In vitro characterisation of the neutrophil in respiratory syncytial virus bronchiolitis

Abstract

Background: Respiratory Syncytial Virus (RSV) is a common cause of bronchiolitis during infancy; virtually all children are infected with this virus by the age of 2 years. Although the majority have a self-limiting, mild infection, 1- 3% have disease severe enough to warrant hospital admission. These children have an abundance of neutrophils (>80% of bronchoalveolar lavage cells) within their airways. There is however, a lack of detailed investigation into their role in viral infection. Our group has previously shown RSV associated with ex vivo neutrophils from infected infants. To explore this observation further, I have modelled and visualised the RSV-neutrophil interaction, hypothesising that neutrophils play a role in the innate viral response in the airway. Aims: The aims of this thesis were to (i) establish an in vitro model of RSVneutrophil interaction, (ii) determine whether RSV is taken up into the neutrophil, (iii) determine if RSV productively replicates inside the neutrophil, (iv) compare neutrophil-RSV interaction in adult and infant neutrophils and (v) investigate the response of the neutrophil to RSV. Methods: An in vitro model of RSV-neutrophil interaction was established using highly purified adult and cord blood neutrophils. Neutrophils were analysed by quantitative RT-PCR, western blot, and by confocal microscopy using indirect immunocytochemistry. In addition, ex vivo BAL neutrophils from RSV infected infants were examined by confocal microscopy. Following validation of this model, confirming the presence of virus within the neutrophil, a human gene microarray was performed. Results: Interaction of neutrophils with RSV was modelled in vitro and demonstrated by PCR analysis and western blot. RSV N gene expression revealed maximal neutrophil uptake at 4 hours. A time course over 20 hours showed no increase in RSV expression, implying that active RSV replication was not occurring. Confocal microscopy revealed internalisation of RSV, with virus distributed throughout the cytoplasm. Imaging of ex vivo neutrophils extracted from bronchoalveolar lavage of RSV-infected infants showed a similar distribution. RSV induced significant change in neutrophil gene expression with over 1000 differentially expressed genes being identified. Pathway analysis revealed an RNA virus specific transcriptional response. Results for key molecules were subsequently validated by RT-PCR. Conclusions: Neutrophils do not allow RSV to replicate within them. In contrast, following interaction with RSV, they alter their gene expression profiles through the modulation of interferon responses and other pathways, thereby inhibiting viral replication. Evidence presented in this thesis supports a novel role for neutrophils as innate cells at the frontline of antiviral immunity in RSV disease.