Impact of KDO in biological activity of Re-LPS
© Prokhorenko et al.; licensee BioMed Central Ltd. 2014
Published: 3 December 2014
The minimal biological active structure of endotoxins (lipopolysaccharides (LPS)) is Re-LPS (KDO2-lipid A), which consist of lipid A and two (or three) molecules of 3-deoxy-D-manno-2-octulosonic acid (KDO) [1, 2]. Biological activity of endotoxins is defined in general by the number and distribution of acyl residues on the lipid A backbone . Recently it has been reported that KDO-treated RAW 264.7 cells exhibited a gene expression pattern similar to that in LPS-treated cells. These authors revealed that free KDO participated in crosstalk between Toll-like receptors (TLR) and G protein-coupled receptors and so that regulated activators and repressors of immune signaling . LPS-dependent TLR4-triggered activation of target cells leads to specific changes in the levels of surface receptors and induces synthesis of proinflammatory cytokines . However, the dependence of these processes on the structural composition of LPS is not well understood. To extend our knowledge in this field, the effects of free KDO as well as KDO as covalently linked to lipid A constituent of Re-LPS on expression of TLR4, CD11b and CD14 receptors and TNFα synthesis in whole human blood have been investigated.
Human blood was incubated with Re-LPS from Escherichia coli JM103 or Salmonella enterica sv Typhimurium SL1181 (100 ng/ml) or with lipids A from E. coli F583 or S. enterica sv Minnesota R595 (80 ng/ml) or with ammonium salt of KDO (20 ng/ml) at 37°C in 5% CO2-humidified atmosphere for 2 or 6 hours to determine receptor expression or TNFα release, respectively. Receptor expression was monitored by EPICS XL-MCL flow cytometer using Alexa Fluor 488 anti-TLR4 (HTA125), anti-CD11b (ICRF44) and anti-CD14 (HCD14) antibodies. Human TNF-α ELISA Kit II was exploited to TNFα determination.
Free KDO in the used concentration was inactive in regulation of TLR4, CD11b and CD14 expression and did not induce TNFα release but its impact in biological activity was detected when KDO was applied as constituent of Re-LPS. This may be explained by the effect of KDO on the spatial conformation of Re-LPS.
The work was supported by Grant 16.N08.12.1014 established by the Russian Ministry of Education and Science.
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