Pellino-1 Positively Regulates Toll-like Receptor (TLR) 2 and TLR4 Signaling and Is Suppressed upon Induction of Endotoxin Tolerance

J Biol Chem. 2015 Jul 31;290(31):19218-32. doi: 10.1074/jbc.M115.640128. Epub 2015 Jun 16.


Endotoxin tolerance reprograms Toll-like receptor (TLR) 4-mediated macrophage responses by attenuating induction of proinflammatory cytokines while retaining expression of anti-inflammatory and antimicrobial mediators. We previously demonstrated deficient TLR4-induced activation of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, and TANK-binding kinase (TBK) 1 as critical hallmarks of endotoxin tolerance, but mechanisms remain unclear. In this study, we examined the role of the E3 ubiquitin ligase Pellino-1 in endotoxin tolerance and TLR signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in macrophages from mice injected with saline and in medium-pretreated human monocytes, THP-1, and MonoMac-6 cells, whereas endotoxin tolerization abrogated LPS inducibility of Pellino-1. Overexpression of Pellino-1 in 293/TLR2 and 293/TLR4/MD2 cells enhanced TLR2- and TLR4-induced nuclear factor κB (NF-κB) and expression of IL-8 mRNA, whereas Pellino-1 knockdown reduced these responses. Pellino-1 ablation in THP-1 cells impaired induction of myeloid differentiation primary response protein (MyD88), and Toll-IL-1R domain-containing adapter inducing IFN-β (TRIF)-dependent cytokine genes in response to TLR4 and TLR2 agonists and heat-killed Escherichia coli and Staphylococcus aureus, whereas only weakly affecting phagocytosis of heat-killed bacteria. Co-expressed Pellino-1 potentiated NF-κB activation driven by transfected MyD88, TRIF, IRAK1, TBK1, TGF-β-activated kinase (TAK) 1, and TNFR-associated factor 6, whereas not affecting p65-induced responses. Mechanistically, Pellino-1 increased LPS-driven K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and phosphorylation of TBK1 and IFN regulatory factor 3. These results reveal a novel mechanism by which endotoxin tolerance re-programs TLR4 signaling via suppression of Pellino-1, a positive regulator of MyD88- and TRIF-dependent signaling that promotes K63-linked polyubiquitination of IRAK1, TBK1, and TAK1.

Keywords: Toll receptor; innate immunity; lipopolysaccharide (LPS); post-translational modification (PTM); signal transduction; ubiquitin ligase.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • HEK293 Cells
  • Humans
  • Interleukin-1 Receptor-Associated Kinases / metabolism
  • Lipopolysaccharides / pharmacology
  • MAP Kinase Kinase Kinases / metabolism
  • Mice, Inbred C57BL
  • Monocytes / immunology
  • Monocytes / metabolism
  • NF-kappa B / metabolism
  • Nuclear Proteins / physiology*
  • Protein Serine-Threonine Kinases / metabolism
  • Signal Transduction
  • TNF Receptor-Associated Factor 6 / metabolism
  • Toll-Like Receptor 2 / metabolism*
  • Toll-Like Receptor 4 / metabolism*
  • Transcriptional Activation / immunology
  • Ubiquitin-Protein Ligases / physiology*
  • Ubiquitination


  • Lipopolysaccharides
  • NF-kappa B
  • Nuclear Proteins
  • TLR2 protein, human
  • TLR4 protein, human
  • TNF Receptor-Associated Factor 6
  • Toll-Like Receptor 2
  • Toll-Like Receptor 4
  • PELI1 protein, human
  • Ubiquitin-Protein Ligases
  • IRAK1 protein, human
  • Interleukin-1 Receptor-Associated Kinases
  • Protein Serine-Threonine Kinases
  • TBK1 protein, human
  • MAP Kinase Kinase Kinases
  • MAP kinase kinase kinase 7