Strain-specific Intracellular Staphylococcus aureus in resistant chronic rhinosinusitis. Disease mechanisms and potential novel therapies.

Abstract

Chronic rhinosinusitis (CRS) is a chronic inflammatory condition affecting the nose and paranasal sinuses. The disease affects up to 15% of the UK population and is recognised as the second most common chronic condition in Europe after arthritis. There are two well documented phenotypes of the condition; CRS without nasal polyps (CRSsNP) and CRS with polyps (CRSwNP). S. aureus colonises the nasal cavity in 60% of patients with CRSwNP, almost double that of the general population. Recently published research has demonstrated intracellular localisation of S. aureus within epithelial and mast cells in CRSwNP subjects. This appears to promote chronic inflammation, disease recalcitrance and treatment resistance. A prospective study was undertaken to determine if S. aureus identified in CRS had an enhanced ability to internalise and survive within mast cells and to investigate the effectiveness of novel S. aureus-targeted therapies aimed at reducing the bacterial and pro-inflammatory cytokine burden. Nasal swabs of patients undergoing rhinological examination or surgical procedures were taken from non-CRS subjects, and from CRSsNP and CRSwNP patients and grown in S. aureus selective media. The cultured strains underwent short-read whole genome sequencing and were interrogated for the presence of virulence factors, antimicrobial resistance genes and stress proteins. Isolates obtained were used in mast and epithelial intracellular infection models to determine any differences in intracellular survival. Our data demonstrate that there were differences between control, CRSsNP and CRSwNP-cultured S. aureus. There were very few virulence factors seen in CRSsNP patients and many associated with intracellular survival and infection in CRSwNP-cultured S. aureus compared to controls. Our HMC-1 and LAD2 mast cell models confirmed that a representative control strain was less able to internalise and replicate within the intracellular compartment compared to a CRSwNP S. aureus strain. Both strains exhibited similar toxicity levels. The lipophilic statin, simvastatin was able to reduce both the extracellular and intracellular burden of a CRSwNP isolate of S. aureus in LAD2 mast cells in the prodrug, but not the activated form. Simvastatin was able to reduce the number of S. aureus colony-forming units internalised and lactate dehydrogenase release in mast cells in a dose-dependent fashion. Simvastatin appeared to have no effect on substance P signalling in mast cells. In air-liquid interface cultured epithelial cells, simvastatin appeared to reduce the production of pro-inflammatory cytokine expression (TNF, TSLP and CXCL8 (IL-8)) although this failed to reach statistical significance. In summary, our results demonstrate that CRSwNP-cultured S. aureus possesses a number of virulence factors which support intracellular localisation, infection and replication in mast cells. Simvastatin is an inexpensive, safe, and well tolerated oral cholesterol-lowering treatment, and our results indicate it may also reduce the burden of intracellular S. aureus. As such, it has the potential to be re-developed and re-branded for use as an anti-S. aureus adjunctive treatment in patients with S. aureus-related chronic inflammatory conditions such as CRS. Our findings therefore warrant further study of the antibacterial effects of simvastatin in primary cell culture and nasal explant models, and future clinical trials.

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Identifiers

ORCID for Simon Patrick Goldie: orcid.org/0000-0002-3912-9026

ORCID for Rami Salib: orcid.org/0000-0002-6753-7844

ORCID for Andrew Walls: orcid.org/0000-0003-4803-4595

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