Neutrophils in preterm birth: friend or foe?

Abstract

Preterm birth is a serious global health problem that affects 5–18% of pregnancies worldwide. In addition to being the major cause of neonatal mortality and morbidity, preterm birth is associated with short term and long term complications in the offspring. Despite this, the causes and pathogenesis of preterm birth remain unclear. Neutrophils are innate immune cells that infiltrate the maternal-fetal interface during normal parturition and their accumulation is dramatically increased during preterm birth, especially in the presence of an infection. Indeed, a defining feature of chorioamnionitis (inflammation of the chorioamnionic fetal membranes) that is associated with more than 40% of preterm births, is neutrophil accumulation. While these cells may play an important role during normal term parturition as well as preterm birth, their functions at the maternal-fetal interface are unclear. This review will provide a broad overview of the relevant studies to enable a better understanding of the roles of neutrophils during normal parturition and preterm birth.

Preterm Birth

Preterm birth, delivery of the fetus prior to 37 weeks of gestation, affects 5–18% of pregnancies worldwide [1, 2]. It is estimated that preterm birth accounts for over 50% of all perinatal mortality and can also lead to many neurodevelopmental complications including visual and hearing impairments, risk for infections, as well as chronic lung disease [1, 3]. Compounding this is evidence to suggest that those born prematurely are at increased risk of learning disabilities, and cardiovascular and metabolic diseases in adulthood, with more severe outcomes for those born earlier [1]. Mothers who delivered prematurely are also at increased risk of repeat preterm birth [4]. As such, in the United States alone, the annual cost of neonatal care for preterm babies is ~$6 billion while the estimated societal economic impact is ~$27 billion [5]. Clearly, preterm birth is a serious obstetric complication that has many long-lasting implications for both the mother and baby.

In ~30% of cases, preterm birth is medically indicated, for example to treat placenta accreta or preeclampsia; however the remainder are spontaneous and further investigations are warranted to identify the causes and pathogenesis of these cases [6]. Around 40% of spontaneous preterm births are associated with intrauterine infection that can cause significant inflammation and immune cell infiltration at the maternal-fetal interface [3, 7]. This review will present our current knowledge about one key population of innate immune cells, neutrophils, that are recruited to the maternal-fetal interface during both term and preterm parturition.

Neutrophils

Neutrophils are short-lived, terminally differentiated innate immune cells that constitute the body’s first line of defense against infection. Neutrophils can detect low levels of chemoattractants, such as endogenous interleukin (IL)-8 or exogenous factors like bacterial N-formyl peptides. Gradients of these chemoattractants induce neutrophils to emigrate from the circulation to the site of infection where they can neutralize pathogens in one of several mechanisms. Neutrophils are phagocytic and can neutralize pathogens internally. Neutrophils also possess several mechanisms of extracellular killing. A primary mechanism of extracellular killing is degranulation, the specific exocytosis of neutrophil cytoplasmic granules containing proteases and antimicrobial proteins that can neutralize surrounding pathogens. Neutrophils can also secrete reactive oxygen species (ROS) and pro-inflammatory cytokines and chemokines that can propagate the local inflammatory response to recruit more immune cells [8]. Recently, a novel mode of neutrophil extracellular killing has been described whereby neutrophils release extracellular traps (ETs) that consist of DNA decorated with proteases and antimicrobial proteins to entrap and kill pathogens extracellularly [9, 10]. Typically, this is a “suicidal” process as the membrane integrity of the neutrophil is compromised during the extrusion of these NETs and this enables the activated neutrophils to be effectively cleared by surrounding cells [10]. While activated neutrophils can effectively remove pathogens, if the neutrophils themselves are insufficiently cleared, such as in severe sepsis [11] and chronic lung disease [12], they can cause collateral damage to the surrounding tissue which can lead to bystander injury.

Chorioamnionitis: Neutrophil Accumulation in the Fetal Membranes

Pregnancy is an immunological paradox in which the fetus, a semi-allograft, must survive and thrive in the maternal host throughout gestation. As such, there are many immunological adaptations at the maternal-fetal interface to facilitate pregnancy, including many well-characterized mechanisms utilized specifically by the placenta to promote local maternal immune cell regulation and tolerance [13, 14]. However, during an infection or tissue injury, increased numbers of inflammatory innate immune cells, including macrophages and neutrophils, are recruited to the maternal-fetal interface. Neutrophil recruitment and accumulation in the placenta and associated fetal membranes (FMs), that enclose the fetus and amniotic fluid throughout gestation, is termed chorioamnionitis [15]. Signs of chorioamnionitis are observed in more than 40% of spontaneous preterm births, especially those at early gestations, suggesting that this may be a pathological mechanism that can manifest to preterm premature rupture of the fetal membranes (PPROM) and subsequent preterm birth [16, 17]. In addition to PPROM and preterm birth, chorioamnionitis is also associated with the fetal inflammatory response syndrome that may lead to perinatal brain injury, abnormal fetal cardiac function and retinopathies [18, 19]. The more advanced the stage of chorioamnionitis (ie. the deeper the neutrophil infiltration into the amniotic layer), the more severe the fetal inflammatory response [15]. Thus, chorioamnionitis even in the absence of prematurity could be detrimental to fetal development.

Neutrophil recruitment to the FMs

The most commonly recognized cause of chorioamnionitis is infection of the FMs by ascending bacteria from the vagina and cervix [20]. Increased numbers of neutrophils have been reported in the FMs from women with acute chorioamnionitis [21], in mouse models of infection-induced preterm birth [22–25], and in non-human primate models of intraamniotic or uterine infection or inflammation [26–28]. However, whether it is the infectious components or the FMs themselves that recruit neutrophils remains unclear. Recent publications from our group and others have shown that normal unstimulated term human FM explants secrete low levels of chemotactic factors that can recruit neutrophils and this is exacerbated after their exposure to bacterial lipopolysaccharide (LPS) [25, 29] or during labor [30]. Mechanistically, in a model of chronically catheterized non-human primates (Macaca nemestrina), inoculation of Group B Streptococcus (GBS) into the uterus rapidly recruited neutrophils to accumulate in the chorionic blood vessels and infiltrate the chorionic membrane [26]. This was mediated by increased expression of chemotactic and neutrophil adhesive factors by the FMs, such as L-selectin and ICAM-1 [26]. Furthermore, in another model of intraamniotic infection in rhesus macaques, LPS activated IRAK-1 signaling in the amnion in an IL1-dependent manner to increase amniotic secretion of IL-8 and CSF3 that were key for maternal neutrophil recruitment and amniotic inflammation [27].

Neutrophil Function in Term and Preterm Parturition

There is abundant evidence to support that physiological parturition is a pro-inflammatory process. While we still do not understand the signaling mechanisms that lead up to this inflammatory cascade, it is well-established from histological studies that immune cells, especially macrophages and neutrophils, infiltrate the human myometrium, FMs, and cervix around the time of labor [24, 31]. The precise roles of these immune cells at the maternal-fetal interface in the context of parturition is not well-studied, however, their presence is accompanied by increased levels of pro-inflammatory IL-1β, IL-6, IL-8 and TNF-α in the FMs that could be important for signaling parturition [24, 32]. In addition to propagating inflammation, the decidual environment may also induce neutrophils to promote angiogenesis by upregulating their expression of VEGF-A and arginase-1 [33] and neutrophils may also play a role in postpartum tissue remodeling of the uterus [24] and cervix [34].

Since physiological parturition is an inflammatory process, and infection also induces inflammation, pathogens may trigger preterm birth by prematurely activating the same inflammatory pathways that mediate term parturition [35]. Furthermore, additional factors secreted by the recruited immune cells may also cause tissue damage leading to PPROM and preterm birth. Since neutrophils are so abundant in the FMs during infection-associated preterm birth [24, 32], it has been hypothesized that they play a key role in directly mediating PPROM and preterm birth. This hypothesis was challenged in several rodent studies which showed that systemic depletion of neutrophils prior to high dose LPS administration was not able to delay the timing of preterm birth [22, 23, 34]. However, it was observed that neutrophil depletion did reduce the levels of pro-inflammatory factors such as IL-1β at the maternal-fetal interface [23], suggesting that neutrophils may play a more indirect role in propagating local inflammation that could subsequently lead to preterm labor.

Neutrophils and NETs: A Double-edged Sword?

In addition to the production of inflammatory cytokines and chemokines, we recently demonstrated that LPS-stimulated FMs induce neutrophil degranulation and ROS production that could be damaging to the FMs [25]. This neutrophil activation was partially mediated by FM derived TNF-α leading to activation of neutrophil p38 MAP kinase signaling (Figure 1). In these in vitro studies, we also demonstrated that both unstimulated and LPS-stimulated human FMs induced neutrophils to release NETs [25]. NET release was augmented after exposure to LPS-stimulated FMs and this was also dependent on FM derived TNF-α and neutrophil MAP kinase signaling [25] (Figure 1). Moreover, NET release was ROS-dependent, and for both unstimulated and LPS-stimulated FMs, neutrophils utilized a pathway distinct from the classical “suicidal” release pathway that enabled neutrophils to remain viable and functional even after NET release. The release of NETs through such a “vital” pathway may contribute to the extended lifespan of neutrophils at the maternal-fetal interface and could have different implications for neutrophil activity after NET release.

Figure 1: Fetal membranes activate neutrophils and induce vital NET release.

Neutrophils exposed to unstimulated fetal membrane conditioned media (CM) secrete low levels of several cytokines and release vital NETs. In contrast, neutrophils that have been exposed to CM from LPS-stimulated FMs secrete many cytokines at high levels, degranulate and release vital NETs through a p38 MAP kinase- and reactive oxygen species (ROS)-dependent mechanism. This was partially mediated by increased TNF-α secretion by LPS-stimulated FMs.

The ability of resting FMs to induce NET release was an intriguing observation that was confirmed in the mouse [25], as well as in women with acute chorioamnionitis [21]. We propose that these NETs are preemptively generated by neutrophils to play a surveillance role at the maternal-fetal interface so that in the event of infection, mechanisms are already in place for microbial entrapment and neutralization. This hypothesis is supported by our observations that these NETs do not affect FM function, while NETs induced by LPS-stimulated FMs do (Tong et al., Manuscript in Preparation; Figure 1). In contrast, during infection, neutrophil activation (increased neutrophil degranulation, cytokine production and NET release) is likely to be important for neutralizing pathogens and limiting microbial spread as it was reported that hemolytic GBS strains that are more virulent contain a pigment toxin that can subvert killing by NETs and induce neutrophil death [26]. However, we hypothesize that since the release of these NETs do not cause neutrophil death that would typically lead to their clearance by macrophages [36], these activated neutrophils may persist at the maternal-fetal interface past their normal lifespan and lead to uncontrolled inflammation at the FMs that could be detrimental for pregnancy. Indeed, an increase in neutrophil viability, and thus a decrease in their clearance, may explain the accumulation of neutrophils in the FMs in chorioamnionitis. We and others have observed that neutrophil-derived factors can amplify FM inflammation and induce FM weakening [37, 38] (Tong et al., Manuscript in Preparation). Clearly, the roles of neutrophils and NETs in the FMs are complex and further studies are warranted to determine their functional significance in different contexts.

Summary

In conclusion, while neutrophils may not be crucial for the induction of preterm labor, studies from our lab and others indicate that neutrophils may contribute to both the process of physiological and pathological parturition, predominantly by regulating and propagating inflammation at the maternal-fetal interface through the release of cytokines and NETs. We propose that neutrophils normally play a protective surveillance role at the maternal-fetal interface and only in the presence of infection or another yet unstudied stimuli do neutrophils become overactivated and cause FM tissue damage (Figure 1). In support of the protective role of NETs, virulent hemolytic GBS are able to enter the amniotic cavity and cause fetal injury by subverting killing by NETs [26]. In order to better understand the contributions that neutrophils and NETs make to both physiological and pathological parturition, more functional and mechanistic studies at the maternal-fetal interface are needed.

The maternal-fetal interface is a highly complex site both in terms of structure and function. The anatomical location of the infection as well as the strain of pathogen are just two of many factors that can affect the FM response and the incidence of preterm birth [27, 37]. Furthermore, while neutrophils that are found at the chorio-decidua are reported to be maternally-derived, neutrophils at the amnion may be derived from the fetus [39]. It is possible that these fetal-derived neutrophils can also play a role in inducing inflammation from an intraamniotic site [40] though it has been reported that compared to maternal neutrophils, neonatal neutrophils have a reduced capacity to phagocytose pathogens [41], produce ROS [26] and extrude NETs [42, 43]. Thus, future studies will need to consider all of these factors in order to create a physiologically relevant model to determine how neutrophil activation and NETs contribute to both normal physiological and pathological preterm birth.