The role of Interleukin-33 in the development of idiopathic pulmonary fibrosis
Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterised by the excessive deposition of extracellular matrix by fibroblasts and an irreversible loss of lung function. Recently, the interleukin (IL)-1 family member IL-33 has been implicated in...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
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2021
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| Online Access: | https://eprints.nottingham.ac.uk/65595/ |
| _version_ | 1848800246400811008 |
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| author | Stephenson, Katherine |
| author_facet | Stephenson, Katherine |
| author_sort | Stephenson, Katherine |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterised by the excessive deposition of extracellular matrix by fibroblasts and an irreversible loss of lung function. Recently, the interleukin (IL)-1 family member IL-33 has been implicated in the pathogenesis of IPF. Currently however, the cellular and molecular mechanisms by which IL-33 exerts its reported pro-fibrotic effects are poorly understood.
Objective: The overall aim of this thesis was to investigate the role of IL-33 in the development of IPF.
Methods: To assess the pro-fibrotic activity of IL-33 in vitro, primary non-IPF and IPF human lung fibroblasts (HLFs) were stimulated with recombinant IL-33 and the expression of fibrotic genes (ACTA2 and COL1A1) assessed by quantitative real-time PCR (qPCR). To determine the importance of IL-33 during the fibrotic response in vivo, the bleomycin (BLM) mouse model of pulmonary fibrosis was used. Specifically, male C57BL/6 mice were exposed to 60 IU of BLM and 10 mg/kg of either isotype control, anti-ST2 antibody (αST2) or ST2-Fc fusion protein administered during the fibrotic phase of the BLM model. Lung and bronchoalveolar lavage (BAL) samples were collected 28 days post BLM treatment and multiple inflammatory (IL-33 and ST2 levels, IL-4 and IL-5 levels and total and differential BAL cell counts) and fibrotic readouts (lung hydroxyproline (collagen) content, lung weight and modified Ashcroft score) measured. To evaluate the fibrotic potential of IL-33 ex vivo, a method to generate and culture human precision cut lung slices (PCLS) was developed. Using this method, PCLS from normal adjacent and IPF lung tissue were stimulated with recombinant IL-33 and the expression of fibrotic markers (ACTA2, COL1A1, FN1 and fibronectin) quantified by qPCR and western blotting. Finally, to assess the importance of the oxidised form of IL-33 in IPF, non-IPF and IPF HLFs were treated with oxidised IL-33 (oxIL-33) in vitro and the phosphorylation of multiple proteins measured via phospho-kinase array and western blotting.
Results: IL-33 stimulation had no effect on ACTA2 and COL1A1 gene expression by HLFs in vitro, with these cells found to lack the ST2 component of the IL-33 receptor. Despite having effects on inflammatory readouts suggestive of target engagement, neither the αST2 antibody nor the ST2-Fc fusion protein reduced established BLM-induced fibrosis in vivo. Likewise, IL-33 stimulation had no effect on ACTA2, COL1A1, FN1 and fibronectin expression by human PCLS. Interestingly however, oxIL-33 treatment appeared to induce ST2-independent PI3K/AKT/mTOR signalling in HLFs, with significant increases in phospho-AKT and phospho-PRAS40 observed verses unstimulated control.
Conclusion: The data presented in this thesis suggests that, when signalling via ST2, IL-33 is unlikely to play a key pathophysiological role during the development of IPF. However, as oxIL-33 signals via a novel, potentially pro-fibrotic pathway in HLFs, it is possible that, once oxidised, IL-33 may promote pulmonary fibrosis. Further work investigating the role of oxIL-33 in the development of IPF is warranted. |
| first_indexed | 2025-11-14T20:48:31Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-65595 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T20:48:31Z |
| publishDate | 2021 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-655952025-02-28T15:12:26Z https://eprints.nottingham.ac.uk/65595/ The role of Interleukin-33 in the development of idiopathic pulmonary fibrosis Stephenson, Katherine Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterised by the excessive deposition of extracellular matrix by fibroblasts and an irreversible loss of lung function. Recently, the interleukin (IL)-1 family member IL-33 has been implicated in the pathogenesis of IPF. Currently however, the cellular and molecular mechanisms by which IL-33 exerts its reported pro-fibrotic effects are poorly understood. Objective: The overall aim of this thesis was to investigate the role of IL-33 in the development of IPF. Methods: To assess the pro-fibrotic activity of IL-33 in vitro, primary non-IPF and IPF human lung fibroblasts (HLFs) were stimulated with recombinant IL-33 and the expression of fibrotic genes (ACTA2 and COL1A1) assessed by quantitative real-time PCR (qPCR). To determine the importance of IL-33 during the fibrotic response in vivo, the bleomycin (BLM) mouse model of pulmonary fibrosis was used. Specifically, male C57BL/6 mice were exposed to 60 IU of BLM and 10 mg/kg of either isotype control, anti-ST2 antibody (αST2) or ST2-Fc fusion protein administered during the fibrotic phase of the BLM model. Lung and bronchoalveolar lavage (BAL) samples were collected 28 days post BLM treatment and multiple inflammatory (IL-33 and ST2 levels, IL-4 and IL-5 levels and total and differential BAL cell counts) and fibrotic readouts (lung hydroxyproline (collagen) content, lung weight and modified Ashcroft score) measured. To evaluate the fibrotic potential of IL-33 ex vivo, a method to generate and culture human precision cut lung slices (PCLS) was developed. Using this method, PCLS from normal adjacent and IPF lung tissue were stimulated with recombinant IL-33 and the expression of fibrotic markers (ACTA2, COL1A1, FN1 and fibronectin) quantified by qPCR and western blotting. Finally, to assess the importance of the oxidised form of IL-33 in IPF, non-IPF and IPF HLFs were treated with oxidised IL-33 (oxIL-33) in vitro and the phosphorylation of multiple proteins measured via phospho-kinase array and western blotting. Results: IL-33 stimulation had no effect on ACTA2 and COL1A1 gene expression by HLFs in vitro, with these cells found to lack the ST2 component of the IL-33 receptor. Despite having effects on inflammatory readouts suggestive of target engagement, neither the αST2 antibody nor the ST2-Fc fusion protein reduced established BLM-induced fibrosis in vivo. Likewise, IL-33 stimulation had no effect on ACTA2, COL1A1, FN1 and fibronectin expression by human PCLS. Interestingly however, oxIL-33 treatment appeared to induce ST2-independent PI3K/AKT/mTOR signalling in HLFs, with significant increases in phospho-AKT and phospho-PRAS40 observed verses unstimulated control. Conclusion: The data presented in this thesis suggests that, when signalling via ST2, IL-33 is unlikely to play a key pathophysiological role during the development of IPF. However, as oxIL-33 signals via a novel, potentially pro-fibrotic pathway in HLFs, it is possible that, once oxidised, IL-33 may promote pulmonary fibrosis. Further work investigating the role of oxIL-33 in the development of IPF is warranted. 2021-08-04 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en arr https://eprints.nottingham.ac.uk/65595/1/THE%20ROLE%20OF%20IL-33%20IN%20THE%20DEVELOPMENT%20OF%20IPF_CORRECTED_FINAL_VERSION.pdf Stephenson, Katherine (2021) The role of Interleukin-33 in the development of idiopathic pulmonary fibrosis. PhD thesis, University of Nottingham. Idiopathic pulmonary fibrosis; Pathogenesis; Pro-fibrotic effects |
| spellingShingle | Idiopathic pulmonary fibrosis; Pathogenesis; Pro-fibrotic effects Stephenson, Katherine The role of Interleukin-33 in the development of idiopathic pulmonary fibrosis |
| title | The role of Interleukin-33 in the development of idiopathic pulmonary fibrosis |
| title_full | The role of Interleukin-33 in the development of idiopathic pulmonary fibrosis |
| title_fullStr | The role of Interleukin-33 in the development of idiopathic pulmonary fibrosis |
| title_full_unstemmed | The role of Interleukin-33 in the development of idiopathic pulmonary fibrosis |
| title_short | The role of Interleukin-33 in the development of idiopathic pulmonary fibrosis |
| title_sort | role of interleukin-33 in the development of idiopathic pulmonary fibrosis |
| topic | Idiopathic pulmonary fibrosis; Pathogenesis; Pro-fibrotic effects |
| url | https://eprints.nottingham.ac.uk/65595/ |