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Wormald, Peter-John Vreugde, Sarah Cooksley, Clare Shaghayegh, Gohar Adelaide Medical School 2022 https://hdl.handle.net/2440/138288 Chronic rhinosinusitis (CRS) is a serious health problem characterised by chronic inflammation of the sinus mucosa associated with tissue remodelling, dysfunction of the sinuses' natural defence mechanisms, and induction of different inflammatory clusters. Staphylococcus aureus mucosal biofilms are associated with severe recalcitrant CRS. However, S. aureus colonisation of the nose and paranasal sinus mucosa is also frequent in the absence of mucosal inflammation. This questions the relevance of S. aureus biofilms in CRS etiopathogenesis. This research aimed to investigate the strain-specific variations in S. aureus biofilm properties in health and disease in relation to inflammation and CRS severity. We further studied the relationship between S. aureus virulence genes and inflammatory markers in patients' sinonasal tissue. Tissue samples, S. aureus clinical isolates, and matched clinical datasets were collected during endoscopic sinus surgery of CRS patients with (CRSwNP) and without (CRSsNP) nasal polyps and non-CRS controls. S. aureus clinical isolates were sequenced and grown into biofilm in vitro, and their virulence genes and biofilm properties, including metabolic activity, biomass, colony-forming units, and exoprotein concentration, were characterised in relation to the frequency and localisation of key immune cells in corresponding sinonasal mucosa and CRS severity scores. Here, we first found increased S. aureus biofilm metabolic activity, biomass, and exoprotein production in relation to increased disease severity scores with infiltration of antibody-secreting cells and loss of regulatory B cells in recalcitrant CRS. Exoproteins from the high biofilm-producing isolates induced mucosal barrier dysfunction in human nasal epithelial cell cultures, and those isolates were more toxic to the C. elegans worm model compared to low biofilm producers. Next, multiplex immunostaining analysis of patient tissue blocks showed increased infiltration of CD3+, CD68+, CD20+, and CD138+ cells in CRSwNP patients' tissue compared to control. CD3+, CD138+, and MBP+ cells diffused deeper into the tissue in CRSwNP while clustered close to the epithelium in controls. S. aureus biofilm properties, inflammatory cell numbers, and CRS severity scores were also positively correlated. In terms of immune T cell subsets analysis, S. aureus biofilm properties and CRS severity scores correlated positively with total CD4+ T cell frequencies but looking into CD4+ T cell subsets showed an inverse correlation with Th1 and Th17 cell frequencies. CD4+ T cell frequencies were higher in patients harbouring lukF.PV-positive S. aureus whilst its regulatory and Th17 cell subset frequencies were lower in patients carrying sea- and sarT/U-positive S. aureus. Finally, innate immune cell analysis showed increased S. aureus biofilm metabolic activity in relation to increased eosinophil cell frequencies and disease severity scores in recalcitrant CRS cases. Mast cell frequencies were found to be higher in tissue samples of patients carrying S. aureus isolates harbouring lukF.PV, sea, and fnbB genes. Altogether, the findings of this project offer insights into a possible pathogenic link between S. aureus biofilms and recalcitrant CRS and could lead to the development of more targeted therapies. Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2022 en Chronic rhinosinusitis, Inflammatory cells, S. aureus biofilm, Disease severity, Virulence genes The relationship between S. aureus biofilm properties, inflammation, and disease severity in chronic rhinosinusitis Thesis This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals.open.access