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Title: Characterising the role of Staphylococcus aureus and its toxins in chronic rhinosinusitis
Author: Biggs, Timothy Charles
ISNI:       0000 0004 7972 1758
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2019
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Chronic rhinosinusitis (CRS) is a chronic inflammatory condition affecting the lining of the nose and paranasal sinuses. It is the second most common chronic disease worldwide, and impacts significantly on patients' quality of life and healthcare resources. CRS is subdivided into two main disease categories: CRS with and without nasal polyps (CRSwNP and CRSsNP respectively). It is well established that bacteria, most notably Staphylococcus aureus (S. aureus), play an important role in the pathogenesis of CRS. Whilst much emphasis has been placed on surface bacteria, intracellular bacteria are gaining more prominence in relation to resistant disease. The intracellular environment may provide a protective niche for pathogenic bacteria to evade host immunity, and provide a reservoir for re-infection. Recently published findings from the Southampton Upper Airway Research Group reported the novel finding of intracellular S. aureus within nasal polyp mast cells, in addition to epithelial cells. Furthermore, the presence/addition of Staphylococcus aureus Enterotoxin B (SEB) further promoted the internalisation of S. aureus into mast cells. The aim of this work was to further characterise the host response towards S. aureus and its toxins, with particular emphasis on S. aureusmast cell interactions. A prospective study was performed using ex-vivo sinonasal mucosa and nasal polyp tissue from CRS patients, and sinonasal mucosa from non-CRS patients undergoing trans-sphenoidal pituitary surgery as a control. Pro-inflammatory cytokine profiles of these tissue sub-sites were measured using real time quantitative polymerase chain reaction (RT-qPCR). An explant tissue model was developed to study the immune response of nasal polyps, and control samples to SEB, with the resultant host immune response measured using RT-qPCR and Luminex. An in vitro cell culture model was developed to characterise interactions between a CRS-specific S. aureus isolate and the RPMI-2650 epithelial cell line, HMC-1 mast cell line and LAD2 mast cell line. S. aureus was cultured to the mid-log phase and combined with all three cell lines, at set time-points and multiplicity of infection ratios. Intracellular uptake of S. aureus was examined using confocal laser scanning microscopy, intracellular viability and its release was examined using colony forming unit (CFU) enumerations, and the associated host immune response was measured using RT-qPCR and Luminex. Study of the potential phenotypic change to S. aureus, from its continual uptake and release from mast cells, was undertaken in both HMC-1 and LAD2 cell lines, utilising CFUs, RT-qPCR and Luminex. For the study of IgE sensitisation and its effect on the activation of S. aureus infected LAD2 cells, the following techniques were used; LDH assays, degranulation (β-hexosaminidase) assays, protein kinase phosphorylation, RT-qPCR and Luminex. In comparison to control patients, CRSwNP patients display upregulated pro-inflammatory cytokines (IL-5, IL-8) toll-like receptors (TLR-4) and matrix metalloproteinases (MMP-28). In many cases the nonpolypoidal sinonasal mucosa of CRSwNP and nasal polyps display strikingly similar immune profiles, with no statistical difference found between the two sites. SEB was found to significantly upregulate nasal polyp gene expression ratios of IL-5, IL-17A, TNFα, TGF-β, and supernatant protein concentrations of IFNγ, IL-5, IL-17A, and TNFα. In cell line culture experiments, RPMI-2650 epithelial cells, as well as HMC-1 and LAD2 mast cells, readily internalised S. aureus. This intracellular uptake was significantly enhanced in the presence of SEB for both epithelial and mast cells at 24 hours. Intracellular S. aureus was found to be viable and capable of release, rapidly replenishing previously eradicated extracellular bacterial populations. Upon S. aureus exposure, both mast cells (HMC-1) and epithelial cells (RPMI-2650) contributed to inflammation through pro-inflammatory cytokine release vi into the culture supernatant (IFNγ, TNFα, IL-17A, IL-1β and IL-6). S. aureus was able to significantly downregulate both the gene expression (IL-8, IL-1β, TNFα, TGF-β1 and IL-5) and protein release (TNFα) of LAD2 mast cells through the sequential repeated intracellular uptake and release of S. aureus. In the presence of prior IgE sensitisation, S. aureus infected LAD2 mast cells were able to limit degranulation, the host immune response (TNFα gene expression and protein release), and the phosphorylation of Atk2 and GSK-3α/β. These findings demonstrate the importance of S. aureus and its toxins in the development of a chronic inflammatory reaction in CRS patients. Epithelial and mast cells appear to provide a protective niche, shielding S. aureusfrom immune mediated clearance, as well as acting as a reservoir of infection that can replenish eradicated bacterial populations. Through prior IgE sensitisation of mast cells, S. aureus was able to manipulate the subsequent host immune response, favouring its own survival. A similar response was also seen following repeated uptake and release of S. aureus from mast cells, suggesting bacterial phenotypic change. This is of particular relevance in patients with elevated IgE levels, as is seen in patients with S. aureus colonisation where IgE forms against both S. aureus and its enterotoxins. This may well facilitate the ongoing survival and submucosal persistence of S. aureus, and could contribute towards the development of a chronic disease process. These patients are likely to benefit from aggressive medical therapy, particularly in the post-operative period, to eradicate S. aureus, in order to improve long-term treatment outcomes.
Supervisor: Salib, Rami ; Pender, Sylvia Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available