The effect of nuclear hormone receptor stimulation in oral epithelia

Hewitt, Benjamin J. ORCID: 0000-0003-1609-6054 (2024). The effect of nuclear hormone receptor stimulation in oral epithelia. University of Birmingham. Ph.D.

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Abstract

Gingivitis is a plaque-mediated, inflammatory periodontal disease of the gingival mucosa with high prevalence in the modern population. The gingival mucosa is covered by the gingival epithelium, which plays a significant role in the pathogenesis of periodontal diseases by instigating inflammatory signalling and recruiting immune cells in response to bacteria. Importantly, the gingival epithelia act as a barrier, maintaining homeostasis and preventing bacterial ingress into the deeper tissues. Gingivitis itself poses little risk to the patient but may progress to periodontitis in susceptible individuals. This is a hyperactive immune response to the plaque stimulus, leading to destruction of the deeper tissues and a loss of attachment of the tooth. This thesis aimed to study these inflammatory and barrier function responses to bacteria in vitro in monolayer cells and novel organotypic models, and then investigate the nuclear hormone receptor peroxisome proliferator activated receptor gamma (PPARγ) as an anti-inflammatory agent.

RT-qPCR, transepithelial electrical resistance and fluorescent macromolecular tracer assays were employed to identify a suitable inducer of inflammation and barrier function failure for use in successive studies. Additionally, two reporter cell lines were generated to validate PPARγ agonists in epithelial cells, with western blotting and PCR performed to identify differential expression of PPARγ tissue isoforms. Finally, a novel culture device was designed, tested and utilised to generate organotypic gingival epithelial cultures, enabling comparison of the response of monolayer and organotypic cultures to an inflammatory agent and PPARγ agonists.
Bacterial lipopolysaccharide (LPS) was shown to effectively induce pro-inflammatory cytokine expression. Treatment with a validated PPARγ agonist (GW1929) abrogated this effect, reducing the expression of pro-inflammatory cytokines and increasing anti-inflammatory cytokines. Barrier function was also significantly reduced by LPS, with this effect appearing to be partially abrogated by PPARγ treatment.
The buoyant epithelial culture device (BECD) was shown to generate stratified, differentiated gingival epithelial mimics, with minimal user input compared to alternative approaches due to a unique floating design. BECD-cultured cells showed minimal pro-inflammatory cytokine expression following LPS treatment, indicating marked differences in inflammatory signalling compared to monolayer cultures.

In conclusion, this thesis demonstrated the usage of LPS as an inducer of inflammation and barrier function failure, whilst showing that PPARγ agonists may pose significant utility in curbing inflammatory responses in gingival epithelia, with the potential to aid in the maintenance of epithelial barrier function. The efficacy of the novel BECD was also demonstrated by showing the marked difference in response between simple monolayer cultures and more complex organotypic cultures. This system may also find use in further studies of other stratified epithelial tissues.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):

Supervisor(s)	Email	ORCID
Milward, Michael	UNSPECIFIED	UNSPECIFIED
Wiench, Malgorzata	UNSPECIFIED	UNSPECIFIED
Shelton, Richard M.	UNSPECIFIED	UNSPECIFIED
Cooper, Paul	UNSPECIFIED	UNSPECIFIED
Lucas, Robert	UNSPECIFIED	UNSPECIFIED