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Annemieke Schuurhof

Tuesday 6 December 2011

Host response to respiratory syncytial virus: genetic associations and effect on disease severity

Promotor: Prof.dr Jan.L.L. Kimpen
Defence: 6 March 2012

Summary
Respiratory syncytial virus (RSV) is the most frequent cause of severe respiratory tract infections in young children. Almost all children become infected with RSV during the first two years of life, and 1-3% needs hospitalization for supportive therapy. Known risk factors for more severe disease in children are age (younger than three months), preterm birth, chronic lung disease of prematurity, congenital heart disease, Down’s syndrome, immune deficiency, cystic fibrosis, male gender, older siblings, daycare attendance, indoor tobacco smoke and abbreviated breast feeding. However, most children who are admitted to the hospital with RSV lower respiratory tract infection were previously healthy infants without known risk factors. Genetic determinants are believed to play a role in the risk of developing RSV infection in healthy infants. A twin study has shown that up to 22% of susceptibility to RSV infection can be attributed to host genetic factors. After recovering from severe RSV infection, half of the infants develop recurrent wheezing during childhood. Mechanisms underlying the association between severe RSV infection and post-bronchiolitis wheeze are poorly understood. Genetic studies may shed light on the issue why some children do and other children do not develop post-bronchiolitis wheeze.
In this thesis, the functional role of the previously reported associated gene variants during RSV infection is studied.

In chapter 1, RSV infection in infants is introduced and the general aims of this thesis are discussed.

In chapter 2, we systematically reviewed genetic factors influencing susceptibility to RSV lower respiratory tract infection and to post-bronchiolitis wheeze. We showed published genetic associations of RSV infection with both single nucleotide polymorphisms (SNPs) and haplotypes. Particularly SNPs in innate immunity genes were associated with the pathogenesis of severe RSV infection. However, no genetic variants associated with RSV infection have been exactly replicated in other cohorts. A limited number of studies have reported associations between SNPs and post-bronchiolitis wheeze, especially in chemokine and Th2 related genes. Consequently, although severe RSV infection and post-bronchiolitis wheeze show similarities in clinical presentation, different genetic factors are important for the development of these diseases in young children.

Chapter 3 describes the first study focusing on gender-specific genetic factors affecting the risk of severe RSV infection. The predominance of RSV bronchiolitis in boys compared to girls is well known, but its mechanism is not yet understood. Therefore, we used a previously described cohort to determine 347 SNPs in 470 children hospitalized for RSV infection, their parents, and 1,008 random population controls. We tested if these SNPs exerted a different effect in boys and girls by performing statistical interaction tests. Only one SNP (rs2069885) had a gender-specific significant association with RSV infection, severe enough to require hospitalization. The major allele of this structural polymorphism in the interleukin-9 gene is associated with an increased susceptibility to severe RSV infection in boys, while there is a decreased susceptibility in girls. Haplotype analysis of two SNPs in the IL9 gene (rs2069885 and rs1799962) showed overrepresentation of the TT haplotype in girls with severe RSV bronchiolitis requiring hospitalization indicating that this is the haplotype conferring the highest risk in girls. Although so far a difference in IL-9 production in boys and girls has not been reported, this study may help in explaining the different risks of severe RSV bronchiolitis in boys and girls.

In chapter 4, we aimed to determine the in vivo role of interleukin-10 in RSV pathogenesis and recurrent wheeze in a new cohort of 235 infants hospitalized for RSV bronchiolitis. Animal studies have suggested that IL-10 plays a critical role in the pathogenesis of RSV bronchiolitis and subsequent airway hyperresponsiveness. Previously, we showed that ex vivo monocyte IL-10 production is a predictor of PBW. Additionally, heterozygosity of the single-nucleotide polymorphism (SNP) rs1800872 in the IL10 promoter region was associated with protection against RSV bronchiolitis. We measured IL-10 levels in nasopharyngeal aspirates (NPAs) at the time of hospitalization and the IL10 SNP rs1800872 genotype was determined. Follow-up data were available for 185 children (79%). Local IL-10 levels during RSV infection turned out to be higher in infants that later developed physician diagnosed PBW as compared to infants without PBW in the first year after RSV infection (958 vs 692 pg/ml, p = 0.02). Accordingly, the relationship between high local IL-10 levels during the initial RSV infection and physician diagnosed PBW provides further evidence of the importance of the IL-10 response during RSV bronchiolitis. The IL10 promoter SNP rs1800872 was not associated with IL-10 concentration in NPAs.

Chapter 5 studied the local immune response in NPAs during RSV infection. Forty-seven potential markers of disease severity were analyzed in a screening cohort of RSV-infected infants with mild disease at home (n=8), hospitalized infants (n=10), and infants requiring mechanical ventilation (n=7). Results were confirmed in a cohort of infants hospitalized for RSV infection (n=200). Finally, genetic validation was studied in a cohort of infants hospitalized for RSV infection (n=465) and healthy controls (n=930). TIMP metallopeptidase inhibitor (TIMP)-1 was higher in the NPAs of hospitalized infants compared to the NPAs of infants at home (1,199 vs 568 ng/ml, p<0.0001). Similar results were found for matrix metalloproteinase (MMP)-3 (765 vs 370 pg/ml, p=0.004). MMP-3 as a marker of disease severity was confirmed in a larger cohort and MMP3 gene polymorphism rs522616 was associated with severe RSV infection (OR 0.82, p<0.05). Therefore, extracellular matrix proteinases play an important role in the pathogenesis of RSV bronchiolitis.

In chapter 6, we report transcription profiles in the lungs of RSV-infected mice. Vaccine-induced immunity has been shown to alter the course of a RSV infection both in murine models and in humans. To elucidate which mechanisms underlie the effect of vaccine-induced immunity on the course of RSV infection, transcription profiles in the lungs of RSV-infected mice were examined by microarray analysis. Three models were used: RSV reinfection as a model for natural immunity, RSV challenge after formalin-inactivated RSV vaccination as a model for vaccine-enhanced disease, and RSV challenge following vaccination with recombinant RSV lacking the G gene (G-RSV) as a model for vaccine-induced immunity. Gene transcription profiles, histopathology, and viral loads were analyzed at 1, 2, and 5 days after RSV challenge. On the first 2 days after challenge, all mice displayed an expression pattern in the lung similar of that found in primary infection, showing a strong innate immune response. On day 5 after RSV reinfection or after challenge following G-RSV vaccination, the innate immune response was waning. In contrast, in mice with vaccine-enhanced disease, the innate immune response 5 days after RSV challenge was still present even though viral replication was diminished. In addition, only in this group was Th2 gene expression induced. These findings support a hypothesis that vaccine-enhanced disease is mediated by prolonged innate immune responses and Th2 polarization in the absence of viral replication.

Chapter 7 describes the transcription profiles in the blood and bronchial lymph nodes of RSV-infected mice. This study was performed to improve our understanding of systemic host responses to RSV. BALB/c mouse gene expression responses at day 1, 2, and 5 during primary RSV infection were compared in lung, bronchial lymph nodes, and blood. We identified a set of 53 interferon-associated and innate immunity genes that give correlated responses in all three murine tissues. Additionally, we identified blood gene signatures that are indicative of acute infection, secondary immune response, and vaccine-enhanced disease, respectively. Eosinophil-associated ribonucleases were characteristic for the vaccine-enhanced disease blood signature. These results indicate that it may be possible to distinguish protective and unfavorable patient lung responses via blood diagnostics.

Finally, in chapter 8 a general discussion on host response to RSV infection is provided. The most important finding is that extracellular matrix proteinases, including TIMP-1 and MMP-3, play an important role in the pathogenesis of RSV bronchiolitis. Lung development, maturation and airway remodeling in infants is an ongoing process, which can be temporarily interrupted by RSV infection. RSV causes airway remodeling where not only the pneumocytes in the alveoli and the epithelium in the bronchioles are affected, also the extracellular matrix and the surrounding tissue in the lung is disturbed.
Furthermore, high IL-10 levels at the site of infection during RSV bronchiolitis are associated with increased risk of post-bronchiolitis wheeze. An opposite gender-specific association is observed between a SNP in IL9 and severe RSV bronchiolitis requiring hospitalization. Vaccine-enhanced disease is mediated by prolonged innate immune responses and Th2 polarization, in the absence of viral replication in mice. And gene expression in blood are indicative for acute inflammation, protection, and vaccine-enhanced disease in RSV infection in mice.
To conclude, host genetic studies offer a complex but robust method to improve our understanding of disease pathogenesis and develop new targets for intervention for one of the most common diseases during early childhood.

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