Paediatrics (RCH) - Theses
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ItemEpigenetic dysregulation underpinning immune cell dysfunction in food allergy( 2022)Food allergy is a major health concern, particularly in Australia, where some of the highest prevalence has been recorded. Although food allergy is most common in younger age groups (between years 0 to 4), most of these allergies resolve within the first few years of life. However, allergies to peanuts and tree nuts tend to be lifelong, and are more likely to cause severe allergic reactions such as anaphylaxis. These severe allergic reactions occur most frequently in the adolescent age group, the highest risk group for fatalities from such reactions. Moreover, up to 30 percent of food allergic adolescents suffer from multiple food allergies, and there is no current effective treatment for this variable phenotype. Rates of food allergy have risen rapidly in the last few decades, at a pace that cannot be explained by genetics alone. While there is a genetic predisposition in food allergy, numerous studies have shown correlations between specific lifestyle and early life factors and food allergy development. Therefore, the origins of food allergy are multi-factorial, and the study of epigenetic factors may allow us to establish the interface between the role of these environmental exposures and our genomes. Indeed, past studies have shown that immune cells from food allergic individuals show aberrant phenotypes even in the absence of allergens, thereby indicating that these immune cells are fundamentally wired to respond differently upon non-specific activation, and these differences have been linked to epigenetic variation. This thesis aimed to identify immune pathways implicated in adolescent food allergy, using samples from individuals enrolled in the SchoolNuts study, a study profiling the prevalence of food allergy in adolescents aged 10 to 14 years in Melbourne, Australia. This was achieved in three parts, first by establishing the baseline immune response to activation in CD4 T cells from non-allergic adolescents. To do this, we compared the DNA methylation (epigenomic) profiles (DNAme) of purified naive CD4 (nCD4) T cells at quiescence (72 hour culture in media alone) or following activation (72hour culture in media with antiCD3 and antiCD28 beads) in healthy infants (aged 12 months, n 18) and adolescents (aged 10 to 15 years, n 15); using the Infinium MethylationEPIC array (EPIC array). This data was then integrated with transcriptomic (bulk RNAseq) and cytokine profiles obtained from culture media from the same samples (quiescent and activated), and publicly available naive CD4 open chromatin (ATACseq) signatures from unrelated infants. By showing that T cell activation induced widespread changes in DNA methylation, I revealed that T cell activation is intrinsically linked to DNA methylation reprogramming. Multi-omic data analysis of transcriptomic and open chromatin datasets validated these findings by showing that this level of reprogramming was reflected in gene expression patterns and altered chromatin conformation, with upregulated gene expression and increased DNA accessibility. While I found similar responses to activation in nCD4T cells from infants and adolescents, there were selected genomic sites showing age specific methylation patterns following activation. Many of these CpG sites were located within the T cell receptor gene locus, suggesting that age influences activation-associated remodelling of DNA methylation at this region. Analysis of cytokine production of these cells at quiescence and following activation indicated enhanced production of IFNgamma, IL2 and IL10 in adolescents, relative to infants. Correlation of these cytokine levels, and DNA methylation level of probes from the EPIC array located near genes encoding for them, highlighted probes (cg01720533, cg05646531, cg14284394, cg16247264) potentially associated with this altered response. I then characterised the epigenomes of naive CD4 T cells from adolescents with food allergy, by generating genome-wide DNAme data in purified nCD4 T cells at quiescence and following activation in adolescents (aged 10 to 15 years old) with peanut allergy (peanut only or peanut and one or more additional food allergy) (FA, n 29), and age-matched non-food allergic controls (NA, n 18). As with the previous study, I also analysed transcriptome-wide gene expression and cytokine production in these cells following activation. I showed that adolescents with FA exhibit unique DNA methylation signatures at quiescence and post-activation at key genes involved in Th1 and Th2 differentiation (RUNX3, RXRA, NFKB1A, IL4R). Combined analysis of DNA methylation, transcriptomic data and cytokine output in the same samples identified an attenuated interferon response in nCD4T cells from FA individuals following activation, with decreased expression of several interferon genes, including IFNgamma and a DMR at a key downstream gene, BST2. The third part of this thesis involved performing the first combined epigenetic and transcriptomic analysis of purified CD19 B cells from adolescents with FA, comparing single-food allergic (peanut only) (SA; n 10), multi-food allergic (peanut and one or more other food) (MA; n 7) and non-allergic (controls) (NA; n 9) individuals. Using bulk RNAseq data and genome wide DNA methylation data generated using the EPIC array, I identified 116 differentially expressed genes (DEGs) distinguishing B cells of FA (both SA and MA) adolescents from NA controls. Additionally, I showed higher methylation at PM20D1, a key regulator of several inflammatory processes, in FA individuals. This analysis also identified, for the first time, distinct epigenetic variation specific to the MA phenotype, which included differential methylation at a number of S100 genes (S100A1, S100A13, S100A14), which are involved in pathways such as inflammation, energy metabolism and cell cycle regulation. This thesis is the first to characterise nCD4 T cell responses across multiple age groups and highlight the levels of epigenetic regulation involved in T cell activation, which marks the first step in T cell differentiation to a range of downstream lineages, such as Th1 and Th2, previously implicated in FA. In doing so, this presents important findings in this field of research by identifying potential timepoints for intervention. Moreover, characterisation of age-specific responses to activation deepens our understanding of immune responses within each age group, which will help us determine timings for interventions relating to age-related decline in immune function. My thesis also presents novel findings in the field of adolescent food allergy, by showing epigenetic dysregulation at several genes implicated in T cell differentiation, as well as at a gene encoding an inflammatory regulator in B cells. Collectively, these results indicate that the immune dysfunction observed in adolescents with FA is likely mediated by epigenetic variations across multiple cell types, potentially in an age-specific manner. Moreover, I identified common epigenetic signature distinguishing MA adolescents from SA adolescents; an important finding given the heterogeneity of this MA clinical group, and point the way forward towards exploring targets for further research, which may allow us to identify potential therapeutic approaches to address peanut allergy in adolescents, and work towards establishing a molecular therapy for the multi food allergic phenotype.