Journal Article

547 Constitutive differences in adult and infant neutrophil transepithelial migration in an in-vitro model of RSV bronchiolitis

Robinson, Elisabeth, Smith, Claire, Smyth, Rosalind, Sawhney, Shyam

2022

Overview

AimsRespiratory Syncytial Virus (RSV) is the leading infectious cause of hospitalisation in infants. Neutrophilic infiltration of the airways has been observed in infants with severe infection clinically, but whether these neutrophils enhance recovery from disease or contribute to its pathology is unknown. Adults do not suffer the same clinical syndrome of bronchiolitis though are still susceptible to RSV infection. This study aims to examine differences in adult and neonatal neutrophil migration across RSV infected airway epithelial cells (AECs), using a novel sophisticated in vitro model, and measure neutrophil apoptosis, cellular markers and secreted markers of neutrophil activation key to clinical pathology.MethodsCord blood neutrophils were collected following uncomplicated term caesarean deliveries at University College Hospital, London. Adult neutrophils were collected from healthy donors at UCL Institute of Child Health. Full research ethics committee approval was provided by Bloomsbury Research Ethics committee (Project ID Number: 14/LO/0863) and UCL Research Ethics Committee (4735/002). Neutrophils were purified using negative immunoselection (StemCell).Neutrophil trans-epithelial migration was modelled using a novel in-vitro system1 (figure 1). Primary human airway epithelial cells (AECs) were grown at air liquid interface for 28 days then infected with RSV A2 for 72hrs. Uninfected AECs inoculated with sterile media (Mock) and uninfected AECs with RSV infected AEC supernatant apically (RSV Sup) were used as controls. Purified neutrophils were then added to the basolateral side of AECs and allowed to migrate for one hour.Neutrophils adhered to AECs, those basolaterally and apically were collected for analysis. Neutrophil numbers were determined by CD11b-APC positivity absolute count in a measured volume via flow cytometry. Viability, death and apoptosis were determined by co-staining with AnnexinV-FITC and Propidium Iodide. Statistical analysis was performed using RStudio v.1.2.5.ResultsSignificantly greater numbers of cord blood neutrophils (336,684 ±48129) (Mean ±SEM) migrated to the apical compartment in 1 hours migration, in comparison to adult neutrophils (56,586 ±16139) in the same conditions (p=<0.0001). Conversely, greater numbers of adult neutrophils (171,641 ±31632) remained basolaterally in comparison to cord blood neutrophils (23,870 ±3359) in the same conditions (p=<0.05). A greater proportion of apical and adherent cord blood neutrophils were apoptotic (43.3% ±10.68) in comparison to adult (9.49% ±2.7) (p=0.0024), although despite this higher proportion of apoptosis there were still greater numbers of viable cord blood neutrophils apical to RSV infected AECs in comparison to adult. Finally, we showed that although cord blood neutrophils displayed greater levels of CD11b, CD64, NE and MPO expression unstimulated, they did not upregulate expression in response to migration in the same order as adult neutrophils.Abstract 547 Figure 1Schematic of experimental protocol including neutrophil collection, neutrophil purification and the neutrophil trans-epithelial migration model1Abstract 547 Figure 2Numbers and viability of neutrophils after transpithelial migration across RSV infected ciliated AECsConclusionGreater numbers of neonatal neutrophils migrate across RSV infected AECs in comparison to adult, of which a greater proportion undergo apoptosis, and which show limited upregulation of activation markers in comparison to adult. These differences may facilitate a greater neutrophilic infiltrate to the lung in response to RSV infection in infants, compared with adults and suggests neutrophil modulating treatments could be beneficial as novel therapies for RSV infection.• β2 integrin LFA1 mediates airway damage following neutrophil trans-epithelial migration during RSV infection, Herbert et al

• Publisher: BMJ Publishing Group Ltd and Royal College of Paediatrics and Child Health,BMJ Publishing Group LTD
• Subject: Abstracts / Adults / Apoptosis / Bronchopneumonia / CD11b antigen / Cell activation / Child Health / Cord blood / Epithelial cells / Ethics / Flow cytometry / Infants / Infections / Iodides / Leukocyte migration / Leukocytes (neutrophilic) / Neonates / Neutrophils / Pathology / Pediatrics / Propidium iodide / Research ethics / Respiratory syncytial virus / Respiratory tract / Statistical analysis / Young Children