoplasm by binding to Kelch-like ECH-associated protein 1 (KEAP1). It translocates into the nucleus following its activation, for example, by reactive oxygen species where it mediates activation of a variety of genes. A large number of cytoprotective and antioxidant proteins have been identified as Nrf2 targets [12], [13]; the genes typically contain an antioxidant response element (ARE), a cis-acting DNA regulatory element with a core sequence of 5′-TGA(C/T)nnnGC(A/G)-3′. Notably, other targets with wide-ranging set of biological functions have also been identified [14], [15], including genes involved in controlling inflammation [14]. Hypoxic Nrf2 deficient mice, in addition to reduced antioxidant enzyme levels and enhanced oxidative stress, have increased inflammatory cytokine expression [16]. In vitro, silencing of Nrf2 increases TNF-α-induced inflammatory responses in human monocytes [17], LPS-induced pro-inflammatory cytokine gene expression in HepG2 cells [18], oxidative stress-induced pro-inflammatory cytokine gene expression in dermal fibroblast [19], and platelet-derived growth factor (PDGF)-induced inflammation in vascular smooth muscle cell (VSMCs) [20].

To our knowledge, there are no studies on Nrf2 in fetal membranes; however, Nrf2 is expressed in human placenta where its expression is lower with preeclampsia [21] and gestational diabetes [22]. Given the central role of inflammation in the processes of human labour and delivery [23], we hypothesised that Nrf2 would be decreased in fetal membranes after labour, hence permitting increased inflammatory activation in these tissues. Thus, in this study we will determine the effect of spontaneous term and preterm labour, and preterm histologic chorioamnionitis on Nrf2 expression in fetal membranes; the mRNA and nuclear protein expression will be quantified using quantitative real-time-PCR (qRT-PCR) and Western blotting, respectively. To determine if Nrf2 regulates pro-inflammatory cytokines in the presence of inflammation or infection, we will investigate the effect of Nrf2 inhibition in primary amnion cells in the presence of the pro-inflammatory cytokines IL-1β and TNF-α, and TLR ligands, bacterial product flagellin (TLR5) and the viral dsRNA analogue polyinosinic:polycytidilic acid (poly(I:C); TLR3).