Clinical applicability of HMGB1 as biomarker and therapeutic target in Respiratory Syncytial Virus infection

Respiratory syncytial virus (RSV), an enveloped, non-segmented, negative-sense RNA virus belonging to the Paramyxoviridae family, is the most common respiratory pathogen in infants and young children worldwide [1]. Studies have suggested a strong association between RSV and lower respiratory tract infection during infancy, and subsequent development of recurrent airway lability in childhood [2, 3]. Nevertheless, a causal link between RSV infection and chronic airway dysfunction remains a matter of debate. Thus, the identification and validation of novel biomarkers, that would allow to predict and monitor the severity and clinical course of RSV infection, could pave the way for research efforts aimed at establishing a causative relationship between early-life RSV infection and childhood airway dysfunction. On this regard, host-derived "danger-associated molecular patterns" (DAMPs) contribute to innate immune responses and serve as markers of disease progression and severity for inflammatory and infectious diseases. There is accumulating evidence that generation of DAMPs such as oxidized phospholipids and high-mobility-group box 1 (HMGB1) during RSV virus infection leads to acute lung injury [4]. However, changes in systemic HMGB1 kinetics during the course of RSV infection, both in vitro and in vivo studies, have yet to establish an association of HMGB1 release with RSV infection. To this end, we used HMGB1 gene and protein expression in infected human bronchial epithelial cells (HBEC) in vitro and in the lungs of rat pups RSV-infected in the neonatal period. Furthermore, we selectively inhibited HMGB1 activity in RSV-infected cells using glycyrrhizin, a natural HMGB1 antagonist, and studied its effects on viral replication. In an experimental model, RSV was able to spread across the placenta from the respiratory tract of the mother to the rat pups, and it was also detected postnatally in the lungs throughout development and into adulthood [3]. Vertical RSV infection was associated with dysregulation of critical neurotrophic pathways during fetal development, leading to aberrant innervation and increased airway reactivity after postnatal reinfection with RSV [3]. Supporting the idea that HMGB1 could probably be involved in the development of vertically transmitted RSV infection, the HMGB1 behaviour was investigated in pregnant rats inoculated intratracheally at midterm using recombinant RSV expressing red fluorescent protein (RFP). In light of these results, the HMGB1’s role has been evaluated in serum cord blood in a population of neonates, assessing the potential utility of this alarmin also in humans. Following the description of the first neonatal case of human RSV infection consistent with vertical transmission from a previously infected mother to her unborn son, we have determined the serologic evidence of anti-RSV immunity in fetal cord blood of offspring with a maternal history of respiratory illness occurring during the third trimester of pregnancy, and also characterized the postnatal clinical outcomes associated with RSV seropositivity. Finally, the RSV-HMGB1 relationship in vertically-infected neonates was also investigated. The last part of this PhD thesis attempts to summarize the clinical manifestations of this infection in order to provide the reader with the background information necessary to fully appreciate the many challenges presented by the clinical management of young children with bronchiolitis. Also, an evidenced-based review of the pharmacologic strategies has been provided, intentionally omitting highly experimental approaches not yet tested in clinical trials and, therefore, not likely to become available in the foreseeable future. Aims The following were specific aims of the study: 1. To investigate the role of HMGB1 for the establishment of productive RSV infection. To this end, we studied its gene and protein expression in human infected bronchial epithelial cells infected in vitro and in the lungs of rat pups RSV-infected in the neonatal period. Furthermore, we selectively inhibited HMGB1 activity in RSV-infected cells using glycyrrhizin and studied its effects on viral replication (Paper 1). 2. We described a case of RSV infection documented at birth in the peripheral blood of a newborn with onset of severe respiratory distress immediately after delivery from a mother with serological and clinical evidence of RSV infection during pregnancy (Paper 2). 3. In order to further investigate the HMGB1’s role, this study evaluated serum cord blood HMGB1 levels in a population of neonates, to investigate the potential utility of alarmin as a novel marker, and its connection with mode of delivery (Paper 3). 4. To determine serologic evidence of anti-RSV immunity in fetal cord blood of offspring with a maternal history of respiratory illness occurring during the third trimester of pregnancy, and also characterized the postnatal clinical outcomes associated with RSV seropositivity (Paper 4). 5. To systematically suggest current guidelines on the management of bronchiolitis, considering the differences and similarities between them and offering recommendations for further research (Paper 5).