DS0408 - Étude des mécanismes de défense de l’organisme

The human body facing and defending from chemical skin allergens reacting by alternative mechanisms: understanding from the molecule to the tissue – DEFCHEMSKALL

DEFCHEMSKALL

The human body facing and defending from chemical skin allergens reacting by alternative mechanisms: understanding from the molecule to the tissue

Understanding how reactivity of allergens through radical mechanisms can modify the epidermal environment providing information to the immune cells activating the innate immune system

Exposure to allergens and the risk of skin sensitization are an essential regulatory issue within industry. It is thus crucial to predict the sensitization potential of chemicals before their use in consumer products. To perform risk assessment, studies helping to understand all mechanisms leading to allergic-inflammatory reactions by sensitizers, and the related cellular-immunological answers, are essential to cover all categories of molecules. This project aims at understanding how chemical reactivity of allergens through radical mechanisms can specifically modify the epidermal environment providing information to the immune cells that will then activate the innate immune system resulting into disease.<br />An integrated strategy approach of three actions is proposed:<br />In chemico by conducting reactivity studies to establish a complete reactivity profile towards amino acids prompt to radical reactions; in situ by studying the behaviour and decay of the formed radical intermediates in reconstructed human epidermis tissue models; in cellulo by examination of their properties to activate the innate immune system, namely dendritic cells (DCs) in their natural context of keratinocytes, and of individual susceptibility factors influencing this process, investigated by analysis of diseased versus healthy keratinocytes.<br />Specific objectives:<br />• Complete chemical reactivity profile towards amino acids through radical mechanisms and in a peptide environment<br />• Production of danger signals in reconstructed human epidermis tissue by electron paramagnetic resonance (EPR)<br />• Impact on the activation of DCs. Impact of DC cross-talk with epithelial tissue environment, namely keratinocytes<br />• Susceptibility factors arising from “diseased keratinocytes” implicated in their activation<br />• To establish correlations between reactivity and DCs activation<br />• Develop a mode of action to assess the sensitizing potential<br />Work scheduled in 5 Tasks

Synthesis of target compounds; Reactivity studies by NMR and LC-MS; In situ production of radicals in reconstructed human epidermis by electron paramagnetic resonance EPR combined to spin-trapping; Measure of redox potentials by cyclic voltamperometry; Dendritic cells activation in co-culture THP-1/HaCaT by PCR, Western Blot and flow cytometry; Use of different keratinocytes cell lines.

Task 1. Chemical reactivity profile through radical mechanisms
Sub-task 1a: We obtained hydroperoxides via the ene-reaction or addition of singlet oxygen to terpenic double bonds. Ascaridole was obtained this way from a-terpinene. The method was unsuccessful for the other target compounds. The expected mixture of hydroperoxides was obtained. However, isolation of pure compounds with good yields was very difficult and non reproducible. Thus, synthetic multi-step pathways were developed.
Sub-task 1b: We identified the intermediates issued from these compounds, using the spin trapping technique combined with EPR. Carbon and oxygen-centred free radicals were successfully trapped by DEPMPO, which appeared to be the best spin trap. Experiments are carried out in solution to optimize the experimental conditions with the ultimate aim of reproducing these studies on reconstructed human epidermis 3D models (RHE).
Sub-task 1c: For the reactivity studies, we have started working with amino acids that are prompt to radical reactions (cysteine, tyrosine, tryptophan, histidine). Adducts have been observed and structure elucidation is undergoing by LC-MS.
Task 2. We started the transfer of EPR technology to the use of RHE.
Task 3. Activation of the innate immune response
Sub-task 3a: Activation of DCs by target compounds by using THP-1/HaCaT co-culture is completed with positive activation results.
Sub-task 3b: Nrf2 activation and analysis of target gene expression is running
Sub-task 3c: Mediator analysis of cell culture supernatants is currently performed
Sub-task 3d: Activation of DCs by target compounds by comparative analysis of MoDCs was started after approval by the ethics committee of Trier University
Task 4: THP-1 co-culture experiments with primary keratinocytes of healthy donors (n=5) is already established and preparation (ethical approval is pending) of analysis of the impact of disease status (allergic contact dermatitis, psoriasis) is ongoing.

Continue to achieve the tasks presented in the project with the following expectations in a near future:
1. Synthesis of target compounds is new, especially Lim-1-OOH, Citr-7-OOH and Citr-6-OOH. Once achieved, they will be published together with the identification of their reactive radicals formed in solution.
2. If the reactivity studies give new information on adducts formed with amino acids, a publication will follow.
3. The use of RHE 3D models for the study of in situ formation of radicals issued from skin allergens is a very important new challenge. We believe in a very near future, we will be able to prove the formation of radicals from target compounds in the reconstructed epidermis model. This of course will be an important subject of publication.
4. THP-1/HaCaT co-culture biological studies of DCs positive activation by target compounds will also be a matter of publication in complete agreement with the Department of Environmental Toxicology of the University of Trier.

Final objective is to interpretate all data together in order to find a method to evaluate the sensitizing potential of allergens reacting through radical intermediates.

1. Non-classical mechanisms in skin sensitization: contribution of radical reactions to protein modifications by chemical allergens.
E. Giménez-Arnau.
Seminar Chemistry Department University of Trier. Trier, Germany (15th february 2016).
2. Non classical mechanisms in skin sensitization: the case of hydroperoxides derived from autoxidation of terpenes.
E. Giménez-Arnau.
13th Congress of the European Society of Contact Dermatitis.
Ibero-Latinoamerican Symposium.
Manchester, United Kingdom (14th-17th september 2016).
3. Fragrance terpenes sensitizing hydroperoxides: radical reactivity and Nrf2 pathway immune response.
E. Giménez-Arnau.
Cutaneous Allergy Research Group Organisation, CARGO Meeting
Stockholm, Sweden (19th november 2016).
4. An abstract will be submitted to the 57th Annual Meeting of the Society of Toxicology, San Antonio, Texas, US (11th-15th march 2018) by partner University of Trier.

Skin allergy is a common occupational and environmental issue. It is a delayed-type hypersensitivity reaction caused by a wide range of reactive chemicals referred to as allergens, natural or synthetic man-made, after repeated contact with the skin. It is regarded as the most frequent expression of immunotoxicity in humans. Prevalence is rising worldwide, and is today a prominent threat to public health. Social and regulatory pressures have strengthened with EU legislation, in a way that exposure to allergens and the risk of skin sensitization have become an essential regulatory issue within industry. It is crucial to predict the sensitization potential of chemicals before their use in consumer products in order to perform risk assessment. In this context, studies helping to understand all mechanisms leading to allergic-inflammatory reactions by sensitizers, and the related cellular-immunological answers, are essential to cover all categories of molecules for risk assessment.
Skin allergy to a chemical is the result of a complex multifactorial series of events. The first critical step is chemical. Allergens (haptens) react with skin proteins forming stable antigenic conjugates that will be recognised and processed for presentation to the immune system. The general mechanism for the hapten-protein interaction is the formation of a covalent bond between the hapten and proteins via a nucleophile-electrophile pathway. However, many allergens do not fit this model. Biological key events are the activation of keratinocytes, main cell population of the epidermis and first cells to come into contact with the allergen, and of dendritic cells (DCs), immunocompetent skin antigen-presenting cells that take up and process the hapten-protein complex ensuring the presentation of altered peptides to T-cells.
DEFCHEMSKALL aims at understanding how chemical reactivity of allergens through alternative mechanisms, involving radical intermediates, can alter proteins in the skin and specifically modify the cellular environment providing information to the immune cells that will then activate the immune system inducing the disease. Our hypothesis is that chemical reactivity translates into specific biological responses in keratinocytes and DCs that dictate the resulting immune responses to allergens reacting through radical intermediates.
In the light of the above considerations, the success of DEFCHEMSKALL will be measured by its scientific breakthroughs related to the mechanisms of action for allergens reacting through radical mechanisms “from the molecule to the tissue”. An integrated strategy approach of three actions is proposed: in chemico by conducting reactivity studies to establish a complete reactivity profile towards amino acids prompt to radical reactions; in situ by studying the behaviour and decay of the formed radical intermediates in reconstructed human epidermis tissue models; in cellulo by examination of their properties to activate the innate immune system, namely DCs in their natural context of keratinocytes, and of individual susceptibility factors influencing this process, investigated by analysis of diseased versus healthy keratinocytes.
For fulfilling the objectives the project is divided into 5 interlinked chemical-biological specific tasks, carried out by a unique French-German consortium. It is built around two major interdisciplinary lines of research. One concerning all chemistry studies, performed by the Dermatochemistry Laboratory (University of Strasbourg, CNRS UMR 7177), and another concerning all biological and cellular studies, carried out by the Department of Environmental Toxicology (University of Trier). After integration of all obtained data and with our expertise, the intention is to establish a strategy allowing the evaluation of the sensitizing potential of allergens precursors of radical intermediates based on their chemical reactivity and using the activation of DCs in their natural context of keratinocytes.

Project coordination

Elena GIMENEZ-ARNAU (Laboratoire de Dermatochimie - Université de Strasbourg)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

University of Trier - Germany Department of Environmental Toxicology
Institut de Chimie - UNISTRA Laboratoire de Dermatochimie - Université de Strasbourg

Help of the ANR 194,747 euros
Beginning and duration of the scientific project: March 2016 - 36 Months

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