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Abnormalities in bacterial sensing and inflammatory response to pathogens in the lung parenchyma of chronic obstructive pulmonary disease patients

Abnormalities in bacterial sensing and inflammatory response to pathogens in the lung parenchyma of chronic obstructive pulmonary disease patients
Abnormalities in bacterial sensing and inflammatory response to pathogens in the lung parenchyma of chronic obstructive pulmonary disease patients
Abnormalities in bacterial sensing and inflammatory response to pathogens in the lung parenchyma of chronic obstructive pulmonary disease patients Chronic obstructive pulmonary disease (COPD) is a progressive disease that results in loss of lung function. COPD patients are prone to periods of increased systemic and localised inflammation and elevation of symptom, termed exacerbations, increased morbidity and mortality. Smoking is the main risk factor. Most exacerbations are triggered following infection with viruses and bacteria such as non-typeable Haemophilus influenza, Moraxella cattarhalis and Streptococcus pneumonia. Colonisation by new bacterial species and a shift in existing colonising strains can also induce exacerbation.

This study investigated inflammatory responses to bacterial exposure in lung parenchyma explants from surgical patients (COPD and controls) in relation to their disease status. Stimuli included Escherischia coli endotoxin (lipopolysaccharide, LPS) and heat killed H. influenza intended to mimic pulmonary response to the multiple ligands present during bacterial colonisation. Potential contribution of the preRexisting cellular inflammatory environment to the excessive inflammation seen in COPD was examined by compared inflammatory cell load (control/COPD lung parenchyma) using immunohistochemistry (IHC) and flow cytometry (FC). Little difference was observed in the inflammatory cell load between control and COPD lungs; although a modest reduction in lung macrophages was observed in the COPD lungs by FC (P = 0.09). Since COPD is characterised by excessive inflammatory responses to smoke particulates, we hypothesised that similar responses would be observed following exposure to bacterial ligands; providing an explanation why COPD patients suffer exacerbations often requiring hospitalisation, whereas control individuals do not.

An ex vivo stimulation model was optimised to determine the peak time-point for mediator secretion and the optimum ligand dose using LPS and H. influenzae as stimuli. Time course study showed that TNF-α peaked at 24 h for both stimuli. Using optimised conditions, the inflammatory mediator response to both stimuli was compared between lung explants (control/COPD). Stimulation with H. influenzae and LPS led to significant release of the majority of cytokines/chemokines (TNF-α, IL-1β, -6, -8, -10, -17, MCP-1, MIP-1Lβ) measured. There was a significantly elevated release of IL-6 and MCP-1 in the COPD group. IL-8 secretion appeared to be depressed by exposure to bacterial ligands in the controls, but unaltered in the COPD group. Since the inflammatory cell load in lung tissues from control/COPD subjects did not give us an answer to account for the excessive inflammatory response observed in COPD patients. We, therefore, measured the expression of two bacterial sensing ligands (TLR 2 and 4) on the surface of epithelial and inflammatory cells resident in the lung tissues at baseline in these two cohorts using FC. A decrease in TLR 4 expression on CD8+ T-lymphocytes in lung tissues from the COPD group was observed. This difference was suggestive of abnormal bacterial sensing in the COPD lung, but the observed reduction is contrary to our prediction of increased levels in COPD. It may be that suppressed bacterial recognition prevents bacterial clearance through cell-mediated immunity, pre-disposing COPD patients to bacterial colonisation, while the overall mediator responses are only modestly elevated.

In term of exacerbation treatment, we looked at the efficacy of two novels inhibitors of an inflammatory cascade namely p38MAPkinase pathway and compared them to corticosteroids (CS). Inhaled (PF-0371545) and oral (PHA-797804) inhibitors were applied to our bacterial ligand stimulation model in dose-response studies; Fluticasone propionate (FP) was the positive control. TNF-α at 24 h was used as the primary outcome. PHA-797804 was more effective than PF- 0371545 as an anti-inflammatory agent in our model, possibly reflecting its solubility characteristics. Despite requiring a higher dose than FP, this inhibitor may be useful as an adjunct to existing corticosteroid therapy, enabling a reduction in dose of both inhibitors to limit side-effects. In summary, this model was able to cause an inflammatory reaction in lung tissue and lung tissue from COPD patients was found to be more inflammatory.



University of Southampton
Moreau, Achika
aad59a62-f5f9-44bc-8b50-eb0c6c0d3807
Moreau, Achika
aad59a62-f5f9-44bc-8b50-eb0c6c0d3807
Djukanovic, Ratko
d9a45ee7-6a80-4d84-a0ed-10962660a98d
Nicholas, Benjamin
785c44fb-6536-4189-803b-4545425e9385

Moreau, Achika (2017) Abnormalities in bacterial sensing and inflammatory response to pathogens in the lung parenchyma of chronic obstructive pulmonary disease patients. University of Southampton, Masters Thesis, 261pp.

Record type: Thesis (Masters)

Abstract

Abnormalities in bacterial sensing and inflammatory response to pathogens in the lung parenchyma of chronic obstructive pulmonary disease patients Chronic obstructive pulmonary disease (COPD) is a progressive disease that results in loss of lung function. COPD patients are prone to periods of increased systemic and localised inflammation and elevation of symptom, termed exacerbations, increased morbidity and mortality. Smoking is the main risk factor. Most exacerbations are triggered following infection with viruses and bacteria such as non-typeable Haemophilus influenza, Moraxella cattarhalis and Streptococcus pneumonia. Colonisation by new bacterial species and a shift in existing colonising strains can also induce exacerbation.

This study investigated inflammatory responses to bacterial exposure in lung parenchyma explants from surgical patients (COPD and controls) in relation to their disease status. Stimuli included Escherischia coli endotoxin (lipopolysaccharide, LPS) and heat killed H. influenza intended to mimic pulmonary response to the multiple ligands present during bacterial colonisation. Potential contribution of the preRexisting cellular inflammatory environment to the excessive inflammation seen in COPD was examined by compared inflammatory cell load (control/COPD lung parenchyma) using immunohistochemistry (IHC) and flow cytometry (FC). Little difference was observed in the inflammatory cell load between control and COPD lungs; although a modest reduction in lung macrophages was observed in the COPD lungs by FC (P = 0.09). Since COPD is characterised by excessive inflammatory responses to smoke particulates, we hypothesised that similar responses would be observed following exposure to bacterial ligands; providing an explanation why COPD patients suffer exacerbations often requiring hospitalisation, whereas control individuals do not.

An ex vivo stimulation model was optimised to determine the peak time-point for mediator secretion and the optimum ligand dose using LPS and H. influenzae as stimuli. Time course study showed that TNF-α peaked at 24 h for both stimuli. Using optimised conditions, the inflammatory mediator response to both stimuli was compared between lung explants (control/COPD). Stimulation with H. influenzae and LPS led to significant release of the majority of cytokines/chemokines (TNF-α, IL-1β, -6, -8, -10, -17, MCP-1, MIP-1Lβ) measured. There was a significantly elevated release of IL-6 and MCP-1 in the COPD group. IL-8 secretion appeared to be depressed by exposure to bacterial ligands in the controls, but unaltered in the COPD group. Since the inflammatory cell load in lung tissues from control/COPD subjects did not give us an answer to account for the excessive inflammatory response observed in COPD patients. We, therefore, measured the expression of two bacterial sensing ligands (TLR 2 and 4) on the surface of epithelial and inflammatory cells resident in the lung tissues at baseline in these two cohorts using FC. A decrease in TLR 4 expression on CD8+ T-lymphocytes in lung tissues from the COPD group was observed. This difference was suggestive of abnormal bacterial sensing in the COPD lung, but the observed reduction is contrary to our prediction of increased levels in COPD. It may be that suppressed bacterial recognition prevents bacterial clearance through cell-mediated immunity, pre-disposing COPD patients to bacterial colonisation, while the overall mediator responses are only modestly elevated.

In term of exacerbation treatment, we looked at the efficacy of two novels inhibitors of an inflammatory cascade namely p38MAPkinase pathway and compared them to corticosteroids (CS). Inhaled (PF-0371545) and oral (PHA-797804) inhibitors were applied to our bacterial ligand stimulation model in dose-response studies; Fluticasone propionate (FP) was the positive control. TNF-α at 24 h was used as the primary outcome. PHA-797804 was more effective than PF- 0371545 as an anti-inflammatory agent in our model, possibly reflecting its solubility characteristics. Despite requiring a higher dose than FP, this inhibitor may be useful as an adjunct to existing corticosteroid therapy, enabling a reduction in dose of both inhibitors to limit side-effects. In summary, this model was able to cause an inflammatory reaction in lung tissue and lung tissue from COPD patients was found to be more inflammatory.



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final June 26 2017 MPhil Thesis - Version of Record
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Published date: 26 June 2017

Identifiers

Local EPrints ID: 422655
URI: https://eprints.soton.ac.uk/id/eprint/422655
PURE UUID: 8a3e7d8b-a379-450a-8021-af1fcfc626ba
ORCID for Ratko Djukanovic: ORCID iD orcid.org/0000-0001-6039-5612

Catalogue record

Date deposited: 27 Jul 2018 16:30
Last modified: 14 Mar 2019 01:55

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Contributors

Author: Achika Moreau
Thesis advisor: Ratko Djukanovic ORCID iD
Thesis advisor: Benjamin Nicholas

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