Assistant Professor University of Virginia, United States
Introduction: Idiopathic pulmonary fibrosis (IPF) is a fatal disease marked by the dense buildup of scar tissue in the lungs with no known cause or lasting cure. The prevalence of this disease continues to rise globally, with up to 45.1 cases per 100,000 persons and a poor prognosis with a 3–4-year median survival time following diagnosis. Fibrosis is a downstream response commonly triggered by dysregulated wound healing and chronic inflammation which leads to excessive amounts of extracellular matrix (ECM) deposition and collagen secretion. Common histopathological indicators of IPF are marked by areas of active fibrogenesis known as fibroblastic foci which comprise concentrated regions of myofibroblasts and newly synthesized ECM. Various immune and inflammatory agents are implicated to be highly expressed in these fibrotic regions, including inflammatory cytokine IL-1β and its receptor IL-1R. However, the mechanistic role of IL-1R in initiating lung fibrosis remains unclear. We seek to understand the pathological role of IL-1R signaling in IPF progression and strive to reveal more biomolecular insight into IL-1R as a therapeutic target for IPF.
Materials and
Methods: Human tissue samples of IPF lungs were obtained from lung tissue explants of patients undergoing lung transplantation. Samples were acquired from clinical collaborators at the University of Minnesota, flash-frozen at –80C, and preserved in optimal cutting temperature (OCT) compound. These samples were sectioned and stained with fluorescent antibodies for α-smooth muscle actin (αSMA) and IL-1R to assess the extent of fibrosis. Non-fibrotic regions within IPF lungs were also identified through histological staining and fluorescent imaging.
To investigate IL-1R's role in fibrosis in vivo, we utilized a bleomycin-induced lung fibrosis model in C57BL6/J wildtype (WT) and IL-1R knockout (KO) mice. Bleomycin is a chemotherapeutic known to trigger epithelial injury and consequential fibrosis in the lungs. WT (n=6) and IL-1R KO (n=5) mice were treated with bleomycin via oropharyngeal delivery (1 U/kg). Their lungs were harvested 21 days following a single-dose administration. The effect of bleomycin was controlled by harvesting lungs from WT (n=6) and IL-1R KO (n=4) mice that received no treatment. A saline bronchoalveolar lavage (BAL) was performed on each mouse. BAL fluid was collected to conduct a Sircol assay to assess soluble collagen content in the lungs. Harvested lungs were embedded and frozen in OCT and sectioned for histology. Sectioned lungs were stained with Hematoxylin & Eosin, Picrosirius Red, and Masson’s Trichrome to visually assess the fibrotic response. UVA clinicians conducted Ashcroft fibrosis tests using histological images to score the extent of lung fibrosis per mouse. All lung sections were fluorescently imaged at 10x objective.
Results, Conclusions, and Discussions: In human IPF tissue, αSMA and IL-1R were highly co-expressed via fluorescence in contrast with a dim co-expression in non-fibrotic tissue regions. IL-1R's elevated presence in human IPF tissue compared to the nonfibrotic regions indicates its potential critical role as a key inflammatory agent in lung fibrosis. These results prompted us to investigate IL-1R's role through the bleomycin-induced in vivo lung fibrosis model. We expected bleomycin to inflict a fibrotic response in the lungs when administered to both WT and IL-1R KO mice. As expected, we observed intensified fibrosis in WT mice treated with bleomycin. In contrast with the WT, the IL-1R KO mice treated with bleomycin had a reduced fibrotic response, indicated by histology which demonstrates decreased collagen deposition in the lung. These findings, along with reduced Ashcroft fibrosis scores and soluble collagen content in IL-1R KO mice, can further indicate that IL-1R is necessary for an amplified fibrotic response in the lungs.
Overall, pulmonary fibrosis is clearly reduced in the absence of IL-1R. These findings highlight that IL-1R could be a reputable therapeutic target for IPF treatment. Future steps in our research will investigate the therapeutic role of Anakinra, an FDA-approved IL-1R inhibitor, in the bleomycin lung fibrosis model.
Acknowledgements (Optional): Thank you to our collaborators at the University of Minnesota for providing us with human lung tissue samples.
