UNDERSTANDING CHEMICAL ALLERGEN POTENCY: CONTRIBUTION OF KERATINOCYTES

Repeated exposure to chemical allergens increases the risk of becoming sensitized. Once an individual has become sensitized, any following exposure to the same chemical may result in allergic contact dermatitis (ACD). The risk to develop ACD is considered a serious health issue and the identification of potential sensitizing agents within consumer products is therefore crucial. With the enforcement of the 7th Amendment to the EU Cosmetics Directive (76/768/EEC) in March 2013, currently known as the Cosmetics Regulation (EU 1223/2009), a ban on the use of animals was introduced for identifying repeated dose toxicity endpoints of chemicals used in cosmetic ingredients and products. This ban results in an urgent need for the development of suitable non-animal methods for safety testing. The development of animal alternatives has become even more urgent due to the Registration, Evaluation, Authorisation and Restriction of CHemicals (REACH) regulation, which may demand toxicity tests for chemicals produced in quantities of over 1 ton per year. Over the last years, many in vitro models have been proposed to identify the potential of chemicals to induce skin sensitization to meet current animal welfare, public opinions and legislation constrains. The development of in vitro, in chemico or in silico models for predicting the sensitizing potential of new chemicals is receiving widespread interest. Keratinocytes (KCs) play a key role in skin sensitization, as they provide the essential danger signals, they are involved in the protein haptenation process, and supply enzymes that are necessary for the metabolic activation of prohapten. Human KCs constitutively express several cytokines, including pro-interleukin (IL)-1, pro-IL-1 and pro-IL-18. Evidences provided from our group has shown that IL-18 production in human KCs can be used as a sensitive method to identify contact allergens, discriminating them from respiratory allergens and irritants with a sensitivity of 87%, specificity of 95% and an accuracy of 90%. IL-18 is synthesized as preform, which require proteolytic maturation by cysteine protease caspase-1, which must first be activated by the inflammasome. More recently, we demonstrated the possibility of combining the Reconstituted human Epidermis (RhE) potency assay with the assessment of IL-18 release to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability (Gibbs et al., 2013). In addition to being able to determine whether or not a chemical is a sensitizer (labelling) it is also equally important to determine the potency of a sensitizer (classification) in order to identify a maximum safe concentration for human exposure (risk assessment). The purpose of this thesis was to understand the role of several genes and proteins involved in contact allergen-induced NLRP3 inflammasome activation and IL-18 production, and their possible correlation with allergenic potency. Another objective of this thesis was to extend the list of chemicals tested in the RhE IL-18 potency assay, and to provide a simple method for the in vitro estimation of the expected sensitization induction level. Results obtained during these three years of research activity have shown that several proteins involved in NLRP3 inflammasome activation/regulation were modulated by contact allergens. In particular I focused my attention on the role of NLRP12 and B lymphocyte induced maturation protein-1 (Blimp-1) in IL-18 production. The expression of NLRP3, ASC and caspase-1 activation were investigated by Western blot analysis and the NLRP12 localization characterized by immunoprecipitation. Regarding potency classification the results obtained using RhE IL-18 potency assay are very promising, and further compounds should be tested to better define the applicability and limitation of RhE IL-18 potency test.