Inhibition of HMGB1/RAGE-mediated Endocytosis by HMGB1 Antagonist Box A, anti-HMGB1 Antibodies, and Cholinergic Agonists Suppresses Inflammation

Mol Med. 2019 Apr 11;25(1):13. doi: 10.1186/s10020-019-0081-6.

Abstract

Background: Extracellular high mobility group box 1 protein (HMGB1) serves a central role in inflammation as a transporter protein, which binds other immune-activating molecules that are endocytosed via the receptor for advanced glycation end-products (RAGE). These pro-inflammatory complexes are targeted to the endolysosomal compartment, where HMGB1 permeabilizes the lysosomes. This enables HMGB1-partner molecules to avoid degradation, to leak into the cytosol, and to reach cognate immune-activating sensors. Lipopolysaccharide (LPS) requires this pathway to generate pyroptosis by accessing its key cytosolic receptors, murine caspase 11, or the human caspases 4 and 5. This lytic, pro-inflammatory cell death plays a fundamental pathogenic role in gram-negative sepsis. The aim of the study was to identify molecules inhibiting HMGB1 or HMGB1/LPS cellular internalization.

Methods: Endocytosis was studied in cultured macrophages using Alexa Fluor-labeled HMGB1 or complexes of HMGB1 and Alexa Fluor-labeled LPS in the presence of an anti-HMGB1 monoclonal antibody (mAb), recombinant HMGB1 box A protein, acetylcholine, the nicotinic acetylcholine receptor subtype alpha 7 (α7 nAChR) agonist GTS-21, or a dynamin-specific inhibitor of endocytosis. Images were obtained by fluorescence microscopy and quantified by the ImageJ processing program (NIH). Data were analyzed using student's t test or one-way ANOVA followed by the least significant difference or Tukey's tests.

Results: Anti-HMGB1 mAb, recombinant HMGB1 antagonist box A protein, acetylcholine, GTS-21, and the dynamin-specific inhibitor of endocytosis inhibited internalization of HMGB1 or HMGB1-LPS complexes in cultured macrophages. These agents prevented macrophage activation in response to HMGB1 and/or HMGB1-LPS complexes.

Conclusion: These results demonstrate that therapies based on HMGB1 antagonists and the cholinergic anti-inflammatory pathway share a previously unrecognized molecular mechanism of substantial clinical relevance.

Keywords: Cytokine; Endocytosis sepsis; HMGB1; LPS; RAGE.

Publication types

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

MeSH terms

  • Acetylcholine / pharmacology
  • Animals
  • Cells, Cultured
  • Cholinergic Agonists / pharmacology
  • Endocytosis / drug effects
  • Enzyme-Linked Immunosorbent Assay
  • HMGB1 Protein / metabolism*
  • Inflammation / metabolism
  • Lipopolysaccharides / pharmacology*
  • Macrophages / drug effects
  • Macrophages / metabolism
  • Mice
  • Mice, Inbred BALB C
  • Microscopy, Fluorescence
  • RAW 264.7 Cells
  • Receptor for Advanced Glycation End Products / metabolism*

Substances

  • Cholinergic Agonists
  • HMGB1 Protein
  • Lipopolysaccharides
  • Receptor for Advanced Glycation End Products
  • Acetylcholine