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Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide

Glucocorticoids (GCs) are widely prescribed therapeutics for the treatment of inflammatory diseases, and endogenous GCs play a key role in immune regulation. Toll-like receptors (TLRs) enable innate immune cells, such as macrophages, to recognize a wide variety of microbial ligands, thereby promotin...

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Autores principales: Hoppstädter, Jessica, Dembek, Anna, Linnenberger, Rebecca, Dahlem, Charlotte, Barghash, Ahmad, Fecher-Trost, Claudia, Fuhrmann, Gregor, Koch, Marcus, Kraegeloh, Annette, Huwer, Hanno, Kiemer, Alexandra K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664002/
https://www.ncbi.nlm.nih.gov/pubmed/31396208
http://dx.doi.org/10.3389/fimmu.2019.01634
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author Hoppstädter, Jessica
Dembek, Anna
Linnenberger, Rebecca
Dahlem, Charlotte
Barghash, Ahmad
Fecher-Trost, Claudia
Fuhrmann, Gregor
Koch, Marcus
Kraegeloh, Annette
Huwer, Hanno
Kiemer, Alexandra K.
author_facet Hoppstädter, Jessica
Dembek, Anna
Linnenberger, Rebecca
Dahlem, Charlotte
Barghash, Ahmad
Fecher-Trost, Claudia
Fuhrmann, Gregor
Koch, Marcus
Kraegeloh, Annette
Huwer, Hanno
Kiemer, Alexandra K.
author_sort Hoppstädter, Jessica
collection PubMed
description Glucocorticoids (GCs) are widely prescribed therapeutics for the treatment of inflammatory diseases, and endogenous GCs play a key role in immune regulation. Toll-like receptors (TLRs) enable innate immune cells, such as macrophages, to recognize a wide variety of microbial ligands, thereby promoting inflammation. The interaction of GCs with macrophages in the immunosuppressive resolution phase upon prolonged TLR activation is widely unknown. Treatment of human alveolar macrophages (AMs) with the synthetic GC dexamethasone (Dex) did not alter the expression of TLRs −1, −4, and −6. In contrast, TLR2 was upregulated in a GC receptor-dependent manner, as shown by Western blot and qPCR. Furthermore, long-term lipopolysaccharide (LPS) exposure mimicking immunosuppression in the resolution phase of inflammation synergistically increased Dex-mediated TLR2 upregulation. Analyses of publicly available datasets suggested that TLR2 is induced during the resolution phase of inflammatory diseases, i.e., under conditions associated with high endogenous GC production. TLR2 induction did not enhance TLR2 signaling, as indicated by reduced cytokine production after treatment with TLR2 ligands in Dex- and/or LPS-primed AMs. Thus, we hypothesized that the upregulated membrane-bound TLR2 might serve as a precursor for soluble TLR2 (sTLR2), known to antagonize TLR2-dependent cell actions. Supernatants of LPS/Dex-primed macrophages contained sTLR2, as demonstrated by Western blot analysis. Activation of metalloproteinases resulted in enhanced sTLR2 shedding. Additionally, we detected full-length TLR2 and assumed that this might be due to the production of TLR2-containing extracellular vesicles (EVs). EVs from macrophage supernatants were isolated by sequential centrifugation. Both untreated and LPS/Dex-treated cells produced vesicles of various sizes and shapes, as shown by cryo-transmission electron microscopy. These vesicles were identified as the source of full-length TLR2 in macrophage supernatants by Western blot and mass spectrometry. Flow cytometric analysis indicated that TLR2-containing EVs were able to bind the TLR2 ligand Pam(3)CSK(4). In addition, the presence of EVs reduced inflammatory responses in Pam(3)CSK(4)-treated endothelial cells and HEK Dual reporter cells, demonstrating that TLR2-EVs can act as decoy receptors. In summary, our data show that sTLR2 and full-length TLR2 are released by macrophages under anti-inflammatory conditions, which may contribute to GC-induced immunosuppression.
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spelling pubmed-66640022019-08-08 Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide Hoppstädter, Jessica Dembek, Anna Linnenberger, Rebecca Dahlem, Charlotte Barghash, Ahmad Fecher-Trost, Claudia Fuhrmann, Gregor Koch, Marcus Kraegeloh, Annette Huwer, Hanno Kiemer, Alexandra K. Front Immunol Immunology Glucocorticoids (GCs) are widely prescribed therapeutics for the treatment of inflammatory diseases, and endogenous GCs play a key role in immune regulation. Toll-like receptors (TLRs) enable innate immune cells, such as macrophages, to recognize a wide variety of microbial ligands, thereby promoting inflammation. The interaction of GCs with macrophages in the immunosuppressive resolution phase upon prolonged TLR activation is widely unknown. Treatment of human alveolar macrophages (AMs) with the synthetic GC dexamethasone (Dex) did not alter the expression of TLRs −1, −4, and −6. In contrast, TLR2 was upregulated in a GC receptor-dependent manner, as shown by Western blot and qPCR. Furthermore, long-term lipopolysaccharide (LPS) exposure mimicking immunosuppression in the resolution phase of inflammation synergistically increased Dex-mediated TLR2 upregulation. Analyses of publicly available datasets suggested that TLR2 is induced during the resolution phase of inflammatory diseases, i.e., under conditions associated with high endogenous GC production. TLR2 induction did not enhance TLR2 signaling, as indicated by reduced cytokine production after treatment with TLR2 ligands in Dex- and/or LPS-primed AMs. Thus, we hypothesized that the upregulated membrane-bound TLR2 might serve as a precursor for soluble TLR2 (sTLR2), known to antagonize TLR2-dependent cell actions. Supernatants of LPS/Dex-primed macrophages contained sTLR2, as demonstrated by Western blot analysis. Activation of metalloproteinases resulted in enhanced sTLR2 shedding. Additionally, we detected full-length TLR2 and assumed that this might be due to the production of TLR2-containing extracellular vesicles (EVs). EVs from macrophage supernatants were isolated by sequential centrifugation. Both untreated and LPS/Dex-treated cells produced vesicles of various sizes and shapes, as shown by cryo-transmission electron microscopy. These vesicles were identified as the source of full-length TLR2 in macrophage supernatants by Western blot and mass spectrometry. Flow cytometric analysis indicated that TLR2-containing EVs were able to bind the TLR2 ligand Pam(3)CSK(4). In addition, the presence of EVs reduced inflammatory responses in Pam(3)CSK(4)-treated endothelial cells and HEK Dual reporter cells, demonstrating that TLR2-EVs can act as decoy receptors. In summary, our data show that sTLR2 and full-length TLR2 are released by macrophages under anti-inflammatory conditions, which may contribute to GC-induced immunosuppression. Frontiers Media S.A. 2019-07-23 /pmc/articles/PMC6664002/ /pubmed/31396208 http://dx.doi.org/10.3389/fimmu.2019.01634 Text en Copyright © 2019 Hoppstädter, Dembek, Linnenberger, Dahlem, Barghash, Fecher-Trost, Fuhrmann, Koch, Kraegeloh, Huwer and Kiemer. 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) and the copyright owner(s) 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
Hoppstädter, Jessica
Dembek, Anna
Linnenberger, Rebecca
Dahlem, Charlotte
Barghash, Ahmad
Fecher-Trost, Claudia
Fuhrmann, Gregor
Koch, Marcus
Kraegeloh, Annette
Huwer, Hanno
Kiemer, Alexandra K.
Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
title Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
title_full Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
title_fullStr Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
title_full_unstemmed Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
title_short Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
title_sort toll-like receptor 2 release by macrophages: an anti-inflammatory program induced by glucocorticoids and lipopolysaccharide
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664002/
https://www.ncbi.nlm.nih.gov/pubmed/31396208
http://dx.doi.org/10.3389/fimmu.2019.01634
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