Investigation of cellular responses to specific protein haptenation by low molecular weight chemical sensitisers

Abstract

Allergies resulting from exposure to small (low MW) organic chemicals are a public health concern, manifesting as varied clinical outcomes. Skin sensitiser chemicals generally induce relatively mild T cell-mediated hypersensitivity, whereas chemical respiratory allergies are often IgE-mediated and potentially life-threatening. Mechanistic understanding of sensitisation to these low molecular weight chemicals is such that all involve a protein haptenation molecular initiation event (MIE). In the MIE, the electrophilic chemical sensitisers covalently modify nucleophilic amino acid residues of endogenous proteins, thereby becoming immunogenic. The key events following the MIE form the basis of Adverse Outcome Pathways (AOPs) for skin and respiratory sensitisation1–3. This thesis uses proteomics-based methods to characterise the differences in reactivity of chemical sensitisers in the context of the haptenome, proteome and immunopeptidome for the identification of potential molecular drivers of downstream immunological divergence.
The proliferative and cytotoxic effects of skin sensitisers (Dinitrochlorobenzene - DNCB, Diphenylcyclopropenone - DPCP) and respiratory sensitisers (Phthalic anhydride - PA, Toluene diisocyanate - 2,4-TDI) on human epithelial cell lines at different concentrations in vitro were initially investigated. Acute changes in cell viability were observed following exposure to skin sensitisers, whereas no such change was observed following exposure to respiratory sensitiser chemicals. The same concentrations of chemical sensitisers were investigated in terms of reactivity and peptide adduct formation with synthetic hexapeptides in chemico using a targeted mass spectrometry method. The resulting peptide reactivity data confirmed specificity for each chemical sensitiser predominantly for cysteine- or lysine-containing peptides, consistent with existing evidence in the literature4–7. These results also provided further understanding of haptenation reaction mechanisms for each individual chemical.
Following optimisation of sub-cytotoxic chemical sensitiser concentrations demonstrating maximum peptide reactivity, living alveolar (A549) and bronchial epithelial cells (16HBE14o-) were exposed to equimolar concentrations of deuterated and un-deuterated chemical sensitisers in vitro using established dual isotope labelling and tandem mass spectrometry methods8. Label-free quantitation of the proteome reproducibly identified significant differences in protein abundance observed between different sensitiser exposure groups. Exposure to respiratory sensitiser chemicals induced a greater magnitude of differential protein expression relative to skin sensitiser chemical exposure. Further, proteins and pathways related to cellular stress (particularly oxidative stress), genotoxicity & proteotoxicity, the ubiquitin proteasome system, and several other potential drivers of immunological divergence were a significant feature of this cellular response. Additionally, small proportions of the proteome were observed to be haptenated, generating haptenomes of sizes similar to previous experiments in our laboratory8–10. The haptenomes were further found to demonstrate more haptenated cysteine residues following skin sensitiser exposure compared to more haptenated lysine residues following exposure to respiratory sensitisers.
Finally, A549 cell cultures exposed to DNCB or PA were subjected to immunoprecipitation of antigen-presenting major histocompatibility complex I (MHC class I) proteins. An immunopeptidomics workflow and analysis of the presented peptides followed for comparison to similar experiments on human keratinocytes performed in our laboratory11. These results did not provide compelling support for hapten-protein conjugates identified previously forming peptide antigens on MHC class I complexes under these experimental conditions. However, methodological insight was provided for improving the efficiency of experiments investigating haptenation of the immunopeptidome by respiratory sensitiser chemicals.
Overall, the results in this thesis characterise haptenation and the associated cellular response based on the specific reactivity of select skin and respiratory chemical sensitisers. The insights presented here also demonstrate a progression from the initial hypotheses regarding interplay between these aspects. Furthermore, the data provides a contribution to the weight-of-evidence approach of the AOPs, as well as generation of new hypotheses for further research in this area.

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Identifiers

ORCID for Paul Skipp: orcid.org/0000-0002-2995-2959

ORCID for Benjamin Nicholas: orcid.org/0000-0003-1467-9643

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