The Distinctive Activation of Toll-Like Receptor 4 in Human Samples with Sepsis

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 phosphory...

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Published in:	Cells (Basel, Switzerland) Vol. 11; no. 19; p. 3020
Main Authors:	Thon, Patrick, Rump, Katharina, Knorr, Annika, Dyck, Birte, Ziehe, Dominik, Unterberg, Matthias, Nowak, Hartmuth, Bergmann, Lars, Wolf, Alexander, Bazzi, Maha, Orlowski, Jennifer, Peters, Marcus, Zarbock, Alexander, Brenner, Thorsten, Adamzik, Michael, Rahmel, Tim, Koos, Björn
Format:	Journal Article
Language:	English
Published:	Basel MDPI AG 27-09-2022 MDPI
Subjects:	Antibodies CD14 antigen Cell culture Cell lines Coronaviruses COVID-19 Cytokines Development and progression Ethics Health aspects Kinases Leukocytes (mononuclear) Lipopolysaccharides Patients Peripheral blood mononuclear cells Phosphorylation proximity ligation assay Registration Sepsis TLR4 protein Toll-Like Receptor 4 Toll-like receptors Germany United States > US Europe
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors contributed equally to this work.
ISSN:	2073-4409 2073-4409
DOI:	10.3390/cells11193020