Analysing the potential anti-inflammatory effects of vitamin d on human corneal epithelial cells

Appleby, Lucy (2022) Analysing the potential anti-inflammatory effects of vitamin d on human corneal epithelial cells. Doctoral thesis, Northumbria University.

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Inflammation of the ocular surface is required to remove a range of pathogens which come into contact with the anterior surface of the eye, lowering the risk of further pathological complications, such as diminished sight or blindness. The cornea is responsible for refracting light into the eye and must remain clear to ensure functionality. However, unregulated immune responses are associated with corneal tissue damage in a range of inflammatory ocular diseases, which manifests as a loss of visual capability. Previous studies have shown vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25D3) suppresses inflammatory pathways by modulating innate immune responses, including the production of pro-inflammatory mediators and antimicrobial peptides.
This research used an in vitro model of corneal inflammation comprising of the human telomerase-immortalized corneal epithelial cell line (hTCEpi) together with various agonists to stimulate toll-like receptors (TLR) which are pattern recognition receptors. The TLR agonists produce inflammatory responses similar to the pathogens they represent. The effects of 10-7M 1,25D3 treatment on these responses were investigated. Using qRT-PCR, the study confirmed that 1,25D3 treatment significantly attenuated the expression of hTCEpi cell TLR3 (p<0.001), TLR5 (p<0.001) and TLR9 (p<0.001) stimulated by their agonists as well as expression of associated inflammatory molecules, including IL-6, IL-8 and IL-1b. Interestingly, IL-1b expression was shown to be significantly attenuated by 4h during TLR3 suppression (p<0.001), which may have suppressed further inflammation. Although there was an overall attenuation in pro-inflammatory cytokine expression following 1,25D3 treatment, 24h of 1,25D3 treatment alongside TLR3 activation led to a significant increase in the expression of cyclooxygenase-2 (COX-2) (p<0.01). This suggested that 1,25D3 is not entirely suppressive towards inflammatory responses, and may exacerbate inflammation.
When hTCEpi cells were stimulated with TLR5 ligands, bacterial flagellin from Pseudomonas aeruginosa and Salmonella typhimurium, 1,25D3 treatment reduced the expression of pro-inflammatory mediators IL-6, IL-8 and IL-1b (p<0.001). The study also used various classes of CpG oligodeoxynucleotide (CpG-ODN), TLR9 ligands which possess different levels of unmethylated CpG motifs, where interestingly, increasing CpG motifs led to a weaker pro-inflammatory response by hTCEpi cells. Addition of 1,25D3 to hTCEpi cells stimulated with either CpG-ODN or Escherichia coli (E. coli) DNA significantly decreased expression of a range of pro-inflammatory mediators, including IL-6 (p<0.001) and IL-8 (p<0.001).
Micro-RNA (miR), which regulate inflammatory protein expression, were investigated for their role in the mechanism of action of 1,25D3 during its attenuation of responses to TLR3, TLR5 and TLR9 stimulation. Data showed that inhibition of miR-93-5p and miR-181-3p led to a significant increase in the expression IL-6, IL-8 and IL-1b following all TLR3 and TLR5 stimulations, with some TLR9 stimulations. These data implicate a regulatory role for these miR in ocular inflammation. Finally, the study failed to identify significant cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/stimulator of IFN Gene (STING) activity in hTCEpi cells.
In conclusion, this study provides evidence that 1,25D3 inhibits inflammation caused by a range of pathogens, whilst certain miR may regulate pro-inflammatory mediators following a range of TLR activations. Hence both 1,25D3 and miR are candidates for treatment of ocular inflammatory diseases.

Item Type: Thesis (Doctoral)
Uncontrolled Keywords: micro-rna, ocular inflammation, the eye, cornea, IL-8 IL-6
Subjects: A300 Clinical Medicine
B500 Ophthalmics
Department: Faculties > Health and Life Sciences > Applied Sciences
University Services > Graduate School > Doctor of Philosophy
Depositing User: Rachel Branson
Date Deposited: 28 Jul 2022 14:10
Last Modified: 28 Jul 2022 14:15

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