Dataset Information

Comparative and temporal characterization of LPS and blue light induced TLR4 signal transduction and gene expression in optogenetically manipulated endothelial cells

ABSTRACT: 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 TLR4 signaling induction is difficult to achieve by LPS due to its poorly controllable binding 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 LEC and opto-TLR4-LOV HUVEC) that allow 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 LEC) revealed high basal activity with fast exploitation of 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.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lymphatic Endothelial Cell

SUBMITTER: Benjamin Neuditschko

LAB HEAD: Franz Herzog

PROVIDER: PXD038764 | Pride | 2023-05-10

REPOSITORIES: Pride

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			Action	DRS
	LEC_TERT_TLR4_LOV_LPS_SN_16h_1_1.mzML	Mzml
	LEC_TERT_TLR4_LOV_LPS_SN_16h_1_1.mzid.gz	Mzid
	LEC_TERT_TLR4_LOV_LPS_SN_16h_1_1.raw	Raw
	LEC_TERT_TLR4_LOV_LPS_SN_16h_1_2.mzML	Mzml
	LEC_TERT_TLR4_LOV_LPS_SN_16h_1_2.mzid.gz	Mzid

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Publications

Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells.

Stierschneider Anna A Neuditschko Benjamin B Colleselli Katrin K Hundsberger Harald H Herzog Franz F Wiesner Christoph C

Cells 20230222 5

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 enginee ...[more]

PMID: 36899833

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