Investigation of the role of immunomodulators oftoll-like receptor 3 (TLR3) function and associated genetic variants in the pathogenesis and progression of idiopathic pulmonary fibrosis (IPF)

Abstract

IPF is a fatal interstitial lung disease of unknown aetiology, with a lack of valid biomarkers and satisfactory therapeutic solutions available. Studies have shown an association between inflammation, viral and bacterial infection and the development and progression of IPF. Previous studies have suggested that almost 15% of IPF patients develop rapidly progressive disease which results in death within 12 months. In 2014, Pirfenidone and Nintedanib were FDA approved for the treatment of IPF. These agents have been shown to reduce the decline of lung function in IPF patients. Therefore, currently there is a significant and unmet clinical need in IPF to identify biomarkers which will facilitate disease stratification in IPF at diagnosis in order to identify patients most at risk of developing rapidly progressive disease, to facilitate their early referral for lung transplant. We previously demonstrated a role for defective TLR3 function in accelerated disease progression in IPF. Specifically, we demonstrated that TLR3 L412F, which is a TLR3 SNP associated with reduced TLR3 function, caused an acceleration, and decline in lung function and increased mortality in IPF patients. We also demonstrated that TLR3 L412F was significantly associated with acute exacerbation (AE)-induced IPF death (AE-IPF) in patients. Furthermore, defective TLR3 function in 412F-variant primary human lung IPF fibroblasts resulted in dysregulated fibroproliferation and reduced IFN-β expression, which exacerbated fibrosis in IPF patients. The role of bacterial pathogens has been implicated in the pathology of IPF. Studies have shown that both the bacterial burden as well bacterial strain present in the BAL fluid of IPF patients were predictive of worsen clinical outcome of the disease (82-85). We previously established that 412F-heterozygous IPF lung fibroblasts have reduced anti- bacterial TLR responses to LPS (TLR4), Pam3CYSK4 (TLR1/2), flagellin (TLR5) and FSL-1 (TLR6/1) and have reduced responses to live Pseudomonas aeruginosa infection. Hence, we have established that TLR3 L412F represents a candidate prognostic biomarker in IPF. In this PhD project, we built on our previous findings which established that defective TLR3 function is pathogenic in IPF patients and drives disease progression. Specifically, in these PhD studies we hypothesised that immunomodulators, such as itaconate or IL-17A, which alter TLR3 function in IPF patients during viral or bacterial infection may accelerate IPF disease progression. In addition, we hypothesised that genetic variants of such immunomodulators, including the IL-17A G197A (rs2275913) promoter polymorphism, may also drive disease progression in IPF. xi Itaconate is a metabolite generated during the Krebs cycle which has emerged as a potent immunomodulator with anti-bacterial and anti-viral properties. To date, the role of itaconate in viral infection in IPF is unreported. In this study, we investigated the effect of 4- octyl itaconate (4-OI), a synthetic analogue of itaconate, on TLR3 function in primary lung fibroblasts from IPF patients. Here, we demonstrated that 4-OI decreased NF-κB and IRF3 activity in IPF lung fibroblasts stimulated with TLR3 ligand. Moreover, we reported that human IPF lung fibroblasts presented with decreased Poly(I:C)-induced transcription of pro-fibrotic markers, and increased transcription of anti-oxidants, following 4-OI treatment. We predict that the dampened, IRF3-dependant anti-viral response observed in our study may be detrimental to disease progression in patients with IPF. In addition, we are the first group to confirm the expression or immune responsive gene 1 (IRG1), a gene involved in itaconate synthesis, in Poly(I:C)-stimulated human lung fibroblasts, highlighting the importance of itaconate during infection. In this PhD project, we also investigated the effects of IL-17A on TLR3 function in terms of IPF pathology. IL-17A is a cytokine with pro-inflammatory and anti-viral properties. Increased levels of IL-17A were shown to confer protection to virally infected cells and to promote viral persistence. We and others have previously demonstrated increased levels of IL-17A in bronchoalveolar lavage (BAL) fluid and pulmonary tissue of IPF patients. Here, we demonstrated that Poly(I:C)-induced IPF lung fibroblasts treated with IL-17A presented with increased NF-κB response and decreased IRF3 response, which we predict may have pathogenic effects on IPF disease progression. Moreover, we demonstrated a significant association between the IL-17A G197A (rs2275913) polymorphism with development of IPF in UK patients. In addition, we demonstrated that IPF patients positive for 197A-variant had an increased risk of IPF-pneumonia death that approached statistical significance (p=0.07). Furthermore, IPF patients variant for 197A allele had less time to death compared to their G197-WT IPF patient counterparts. This effect was not due to a significant difference in pulmonary function in G197-WT IPF patients at diagnosis. These IL-17A G197A studies were carried out in an IPF cohort of modest size due to sample availability. These findings warrant further investigation into the potential use of IL-17A G197A SNP as a prognostic biomarker for IPF patients. In the final chapter of this PhD thesis, we investigated the functionality of the TLR3 L412F polymorphism in vivo. Firstly, we demonstrated that IPF patients who are 412F-variant exhibit a dysregulated lung microbiome, a reduction in overall pulmonary bacterial load and have xii increased frequencies of the bacteria, Streptococcus and S. aureus compared to 413-WT IPF patients. Secondly, we investigated the functionality of the TLR3 L412F polymorphism in vivo using the novel TLR3 L413F CRISPR-Cas-9 knock-in mice which are unique to our lab. In these studies, we demonstrated that both female and male 413F-HET mice have higher constitutive lung production of RANTES and KC compared to L413-WT mice. In contrast, female L413-WT mice have increased Poly(I:C)-induced pro-inflammatory and anti-viral responses in the lung compared to 413F-HET mice. However, the lungs of male 413F-HET mice have increased levels of Poly(I:C)-induced RANTES and KC protein production compared to 413-WT female mice. These data confirm that the TLR3 L412F polymorphism is functional in our TLR3 L413F CRISPR- Cas-9 knock-in mice. These data also suggest that there may be a sex-related difference in the function of TLR3 L412F. The novel findings reported in this PhD thesis warrant further investigation in order to elucidate their role in the pathogenesis and development of IPF, and their relevance in the subsequent quest for novel therapies and prognostic biomarkers.