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Prediction of Chemical Respiratory and Contact Sensitizers by OX40L Expression in Dendritic Cells Using a Novel 3D Coculture System

The use of animal models in chemical safety testing will be significantly limited due to the recent introduction of the 3Rs principle of animal experimentation in research. Although several in vitro assays to predict the sensitizing potential of chemicals have been developed, these methods cannot di...

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Autores principales: Mizoguchi, Izuru, Ohashi, Mio, Chiba, Yukino, Hasegawa, Hideaki, Xu, Mingli, Owaki, Toshiyuki, Yoshimoto, Takayuki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543289/
https://www.ncbi.nlm.nih.gov/pubmed/28824649
http://dx.doi.org/10.3389/fimmu.2017.00929
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author Mizoguchi, Izuru
Ohashi, Mio
Chiba, Yukino
Hasegawa, Hideaki
Xu, Mingli
Owaki, Toshiyuki
Yoshimoto, Takayuki
author_facet Mizoguchi, Izuru
Ohashi, Mio
Chiba, Yukino
Hasegawa, Hideaki
Xu, Mingli
Owaki, Toshiyuki
Yoshimoto, Takayuki
author_sort Mizoguchi, Izuru
collection PubMed
description The use of animal models in chemical safety testing will be significantly limited due to the recent introduction of the 3Rs principle of animal experimentation in research. Although several in vitro assays to predict the sensitizing potential of chemicals have been developed, these methods cannot distinguish chemical respiratory sensitizers and skin sensitizers. In the present study, we describe a novel in vitro assay that can discriminate respiratory sensitizers from chemical skin sensitizers by taking advantage of the fundamental difference between their modes of action, namely the development of the T helper 2 immune response, which is critically important for respiratory sensitization. First, we established a novel three-dimensional (3D) coculture system of human upper airway epithelium using a commercially available scaffold. It consists of human airway epithelial cell line BEAS-2B, immature dendritic cells (DCs) derived from human peripheral blood CD14(+) monocytes, and human lung fibroblast cell line MRC-5. Respective cells were first cultured in individual scaffolds and subsequently assembled into a 3D multi-cell tissue model to more closely mimic the in vivo situation. Then, three typical chemicals that are known respiratory sensitizers (ortho-phthaldialdehyde, hexamethylene diisocyanate, and trimellitic anhydride) and skin sensitizers (oxazolone, formaldehyde, and dinitrochlorobenzene) were added individually to the 3D coculture system. Immunohistochemical analysis revealed that DCs do not migrate into other scaffolds under the experimental conditions. Therefore, the 3D structure was disassembled and real-time reverse transcriptase-PCR analysis was performed in individual scaffolds to analyze the expression levels of molecules critical for Th2 differentiation such as OX40 ligand (OX40L), interleukin (IL)-4, IL-10, IL-33, and thymic stromal lymphopoietin. Both sensitizers showed similarly augmented expression of DC maturation markers (e.g., CD86), but among these molecules, OX40L expression in DCs was most consistently and significantly enhanced by respiratory sensitizers as compared to that by skin sensitizers. Thus, we have established a 3D coculture system mimicking the airway upper epithelium that may be successfully applied to discriminate chemical respiratory sensitizers from skin sensitizers by measuring the critical molecule for Th2 differentiation, OX40L, in DCs.
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spelling pubmed-55432892017-08-18 Prediction of Chemical Respiratory and Contact Sensitizers by OX40L Expression in Dendritic Cells Using a Novel 3D Coculture System Mizoguchi, Izuru Ohashi, Mio Chiba, Yukino Hasegawa, Hideaki Xu, Mingli Owaki, Toshiyuki Yoshimoto, Takayuki Front Immunol Immunology The use of animal models in chemical safety testing will be significantly limited due to the recent introduction of the 3Rs principle of animal experimentation in research. Although several in vitro assays to predict the sensitizing potential of chemicals have been developed, these methods cannot distinguish chemical respiratory sensitizers and skin sensitizers. In the present study, we describe a novel in vitro assay that can discriminate respiratory sensitizers from chemical skin sensitizers by taking advantage of the fundamental difference between their modes of action, namely the development of the T helper 2 immune response, which is critically important for respiratory sensitization. First, we established a novel three-dimensional (3D) coculture system of human upper airway epithelium using a commercially available scaffold. It consists of human airway epithelial cell line BEAS-2B, immature dendritic cells (DCs) derived from human peripheral blood CD14(+) monocytes, and human lung fibroblast cell line MRC-5. Respective cells were first cultured in individual scaffolds and subsequently assembled into a 3D multi-cell tissue model to more closely mimic the in vivo situation. Then, three typical chemicals that are known respiratory sensitizers (ortho-phthaldialdehyde, hexamethylene diisocyanate, and trimellitic anhydride) and skin sensitizers (oxazolone, formaldehyde, and dinitrochlorobenzene) were added individually to the 3D coculture system. Immunohistochemical analysis revealed that DCs do not migrate into other scaffolds under the experimental conditions. Therefore, the 3D structure was disassembled and real-time reverse transcriptase-PCR analysis was performed in individual scaffolds to analyze the expression levels of molecules critical for Th2 differentiation such as OX40 ligand (OX40L), interleukin (IL)-4, IL-10, IL-33, and thymic stromal lymphopoietin. Both sensitizers showed similarly augmented expression of DC maturation markers (e.g., CD86), but among these molecules, OX40L expression in DCs was most consistently and significantly enhanced by respiratory sensitizers as compared to that by skin sensitizers. Thus, we have established a 3D coculture system mimicking the airway upper epithelium that may be successfully applied to discriminate chemical respiratory sensitizers from skin sensitizers by measuring the critical molecule for Th2 differentiation, OX40L, in DCs. Frontiers Media S.A. 2017-08-04 /pmc/articles/PMC5543289/ /pubmed/28824649 http://dx.doi.org/10.3389/fimmu.2017.00929 Text en Copyright © 2017 Mizoguchi, Ohashi, Chiba, Hasegawa, Xu, Owaki and Yoshimoto. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Mizoguchi, Izuru
Ohashi, Mio
Chiba, Yukino
Hasegawa, Hideaki
Xu, Mingli
Owaki, Toshiyuki
Yoshimoto, Takayuki
Prediction of Chemical Respiratory and Contact Sensitizers by OX40L Expression in Dendritic Cells Using a Novel 3D Coculture System
title Prediction of Chemical Respiratory and Contact Sensitizers by OX40L Expression in Dendritic Cells Using a Novel 3D Coculture System
title_full Prediction of Chemical Respiratory and Contact Sensitizers by OX40L Expression in Dendritic Cells Using a Novel 3D Coculture System
title_fullStr Prediction of Chemical Respiratory and Contact Sensitizers by OX40L Expression in Dendritic Cells Using a Novel 3D Coculture System
title_full_unstemmed Prediction of Chemical Respiratory and Contact Sensitizers by OX40L Expression in Dendritic Cells Using a Novel 3D Coculture System
title_short Prediction of Chemical Respiratory and Contact Sensitizers by OX40L Expression in Dendritic Cells Using a Novel 3D Coculture System
title_sort prediction of chemical respiratory and contact sensitizers by ox40l expression in dendritic cells using a novel 3d coculture system
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543289/
https://www.ncbi.nlm.nih.gov/pubmed/28824649
http://dx.doi.org/10.3389/fimmu.2017.00929
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