This project has been designed to investigate the mechanisms of susceptibility to respiratory syncytial virus (RSV) infection and disease progression. RSV is the leading viral respiratory cause of hospitalization in infants and young children in the United States and in the world. The reason why some previously healthy infants develop LRI (bronchiolitis and pneumonia) while others remain asymptomatic or only develop upper respiratory tract symptoms after RSV infection is not well understood. Evidence exists that the degree of previous injury of the lung parenchyma in small infants could play a role in disease severity, as children with chronic lung disease are at high risk of RSV LRI. However, the majority of hospitalizations occur in previously healthy infants. Another potentially important factor that can cause lung injury during RSV LRI is innate immunity. The pulmonary infiltration during RSV LRI is composed overwhelmingly by neutrophils and macrophages and damage to the small airways (10-300 microns) affected by the virus could easily cause debris accumulation in the lumen, inflammation and edema of the small airways, and compromise ventilation. Further, most infants with RSV-associated wheezing do not respond to b2-bronchodilators, but may benefit from inhaled a-agonists that decrease edema/inflammation. Additionally, high levels of CXC chemokines (particularly MIP-1a, MCP-1 and IL-8) have been associated with increased RSV disease severity. ? ? Several studies have determined that RSV stimulates ROS production and lipid peroxidation in lung cells and tissues, and more importantly, antioxidant treatment has been suggested to protect against the pathogenesis of RSV disease by a recent study. Considering that airway epithelium is not only the major source of antioxidant enzymes/defense proteins but also the primary host target for RSV, it is important to determine the role of cellular antioxidant defenses in RSV pathogenesis. Suppression or lack of ARE-dependent antioxidant expression in Nrf2-/- mice relative to Nrf2+/+ mice has exacerbated lung inflammation and injury induced by a number of pro-inflammatory agents. Based on these findings, we performed a study to test the hypothesis that Nrf2-/- mice have increased susceptibility to airway oxidative injury and inflammation caused by RSV, relative to Nrf2+/+ mice. We found that upper airway (nasal) epithelial injury and lower airway bronchopulmonary inflammation, epithelial cell loss and hyperplasia/mucous cell metaplasia were significantly greater in Nrf2-/- mice than in Nrf2+/+ mice. Significantly attenuated lung viral clearance correlated with elevated protein and lipid oxidation, potentiated nuclear AP-1 and NF-kB, and suppressed antioxidant induction demonstrated increased susceptibility and oxidative stress of Nrf2-/- mice in response to RSV. To support further the anti-viral activity for Nrf2, Nrf2+/+ mice were pretreated orally with a potent phytochemical Nrf2-ARE inducer (sulforaphane) or PBS before RSV infection. We found that mice pretreated with sulforaphane had significantly lower RSV-induced inflammation and disease probably through pre-induction of Nrf2 and ARE responsive genes before infection. Results support a key role for oxidant stress in the pathogenesis of RSV-induced airway disease and the importance of Nrf2-ARE pathway in host defense against RSV.? ? In collaboration with Dr. Fernando Polack (Johns Hopkins University) and the Infant Foundation in Buenos Aires, Argentina we recruited a prospective cohort of children aged 0 9 months of age from 5 hospitals in Buenos Aires. This research was supported through a contract funded by the NIEHS intramural program. The primary clinical outcome is severity of disease (O2 saturation < 93%) and the secondary outcomes are RSV titer and Th2 polarization (blood lymphocytes are isolated and challenged with pro-inflammatory agonists including LPS). From 2003-2005, we recruited approximately 800 children to the study: 400 infected with RSV (240 with severe disease, 160 with mild disease) and 400 controls (uninfected). Our initial genetic analyses have focused on the role of functional SNPs in TLR4 (Asp299Gly and Thr399Ile), and NRF2 (-653A/G, -651G/A and -617C/A). These genes have been identified as candidate susceptibility genes for RSV infection in mice, and TLR4 has also been associated with RSV infection in children. In our initial analyses, we found that the -651G/A SNP was only found in severe RSV cases and conferred greater risk of disease relative to the wild type (OR: 1.9; CI: 1.4, 2.7). We also found that children heterozygous for the Asp299Gly/Thr399Ile mutations in TLR4 were at higher risk of severe RSV disease relative to children with wild-type TLR4 genotype (OR: 1.7; CI: 1.2, 2.6). However, when socioeconomic status (SES) was considered, we found children from low SES families and heterozygous for TLR4 Asp299Gly/Thr399Ile mutations had significantly less severe disease compared to wild type (4% severe disease vs 11% severe disease, p<0.05) compared with children from middle/high SES (9% severe disease vs 2% mild disease, p<0.05). These initial results are consistent with a role for TLR4 in RSV disease severity, but interaction of TLR4 genotype with environment must be considered when evaluating risk of these mutations. ? ? We have also begun to evaluate other susceptibility factors that may impact on disease severity in high risk infants. The protective role of breastfeeding against severe acute lung disease in infants is well established, but its mechanism is unclear. Most hypotheses assume that breastfeeding confers similar passive protection to every infant; however, a few observations have suggested that the benefits of breast milk against severe lung disease may differ according to gender. We have evaluated whether the effect of breastfeeding on susceptibility to severe acute lung disease among infants at high risk is different for girls and boys. Our RSV cohort was analyzed prospectively by use of 2 different strategies: (1) predictors of first episode of rehospitalization by univariate and multivariate analyses using robust Poisson regression and (2) mean number of rehospitalizations between groups using multiple regression negative binomial models. A total of 119 high-risk, very low birth weight infants were enrolled. Breast milk protected girls but not boys against severe acute lung disease. The interaction between breastfeeding and gender was clinically and statistically significant, even after adjustment for variables that can affect severity of acute lung disease. Disease was most severe in formula-fed girls (versus formula-fed boys). We concluded that breastfeeding decreased the risk for severe acute lung disease in girls but not in boys. These findings suggest that breast milk protection is not universally conferred by passive transfer of humoral immunity (which should be gender indifferent), show that respiratory symptoms may be amenable to nonspecific modulation, and identify nonbreastfed preterm infant girls as an at-risk group for severe acute lung disease
