Cargando…
Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells
In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators, beneficial in controlling bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflamm...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10000987/ https://www.ncbi.nlm.nih.gov/pubmed/36899833 http://dx.doi.org/10.3390/cells12050697 |
_version_ | 1784904020477870080 |
---|---|
author | Stierschneider, Anna Neuditschko, Benjamin Colleselli, Katrin Hundsberger, Harald Herzog, Franz Wiesner, Christoph |
author_facet | Stierschneider, Anna Neuditschko, Benjamin Colleselli, Katrin Hundsberger, Harald Herzog, Franz Wiesner, Christoph |
author_sort | Stierschneider, Anna |
collection | PubMed |
description | In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators, beneficial in controlling bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflammatory diseases. Since distinct and rapid induction of TLR4 signaling is difficult to achieve with LPS due to the specific and non-specific affinity to other surface molecules and receptors, we engineered new light-oxygen-voltage-sensing (LOV)-domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LECs and opto-TLR4-LOV HUVECs) that allow fast, precise temporal, and reversible activation of TLR4 signaling pathways. Using quantitative mass-spectrometry, RT-qPCR, and Western blot analysis, we show that pro-inflammatory proteins were not only expressed differently, but also had a different time course when the cells were stimulated with light or LPS. Additional functional assays demonstrated that light induction promoted chemotaxis of THP-1 cells, disruption of the EC monolayer and transmigration. In contrast, ECs incorporating a truncated version of the TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LECs) revealed high basal activity with fast depletion of the cell signaling system upon illumination. We conclude that the established optogenetic cell lines are well suited to induce rapid and precise photoactivation of TLR4, allowing receptor-specific studies. |
format | Online Article Text |
id | pubmed-10000987 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-100009872023-03-11 Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells Stierschneider, Anna Neuditschko, Benjamin Colleselli, Katrin Hundsberger, Harald Herzog, Franz Wiesner, Christoph Cells Article In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators, beneficial in controlling bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflammatory diseases. Since distinct and rapid induction of TLR4 signaling is difficult to achieve with LPS due to the specific and non-specific affinity to other surface molecules and receptors, we engineered new light-oxygen-voltage-sensing (LOV)-domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LECs and opto-TLR4-LOV HUVECs) that allow fast, precise temporal, and reversible activation of TLR4 signaling pathways. Using quantitative mass-spectrometry, RT-qPCR, and Western blot analysis, we show that pro-inflammatory proteins were not only expressed differently, but also had a different time course when the cells were stimulated with light or LPS. Additional functional assays demonstrated that light induction promoted chemotaxis of THP-1 cells, disruption of the EC monolayer and transmigration. In contrast, ECs incorporating a truncated version of the TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LECs) revealed high basal activity with fast depletion of the cell signaling system upon illumination. We conclude that the established optogenetic cell lines are well suited to induce rapid and precise photoactivation of TLR4, allowing receptor-specific studies. MDPI 2023-02-22 /pmc/articles/PMC10000987/ /pubmed/36899833 http://dx.doi.org/10.3390/cells12050697 Text en © 2023 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 Stierschneider, Anna Neuditschko, Benjamin Colleselli, Katrin Hundsberger, Harald Herzog, Franz Wiesner, Christoph Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells |
title | Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells |
title_full | Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells |
title_fullStr | Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells |
title_full_unstemmed | Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells |
title_short | Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells |
title_sort | comparative and temporal characterization of lps and blue-light-induced tlr4 signal transduction and gene expression in optogenetically manipulated endothelial cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10000987/ https://www.ncbi.nlm.nih.gov/pubmed/36899833 http://dx.doi.org/10.3390/cells12050697 |
work_keys_str_mv | AT stierschneideranna comparativeandtemporalcharacterizationoflpsandbluelightinducedtlr4signaltransductionandgeneexpressioninoptogeneticallymanipulatedendothelialcells AT neuditschkobenjamin comparativeandtemporalcharacterizationoflpsandbluelightinducedtlr4signaltransductionandgeneexpressioninoptogeneticallymanipulatedendothelialcells AT collesellikatrin comparativeandtemporalcharacterizationoflpsandbluelightinducedtlr4signaltransductionandgeneexpressioninoptogeneticallymanipulatedendothelialcells AT hundsbergerharald comparativeandtemporalcharacterizationoflpsandbluelightinducedtlr4signaltransductionandgeneexpressioninoptogeneticallymanipulatedendothelialcells AT herzogfranz comparativeandtemporalcharacterizationoflpsandbluelightinducedtlr4signaltransductionandgeneexpressioninoptogeneticallymanipulatedendothelialcells AT wiesnerchristoph comparativeandtemporalcharacterizationoflpsandbluelightinducedtlr4signaltransductionandgeneexpressioninoptogeneticallymanipulatedendothelialcells |