Early administration of umbilical cord blood cells following brief high tidal volume ventilation in preterm sheep: a cautionary tale.

BACKGROUND: Umbilical cord blood (UCB) cells are a promising treatment for preterm brain injury. Access to allogeneic sources of UCB cells offer the potential for early administration to optimise their therapeutic capacities. As preterm infants often require ventilatory support, which can contribute to preterm brain injury, we investigated the efficacy of early UCB cell administration following ventilation to reduce white matter inflammation and injury. METHODS: Preterm fetal sheep (0.85 gestation) were randomly allocated to no ventilation (SHAM; n = 5) or 15 min ex utero high tidal volume ventilation. One hour following ventilation, fetuses were randomly allocated to i.v. administration of saline (VENT; n = 7) or allogeneic term-derived UCB cells (24.5 ± 5.0 million cells/kg; VENT + UCB; n = 7). Twenty-four hours after ventilation, lambs were delivered for magnetic resonance imaging and post-mortem brain tissue collected. Arterial plasma was collected throughout the experiment for cytokine analyses. To further investigate the results from the in vivo study, mononuclear cells (MNCs) isolated from human UCB were subjected to in vitro cytokine-spiked culture medium (TNFα and/or IFNγ; 10 ng/mL; n = 3/group) for 16 h then supernatant and cells collected for protein and mRNA assessments respectively. RESULTS: In VENT + UCB lambs, systemic IFNγ levels increased and by 24 h, there was white matter neuroglial activation, vascular damage, reduced oligodendrocytes, and increased average, radial and mean diffusivity compared to VENT and SHAM. No evidence of white matter inflammation or injury was present in VENT lambs, except for mRNA downregulation of OCLN and CLDN1 compared to SHAM. In vitro, MNCs subjected to TNFα and/or IFNγ displayed both pro- and anti-inflammatory characteristics indicated by changes in cytokine (IL-18 & IL-10) and growth factor (BDNF & VEGF) gene and protein expression compared to controls. CONCLUSIONS: UCB cells administered early after brief high tidal volume ventilation in preterm fetal sheep causes white matter injury, and the mechanisms underlying these changes are likely dysregulated responses of the UCB cells to the degree of injury/inflammation already present. If immunomodulatory therapies such as UCB cells are to become a therapeutic strategy for preterm brain injury, especially after ventilation, our study suggests that the inflammatory state of the preterm infant should be considered when timing UCB cells administration.

Postnatal steroids as lung protective and anti-inflammatory in preterm lambs exposed to antenatal inflammation.

BACKGROUND: Lung inflammation and impaired alveolarization precede bronchopulmonary dysplasia (BPD). Glucocorticoids are anti-inflammatory and reduce ventilator requirements in preterm infants. However, high-dose glucocorticoids inhibit alveolarization. The effect of glucocorticoids on lung function and structure in preterm newborns exposed to antenatal inflammation is unknown. We hypothesise that postnatal low-dose dexamethasone reduces ventilator requirements, prevents inflammation and BPD-like lung pathology, following antenatal inflammation. METHODS: Pregnant ewes received intra-amniotic LPS (E.coli, 4 mg/mL) or saline at 126 days gestation; preterm lambs were delivered 48 h later. Lambs were randomised to receive either tapered intravenous dexamethasone (LPS/Dex, n = 9) or saline (LPS/Sal, n = 10; Sal/Sal, n = 9) commencing <3 h after birth. Respiratory support was gradually de-escalated, using a standardised protocol aimed at weaning from ventilation towards unassisted respiration. Tissues were collected at day 7. RESULTS: Lung morphology and mRNA levels for inflammatory mediators were measured. Respiratory support requirements were not different between groups. Histological analyses revealed higher tissue content and unchanged alveolarization in LPS/Sal compared to other groups. LPS/Dex lambs exhibited decreased markers of pulmonary inflammation compared to LPS/Sal. CONCLUSION: Tapered low-dose dexamethasone reduces the impact of antenatal LPS on ventilation requirements throughout the first week of life and reduces inflammation and pathological thickening of the preterm lung IMPACT: We are the first to investigate the combination of antenatal inflammation and postnatal dexamethasone therapy in a pragmatic study design, akin to contemporary neonatal care. We show that antenatal inflammation with postnatal dexamethasone therapy does not reduce ventilator requirements, but has beneficial maturational impacts on the lungs of preterm lambs at 7 days of life. Appropriate tapered postnatal dexamethasone dosing should be explored for extuabtion of oxygen-dependant neonates.

Airway smooth muscle thickness and contraction are enhanced by intra-amniotic lipopolysaccharide in an ovine model of premature birth.

Abnormalities of the airway smooth muscle (ASM) layer in asthma may develop before birth. We hypothesize that antenatal inflammation causes physiological abnormalities of the ASM that predisposes asthma. This study determined the short-term effects of antenatal inflammation on the developing ASM. Fourteen pregnant ewes were randomly assigned to one of three groups. Fetal lambs were exposed to intra-amniotic injections of lipopolysaccharide (LPS, n = 4) or saline (controls; n = 5) at 127 days' gestational age (GA). Preterm lambs were surgically delivered at 129 days' GA and received intensive care for 7 days before euthanasia. Naïve fetal controls (n = 5) were delivered and euthanized at 136 days' GA. ASM force to acetylcholine was measured in bronchial rings and normalized to ring length (tension) and ASM cross-sectional area (stress). Airway narrowing (% volume) to acetylcholine was assessed in bronchial segments. Fetal controls were structurally and functionally similar to saline-exposed lambs. Compared with saline, LPS-exposed lambs had increased macrophages in lung tissue (P = 0.0002) and interleukin-8 in alveolar wash (P = 0.003). LPS exposure increased ASM thickness (P = 0.005), airway narrowing (P = 0.003), ASM tension (P = 0.0002), and contractile stress (P < 0.0001). Notably, LPS-exposed lambs were more dependent on mechanical ventilation, and both LPS (P < 0.001) and ventilation (P = 0.012) were independent factors in increasing ASM stress. Only LPS independently increased ASM thickness (P = 0.045). Results indicate that antenatal exposure to LPS and subsequent mechanical ventilation promotes intrinsic changes to the ASM that enhances bronchoconstriction. If persistent into postnatal life, these developmental abnormalities may contribute to the known association between chorioamnionitis and asthma.NEW & NOTEWORTHY Abnormalities of the airway smooth muscle (ASM) layer in asthma may develop before birth. Using an ovine model of antenatal inflammation, we demonstrate thickening and increased contraction of the premature ASM layer. If such physiological abnormalities persist throughout postnatal life, this represents a predisposition to an asthma diagnosis.

Investigating Pathways of Ventilation Induced Brain Injury on Cerebral White Matter Inflammation and Injury After 24 h in Preterm Lambs.

Initiation of respiratory support in the delivery room increases the risk and severity of brain injury in preterm neonates through two major pathways: an inflammatory pathway and a haemodynamic pathway. The relative contribution of each pathway on preterm brain injury is not known. We aimed to assess the role of the inflammatory and haemodynamic pathway on ventilation-induced brain injury (VIBI) in the preterm lamb. Fetal lambs (125 ± 1 day gestation) were exteriorised, instrumented and ventilated with a high tidal-volume (VT) injurious strategy for 15 min either with placental circulation intact to induce the inflammatory pathway only (INJINF; n = 7) or umbilical cord occluded to induce both the inflammatory and haemodynamic pathways (INJINF+HAE; n = 7). Sham controls were exteriorised but not ventilated (SHAM; n = 5) while unoperated controls (UNOP; n = 7) did not undergo fetal instrumentation. Fetuses were returned in utero following intervention and the ewe allowed to recover. Arterial blood gases and plasma were sampled periodically. Twenty-four hours following intervention, lambs were delivered and maintained on non-injurious ventilation for ∼40 min then brains were collected post-mortem for immunohistochemistry and RT-qPCR to assess inflammation, vascular pathology and cell death within white matter regions. Compared to INJINF lambs, INJINF+HAE lambs achieved a consistently higher VT during injurious ventilation and carotid blood flow was significantly lower than baseline by the end of ventilation. Throughout the 24 h recovery period, systemic arterial IL-6 levels of INJINF+HAE lambs were significantly higher than SHAM while there was no difference between INJINF and SHAM animals. At 24 h, mRNA expression levels of pro-inflammatory cytokines, tight junction proteins, markers of cell death, and histological injury indices of gliosis, blood vessel protein extravasation, oligodendrocyte injury and cell death were not different between groups. Injurious ventilation, irrespective of strategy, did not increase brain inflammation or injury 24 h later when compared to control animals. However, the haemodynamic pathway did influence carotid blood flow adaptations during injurious ventilation and increased systemic arterial IL-6 that may underlie long-term pathology. Future studies are required to further characterise the pathways and their long-term effects on VIBI.

Therapeutic Targeting of Endosome and Mitochondrial Reactive Oxygen Species Protects Mice From Influenza Virus Morbidity.

There is an urgent need to develop effective therapeutic strategies including immunomodulators to combat influenza A virus (IAV) infection. Influenza A viruses increase ROS production, which suppress anti-viral responses and contribute to pathological inflammation and morbidity. Two major cellular sites of ROS production are endosomes via the NOX2-oxidase enzyme and the electron transport chain in mitochondria. Here we examined the effect of administration of Cgp91ds-TAT, an endosome-targeted NOX2 oxidase inhibitor, in combination with mitoTEMPO, a mitochondrial ROS scavenger and compared it to monotherapy treatment during an established IAV infection. Mice were infected with IAV (Hkx31 strain; 104PFU/mouse) and 24 h post infection were treated with Cgp91ds-TAT (0.2 mg/kg), mitoTEMPO (100 µg) or with a combination of these inhibitors [Cgp91ds-TAT (0.2 mg/kg)/mitoTEMPO (100 µg)] intranasally every day for up to 2 days post infection (pi). Mice were euthanized on Days 3 or 6 post infection for analyses of disease severity. A combination of Cgp91ds-TAT and mitoTEMPO treatment was more effective than the ROS inhibitors alone at reducing airway and neutrophilic inflammation, bodyweight loss, lung oedema and improved the lung pathology with a reduction in alveolitis following IAV infection. Dual ROS inhibition also caused a significant elevation in Type I IFN expression at the early phase of infection (day 3 pi), however, this response was suppressed at the later phase of infection (day 6 pi). Furthermore, combined treatment with Cgp91ds-TAT and mitoTEMPO resulted in an increase in IAV-specific CD8+ T cells in the lungs. In conclusion, this study demonstrates that the reduction of ROS production in two major subcellular sites, i.e. endosomes and mitochondria, by intranasal delivery of a combination of Cgp91ds-TAT and mitoTEMPO, suppresses the severity of influenza infection and highlights a novel immunomodulatory approach for IAV disease management.

Influenza A virus causes maternal and fetal pathology via innate and adaptive vascular inflammation in mice.

Influenza A virus (IAV) infection during pregnancy causes severe maternal and perinatal complications, despite a lack of vertical transmission of IAV across the placenta. Here, we demonstrate a significant alteration in the maternal vascular landscape that underpins the maternal and downstream fetal pathology to IAV infection in mice. In IAV infection of nonpregnant mice, the local lung inflammatory response was contained to the lungs and was self-resolving, whereas in pregnant mice, virus dissemination to major maternal blood vessels, including the aorta, resulted in a peripheral "vascular storm," with elevated proinflammatory and antiviral mediators and the influx of Ly6Clow and Ly6Chigh monocytes, plus neutrophils and T cells. This vascular storm was associated with elevated levels of the adhesion molecules ICAM and VCAM and the pattern-recognition receptors TLR7 and TLR9 in the vascular wall, resulting in profound vascular dysfunction. The sequalae of this IAV-driven vascular storm included placental growth retardation and intrauterine growth restriction, evidence of placental and fetal brain hypoxia, and increased circulating cell free fetal DNA and soluble Flt1. In contrast, IAV infection in nonpregnant mice caused no obvious alterations in endothelial function or vascular inflammation. Therefore, IAV infection during pregnancy drives a significant systemic vascular alteration in pregnant dams, which likely suppresses critical blood flow to the placenta and fetus. This study in mice provides a fundamental mechanistic insight and a paradigm into how an immune response to a respiratory virus, such as IAV, is likely to specifically drive maternal and fetal pathologies during pregnancy.

Bronchopulmonary dysplasia: Pathophysiology and potential anti-inflammatory therapies.

Inflammation of the preterm lungs is key to the pathogenesis of bronchopulmonary dysplasia (BPD), whether it arises as a consequence of intrauterine inflammation or postnatal respiratory management. This review explores steroidal and non-steroidal therapies for reducing neonatal pulmonary inflammation, aimed at treating or preventing BPD.