Research into the respiratory immunodisturbing mechanisms of chemical allergens by means of in vitro test systems and transcriptomics

Abstract

Chemical-induced respiratory allergy poses significant challenges for the toxicologist and regulator, not least because there are, as yet, no validated or widely accepted methods available for the identification of chemicals that have the potential to cause respiratory sensitization. This may be attributable to the fact that there remains uncertainty about the complete mechanisms through which respiratory sensitization to chemicals can be acquired. Toxicological assessment of most xenobiotics mainly relies on in vivo animal tests, having their economical and ethical drawbacks. Therefore an in vitro screening model for the identification of respiratory allergens is needed. Besides the choice of an appropriate cell model, it is also important to study relevant mechanisms and derive endpoints in the context of respiratory sensitization. For this purpose, transcriptomics, proteomics, and metabolomics are promising tools to better understand the underlying molecular and biological mechanisms through which allergic sensitization to chemicals is induced and regulated. The major goal of this thesis was to investigate the alterations in gene expression of human bronchial (BEAS-2B) and alveolar (A549) epithelial cells, and THP-1 macrophages after exposure to respiratory sensitizers and respiratory non-sensitizing chemicals, and to identify genes that are able to discriminate between both groups of compounds. The cells were exposed during 6, 10, and 24 hours to the respiratory sensitizers ammonium hexachloroplatinate IV, hexamethylene diisocyanate, and trimellitic anhydride, the irritants acrolein and methyl salicylate, and the skin sensitizer 1-chloro-2,4-dinitrobenzene at subcytotoxic levels. Overall changes in gene expression were evaluated using Agilent Whole Human Genome 4x44K oligonucleotide arrays. In each cell model, a Fisher Linear Discriminant Analysis was used to obtain a ranking of genes that reflected the potential to discriminate between respiratory sensitizers and respiratory non-sensitizing chemicals. In a first attempt to develop an in vitro respiratory sensitization assay, the 20 most discriminative genes that were able to distinguish between both groups of chemicals were discussed for each cell model. However, the exact role of these marker genes in the respiratory sensitization process and how they are influenced in vivo after exposure to respiratory allergens is yet unknown, and it is likely that different pathways are involved in sensitization of the respiratory tract. Secondly, the 1000 most discriminating genes for each cell model were used to identify canonical pathways which may contribute to a better understanding of the underlying mechanisms of respiratory sensitization. Using pathway analysis, platelet-derived growth factor signaling was found as being a possible important pathway involved in the respiratory sensitization process in THP-1 macrophages. Within the BEAS-2B cell model, the phosphatase and tensin homolog (PTEN) signaling pathway was identified and might be specific for respiratory sensitization. None of the canonical signaling pathways activated in A549 cells were specific for respiratory sensitization. In conclusion, this study demonstrated the feasibility and utility of transcriptomics approaches to identify selective markers that can discriminate respiratory sensitizers from respiratory nonsensitizers. Canonical pathways which might be specific for respiratory sensitization were identified. For the development of an in vitro assay for respiratory sensitization, these findings will need to be expanded using a larger set of chemicals, with priority on gene expression studies and analysis of mechanisms of respiratory sensitization in the BEAS-2B cell model