Aoife N McElroy1, Rachele Invernizzi2, Joanna W Laskowska1, Andrew O'Neill1, Mohammad Doroudian3, Mohsen Moghoofei4, Shayan Mostafaei5, Feng Li6, Alexander A Przybylski6, David N O'Dwyer7, Andrew G Bowie8, Padraic G Fallon1,9,10, Toby M Maher2,11, Cory M Hogaboam12, Philip L Molyneaux2,11, Nik Hirani6,13, Michelle E Armstrong1, Seamas C Donnelly1,14. 1. School of Medicine, Trinity Biomedical Sciences Institute, Trinity College, Dublin, Ireland. 2. National Heart and Lung Institute, Imperial College London, London, United Kingdom. 3. Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran. 4. Infectious Diseases Research Centre and. 5. Department of Biostatistics, School of Health, Kermanshah University of Medical Sciences, Kermanshah, Iran. 6. MRC Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom. 7. Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, Michigan. 8. School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, and. 9. Trinity Translational Medicine Institute, St. James's Hospital, Trinity College Dublin, Dublin, Ireland. 10. National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland. 11. Royal Brompton Hospital, London, United Kingdom. 12. Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California. 13. Edinburgh Lung Fibrosis Clinic, Royal Infirmary Edinburgh, Edinburgh, United Kingdom; and. 14. Department of Clinical Medicine, Trinity Centre for Health Sciences, Tallaght University Hospital, Tallaght, Dublin, Ireland.
Abstract
Rationale: The Toll-like receptor 3 Leu412Phe (TLR3 L412F) polymorphism attenuates cellular antiviral responses and is associated with accelerated disease progression in idiopathic pulmonary fibrosis (IPF). The role of TLR3 L412F in bacterial infection in IPF or in acute exacerbations (AE) has not been reported. Objectives: To characterize the association between TLR3 L412F and AE-related death in IPF. To determine the effect of TLR3 L412F on the lung microbiome and on antibacterial TLR responses of primary lung fibroblasts from patients with IPF. Methods: TLR-mediated antibacterial and antiviral responses were quantitated in L412F wild-type and 412F-heterozygous primary lung fibroblasts from patients with IPF using ELISA, Western blot analysis, and quantitative PCR. Hierarchical heatmap analysis was employed to establish bacterial and viral clustering in nasopharyngeal lavage samples from patients with AE-IPF. 16S ribosomal RNA quantitative PCR and pyrosequencing were used to determine the effect of TLR3 L412F on the IPF lung microbiome. Measurements and Main Results: A significant increase in AE-related death in patients with 412F-variant IPF was reported. We established that 412F-heterozygous IPF lung fibroblasts have reduced antibacterial TLR responses to LPS (TLR4), Pam3CYSK4 (TLR1/2), flagellin (TLR5), and FSL-1 (TLR6/1) and have reduced responses to live Pseudomonas aeruginosa infection. Using 16S ribosomal RNA sequencing, we demonstrated that 412F-heterozygous patients with IPF have a dysregulated lung microbiome with increased frequencies of Streptococcus and Staphylococcus spp. Conclusions: This study reveals that TLR3 L412F dysregulates the IPF lung microbiome and reduces the responses of IPF lung fibroblasts to bacterial TLR agonists and live bacterial infection. These findings identify a candidate role for TLR3 L412F in viral- and bacterial-mediated AE death.
Rationale: The Toll-like receptor 3 Leu412Phe (TLR3 L412F) polymorphism attenuates cellular antiviral responses and is associated with accelerated disease progression in idiopathic pulmonary fibrosis (IPF). The role of TLR3 L412F in bacterial infection in IPF or in acute exacerbations (AE) has not been reported. Objectives: To characterize the association between TLR3 L412F and AE-related death in IPF. To determine the effect of TLR3 L412F on the lung microbiome and on antibacterial TLR responses of primary lung fibroblasts from patients with IPF. Methods: TLR-mediated antibacterial and antiviral responses were quantitated in L412F wild-type and 412F-heterozygous primary lung fibroblasts from patients with IPF using ELISA, Western blot analysis, and quantitative PCR. Hierarchical heatmap analysis was employed to establish bacterial and viral clustering in nasopharyngeal lavage samples from patients with AE-IPF. 16S ribosomal RNA quantitative PCR and pyrosequencing were used to determine the effect of TLR3 L412F on the IPF lung microbiome. Measurements and Main Results: A significant increase in AE-related death in patients with 412F-variant IPF was reported. We established that 412F-heterozygous IPF lung fibroblasts have reduced antibacterial TLR responses to LPS (TLR4), Pam3CYSK4 (TLR1/2), flagellin (TLR5), and FSL-1 (TLR6/1) and have reduced responses to live Pseudomonas aeruginosa infection. Using 16S ribosomal RNA sequencing, we demonstrated that 412F-heterozygous patients with IPF have a dysregulated lung microbiome with increased frequencies of Streptococcus and Staphylococcus spp. Conclusions: This study reveals that TLR3 L412F dysregulates the IPF lung microbiome and reduces the responses of IPF lung fibroblasts to bacterial TLR agonists and live bacterial infection. These findings identify a candidate role for TLR3 L412F in viral- and bacterial-mediated AE death.