Abstract
Purpose: :
Human amniotic membrane (AM) and AM extract (AME)exert anti-inflammatory actions by inducing neutrophil apoptosis andsuppressing macrophage activation. We attempt to elucidate the mechanism by comparing HC-HA complex purifiedfrom AME to high molecular weight hyaluronic acid (HA).
Methods: :
LPS-stimulated RAW264.7 cells were treated with soluble or immobilizedHC-HA. The cell viability, proliferation, apoptosis, and adhesion weredetermined. Macrophage M1 and M2 phenotypes were assessed by qPCR, proteomicarrays, cytokine ELISAs, immunostaining, and Western blot. The effects of HC-HAon apoptosis of activated neutrophils and phagocytosis of apoptotic neutrophilsby macrophages were also determined.
Results: :
In solution, the cell viability was dose-dependentlyinhibited by HC-HA at 5-100 μg/ml but by HA only at 100 μg/ml (p <0.05). Thedecreased viability was attributed to reduced cell adhesion and elevatedapoptosis. Cells adhered on immobilized HC-HA but poorly on immobilized HA viaCD44 and TLR4 receptors, and expressed significant lower pro-inflammatory M1markers (TNF-α, IL-12,) but higher anti-inflammatory M2 markers (IL-10, SPHK1,LIGHT). IRF5, a transcriptional factor essential for polarizing toward M1phenotype, is downregulated and excluded from nuclei. Compared to PBS and HA, bothsoluble and immobilized HC-HA significantly promoted apoptosis of activatedneutrophils and phagocytosis of apoptotic neutrophils. Antibody blocking of CD44binding eliminated the M2 polarization on immobilized HC-HA.
Conclusions: :
Polarization of LPS-stimulated macrophages byimmobilized HC-HA toward M2 phenotype is mediated by CD44 receptor resulting insuppression of TLR4 signaling.
Keywords: differentiation • apoptosis/cell death • signal transduction