The impact of asthma, toll-like receptors 7 and 8, genetic and clinical determinants on antiviral immune response

Abstract

People with asthma suffer from worse respiratory infections. The underlying causes have been suggested to arise from type 2-biased inflammation, deficiencies in antiviral immune response, epithelial damage, or a combination of all three. The antiviral immune response is activated by pathogen recognition receptors that include the viral ssRNA detecting toll-like receptor (TLR)7 and its paralogue TLR8. TLR7 induces the production of the antiviral cytokine type I interferon (IFN) that has been reported to be deficient in people with asthma. Additional reports that asthma is associated with reduced TLR7 function and genetic variation in TLR7/8 gene region warrants investigation of the roles of TLR7/8 in antiviral immune response in healthy people and those with asthma. Three primary aims of this study are:1. To determine what clinical and immunological parameters are associated with self-reported respiratory infection frequency, the presence of asthma, asthma severity and control, with a specific focus on the role of TLR7/8 gene expression and function, and the extent to which this differs between women and men.2. To examine what host and transcriptional variations predict variation in antiviral type I IFN responses in vitro and determine how transcriptome patterns vary between individuals with high and low antiviral type I IFN responses.3. To examine the functional impact that genetic variations in the TLR7/8 region has on TLR7/8 gene expression, downstream cytokine production, antiviral immunity, and immune cell counts in peripheral blood.To address the study aims, I recruited 150 asthma cases and 151 controls, documented self-reported respiratory infection frequency by questionnaire, and measured baseline TLR7/8 gene expression in whole blood, and cytokine responses using rhinovirus (RV)16-stimulated or TLR7/8-ligand stimulated peripheral blood mononuclear cells (PBMC).Plasmacytoid dendritic cells (pDC) are of interest to the current study because they are the primary producers of type I IFN and express TLR7. In Chapter 2, we show that measuring CLEC4C gene expression provides a useful alternative method for quantifying pDC numbers that may be particularly relevant to large cohort studies where flow cytometry is impractical or inaccessible.Chapter 3 addresses the first aim of the study and we report that CLEC4C gene expression (beta = 0.90, p = 0.01) together with TLR7 gene expression (beta = -1.13, p = 0.02) are associated with self-reported respiratory infection frequency in men. In women, age (beta = -0.02, p < 0.001) and body mass index (BMI; beta = 0.03, p < 0.001) are associated with respiratory infection frequency. Asthma cases reported significantly more respiratory infections per year than controls (median 3 vs 2, p < 0.001) and reduced TLR7 gene expression (odds ratio (OR) = 0.12, p = 0.02) and increased BMI (OR = 1.1, p < 0.001). Asthma symptom control was associated with reduced TLR8 gene expression (beta = -1.4, p = 0.036) and increased BMI (beta = 0.04, p = 0.004), whereas, increased age (OR = 1.0, p = 0.005) and BMI (OR = 1.1, p = 0.004) were associated with asthma severity.To address the second aim, we sequenced transcriptomes of baseline and RV16-stimulated PBMC samples from subsets of participants with the highest and lowest 15% of IFNα response determined in Chapter 3. In Chapter 4, we report that at baseline in comparison to the low IFNα producers, high IFNα producers have a higher gene expression of innate immune variables including the pDC gene CLEC4C and complement genes; and lower gene expression genes, which may be markers of oxidative stress. During antiviral immune response, in comparison to the low IFNα producers, the high IFNα producers express IFNα genes and IFNα response genes at a higher level, as expected, and lower levels of genes linked to antibacterial activity.For Chapter 5, we genotyped the TLR7/8 gene region in participants with European ancestry to address aim three. This revealed associations between an asthma risk SNP rs850637 in the TLR7/8 gene region and basophil count in our study population. Additional analyses using publicly available external databases revealed associations between rs850637 and TLR7 and TLR8-AS1 gene expression. TLR8-AS1 is a type of antisense gene, which are thought to have a function in regulating the gene expression of their complementary gene product. However, we did not find any genetic variants linked to respiratory infection frequency or capacity for IFNα production in vitro.These findings indicate that multiple risk factors are associated with antiviral immune responses and susceptibility to respiratory infections. These risk factors differ between women and men, with age and BMI prominent in women, whereas innate immune variations were more prominent in men. Decreased TLR7/8 expression in asthma patients may also contribute towards the respiratory infection susceptibility. The asthma-related SNP rs850637 was associated with lower TLR7 gene expression and increased basophil count that is associated with asthma inflammation. Considering these findings, TLR7/8 appear to be crucial for the antiviral immune response and asthma inflammation.