Nitric oxide and Staphylococcus aureus biofilms: defining their intricate relationship in chronic rhinosinusitis

Abstract

This thesis aims to address the relationship of Staphylococcus aureus (S. aureus) biofilms to the endogenously produced gas nitric oxide (NO) in chronic rhinosinusitis (CRS). While S. aureus biofilms are associated with recalcitrance and high severity in CRS, the naturally elevated NO gas is significantly lower in sinuses of CRS patients. However, the relationship of these 3 important factors in CRS aetiopathogenesis is poorly defined. To further clarify this host-microbe-environment (NO) relationship, this thesis first looks into the history of each factor, the roles they play in other disease processes, and the most recent clinical findings and applications in current literature. Building on this foundation, the projects emanating from this thesis hoped to fill in some gaps in knowledge of these 3 components, identifying that all are linked to disease manifestation, and that each can mutually contribute to CRS pathogenesis. The first project was designed to establish a clearer description of the relationship between NO and S. aureus biofilms. Utilizing S. aureus strains from CRS patients, these were grown as biofilms and exposed to various NO concentrations mimicking NO levels measured in healthy sinuses vs. CRS patients. We demonstrated the dualistic effects of NO on biofilm growth: increased at lower NO concentrations mimicking diseased sinuses, and anti-biofilm effects at higher concentrations similar to measurements in healthy sinuses. These findings became a stepping stone for the potential design of NO as a therapeutic agent in S. aureus-associated CRS. But first, further characterization of NO’s role on the host immune response was needed. The 2nd and 3rd projects aimed to define the host–NO relationship, focusing on the genes involved in NO regulation within the sinonasal mucosa. Because NO is considered one of the reactive oxygen species (ROS), major players of the innate immune response, genes involved in ROS/innate immunity were investigated. CRS patients, with or without polyps, were sub-classified as either with or without S. aureus biofilms, allowing a separate analysis of the role S. aureus biofilms play in the alteration of gene expression. The results showed that S. aureus biofilm presence associates with a significant difference in the certain gene expressions which have specific roles in NO regulation. This indicates that the microorganism may alter or contribute to an impaired localized innate immune response in the sinuses, or alternatively favor growth in genetically susceptible individuals. Although the cause-effect timeline was not established, these results will serve as baseline for future gene and protein studies that will further increase our understanding of the NO-CRS pathophysiology. Lastly, building on the therapeutic potential of NO as an anti-biofilm agent, we aimed to design a suitable NO-based topical agent against S. aureus biofilms. The 4th project tested a multitude of liposome-encapsulated NO formulations in-vitro with the best formulation tested for safety and efficacy in a sheep model of rhinosinusitis. These projects were designed with an aim for future clinical trials, to test a novel NO-based topical agent, which can be used as a safe and efficacious topical sinus rinse to benefit CRS patients.