Hopkins, Georgina
(2023)
The role of invariant natural killer T cells and lipids in the development of allergic sensitisation.
PhD thesis, University of Nottingham.
Abstract
Introduction
IgE-mediated food allergies are increasing in prevalence, currently affecting 2.6% of infants in the UK. However, the mechanisms underpinning the first phase of IgE-mediated allergy, allergic sensitisation, are still not clear. Recently, the potential involvement of lipids in allergic sensitisation has been proposed, with reports that they can activate invariant natural killer T (iNKT) cells, to secrete Th1 and Th2 cytokines. However, the existing research in this area is limited and predominantly use murine models. Thus, this research developed a human in vitro method to study the role of lipids and iNKT cells in a model of allergic sensitisation. This method was applied to peanut allergy, one of the most common food allergies in children and adults. Thus, the total lipid fraction from peanuts (peanut oil) was utilised with and without the lipophilic peanut allergen, Ara h 8, to examine any influence on iNKT cell cytokine production, comparing between peanut-allergic and non-allergic individuals.
Methods
Due to low abundance of iNKT cells in human peripheral blood, iNKT cells were expanded over 14 days by stimulation with the glycolipid, α-Galactosylceramide (α-GalCer), which is a potent activator of iNKT cells. Autologous dendritic cells (DCs) were generated from monocytes and stimulated with either peanut oil, Ara h 8, or both peanut oil and Ara h 8. The expanded iNKT cells were then immunomagnetically isolated and co-cultured with autologous DCs to allow lipid and/or allergen presentation to iNKT cells. This co-culture was first optimised using α-GalCer-pulsed DCs before applying to peanut oil and Ara h 8. Th1 and Th2 iNKT cell cytokine expression was then measured during iNKT-DC co-culture by flow cytometry.
Results
Flow cytometry staining of iNKT cells from peanut-allergic and non-allergic subject’s peripheral blood found a 5-fold higher iNKT cell population in peanut-allergic subjects compared to non-allergic subjects. The iNKT cells from both subject groups were then successfully expanded, with iNKT cell populations increasing by 133-fold in peanut-allergic subjects and 122-fold in non-allergic subjects after 14 days of culture with α-GalCer. A shift in iNKT cell phenotype to CD4+ iNKT cells was observed in both subject groups after expansion. Also, DCs were successfully generated at high purities from monocytes, and imaging flow cytometry found the immature DCs can internalise lipids and allergens. Finally, iNKT cell co-culture with α-GalCer-pulsed DCs showed increases in iNKT cell production of IFNγ-only and IFNγ+IL4+ after 5 hours, confirming this in vitro, human, cell-based assay is functional. However, when the method was applied to peanut allergy, utilising peanut oil and Ara h 8, the results showed peanut oil and/or Ara h 8 did not have an influence on cytokine production by iNKT cells.
Conclusion
Overall, this study establishes a human model system where allergen-associated lipids can be assessed to determine whether they enhance iNKT cell Th2 cytokine secretion, shifting towards a state of allergic sensitisation. However, peanut oil had no effect on iNKT cell cytokine production. Future research could focus on a specific lipid class from peanut oil, such as the fatty acid oleic acid, to investigate any influence on iNKT cell cytokine production.
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