Evaluation of 5-methylcytosine and 5-hydroxymethylcytosine as potential biomarkers for characterisation of chemical allergens.

Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, and chemicals that cause sensitization of the respiratory tract and occupational asthma. In mice these are characterized by different T helper (Th) cell responses. Changes in DNA methylation in particular have been implicated in the in vivo responses to chemical allergy. As such it was hypothesised that differentially methylated regions (DMR) may provide candidates biomarkers of chemical allergy To examine this, mice were exposed to 2,4-dinitrochlorobenzene (DNCB; a contact allergen) or trimellitic anhydride (TMA; a respiratory allergen). DNA from draining lymph nodes was processed for methylated (5mC) and hydroxymethylated (5hmC) DNA immunoprecipitation (MeDIP/hMeDIP) then selected DMR analysed by qPCR. We describe a number of DMRs which, by combined analysis of 5mC and 5hmC, differentiate between responses induced by DNCB and those by TMA. Furthermore, these changes in methylation are specific to the draining lymph node. The Gmpr DMR is suggested as a possible biomarker for contact allergen-induced immune responses; it is characterised by divergent levels of 5mC and 5hmC DNCB-treated mice only. In contrast, the Nwc DMR was characterised by divergent 5mC and 5hmC specifically in response to TMA, highlighting its possible utility as a biomarker for responses induced by chemical respiratory allergens. These data not only represent novel analysis of 5hmC in response to chemical allergy in vivo, but with further investigation, may also provide a possible basis for differentiation between classes of chemical allergens.