Inhibition of HMGB1/RAGE-mediated endocytosis by HMGB1 antagonist box A, anti-HMGB1 antibodies, and cholinergic agonists suppresses inflammation.

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.

• Authors: Huan Yang, Hui Liu, Qiong Zeng, Gavin H Imperato, Meghan E Addorisio, Jianhua Li, Mingzhu He, Kai Fan Cheng, Yousef Al-Abed, Helena E Harris, Sangeeta S Chavan, Ulf Andersson, Kevin J Tracey
• Journal: Molecular medicine (Cambridge, Mass.) (Mol Med) Vol. 25 Issue 1 Pg. 13 (04 11 2019) ISSN: 1528-3658 [Electronic] England
• PMID: 30975096 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

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

Topics

• 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, metabolism)
• Mice
• Mice, Inbred BALB C
• Microscopy, Fluorescence
• RAW 264.7 Cells
• Receptor for Advanced Glycation End Products (metabolism)