Supplementary MaterialsS1 Table: Plasma TNF and IL-6 concentrations. Ventilation-induced pulmonary leukocyte recruitment was greater in hAEC-treated lambs than in vehicle-treated lambs. Lung IL-1 and IL-6 mRNA expression was higher in vehicle- and hAEC-treated ventilated lambs than in controls but IL-8 mRNA levels were greater than control only in vehicle-treated ventilated lambs. Rabbit Polyclonal to Caspase 7 (Cleaved-Asp198) Numbers of CD44+ and CD21+ lymphocytes and macrophages from the lungs were altered in vehicle- and hAEC-treated ventilated lambs. Numbers of CD8+ macrophages were lower in hAEC-treated ventilated lambs than in vehicle-treated ventilated lambs. Indices of systemic inflammation were not different between vehicle- and hAEC-treated lambs. Human amnion epithelial cells modulate the pulmonary inflammatory response to ventilation in preterm lambs, and reduce acute lung injury. Immunomodulatory effects of hAECs reduce lung injury in preterm neonates and may protect against longer-term respiratory disease. CI-1040 inhibitor Introduction Assisted or mechanical ventilation at birth is required by about 1 in 10 neonates [1], and is often provided in the form of intermittent positive pressure ventilation [2]. Such ventilation, while needed, causes inadvertent lung injury and long-term respiratory disease in some babies [3]. Such injury may contribute to development of bronchopulmonary dysplasia (BPD) [4]. Mechanical ventilation can result in airway inflammation and elevated pulmonary mRNA expression of inflammatory mediators, including serum amyloid A3 (SAA-3), interleukin (IL)-1 and IL-6, within 1 h of ventilation [5, 6]. Activation of circulating CD4+ and CD8+ T cells occurs in preterm infants with BPD [7], however, the absolute lymphocyte number is usually decreased, owing to a decrease in circulating CD4+ cells [8]. This initial inflammatory response to ventilation may influence development of lung injury [9, 10]. Prevention or attenuation of early detrimental responses to mechanical ventilation, through immunomodulation, may therefore reduce the risk of development of BPD [11]. One potential immunomodulatory therapy for BPD is usually administration of stem cellClike human amnion epithelial cells (hAECs). Epithelial cells of the amnion can differentiate into ectodermal, mesodermal and endodermal lineages [12C14], and into lung epithelialClike cells [15, 16] and [17]. Administration of hAECs reduces inflammatory gene expression in lung tissue and prevent inflammation-induced changes in fetal lung development induced by either intra-amniotic injection of lipopolysaccharide [18] or a 12-h period of mechanical ventilation [17] in sheep. Human AECs can also moderate abnormal lung development in hyperoxic neonatal mice [19], but their ability to influence ventilation-induced lung inflammation and injury in neonates has not been examined. We hypothesised that administration of hAECs to ventilated preterm lambs CI-1040 inhibitor would reduce lung injury, and pulmonary and systemic inflammatory responses. Materials and methods Human amnion epithelial cell isolation and preparation Human procedures (including the consent process) were approved by the Monash Health Human Research and Ethics Committee (ref #: MUHREC-CF13/2144-2013001109). Placentae were obtained from women with uncomplicated pregnancies who provided written consent before elective caesarean section at term (37C40 weeks). Amnion epithelial cells were isolated as previously described [20]. Cell counts and viability were assessed by trypan blue exclusion prior to and after cryopreservation. For treatment of preterm lambs, hAECs from three donors were thawed and combined, washed, counted and assessed for viability, then resuspended at 30×106 cells/ml in sterile phosphate-buffered saline (PBS) for administration. Animal experiments Animal experimentation was approved by the relevant Monash University Animal Ethics Committee (ref #: MMCA/2012/10). Experimental animals were obtained from a timed CI-1040 inhibitor mating program managed by the Monash Animal Research Platform, Monash University. They were transported from an open filed environment to indoor housing (12 h light/dark cycle) in individual pens at least 1 week before any experimental intervention. Ewes remained in constant visual contact of other sheep, had continual access to water and were fed a pelleted diet, supplemented with chaff and/or lucerne hay, twice daily. For the laparotomy procedure, ewes bearing twins at 126 1 (mean SD) days of gestation (term ~147 days) were anaesthetised (2% isoflurane in O2, delivered by positive-pressure ventilation, after induction by IV injection of 20mg/kg sodium thiopentone)..