Davis, Lauren C. 
ORCID: 0000-0002-8597-7747
(2024).
The role of hypoxia in idiopathic pulmonary fibrosis.
University of Birmingham.
Ph.D.
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Davis2024PhD.pdf
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Abstract
Idiopathic pulmonary fibrosis (IPF) is a devastating condition of unknown cause that results in progressive, irreversible scarring of the lung. Resultant lung function decline is currently poorly controlled by antifibrotic therapies. The mechanisms driving pathology remain poorly understood. Current evidence suggests that repetitive epithelial injury, in genetically susceptible individuals, escalates to an aberrant wound healing response. Positive feedback potentiates fibroblast ECM secretion. Lung resident macrophages are implicated as the link between injury and fibroblast activation, as key orchestrators of normal wound healing.
Various genetic and environmental factors likely contribute to disease progression. Evidence indicates that some patients’ lung tissue contains areas of hypoxia. Nocturnal hypoxemia correlates with worse outcomes in IPF patients, suggesting a pathological role for hypoxia in IPF. Accordingly, hypoxia drives fibrotic changes in human stromal cells in vitro.
This thesis hypothesises that hypoxia can cause and/or potentiate profibrotic effects in the IPF lung, altering alveolar macrophage (AM) phenotype and function and causing multi-cellular effects in precision cut lung slices (PCLS). This hypothesis is assessed in using cells and PCLS derived from human lung tissue.
A 48-hour 1% oxygen hypoxic exposure was validated in AMs, to assess phenotypic and functional effects. After demonstrating HIF1α induced transcriptional responses, key mediators implicated in AM contribution to fibrosis were assessed, detecting increased matrix metalloproteinase (MMP)-7 secretion and a trend towards increased MMP-1 secretion. Additionally, hypoxia impaired AM efferocytosis of apoptotic neutrophils, but not phagocytosis of heat-killed Streptococcus pneumoniae. No changes in pro-efferocytic (CD163 and CD260) or anti-efferocytic (SIRPα) receptor levels corresponded to this difference.
As efferocytosis introduces more complex cargo than phagocytosis, differences in metabolic demand were a possible distinguishing factor. The role of HIF1α was assessed, given its role in glycolytic shift in hypoxia. Artificial HIF1α stabilisation did not alter efferocytosis, however trends indicate it increased phagocytosis. This distinction suggests that HIF1α activation has distinct effects on the two processes. Further work is required to delineate this difference.
AMs underutilise glycolysis due to adaptation to the lung environment. Therefore, glycolysis was assessed by measuring lactate release in hypoxia with and without lipopolysaccharide (LPS) stimulation. Lactate secretion was increased in hypoxia, although LPS and hypoxia did not have additive effects. This preliminary data on AM glycolytic capacity in hypoxia provides the rationale for real-time analysis of AM metabolism in hypoxia and during efferocytosis.
A 48-hour 1% oxygen challenge was also validated in PCLS, to assess whether profibrotic effects extended beyond effects on human monocultures. HIF1α signalling was again activated and fibrotic markers collagen I and α smooth muscle actin were assessed. Although no changes could be detected at the RNA level, collagen I protein was increased by hypoxia after 48 hours. There was no additive effect of a previously published profibrotic cocktail on changes induced by hypoxia. Future work should explore these findings using spatial techniques to discern cellular contributions.
Overall, this data shows that AMs may contribute to the profibrotic effects of hypoxia, as hypoxia causes increased release of damaging MMP enzymes and reduces efferocytic capacity. Additionally, a human PCLS model supports previous literature describing increased collagen secretion in hypoxia.
| Type of Work: | Thesis (Doctorates > Ph.D.) | ||||||||||||
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| Award Type: | Doctorates > Ph.D. | ||||||||||||
| Supervisor(s): |
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| Licence: | All rights reserved | ||||||||||||
| College/Faculty: | Colleges (2008 onwards) > College of Medical & Dental Sciences | ||||||||||||
| School or Department: | Institute of Inflammation and Ageing | ||||||||||||
| Funders: | Wellcome Trust | ||||||||||||
| Subjects: | Q Science > Q Science (General) Q Science > QH Natural history > QH301 Biology |
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| URI: | http://etheses.bham.ac.uk/id/eprint/13994 |
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