Related Experiment Video
Updated: Aug 25, 2025
Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach
Published on: May 2, 2018
The Distinctive Activation of Toll-Like Receptor 4 in Human Samples with Sepsis
Patrick Thon1, Katharina Rump1, Annika Knorr1
1Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum, 44892 Bochum, Germany.
Abstract:
Clinical success of Toll-Like receptor-4 (TLR-4) antagonists in sepsis therapy has thus far been lacking. As inhibition of a receptor can only be useful if the receptor is active, stratification of patients with active TLR-4 would be desirable. Our aim was to establish an assay to quantify phosphorylated TLR-4 using the proximity ligation assay (PLA). HEK293 TLR4/MD2/CD14 as well as THP-1 cells were stimulated with LPS and the activation of TLR-4 was measured using the PLA. Furthermore, peripheral blood mononuclear cells (PBMCs) from 25 sepsis patients were used to show the feasibility of this assay in clinical material. Activation of TLR-4 in these samples was compared to the PBMCs of 11 healthy individuals. We could show a transient activation of TLR-4 in both cell lines. Five min after the LPS stimulation, the signal increased 6.7-fold in the HEK293 cells and 4.3-fold in the THP-1 cells. The assay also worked well in the PBMCs of septic patients. Phosphorylation of TLR-4 at study inclusion was 2.9 times higher in septic patients compared to healthy volunteers. To conclude, we established a diagnostic assay that is able to quantify the phosphorylation of TLR-4 in cell culture and in clinical samples of sepsis patients. This makes large-scale stratification of sepsis patients for their TLR-4 activation status possible.
Insights
A new assay quantifies phosphorylated Toll-Like Receptor-4 (TLR-4) activation. This method enables patient stratification for sepsis therapy, identifying those with active TLR-4 for targeted treatment.
Area of Science:
- Immunology
- Biochemistry
- Molecular Biology
Background:
- Clinical trials of Toll-Like Receptor-4 (TLR-4) antagonists in sepsis have not been successful.
- Patient stratification based on TLR-4 activation is crucial for effective targeted therapy.
Purpose of the Study:
- To develop a proximity ligation assay (PLA) for quantifying phosphorylated TLR-4.
- To assess the feasibility of this assay in clinical samples from sepsis patients.
Main Methods:
- HEK293 and THP-1 cell lines were stimulated with LPS to measure TLR-4 activation via PLA.
- Peripheral blood mononuclear cells (PBMCs) from sepsis patients and healthy individuals were analyzed.
Main Results:
- A transient increase in TLR-4 activation was observed in cell lines post-LPS stimulation (6.7-fold in HEK293, 4.3-fold in THP-1).
- Phosphorylation of TLR-4 was 2.9 times higher in sepsis patients compared to healthy controls.
- The PLA assay demonstrated efficacy in clinical PBMCs.
Conclusions:
- A novel diagnostic assay effectively quantifies TLR-4 phosphorylation in both cell cultures and clinical sepsis samples.
- This assay facilitates large-scale patient stratification based on TLR-4 activation status for potential therapeutic interventions.
