Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells.
Anna StierschneiderBenjamin NeuditschkoKatrin ColleselliHarald HundsbergerFranz HerzogChristoph WiesnerPublished in: Cells (2023)
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.
Keyphrases
- toll like receptor
- inflammatory response
- nuclear factor
- immune response
- endothelial cells
- lps induced
- gene expression
- induced apoptosis
- high throughput
- intensive care unit
- oxidative stress
- stem cells
- mesenchymal stem cells
- magnetic resonance
- endoplasmic reticulum stress
- liquid chromatography
- cell therapy
- epithelial mesenchymal transition
- bone marrow
- high performance liquid chromatography