March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
HC-HA but not High Molecular Weight HA Polarizes LPS-Activated Macrophages toward M2 Phenotype via CD44-Mediated Suppression of TLR4 Signaling
Author Affiliations & Notes
  • Hua He
    TissueTech and Ocular Surface Center, Miami, Florida
  • Scheffer C. Tseng
    Ocular Surface Center, Ocular Surface Res & Educ Fndtn, Miami, Florida
  • Footnotes
    Commercial Relationships  Hua He, TissueTech, Inc. (E); Scheffer C. Tseng, TissueTech, Inc. (I, P)
  • Footnotes
    Support  NIH, NEI, R44 EY017497 and R43 EY021045
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 6281. doi:
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      Hua He, Scheffer C. Tseng; HC-HA but not High Molecular Weight HA Polarizes LPS-Activated Macrophages toward M2 Phenotype via CD44-Mediated Suppression of TLR4 Signaling. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6281.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 

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