The environment and the host in chronic Rhinosinusitis.

The research described herein follows an extensive literature review of the role of environmental agents and the host immune system in the manifestation of CRS. There are clear deficiencies in our understanding regarding the microbial flora of CRS patients and non-diseased sinuses. Chapter two describes a detailed study of the fungal and bacterial microbiome of diseased and healthy sinuses and forms a basis on which to build the subsequent research projects. The third and fourth chapters describe the development of two animal models to determine the environmental and host factors, which are associated with sinonasal fungal biofilm formation. The final chapter seeks to determine the relevance of sinonasal microorganisms by detecting them on host surfaces and correlating these with specific host immune responses. The interaction of bacteria and host hypersensitivities to allergens is also explored. The initial investigation focused on understanding the microbial flora in CRS patients. This study forms a foundation for the thesis, and was critical to address the many deficiencies and contradictions in the published literature regarding the microbiome of CRS patients. We used state of the art microbial detection techniques to determine the presence and abundance of fungi and bacteria on the mucosa of CRS patients, and appropriate healthy control mucosa. This highlighted some cornerstones of microbial variability between healthy and diseased sinuses. We have shown that the healthy sinus is clearly not sterile, and that prevalence, but more importantly, species composition and population density are critical factors in determining the disease state. Comparisons between various detection techniques such as molecular analysis, Fluorescence in situ hybridization (FISH), and conventional culture showed FISH to be highly sensitive and specific, with a detection threshold related to organism abundance, whereas culture has a tendency to select for rapidly growing organisms. The subsequent study is detailed in chapter three, and addresses two of the most contentious, environment versus host issues in the CRS research community – the interaction between fungal organisms, and the host with type I hypersensitivity to fungi. We developed a large animal (sheep) model of fungal sinusitis to investigate these factors and successfully sensitized 45% of animals to fungal antigens, as evidenced by positive skin prick tests. Despite the presence of fungal hypersensitivity, we were unable to produce fungal biofilms in the occluded frontal sinus. Following our clinical observations of fungi frequently co-habiting with bacteria, particularly Staphylococcus aureus, we co-inoculated fungi with this bacterium and florid fungal biofilm formed on sinus mucosa. Type I hypersensitivity to fungi had no correlation with fungal biofilm or inflammation. These results suggested that fungi may not be able to form biofilm on mucosa with intact immune defences and a primary insult from the bacteria was requisite for fungal adhesion and proliferation. A follow up study addressed the factors, which contribute to fungal biofilm establishment on sinus mucosa. An animal model was again developed to determine if co-inoculation of fungi with other bacterial species would allow fungi to proliferate. Four bacterial species commonly detected in CRS patients were utilized. We hypothesized that bacterial induced cilial injury may have a role in allowing early fungal adhesion, and a cilia toxin was utilized to assess the effect of isolated cilial impairment on fungal proliferation. Cilia were assessed using transmission electron microscopy. Again, no fungal biofilm formed when fungi was inoculated in isolation. Three of the bacterial species formed bacterial biofilms in >75% of sinuses, and this was associated with significant cilial damage, and fungal biofilm formation. One of the bacterial species did not form biofilm, and no fungal biofilm formed in co-inoculated sinuses. Cilia toxin caused significant cilial injury, and was also associated with fungal proliferation. This study demonstrates the importance of the physiochemical barrier in defence against fungal organisms. This led to the question of the role of fungi in CRS patients – are they contributing to the inflammation or merely saprophytic colonizers of the impaired mucosa? The final study addressed this question in a human subject cohort. To determine if microorganisms have a role in inflammatory processes, we need to be able to display an organism specific immune response in the host. We measured the organism specific IgE levels in the serum and mucosa of 48 CRS patients and 10 controls. We also determined the presence of these microorganisms on the mucosa using conventional culture, and FISH using specific probes. We showed that in CRSwNP patients, the presence of S. aureus and fungi on the mucosa was related to elevated organism specific IgE within the mucosa. This phenomenon was specific to nasal polyp patients, and was not observed in nonpolyp CRS or control patients. This demonstrates that these organisms have the capacity to incite specific immune responses in the host, potentially contributing to mucosal inflammation in CRS. Additionally we determined that the presence of S. aureus on the mucosa also exacerbates mucosal fungal allergy, potentially enhancing hypersensitivity to ubiquitous airborne fungal allergens. Although this mechanism has been observed in other atopic diseases, this is the first study to document the phenomenon in CRS. It adds to the mounting evidence that S. aureus has an important role in the pathogenesis of CRS.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 201