Elucidation of the roles of PTN and ISG15 in RSV cytopathogenesis: possible biomarkers of severe disease?
Respiratory syncytial virus (RSV) infection is the most common cause of severe lower respiratory tract infection in infants under two years old. Sparking seasonal epidemics, RSV contributes to around 20,000 admissions to hospital in the UK annually and many require intensive care support. Peak incidences of severe RSV disease occur between 6 weeks and 6 months of age. Despite over 60 years of research since its discovery, no RSV vaccine or specific therapy exists. The only preventative strategy against RSV is the monoclonal antibody palivizumab, which is very expensive and, accordingly, only available to those infants at known high risk of severe RSV disease, including preterm infants and those with congenital heart conditions or bronchpulmonary dysplasia. As the majority of infants hospitalised with RSV have no known predisposing risk factor for severe illness, the benefits of palivizumab for managing the impact of RSV is very restricted. Considerable challenges in performing research in vulnerable young infants has slowed progress in RSV research. Animal models only partially reflect RSV‐human host interactions and thus extrapolation of results from these models to human responses is of limited value. Study of autopsy specimens from infants who succumbed to RSV infection identified that the infection is restricted primarily to the airway epithelium. As such, research aimed at detecting innate immune responses of human airway epithelium to RSV is likely to yield crucial insights into the cytopathogenesis of this infection. It has previously been demonstrated that an ex‐vivo/in‐vitro model of human airway epithelium/RSV interactions, termed well‐differentiated primary paediatric nasal epithelial cell cultures (WD‐PNECs), provides a reasonable surrogate for in vivo responses. Because of the strong association of severe RSV disease with prematurity and young age, the first part of this thesis describes the utilisation of the WD‐PNEC model to establish and 6 characterise unique nasal samples from preterm and term infants at birth and repeated at one‐year old. This work is the first description of morphologically and physiologically authentic WD‐PNEC cultures generated from term and preterm newborn infants and as such represents an exceptional opportunity to study RSV‐human host interactions in early life. We found that newborn term‐ and preterm‐derived WD‐PNECs were morphologically indistinguishable under light or fluorescent microscopy analysis. However, interestingly, newborn WD‐PNECs demonstrated significantly higher proportions of goblet cells compared to one‐year repeat WD‐PNECs. This finding indicates the possibility of increased mucous production in newborn infants, which may, in part, explain their susceptibility to more severe RSV disease. Importantly, we demonstrated nasal sampling to be a safe, minimally invasive method performed consistently with high rates of success. Furthermore, we were also able to successfully freeze, thaw, and subsequently differentiate the nasal epithelial cells. This confirmed the exciting possibility of storing newborn ‘‘naive’’ airway epithelial cells (AECs) indefinitely for use in subsequent experimentation, e.g., once clinical phenotypes, like severe RSV or asthma, have been established. We next sought to establish if differential RSV‐induced innate immune responses of airway epithelial cells could account, at least in part, for the increased susceptibility of preterm and very young infants to severe RSV disease. To investigate this, we infected (or mock‐infected) WD‐PNEC cultures established from term and preterm infants at birth and repeated at one‐year‐old. No significant differences in cytopathology or viral growth kinetics were evident in WD‐PNECs derived from any cohort following RSV infection. However, crucially, we observed significantly higher secretion of interferon 1 (IL‐29) (P<0.01), IP‐10 (CXCL‐10) (P<0.05) and RANTES (CCL‐5) (P<0.05) following RSV infection of one‐year‐derived WD‐PNEC cultures compared to newborn‐derived cultures. These novel findings suggest airway 7 epithelium innate immune responses to RSV are less robust in newborn infants compared to older infants, which may contribute to the increased susceptibility of very young infants to severe RSV‐related disease. This is the first report of age‐related differences in airway epithelial cell innate immune responses. Previous work performed in RSV‐infected WD‐PNECs derived from infants with histories of severe versus mild RSV disease identified differential expression in a number of genes. Two of these genes, interferon‐stimulate gene 15 (isg15) and pleiotrophin (ptn), were of particular interest based on published work identifying the anti‐viral role of ISG15 in a number of viral illnesses and the established interaction of PTN with the recently identified RSV co‐receptor/entry factor, nucleolin. Our findings on the role of PTN are particularly exciting. We found differential expression of PTN in infants at greater risk of severe RSV disease compared to older infants and in those with a history of severe compared to mild RSV disease. These findings suggest a potentially vital role for PTN in innate immune defence against RSV. Consistent with such a role, we demonstrated that PTN has anti‐RSV activity in vitro, mediated via interaction with cell surface nucleolin. Taken together, these novel findings suggest that relatively lower endogenous expression of PTN expression may explain, in part, the increased susceptibility of some infants to severe RSV disease. Finally, we established that isg15 mRNA expression is increased in both newborn‐ and 1 year old infant‐derived WD‐PNECs following RSV infection, with similar levels of expression between term‐ versus preterm‐derived WD‐PNECs. We also demonstrated an anti‐viral effect of ISG15 in vitro, in which increased RSV infection was evident following isg15knockdown in BEAS‐2B cells pre‐treated with IFN‐1 (IL 29) compared to control siRNA‐transfected cells. In addition, we found that RSV antagonizes ISG15 expression in individual infected cells compared to surrounding non‐infected cells within WD‐PBECs. Our data suggest that ISG15 acts as an innate antiviral molecule against RSV and differential 8 expression of ISG15 in infant airways following infection may contribute to susceptibility to severe RSV disease. In summary, this thesis reports the exciting discovery of an anti‐viral role for PTN in RSV infection and highlights the biomarker potential for both PTN and ISG15 in identifying individuals at increased risk of severe RSV disease. This work also adds to our understanding of early life innate immune responses to RSV infection and provides an innovative model for identifying new potential therapeutic targets and strategies in the management of RSV.
RSV, Nasal epithelium, innate immunity, neonates
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