- Title
- The fibrogenic actions of IL-25 in Idiopathic Pulmonary Fibrosis (IPF)
- Creator
- Kanwal, Amama
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2023
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Idiopathic pulmonary fibrosis (IPF) is the most prevalent form of idiopathic interstitial pneumonia (IIP) and continues to increase in incidence in an ageing population. IPF remains a detrimental disease with a poor prognosis, and life expectancy is 3 to 5 years from diagnosis. The underlying pathogenesis of IPF remains elusive; however, the current consensus is that repetitive injuries to an ageing alveolar epithelium and impaired repair mechanisms play a central role in the development of IPF. Two pharmacological therapies (Pirfenidone and Nintedanib) have been shown to slow down lung function decline in patients with this disease. Despite this effect, the disease remains non-reversible and progressive in nature whilst the side effect profiles of these drugs are problematic. There is still no cure for IPF, and lung transplant remains the only viable option for prolonging the survival of these patients. Due to this unmet medical need, it is important to identify additional therapeutic targets secreted by the injured alveolar epithelium (such as IL-25) involved in the initiation and progression of IPF. Recent studies have shown increased levels of IL-25 in the lungs of IPF patients; however, the precise fibrogenic role of IL-25 in IPF remains unclear. This thesis aimed to investigate the fibrogenic action of IL-25 released by injured alveolar epithelial cells (AECs) in IPF. In Chapter 3, histological analysis confirmed increased IL-25 and IL-17RB receptor subunit expression in fibrotic lung tissue obtained from IPF patients. Immunohistochemistry (IHC) staining revealed that type II AECs and fibroblasts express IL-17RB in the area of fibrotic foci. Dual immunofluorescence (IF) staining provided evidence of distinct cell populations of IL-25 and IL-17RB-positive cells in fibrotic lung tissue. IL-25 responsive cells (IL-17RB-positive cells) were identified as type II AECs and CRTH2-positive cells (CD45-negative), potentially fibroblasts within fibrotic lung tissue. A subset of IL-25-positive cells within fibrotic lung tissue demonstrated co-expression with surfactant protein C (a marker of type II alveolar epithelial cells) and CRTH2-expressing cells, primarily observed in close proximity to the epithelium. However, majority of IL-25-positive cells within the fibrotic lung tissue remained unidentified. In Chapter 4, results demonstrated baseline gene and protein expression of lL-25 and IL-17RB in primary lung fibroblasts (LFs) derived from IPF and non-diseased human lung tissue. Treatment with recombinant IL-25 (1, 10, and 100ng) resulted in increased fibronectin and α-SMA gene expression in primary LFs obtained from IPF and non-disease control lung tissue compared to untreated LFs. Like primary LFs, MRC-5 fibroblasts also express receptor for IL-25 (IL-17RB). Therefore, experiments included the MRC-fibroblast cell line to extend evidence of the fibrogenic effect of IL-25 and determine the utility of this fibroblast cell line to study IL-25 biology in lung fibrosis. Treatment with recombinant IL-25 induced MRC-5 fibroblast proliferation and expression of pro-fibrotic mediators (fibronectin and collagen I) at different time points (gene and protein). Combining IL-25 with TGF-β1 had an additive effect on the expression of pro-fibrotic mediators by MRC-5 fibroblasts (gene and protein). However, the addition of IL-13 to combinations of IL-25 and TGF-β1 only induced collagen I expression (gene and protein). These results show that IL-25 is sufficient to activate fibroblasts with evidence of interaction with other pro-fibrotic mediators. In Chapter 5, an in vitro model of oxidative stress and DNA damage of AEC was established using A549 cell line (a surrogate for primary type II AECs). Treatment with H2O2 (100, 300μM, and 1mM) and etoposide (1, 3, and 10μM), alone and in combination, resulted in increased release of IL-25 protein by AECs. To generate conditioned media (CM) from injured AECs, the highest doses of H2O2 (1mM) and etoposide (10μM) were selected and later used to stimulate MRC-5 and primary LFs. IL-25 CM from injured AECs induced fibroblast proliferation and expression of pro-fibrotic mediators in MRC-5 fibroblasts and primary LFs (isolated from fibrotic and non-fibrotic lung tissue). Later, the inhibitory effect of anti-IL-25 monoclonal antibody (LNR125) was assessed in the same experimental model. These results demonstrated that blocking IL-25 in CM with LNR125 significantly reduced the fibrogenic response induced by IL-25. In conclusion, this work provides evidence of increased IL-25-and IL-17RB-expressing cells in the fibrotic lung tissue of IPF patients. The majority of IL-17RB-positive cells were identified as non-immune cells (CD45-negative) and were co-expressed with surfactant protein C (type II AECs) and CRTH2-positive cells (potentially fibroblasts). In vitro, CM derived from injured AEC stimulated fibroblast proliferation and expression of pro-fibrotic mediators. Inhibition of IL-25 attenuated fibrogenic response mediated by CM from injured AECs. These findings suggest that targeting IL-25 with a monoclonal anti-IL-25 antibody (LNR125) could be a promising therapeutic approach to suppress lung fibrotic diseases such as IPF.
- Subject
- IPF; IL-25; fibroblasts; alveolar epithelial cells (AECs); A549 cells; MRC-5 fibroblasts; fibrosis; fibrotic mediators
- Identifier
- http://hdl.handle.net/1959.13/1506687
- Identifier
- uon:55919
- Rights
- Copyright 2023 Amama Kanwal
- Language
- eng
- Full Text
- Hits: 95
- Visitors: 108
- Downloads: 15
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 6 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 528 KB | Adobe Acrobat PDF | View Details Download |