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A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage

Established in vitro assays for regulatory testing of skin sensitisation partly suffer from only moderate sensitivity, specificity, and predictivity when testing specific groups of chemicals. This may be due to limited biomarker response in vitro in cell types that interact as crucial players of in...

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Autores principales: Sonnenburg, Anna, Stahlmann, Ralf, Kreutz, Reinhold, Peiser, Matthias
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10182954/
https://www.ncbi.nlm.nih.gov/pubmed/37147507
http://dx.doi.org/10.1007/s00204-023-03506-3
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author Sonnenburg, Anna
Stahlmann, Ralf
Kreutz, Reinhold
Peiser, Matthias
author_facet Sonnenburg, Anna
Stahlmann, Ralf
Kreutz, Reinhold
Peiser, Matthias
author_sort Sonnenburg, Anna
collection PubMed
description Established in vitro assays for regulatory testing of skin sensitisation partly suffer from only moderate sensitivity, specificity, and predictivity when testing specific groups of chemicals. This may be due to limited biomarker response in vitro in cell types that interact as crucial players of in vivo skin sensitisation pathogenesis. Here, we propose a molecular approach to overcome this limitation. In our model, we apply genome editing and blocking of immunoregulatory molecules to increase the range of biomarker modulation by sensitising chemicals. To this end, aryl hydrocarbon receptor (AhR) knockout was done by CRISPR/Cas9 technology in THP-1 cells and combined with Programmed Cell Death-Ligand (PD-L)1 blockade. AhR-knockout THP-1 in coculture with HaCaT keratinocytes showed increased CD54 expression compared to wild type cells after stimulation with 10 µmol/L dinitrochlorobenzene (DNCB) that was further enhanced by anti-PD-L1. After stimulation of AhR-knockout THP-1 with 200 µmol/L mercaptobenzothiazol or 10 µmol/L DNCB, cocultivated Jurkat T cells significantly increased expression of T cell receptor-associated CD3. No such increase was detected after prior treatment of THP-1 with 150 µmol/L of irritant sodium lauryl sulphate. Additionally, higher levels of inflammatory cytokines MIP-3α, MIP-1β, TNF-α, and IL-8 were found in supernatants of enhanced loose-fit co-culture based sensitisation assay (eLCSA) after substance treatment. Hence, eLCSA allowed to discriminate between sensitisers and non-sensitisers. Thus, inhibition of immunoinhibitory pathway signalling by combining AhR knockout and PD-L1 antibody blockage into an assay involving main acting cell types in skin sensitisation may increase sensitivity and specificity of such assays and allow potency derivation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00204-023-03506-3.
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spelling pubmed-101829542023-05-15 A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage Sonnenburg, Anna Stahlmann, Ralf Kreutz, Reinhold Peiser, Matthias Arch Toxicol In Vitro Systems Established in vitro assays for regulatory testing of skin sensitisation partly suffer from only moderate sensitivity, specificity, and predictivity when testing specific groups of chemicals. This may be due to limited biomarker response in vitro in cell types that interact as crucial players of in vivo skin sensitisation pathogenesis. Here, we propose a molecular approach to overcome this limitation. In our model, we apply genome editing and blocking of immunoregulatory molecules to increase the range of biomarker modulation by sensitising chemicals. To this end, aryl hydrocarbon receptor (AhR) knockout was done by CRISPR/Cas9 technology in THP-1 cells and combined with Programmed Cell Death-Ligand (PD-L)1 blockade. AhR-knockout THP-1 in coculture with HaCaT keratinocytes showed increased CD54 expression compared to wild type cells after stimulation with 10 µmol/L dinitrochlorobenzene (DNCB) that was further enhanced by anti-PD-L1. After stimulation of AhR-knockout THP-1 with 200 µmol/L mercaptobenzothiazol or 10 µmol/L DNCB, cocultivated Jurkat T cells significantly increased expression of T cell receptor-associated CD3. No such increase was detected after prior treatment of THP-1 with 150 µmol/L of irritant sodium lauryl sulphate. Additionally, higher levels of inflammatory cytokines MIP-3α, MIP-1β, TNF-α, and IL-8 were found in supernatants of enhanced loose-fit co-culture based sensitisation assay (eLCSA) after substance treatment. Hence, eLCSA allowed to discriminate between sensitisers and non-sensitisers. Thus, inhibition of immunoinhibitory pathway signalling by combining AhR knockout and PD-L1 antibody blockage into an assay involving main acting cell types in skin sensitisation may increase sensitivity and specificity of such assays and allow potency derivation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00204-023-03506-3. Springer Berlin Heidelberg 2023-05-05 2023 /pmc/articles/PMC10182954/ /pubmed/37147507 http://dx.doi.org/10.1007/s00204-023-03506-3 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle In Vitro Systems
Sonnenburg, Anna
Stahlmann, Ralf
Kreutz, Reinhold
Peiser, Matthias
A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage
title A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage
title_full A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage
title_fullStr A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage
title_full_unstemmed A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage
title_short A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage
title_sort new cell line based coculture system for skin sensitisation testing in one single assay using t cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage
topic In Vitro Systems
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10182954/
https://www.ncbi.nlm.nih.gov/pubmed/37147507
http://dx.doi.org/10.1007/s00204-023-03506-3
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