The role of NF-κB in the synergistic response of human macrophages to combined stimulation of NOD1 and TLR4 receptors in vitro

• Nina E. Murugina
• Anna S. Budikhina
• Vladimir V. Murugin
• Selezneva E.M.
• Chkadua G.Z.
• Mikhail V. Pashenkov

Abstract

Introduction. Stimulation of macrophages by a combination of NOD1 and TLR4 receptor agonists results in a synergistic enhancement of cytokine expression: cellular response to the combination of agonist is greater than the sum of responses to each individual agonist. A central role in NOD1- and TLR4-dependent cytokine expression belongs to the NF-κB family of transcription factors.

Aims - to investigate the mode of activation of NF-κB-dependent signaling pathway in macrophages upon their treatment with a combination of NOD1 and TLR4 agonists, to compare kinetics of NF-κB pathway activation with kinetics of TNF and IL6 mRNA expression.

Material and methods. Macrophages were obtained from blood monocytes of healthy donors and stimulated with NOD1 agonist (N-acetyl-D-muramyl-L-alanyl-D-isoglutamyl-meso-diaminopimelic acid), TLR4 agonist (lipopolysaccharide) and their combination. TNF and IL6 mRNA were determined by reverse-transcription real-time PCR, levels of NF-kB proteins in the nucleus and of IκBα in the cytoplasm and in the nucleus by Western blotting.

Results. Translocation of NF-κB proteins (p65/RelA, c-Rel, p50) to the nucleus during the first 4 h of combined NOD1 and TLR4 stimulation is not synergistic. However, at the output of the nucleus, a synergistic enhancement of TNF and IL6 mRNA expression is observed after 150 minutes of combined receptor stimulation. We consider possible role of relative deficiency of nuclear IκBα in the development of the synergistic response.

Keywords:macrophages; NOD1; TLR4; synergy; NF-κB; tumor necrosis factor

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