Investigating the impact of hypoxia on the host-pathogen interactions in the lung

Abstract

Chronic Obstructive Pulmonary Disease (COPD) is a debilitating respiratory disorder characterised by persistent airflow limitation and heightened susceptibility to bacterial infections, such as those caused by nontypeable Haemophilus influenzae (NTHi). Despite the prevalence of NTHi during exacerbations, the mechanisms underlying its impact on COPD progression remain elusive. The airway epithelium plays a crucial role in the host defence against pathogens, and its responses are influenced by various microenvironmental factors, including oxygen levels. Hypoxia, a common feature in the mucus-plugged COPD airways, has been suggested to modulate cellular responses and exacerbate disease progression. However, it is unclear what impact hypoxia has on host-pathogen interactions in the airways. Therefore, the aim of this thesis was to explore the influence of hypoxia on immune responses to NTHi infection, with the goal of uncovering its role in shaping airway epithelial responses within the context of COPD. To achieve this objective, a two-pronged approach was adopted. Transcriptomic analysis was conducted using bulk RNA sequencing on both a BCi-NS1.1 airway epithelium-NTHi infection model and a human lung tissue explant-NTHi infection model under hypoxic conditions. The transcriptomic examination of NTHi-infected BCi-NS1.1 cells revealed a marked upregulation of proinflammatory cytokine responses, with IL1A, IL1B, and TNF emerging as the most differentially expressed genes within the enriched inflammatory response pathway. Subsequent validation of these prominent genes in BCi-NS1.1 cells during hypoxic infection confirmed a significant elevation in the expression of IL1B and TNF. In parallel, the transcriptomic analysis of the lung explant-NTHi infection model unveiled notable alterations in immune responses, characterised by downregulation of anti-inflammatory immune genes (such as IL10 and CD163) as well as a reduction in chemokine response genes (including CCL20 and CCL23). Gene expression validation further illustrated that hypoxia led to a significant increase in TNF expression while causing a significant decrease in IL10 expression. In both the NTHi-infected BCi-NS1.1 model and the NTHi-infected lung explant model, hypoxia potentiated the release of IL-1β and TNF-α, accompanied by a significant reduction in the release of IL-10 cytokine within NTHi-infected lung tissue explants. Exploration into the mechanistic insights of the role of HIF-1α uncovered that, in the presence of a prolyl hydroxylase inhibitor, dimethyloxalylglycine (DMOG), the cytokine release of IL-1β and TNF-α was significantly increased in BCi-NS1.1 cells infected with NTHi. Correspondingly, within NTHi-infected lung explants, the levels of IL-1β exhibited a significant increase, while the levels of IL-10 showed a significant decrease under the influence of DMOG. Finally, an assessment of bacterial load was conducted in infected samples to discern the impact of hypoxia on BCi-NS1.1 cell and lung tissue explant infections. The findings indicated increases in bacterial load in both NTHi-infected BCi-NS1.1 cells and lung tissue explants subjected to hypoxia. Manipulation of the HIF-1 signalling pathway using DMOG demonstrated a further substantial increase in bacterial load in NTHi-infected samples. In conclusion, the data in this thesis indicated that hypoxia dysregulates inflammatory cytokine responses to NTHi infection, contributing to increased bacterial infection in the airway epithelium and whole lung. This thesis sheds light on the interplay between hypoxia and immune responses to NTHi infection, offering insights into the complex mechanisms underlying the airway epithelium's reactions within the context of COPD.

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Identifiers

ORCID for Lee Kevin Page: orcid.org/0000-0001-7435-1813

ORCID for Cosma Mirella Spalluto: orcid.org/0000-0001-7273-0844

ORCID for Karl Staples: orcid.org/0000-0003-3844-6457

ORCID for Jodie Ackland: orcid.org/0000-0003-3120-3620

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