In collaboration with Dr. Fernando Polack (Vanderbilt 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. 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 2003-2006, 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. Th2 polarization of the immune response has been proposed as an explanation for RSV bronchiolitis for decades. From the presence of eosinophils in the aberrant response to RSV infection experienced by infants immunized with a formalin-inactivated vaccine against the virus, to detection of high RSV-specific IgE levels in severe cases and similarities in symptoms to those of pediatric asthma, a link between Th2 and RSV disease has been frequently speculated. Interestingly, while we found no link between systemic Th2 bias and severe disease, RSV illness correlated with a predominance of the Th2 transcription factor GATA3 over the Th1 factor T-bet in the respiratory tract. GATA3 is the master regulator of Th2 responses and has been associated with bronchoconstriction through IL-4, IL-13 and leukotrienes in mice. In fact, several studies have reported higher levels of Th2 cytokines or cysteinil-leukotrienes in RSV-infected infants compared to controls. While our study suggests that GATA3/T-bet ratios are in the pathogenic pathway for RSV disease, other pathway/s associated with mechanism of illness remain to be determined. In support of our observations, the effect of the interaction between TLR4 and the environment on disease severity during RSV infection becomes non-significant when adjusted for GATA3/T-bet. But the statistical effect of GATA3/T-bet is limited, suggesting that other, likely complementary mechanisms account for additional pathogenicity (the partial effect of the adjustment for GATA3/T-bet may also be attributable to our testing only a third of all samples for GATA3 and T-bet, compared to 100% for TLR4). Moreover, TLR4 heterozygosity affects 6.7% of our population, highlighting that there are other genes and risk factors influencing RSV bronchiolitis. In fact, GATA3/T-bet ratios may be part of a downstream pathogenic pathway modified at various levels by different molecules of the immune system and the virus in the context of the environment. This paradigm integrates previous findings, where severe illness was associated with SNPs in other genes that can affect Th2 bias and TLR pathways in other populations, is in line with the modulation of TLR4 by RSV F and GCRR, and encourages the study of environmental factors such as smoking, poverty and crowding, all likely associated through LPS with chronic TLR4 agonism, and consequent more severe disease in >90% infants with WT TLR4 genotypes. Many patients experience mild symptoms, but some individuals are highly susceptible to RSV infection and exhibit more severe symptoms which may result in hospitalization and sometimes death. The mechanism for differential responses to RSV infection is unknown. To develop a cell model to identify genetic determinants of differential susceptibility to RSV infection in humans. Lymphoblasts from the Coriell Institutes human variation panels were used. These panels consist of various human lymphoblast cell lines established from multiple donors from a number of different ethnic and racial groups which can be genotyped and profiled for gene expression patterns to provide information on genetic differences that determine differential responses to RSV. Cells were infected with RSV (strain A2) and analyzed at various time-points for levels of inflammatory cytokines and chemokines using real-time RT-PCR. Infectivity was evaluated by measuring expression of viral genes and proteins using real-time PCR and flow cytometry, respectively. The viral load differed significantly between panels as well as between individuals within panels. Similarly, expression levels of IL-6, TNF-, IFN-, IFN-, and TLR4 differed between panels and individuals within panels in response to infection. Differences were also observed in the kinetics of the cytokine response and viral load. We have developed a novel cell model of RSV disease. Significant inter-individual variation in infectivity and inflammatory response to RSV infection suggests that genetic background is an important determinant of susceptibility to RSV disease. Previous in vivo studies have suggested that RSV susceptibility is a polygenic trait;however, the specific genes regulating this phenotype are not known. The purpose of this study was to use in silico haplotype mapping in mice to identify quantitative trait loci (QTLs) that contain genes that contribute to RSV susceptibility. Thirty-five inbred mouse strains were infected with a single dose of 1x106 plaque forming units of RSV or control and sacrificed 1 and 5 d post-infection (pi). Inflammatory response, lung permeability, pathology, mucus cell metaplasia, and viral load were analyzed and compared among and within all inbred strains. Using in silico mapping, the degree of correlation between the observed phenotypic differences and the genotype of the inbred strains was used to determine candidate QTLs. QTLs with a log P value greater than 3.5 were considered significant. RSV response phenotypes were distributed continuously across the inbred strains. Futher, the strain distribution patterns varied for each phenotype and time point (i.e., not concordant), suggesting that multiple mechanisms influence RSV susceptibility. Correlation analysis of phenotype relatedness also suggests that the response to RSV at 1 and 5 days pi are independent. In silico haplotype mapping identified phenotype-specific significant QTLs. This study confirms that susceptibility to RSV infection is a complex genetic trait. In silico mapping identified possible chromosomal QTLs for multiple phenotypes, and candidate genes for each.
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