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Introduction: Little is known about the interplay between neutrophil heterogeneity in neonates in health and disease states. Olfactomedin-4 (OLFM4) marks a subset of neutrophils that have been described in adults and pediatric patients but not neonates, and this subset is thought to play a role in modulating the host inflammatory response., Methods: This is a prospective cohort of neonates who were born between June 2020 and December 2021 at the University of Cincinnati Medical Center NICU. Olfactomedin-4-positive (OLFM4+) neutrophils were identified in the peripheral blood using flow cytometry., Results: OLFM4+ neutrophil percentage was not correlated with gestational age or developmental age. Neonates with sepsis had a higher percentage than those without the condition, 66.9% (IQR 24.3-76.9%) versus 21.5% (IQR 10.6-34.7%), respectively, p = 0.0003. At birth, a high percentage of OLFM4+ neutrophils was associated with severe chorioamnionitis at 49.1% (IQR 28.2-61.5%) compared to those without it at 13.7% (IQR 7.7-26.3%), p < 0.0001. Among neonates without sepsis, the percentages of OLFM4+ neutrophils were lower in the BPD/early death group compared to those without BPD, 11.8% (IQR 6.3-29.0%) versus 32.5% (IQR 18.5-46.1%), p = 0.003, and this retained significance in a multiple logistic regression model that included gestational age, birthweight, and race., Conclusion: This is the first study describing OLFM4+ neutrophils in neonates and it shows that this neutrophil subpopulation is not influenced by gestational age but is elevated in inflammatory conditions such as sepsis and severe chorioamnionitis, and lower percentage at birth is associated with developing bronchopulmonary dysplasia., (© 2022 S. Karger AG, Basel.)

Background: Acute chorioamnionitis contributes to premature birth, and is associated with postbirth complications. How chorioamnionitis impacts neonate's developing immune system has not been well defined., Methods: Blood from extremely preterm infants (≤28 wk gestation) was drawn at the first, second, and fourth week of life. Blood was either left unstimulated or stimulated for 4 h with PMA/ionomycin. mRNA expression of transcription factors in unstimulated cells (RORC, TBET, GATA3, and Forkhead box protein 3 (FOXP3)) and inflammatory cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-5, and IL-6) in unstimulated and stimulated cells were analyzed. Data were analyzed based on the diagnosis of chorioamnionitis, funisitis and bronchopulmonary dysplasia (BPD)., Results: At 1 wk of life, exposure to funisitis, but not maternal chorioamnionitis was associated with an increased expression of RORC and RORC/FOXP3 ratio. These increases in RORC and RORC/FOXP3 ratio were sustained over the 4 wk of follow-up. Leukocytes from infants who developed BPD had increased stimulated and unstimulated IL-4 at the first week of life, but these increases were not sustained over time. In contrast, infants with mild BPD had a sustained decrease in stimulated IL-2., Conclusion: Chorioamnionitis exposure, in particular to funisitis, lead to enhanced Th17-like responses that persist for 4 wk after birth. Infants who later developed BPD did not exhibit a strikingly distinct immune profile., Competing Interests: There is no conflict of interest to be reported.

Objective: To investigate the relationships between serum cytokine concentrations and chorioamnionitis (CAM) and CAM-related bronchopulmonary dysplasia (BPD) in premature infants., Methods: Serum was collected at 0 and 7 days after birth from 36 premature infants born at <32 weeks of gestation. We examined the relationships between 30 cytokine concentrations and CAM, BPD, and other perinatal factors., Results: On day 0, GM-CSF, IL-15, IL-17, IL-2, IL-2R, VEGF, and MIG concentrations were significantly higher in the CAM group (n = 17) than in the non-CAM group (n = 19). These concentrations had decreased by day 7 and were similar in both groups. The IL-12p70 concentration on day 0 was significantly lower in the BPD group (n = 16) than in the non-BPD group (n = 15). BPD incidence was similar between the CAM and non-CAM groups., Conclusions: These data support the hypothesis that intrauterine inflammation is not a primary risk factor for BPD. The immunological environment at birth or soon after, rather than intrauterine fetal inflammation (e.g., CAM), is a primary risk factor for BPD onset in preterm infants. Decreased inflammatory responses are particularly relevant, as indicated by the relationship between BPD and low serum IL-12p70 concentrations on day 0.

Alterations of pulmonary microbiome have been recognized in multiple respiratory disorders. It is critically important to ascertain if an airway microbiome exists at birth and if so, whether it is associated with subsequent lung disease. We found an established diverse and similar airway microbiome at birth in both preterm and term infants, which was more diverse and different from that of older preterm infants with established chronic lung disease (bronchopulmonary dysplasia). Consistent temporal dysbiotic changes in the airway microbiome were seen from birth to the development of bronchopulmonary dysplasia in extremely preterm infants. Genus Lactobacillus was decreased at birth in infants with chorioamnionitis and in preterm infants who subsequently went on to develop lung disease. Our results, taken together with previous literature indicating a placental and amniotic fluid microbiome, suggest fetal acquisition of an airway microbiome. We speculate that the early airway microbiome may prime the developing pulmonary immune system, and dysbiosis in its development may set the stage for subsequent lung disease.

Background: Prenatal or postnatal systemic inflammation can contribute to the development of bronchopulmonary dysplasia (BPD). We investigated whether prenatal intra-amniotic (i.a.) inflammation or early postnatal systemic inflammation can induce BPD in a rat model., Methods: One microgram of lipopolysaccharide (LPS) or vehicle was injected into the amniotic sacs 2 d before delivery (E20). After birth, 0.25 mg/kg of LPS or vehicle was injected into the peritoneum of pups on postnatal day (P)1, P3, and P5. On P7 and P14, peripheral blood (PB), bronchoalveolar lavage fluid (BALF), and lung tissue were obtained and analyzed., Results: Postnatal i.p. injections of LPS significantly increased neutrophil counts in PB and BALF on P7 and P14. Similarly, proinflammatory cytokine and angiogenic factor transcript levels were increased in the lung by i.p. LPS on P7. Alveolar and pulmonary vascular development was markedly disrupted by i.p. LPS on P14. However, pretreatment with i.a. LPS significantly negated the detrimental effects of postnatal i.p. LPS on PB and BALF neutrophil counts and on lung proinflammatory cytokine expression and histopathological changes., Conclusion: Exposure to early postnatal systemic LPS induces BPD, an arrest in alveolarization, in neonatal rats. Preceding exposure to i.a. LPS protects the lungs against BPD triggered by postnatal systemic inflammation.

Background: Chorioamnionitis (CA) is associated with premature delivery and bronchopulmonary dysplasia (BPD). We hypothesize that preterm infants exposed to CA have reduced suppressive regulatory T cells (Treg) and increased non-regulatory T cell pro-inflammatory cytokines, increasing risk for BPD., Objective: To evaluate cord blood CD4(+) T cell regulatory phenotype and pro-inflammatory cytokine production in CA and BPD groups., Study Design: Cord blood mononuclear cells from infants (GA ⩽32 weeks), with or without placental histological evidence of CA (hChorio), were analyzed by flow cytometry. Clinical information was collected by retrospective chart review. Numbers of putative Treg (CD4(+)FoxP3(+)CD25(+)CD127Dim), CD4(+) non-Tregs, and CD4(+) T cell intracellular cytokine content following in vitro stimulation were compared with CA status and oxygen requirement at 36weeks postmenstrual age., Result: Absolute Treg numbers were not different in CA and non-CA exposed samples. However, the infants who developed BPD had a significant decrease in Treg and non-regulatory T cell numbers. Greater IL-6 production was observed in hCA group., Conclusion: A pro-inflammatory CD4(+) T cell status is noted in CA and BPD but the later disease is also associated with decrease in Tregs, suggesting that the development of BPD is marked by distinct inflammatory changes from those of CA exposed infants., (Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Aim: To assess whether the levels of inflammatory and anti-inflammatory proteins in gastric fluid of premature infants shortly after birth are associated with the development of bronchopulmonary dysplasia (BPD)., Methods: Gastric fluid retrieved within 1 h of birth of premature infants (gestational age <29 weeks) was analysed for interleukin (IL)-8, growth-related oncogene (Gro)-α, epithelial cell-derived neutrophil-activating peptide (ENA)-78, IL-1β and Clara cell secretory protein with ELISA., Results: Of 51 enrolled infants, 86% had BPD. Of these, 54% had mild BPD, 30% had moderate BPD and 16% had severe BPD. Clinical chorioamnionitis was associated with high levels of IL-8, Gro-α, Epithelial cell-derived neutrophil-activating peptide-78 (ENA-78) and IL-1β in gastric fluid. Gastric fluid levels of IL-8, Gro-α, ENA-78 and IL-1β were higher in infants with moderate or severe BPD than in those with no or mild BPD. Ligation of the patent ductus arteriosus was associated with the development of moderate or severe BPD. These associations were no longer significant after adjustment for gestational age., Conclusion: The levels of inflammatory mediators in gastric fluid samples retrieved soon after birth from intubated or nonintubated infants can be used to assess the infants' perinatal exposure to inflammatory mediators and its association with neonatal outcome., (© 2011 The Author(s)/Acta Paediatrica © 2011 Foundation Acta Paediatrica.)

The fetal lung vasculature forms in tandem with developing airways. Whereas saccular airway morphogenesis is arrested in bronchopulmonary dysplasia (BPD), the potential vascular phenotype in BPD at this stage of development is less well-understood. As inflammation increases the risk of BPD and induces arrest of saccular airway morphogenesis, we tested the effects of Escherichia coli LPS on fetal mouse lung vascular development. Injecting LPS into the amniotic fluid of Tie2-lacZ endothelial reporter mice at embryonic day 15 stimulated angiogenesis in the saccular stage fetal lung mesenchyme. LPS also increased the number of endothelial cells in saccular stage fetal mouse lung explants. Inflammation appeared to directly promote vascular development, as LPS stimulated pulmonary microvascular endothelial cell angiogenesis, cell migration, and proliferation in vitro. Whereas LPS did not increase expression of VEGF, angiopoietin-1 (Ang-1), Tie2, fetal liver kinase-1 (Flk-1), fms-like tyrosine kinase-1 (Flt-1), PDGFA, PDGFB, heparin-binding EGF-like growth factor (HB-EGF), or connective tissue growth factor (CTGF), LPS did stimulate the production of the angiogenic CC chemokines macrophage inflammatory protein-1α (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1). Both MIP-1α and MCP-1 increased angiogenesis in fetal mouse lung explants. In addition, inhibitory antibodies against MIP-1α and MCP-1 blocked the effects of LPS on fetal lung vascular development, suggesting these chemokines are downstream mediators of LPS-induced angiogenesis. We speculate that an inflammation-mediated surge in angiogenesis could lead to formation of aberrant alveolar capillaries in the lungs of patients developing BPD.

Chorioamnionitis is associated with increased lung and brain injury in premature infants. Ureaplasma is the microorganisms most frequently associated with preterm birth. Whether Ureaplasma-induced antenatal inflammation worsens lung and brain injury is unknown. We developed a mouse model combining antenatal Ureaplasma infection and postnatal oxygen exposure. Intraamniotic Ureaplasma Parvum (UP) increased proinflammatory cytokines in placenta and fetal lungs. Antenatal exposure to UP or broth caused mild postnatal inflammation and worsened oxygen-induced lung injury. Antenatal UP exposure induced central microgliosis and disrupted brain development as detected by decreased number of calbindin-positive and calretinin-positive neurons in the neocortex. Postnatal oxygen decreased calretinin-positive neurons in the neocortex but combined with antenatal UP exposure did not worsen brain injury. Antenatal inflammation exacerbates the deleterious effects of oxygen on lung development, but the broth effects prohibit concluding that UP by itself is a compounding risk factor for bronchopulmonary dysplasia. In contrast, antenatal UP-induced inflammation alone is sufficient to disturb brain development. This model may be helpful in exploring the pathophysiology of perinatal lung and brain injury to develop new protective strategies.

Preterm delivery is frequently preceded by chorioamnionitis, resulting in exposure of the fetal lung to inflammation. We hypothesized that ventilation of the antenatally inflamed lung would result in amplification of the lung injury. Therefore, we induced fetal lung inflammation with intra-amniotic endotoxin (10 mg of Escherichia coli 055:B5) 4 days before premature delivery at 130 days of gestation. Lung function and lung inflammation after surfactant treatment and 4 h of mechanical ventilation were evaluated. Inflammatory cell numbers in amniotic fluid were increased >10-fold by antenatal endotoxin exposure. Antenatal endotoxin exposure had minimal effects on blood pressure, heart rate, lung compliance, and blood gas values. The endotoxin-exposed lungs required higher ventilation pressures. Ventilation did not increase the number of inflammatory cells or the protein in bronchoalveolar lavage fluid of the endotoxin-exposed animals above that measured in endotoxin-exposed fetuses that were not ventilated. IL-1beta, IL-6, and IL-8 mRNA in cells from bronchoalveolar lavage fluid were increased by antenatal endotoxin exposure but not changed by ventilation. IL-1beta and IL-8 protein was increased in lung tissue by 4 h of ventilation. Very little inflammation was induced by ventilation in this premature lamb model of surfactant treatment and gentle ventilation. After lung inflammation was induced by intra-amniotic endotoxin injection, ventilation did not increase lung injury.

Despite improvement in neonatal care, the incidence of bronchopulmonary dysplasia has not decreased over the last decade. Moreover, chronic lung disease still occurs in very premature infants who do not require ventilatory support at birth. This review presents the growing body of epidemiological, experimental and clinical evidence suggesting that the occurrence of an inflammatory reaction triggered in utero or immediately after birth is associated with the subsequent development of chronic lung disease. However, stimulators of inflammation or specific proinflammatory cytokines may also have beneficial on lung maturation. How proinflammatory mediators interfere with lung maturation and alveolarization needs to be better understood in order to pave the way for new immunomodulatory strategies to prevent chronic lung disease in very preterm infants.