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Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures

The liver with resident tissue macrophages is the site of vivid innate immunity, activated also by pathogen-associated molecular patterns (PAMPs) leaking through the intestinal barrier. As gut-derived inflammatory diseases are of outstanding importance in broiler chickens, the present study aimed to...

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Autores principales: Sebők, Csilla, Tráj, Patrik, Vörösházi, Júlia, Mackei, Máté, Papp, Márton, Gálfi, Péter, Neogrády, Zsuzsanna, Mátis, Gábor
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8394239/
https://www.ncbi.nlm.nih.gov/pubmed/34440679
http://dx.doi.org/10.3390/cells10081910
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author Sebők, Csilla
Tráj, Patrik
Vörösházi, Júlia
Mackei, Máté
Papp, Márton
Gálfi, Péter
Neogrády, Zsuzsanna
Mátis, Gábor
author_facet Sebők, Csilla
Tráj, Patrik
Vörösházi, Júlia
Mackei, Máté
Papp, Márton
Gálfi, Péter
Neogrády, Zsuzsanna
Mátis, Gábor
author_sort Sebők, Csilla
collection PubMed
description The liver with resident tissue macrophages is the site of vivid innate immunity, activated also by pathogen-associated molecular patterns (PAMPs) leaking through the intestinal barrier. As gut-derived inflammatory diseases are of outstanding importance in broiler chickens, the present study aimed to establish a proper hepatic inflammatory model by comparing the action of different PAMPs from poultry pathogens on chicken 2D and 3D primary hepatocyte—non-parenchymal cell co-cultures, the latter newly developed with a magnetic bioprinting method. The cultures were challenged by the bacterial endotoxins lipopolysaccharide (LPS) from Escherichia coli, lipoteichoic acid (LTA) from Staphylococcus aureus and by enterotoxin (ETxB) from Escherichia coli, Salmonella Typhimurium derived flagellin, phorbol myristate acetate (PMA) as a model proinflammatory agent and polyinosinic polycytidylic acid (poly I:C) for mimicking viral RNA exposure. Cellular metabolic activity was assessed with the CCK-8 test, membrane damage was monitored with the lactate dehydrogenase (LDH) leakage assay and interleukin-6 and -8 (Il-6 and -8) concentrations were measured in cell culture medium with a chicken specific ELISA. Both LPS and LTA increased the metabolic activity of the 3D cultures, concomitantly decreasing the LDH leakage, while in 2D cultures ETxB stimulated, PMA and poly I:C depressed the metabolic activity. Based on the moderately increased extracellular LDH activity, LTA seemed to diminish cell membrane integrity in 2D and poly I:C in both cell culture models. The applied endotoxins remarkably reduced the IL-8 release of 3D cultured cells, suggesting the effective metabolic adaptation and the presumably initiated anti-inflammatory mechanisms of the 3D spheroids. Notwithstanding that the IL-6 and IL-8 production of 2D cells was mostly not influenced by the endotoxins used, only the higher LTA dose was capable to evoke an IL-8 surge. Flagellin, PMA and poly I:C exerted proinflammatory action in certain concentrations in both 2D and 3D cultures, reflected by the increased cellular IL-6 release. Based on these data, LTA, flagellin, PMA and poly I:C can be considered as potent candidates to induce inflammation in chicken primary hepatic cell cultures, while LPS failed to trigger proinflammatory cytokine production, suggesting the relatively high tolerance of avian liver cells to certain bacterial endotoxins. These results substantiate that the established 3D co-cultures seemed to be proper tools for testing potential proinflammatory molecules; however, the remarkable differences between 2D and 3D models should be addressed and further studied.
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spelling pubmed-83942392021-08-28 Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures Sebők, Csilla Tráj, Patrik Vörösházi, Júlia Mackei, Máté Papp, Márton Gálfi, Péter Neogrády, Zsuzsanna Mátis, Gábor Cells Article The liver with resident tissue macrophages is the site of vivid innate immunity, activated also by pathogen-associated molecular patterns (PAMPs) leaking through the intestinal barrier. As gut-derived inflammatory diseases are of outstanding importance in broiler chickens, the present study aimed to establish a proper hepatic inflammatory model by comparing the action of different PAMPs from poultry pathogens on chicken 2D and 3D primary hepatocyte—non-parenchymal cell co-cultures, the latter newly developed with a magnetic bioprinting method. The cultures were challenged by the bacterial endotoxins lipopolysaccharide (LPS) from Escherichia coli, lipoteichoic acid (LTA) from Staphylococcus aureus and by enterotoxin (ETxB) from Escherichia coli, Salmonella Typhimurium derived flagellin, phorbol myristate acetate (PMA) as a model proinflammatory agent and polyinosinic polycytidylic acid (poly I:C) for mimicking viral RNA exposure. Cellular metabolic activity was assessed with the CCK-8 test, membrane damage was monitored with the lactate dehydrogenase (LDH) leakage assay and interleukin-6 and -8 (Il-6 and -8) concentrations were measured in cell culture medium with a chicken specific ELISA. Both LPS and LTA increased the metabolic activity of the 3D cultures, concomitantly decreasing the LDH leakage, while in 2D cultures ETxB stimulated, PMA and poly I:C depressed the metabolic activity. Based on the moderately increased extracellular LDH activity, LTA seemed to diminish cell membrane integrity in 2D and poly I:C in both cell culture models. The applied endotoxins remarkably reduced the IL-8 release of 3D cultured cells, suggesting the effective metabolic adaptation and the presumably initiated anti-inflammatory mechanisms of the 3D spheroids. Notwithstanding that the IL-6 and IL-8 production of 2D cells was mostly not influenced by the endotoxins used, only the higher LTA dose was capable to evoke an IL-8 surge. Flagellin, PMA and poly I:C exerted proinflammatory action in certain concentrations in both 2D and 3D cultures, reflected by the increased cellular IL-6 release. Based on these data, LTA, flagellin, PMA and poly I:C can be considered as potent candidates to induce inflammation in chicken primary hepatic cell cultures, while LPS failed to trigger proinflammatory cytokine production, suggesting the relatively high tolerance of avian liver cells to certain bacterial endotoxins. These results substantiate that the established 3D co-cultures seemed to be proper tools for testing potential proinflammatory molecules; however, the remarkable differences between 2D and 3D models should be addressed and further studied. MDPI 2021-07-27 /pmc/articles/PMC8394239/ /pubmed/34440679 http://dx.doi.org/10.3390/cells10081910 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Sebők, Csilla
Tráj, Patrik
Vörösházi, Júlia
Mackei, Máté
Papp, Márton
Gálfi, Péter
Neogrády, Zsuzsanna
Mátis, Gábor
Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures
title Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures
title_full Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures
title_fullStr Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures
title_full_unstemmed Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures
title_short Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures
title_sort two sides to every question: attempts to activate chicken innate immunity in 2d and 3d hepatic cell cultures
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8394239/
https://www.ncbi.nlm.nih.gov/pubmed/34440679
http://dx.doi.org/10.3390/cells10081910
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