Matthew W. Kemp

Preterm birth, infection, and inflammation advances from the study of animal models.

Inflammation is a protective response mediated by both innate and adaptive arms of the immune system following exposure to a range of harmful stimuli. Although inflammation is an essential mechanism in response to challenges including tissue injury and microbiological insult, inappropriate or excessive induction of the inflammatory response is itself a well-characterized cause of morbidity and mortality in adult populations. There is currently a growing appreciation of the potential for inflammation to play an adverse role in fetal health. The expression of cytokines (notably interleukin 1β [IL-1β], IL-6, IL-8, and tumor necrosis factor α [TNF-α]) by either the fetal or maternal tissues has been demonstrated to upregulate the activity of a number of uterine and cervical factors (eg, prostaglandin hormones and their receptors, matrix metalloproteinases, vascular endothelial growth factor [VEGF]), leading to premature initiation of the parturition process. Herein, we review important developments in our understanding of the link between preterm birth and fetal inflammation subsequent to infection, gained from studies undertaken in animal models.

Intra-amniotic LPS and antenatal betamethasone: inflammation and maturation in preterm lamb lungs

The proinflammatory stimulus of chorioamnionitis is commonly associated with preterm delivery. Women at risk of preterm delivery receive antenatal glucocorticoids to functionally mature the fetal lung. However, the effects of the combined exposures of chorioamnionitis and antenatal glucocorticoids on the fetus are poorly understood. Time-mated ewes with singleton fetuses received an intra-amniotic injection of lipopolysaccharide (LPS) either preceding or following maternal intramuscular betamethasone 7 or 14 days before delivery, and the fetuses were delivered at 120 days gestational age (GA) (term = 150 days GA). Gestation matched controls received intra-amniotic and maternal intramuscular saline. Compared with saline controls, intra-amniotic LPS increased inflammatory cells in the bronchoalveolar lavage and myeloperoxidase, Toll-like receptor 2 and 4 mRNA, PU.1, CD3, and Foxp3-positive cells in the fetal lung. LPS-induced lung maturation measured as increased airway surfactant and improved lung gas volumes. Intra-amniotic LPS-induced inflammation persisted until 14 days after exposure. Betamethasone treatment alone induced modest lung maturation but, when administered before intra-amniotic LPS, suppressed lung inflammation. Interestingly, betamethasone treatment after LPS did not counteract inflammation but enhanced lung maturation. We conclude that the order of exposures of intra-amniotic LPS or maternal betamethasone had large effects on fetal lung inflammation and maturation.

Chronic Fetal Exposure to Ureaplasma parvum Suppresses Innate Immune Responses in Sheep

The chorioamnionitis associated with preterm delivery is often polymicrobial with ureaplasma being the most common isolate. To evaluate interactions between the different proinflammatory mediators, we hypothesized that ureaplasma exposure would increase fetal responsiveness to LPS. Fetal sheep were given intra-amniotic (IA) injections of media (control) or Ureaplasma parvum serovar 3 either 7 or 70 d before preterm delivery. Another group received an IA injection of Escherichia coli LPS 2 d prior to delivery. To test for interactions, IA U. parvum-exposed animals were challenged with IA LPS and delivered 2 d later. All animals were delivered at 124 ± 1-d gestation (term = 150 d). Compared with the 2-d LPS exposure group, the U. parvum 70 d + LPS group had 1) decreased lung pro- and anti-inflammatory cytokine expression and 2) fewer CD3+ T lymphocytes, CCL2+, myeloperoxidase+, and PU.1+ cells in the lung. Interestingly, exposure to U. parvum for 7 d did not change responses to a subsequent IA LPS challenge, and exposure to IA U. parvum alone induced mild lung inflammation. Exposure to U. parvum increased pulmonary TGF-β1 expression but did not change mRNA expression of either the receptor TLR4 or some of the downstream mediators in the lung. Monocytes from fetal blood and lung isolated from U. parvum 70 d + LPS but not U. parvum 7 d + LPS animals had decreased in vitro responsiveness to LPS. These results are consistent with the novel finding of downregulation of LPS responses by chronic but not acute fetal exposures to U. parvum. The findings increase our understanding of how chorioamnionitis-exposed preterm infants may respond to lung injury and postnatal nosocomial infections.

Sustained inflation at birth did not protect preterm fetal sheep from lung injury

Sustained lung inflations (SI) at birth may recruit functional residual capacity (FRC). Clinically, SI increase oxygenation and decrease need for intubation in preterm infants. We tested whether a SI to recruit FRC would decrease lung injury from subsequent ventilation of fetal, preterm lambs. The preterm fetus (128±1 day gestation) was exteriorized from the uterus, a tracheostomy was performed, and fetal lung fluid was removed. While maintaining placental circulation, fetuses were randomized to one of four 15-min interventions: 1) positive end-expiratory pressure (PEEP) 8 cmH2O (n=4), 2) 20 s SI to 50 cmH2O then PEEP 8 cmH2O (n=10), 3) mechanical ventilation at tidal volume (VT) 7 ml/kg (n=13), or 4) 20 s SI then ventilation at VT 7 ml/kg (n=13). Lambs were ventilated with 95% N2/5% CO2 and PEEP 8 cmH2O. Volume recruitment was measured during SI, and fetal tissues were collected after an additional 30 min on placental support. SI achieved a mean FRC recruitment of 15 ml/kg (range 8-27). Fifty percent of final FRC was achieved by 2 s, 65% by 5 s, and 90% by 15 s, demonstrating prolonged SI times are needed to recruit FRC. SI alone released acute-phase proteins into the fetal lung fluid and increased mRNA expression of proinflammatory cytokines and acute-phase response genes in the lung. Mechanical ventilation further increased all markers of lung injury. SI before ventilation, regardless of the volume of FRC recruited, did not alter the acute-phase and proinflammatory responses to mechanical ventilation at birth.

Exposure to In Utero Lipopolysaccharide Induces Inflammation in the Fetal Ovine Skin

Inflammation is a defensive process by which the body responds to both localized and systemic tissue damage by the induction of innate and adaptive immunity. Literature from human and animal studies links inappropriate in utero inflammation to preterm parturition and fetal injury. The pathways by which such inflammation may cause labor, however, are not fully understood. Any proinflammatory agonist in the amniotic fluid will contact the fetal skin, in its entirety, but a potential role of the fetal skin in the pathways to labor have not previously been explored. We hypothesized that the fetal skin would respond robustly to the presence of intra-amniotic lipopolysaccharide (LPS) in our ovine model of in utero inflammation. In vitro and in utero exposure of fetal ovine keratinocytes or fetal skin to Escherichia coli LPS reliably induced significant increases in interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), and IL-8 expression. We demonstrate that, in utero, this expression requires direct exposure with LPS suggesting that the inflammation is triggered directly in the skin itself, rather than as a secondary response to a systemic stimuli and that inflammation involves Toll-like receptor (TLR) regulation and neutrophil chemotaxis in concordance with an acute inflammatory reaction. We show that this response involves multiple inflammatory mediators, TLR regulation, and localized inflammatory cell influx characteristic of an acute inflammatory reaction. These novel data strongly suggests that the fetal skin acts as an important mediator of the fetal inflammatory response and as such may contribute to preterm birth.

Inflammation of the fetal ovine skin following in utero exposure to Ureaplasma parvum.

There is increasing evidence linking in utero infection and inflammation to preterm birth. Many commensal urogenital tract microorganisms, including the Mycoplasmas and Ureaplasmas, are commonly detected in association with preterm birth. Using an ovine model of sterile fetal inflammation, we demonstrated previously that the fetal skin generates a robust inflammatory response following in utero exposure to lipopolysaccharides from Escherichia coli. The fetal skin’s response to colonization of the amniotic fluid by viable microorganisms remains unstudied. We hypothesised that in utero infection with Ureaplasma parvum serovar 3 would induce a proinflammatory response in the fetal skin. We found that (1) cultured fetal keratinocytes (the primary cellular constituent of the epidermis) respond to U. parvum exposure in vitro by increasing the expression of the chemotactant monocyte chemoattractant protein 1 (MCP-1) but not interleukin 1β (IL-1β), IL-6, IL-8, or tumor necrosis factor-α (TNF-α); (2) the fetal skin’s response to 7 days of U. parvum exposure is characterized by elevated expression of MCP-1, TNF-α, and IL-10; and (3) the magnitude of inflammatory cytokine/chemokine expression in the fetal skin is dependent on the duration of U parvum exposure. These novel findings provide further support for the role of the fetal skin in the development of fetal inflammation and the preterm birth that may follow.

LPS-induced chorioamnionitis and antenatal corticosteroids modulate Shh signaling in the ovine fetal lung

Chorioamnionitis and antenatal corticosteroids mature the fetal lung functionally but disrupt late-gestation lung development. Because Sonic Hedgehog (Shh) signaling is a major pathway directing lung development, we hypothesized that chorioamnionitis and antenatal corticosteroids modulated Shh signaling, resulting in an altered fetal lung structure. Time-mated ewes with singleton ovine fetuses received an intra-amniotic injection of lipopolysaccharide (LPS) and/or maternal intramuscular betamethasone 7 and/or 14 days before delivery at 120 days gestational age (GA) (term = 150 days GA). Intra-amniotic LPS exposure decreased Shh mRNA levels and Gli1 protein expression, which was counteracted by both betamethasone pre- or posttreatment. mRNA and protein levels of fibroblast growth factor 10 and bone morphogenetic protein 4, which are important mediators of lung development, increased 2-fold and 3.5-fold, respectively, 14 days after LPS exposure. Both 7-day and 14-day exposure to LPS changed the mRNA levels of elastin (ELN) and collagen type I alpha 1 (Col1A1) and 2 (Col1A2), which resulted in fewer elastin foci and increased collagen type I deposition in the alveolar septa. Corticosteroid posttreatment prevented the decrease in ELN mRNA and increased elastin foci and decreased collagen type I deposition in the fetal lung. In conclusion, fetal lung exposure to LPS was accompanied by changes in key modulators of lung development resulting in abnormal lung structure. Betamethasone treatment partially prevented the changes in developmental processes and lung structure. This study provides new insights into clinically relevant prenatal exposures and fetal lung development.

Oral, Nasal and Pharyngeal Exposure to Lipopolysaccharide Causes a Fetal Inflammatory Response in Sheep

Background A fetal inflammatory response (FIR) in sheep can be induced by intraamniotic or selective exposure of the fetal lung or gut to lipopolysaccharide (LPS). The oral, nasal, and pharyngeal cavities (ONP) contain lymphoid tissue and epithelium that are in contact with the amniotic fluid. The ability of the ONP epithelium and lymphoid tissue to initiate a FIR is unknown. Objective To determine if FIR occurs after selective ONP exposure to LPS in fetal sheep. Methods Using fetal recovery surgery, we isolated ONP from the fetal lung, GI tract, and amniotic fluid by tracheal and esophageal ligation and with an occlusive glove fitted over the snout. LPS (5 mg) or saline was infused with 24 h Alzet pumps secured in the oral cavity (n = 7–8/group). Animals were delivered 1 or 6 days after initiation of the LPS or saline infusions. Results The ONP exposure to LPS had time-dependent systemic inflammatory effects with changes in WBC in cord blood, an increase in posterior mediastinal lymph node weight at 6 days, and pro-inflammatory mRNA responses in the fetal plasma, lung, and liver. Compared to controls, the expression of surfactant protein A mRNA increased 1 and 6 days after ONP exposure to LPS. Conclusion ONP exposure to LPS alone can induce a mild FIR with time-dependent inflammatory responses in remote fetal tissues not directly exposed to LPS.

Selective Exposure of the Fetal Lung and Skin/Amnion (but Not Gastro-Intestinal Tract) to LPS Elicits Acute Systemic Inflammation in Fetal Sheep

Inflammation of the uterine environment (commonly as a result of microbial colonisation of the fetal membranes, amniotic fluid and fetus) is strongly associated with preterm labour and birth. Both preterm birth and fetal inflammation are independently associated with elevated risks of subsequent short- and long-term respiratory, gastro-intestinal and neurological complications. Despite numerous clinical and experimental studies to investigate localised and systemic fetal inflammation following exposure to microbial agonists, there is minimal data to describe which fetal organ(s) drive systemic fetal inflammation. We used lipopolysaccharide (LPS) from E.coli in an instrumented ovine model of fetal inflammation and conducted a series of experiments to assess the systemic pro-inflammatory capacity of the three major fetal surfaces exposed to inflammatory mediators in pregnancy (the lung, gastro-intestinal tract and skin/amnion). Exposure of the fetal lung and fetal skin/amnion (but not gastro-intestinal tract) caused a significant acute systemic inflammatory response characterised by altered leucocytosis, neutrophilia, elevated plasma MCP-1 levels and inflammation of the fetal liver and spleen. These novel findings reveal differential fetal organ responses to pro-inflammatory stimulation and shed light on the pathogenesis of fetal systemic inflammation after exposure to chorioamnionitis.

The clinical use of corticosteroids in pregnancy

BACKGROUND The use of antenatal steroid therapy is common in pregnancy. In early pregnancy, steroids may be used in women for the treatment of recurrent miscarriage or fetal abnormalities such as congenital adrenal hyperplasia. In mid-late pregnancy, the antenatal administration of corticosteroids to expectant mothers in anticipation of preterm birth is one of the most important advances in perinatal medicine; antenatal corticosteroids are now standard care for pregnancies at risk of premature delivery in high- and middle-income countries. The widespread uptake of this therapy is due to a compelling body of evidence demonstrating improved neonatal outcomes following antenatal corticosteroid exposure, stemming most notably from corticosteroid-driven maturation of fetal pulmonary function. As we approach the 50th anniversary of landmark work in this area by Liggins and Howie, it is apparent that much remains to be understood with regards to how we might best apply antenatal corticosteroid therapy to improve pregnancy outcomes at both early and mid to late gestation. METHODS Drawing on advances in laboratory science, pre-clinical and clinical studies, we performed a narrative review of the scientific literature to provide a timely update on the benefits, risks and uncertainties regarding antenatal corticosteroid use in pregnancy. Three, well-established therapeutic uses of antenatal steroids, namely recurrent miscarriage, congenital adrenal hyperplasia and preterm birth, were selected to frame the review. RESULTS Even the most well-established antenatal steroid therapies lack the comprehensive pharmacokinetic and dose-response data necessary to optimize dosing regimens. New insights into complex, tissue-specific corticosteroid signalling by genomic-dependent and independent mechanisms have not been used to inform corticosteroid treatment strategies. There is growing evidence that some fetal corticosteroid treatments are either ineffective, or may result in adverse outcomes, in addition to lasting epigenetic changes in a variety of homeostatic mechanisms. Nowhere is the need to better understand the intricacies of corticosteroid therapy better conveyed than in the findings of Althabe and colleagues who recently reported an increase in overall neonatal mortality and maternal morbidity in association with antenatal corticosteroid administration in low-resource settings. CONCLUSIONS New research to clarify the benefits and potential risks of antenatal corticosteroid therapy is urgently needed, especially with regard to corticosteroid use in low-resource environments. We conclude that there is both significant scope and an urgent need for further research-informed refinement to the use of antenatal corticosteroids in pregnancy.