natural aromatase inhibitors br Materials and Methods br Res
Materials and Methods
Discussion The impact of RSV disease, particularly in vulnerable infants, remains a global public health concern (Lozano et al., 2012). Unfortunately, no RSV vaccines are approved for clinical use. Therefore, it is critical to identify host risk factors that lead to development of prevention and intervention strategies to reduce risk of RSV disease morbidity and mortality, as well as natural aromatase inhibitors incidence because RSV disease has been identified as a predisposing risk factor (Dakhama et al., 2005; Edwards et al., 2012; Gelfand, 2012; Feldman et al., 2015). Our study used genome-wide association analyses of RSV disease parameters that model human disease to identify biologically plausible susceptibility gene candidates, including Marco. Moreover, a missense loss of function polymorphism in the human MARCO homolog also associated with severe RSV disease in two populations of infected infants, and illustrates the utility of a genetic mouse model for understanding human disease. Importantly, this study provides additional genetic insight that may lead to a means to identify individuals at risk for severe RSV disease. Anh and Desmecht (2006) reported that pneumonia virus of mice (PMV, the murine form of RSV) infection in a panel of 6 strains caused strain-specific histopathologic inflammatory effects, decreased body weight, and changes in pulmonary function particularly 5–7days PI. The studies are not directly comparable because mice were infected with different viruses and late phase PI times investigated, but the inter-strain variation in phenotypes is consistent with our findings. Others (Stark et al., 2002) compared RSV infection in 8 inbred strains and found significant inter-strain variation in infectivity at 4days PI, but histopathologic changes were minimal and cellular effects were discordant with the magnitude of infection likely due to the time point when acute virus effects on histopathology were no longer present. We leveraged extensive genetic diversity among the 30 strains of mice to perform GWA of multiple RSV disease phenotypes and thus were not guided a priori by hypotheses about the role of specific genes. Significant and suggestive QTLs were identified for inflammatory cells, intraepithelial cell mucus, and RSV N mRNA expression. Several genes that may contribute to viral response, pulmonary inflammation, and cytokine trafficking were found within the identified QTLs [e.g. Ptgs2 (Obata et al., 2013), Ramp1 (Li et al., 2014), Rhbdf2 (Issuree et al., 2013)], and putative functional SNPs in the genes associated with differential RSV disease phenotypes among the strains. SNPs in other QTL genes strongly associated with disease phenotypes and, while little is known about their function in infectious disease, they may be important targets for future investigation of their roles in RSV disease progression. We also found significant QTLs using the first PC as an RSV disease index. Two of these QTLs overlapped those found for lymphocytes, and suggests that further investigation of genes within the QTLs could provide greater understanding of disease susceptibility. Future GWA investigations using PC analyses may provide additional insight to disease pathogenesis beyond using single phenotypes. Interestingly, genome-wide linkage analyses of RSV infectivity in back-cross and F2 populations derived from C57BL/6J and AKR/J mice by Stark et al. (2010) identified a QTL on chromosome 6 that associated with increased susceptibility to infection, though disease phenotypes were not presented. While GWA in the present investigation identified suggestive QTLs on chromosomes 1 and 11 for RSV-N mRNA expression, none were detected on chromosome 6. Differences in study design (e.g. haplotype mapping with many strains versus linkage analysis with 2 strains, PI time differences) may have contributed to this discrepancy. We focused on the gene candidate Marco, which was identified by GWA for monocytes, and has been shown previously to have protective effects in the lung following inhalation of particulate matter, exposure to ozone, and bacterial infection (Arredouani et al., 2005; Dahl et al., 2007; Thakur et al., 2008; Thakur et al., 2009; Ghosh et al., 2011). MARCO gene expression was also upregulated in children infected with RSV (Fjaerli et al., 2006). Our hypothesis that Marco contributes to defense against RSV-induced pulmonary inflammation was supported by two lines of evidence. First, strains of mice with a Marco haplotype containing the T allele of missense coding SNP rs30741725 were at greater risk of developing more severe RSV disease compared to strains with the wild type A allele. Second, numbers of BALF monocytes and PMNs were significantly increased in Marco mice compared to Marco mice. The mechanism through which Marco modulates the inflammatory response to RSV is not completely understood. We initially hypothesized that Marco has a direct role in clearing RSV from the airways, perhaps binding the virus for internal degradation by macrophages. To test this hypothesis, we infected with RSV lung macrophages isolated from Marco and Marco mice but found no differences in infectivity, with and without co-culturing with epithelial cells (data not shown). Marco also has an important role in clearance of apoptotic cells (Rogers et al., 2009; Getts et al., 2014), and recent studies have targeted Marco receptors with immune-modifying particles to resolve monocytic inflammation (Getts et al., 2014). It is plausible that loss of function mutations in Marco compromise clearance of monocytes and apoptotic cells which leads to accumulation of these cells in the airways, further contributing to RSV disease pathology. Impaired resolution of the inflammatory response to RSV infection and more severe disease in Marco mice compared to Marco mice is consistent with this hypothesis.