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Published on: April 29, 2015
Amniotic membrane induces peroxisome proliferator-activated receptor-γ positive alternatively activated macrophages
Dirk Bauer1, Maren Hennig, Susanne Wasmuth
1Department of Ophthalmology, Ophtha-Lab at St. Franziskus Hospital, Muenster, Germany. dirk.bauer@uveitis-zentrum.de
Purpose:
Amniotic membrane transplantation (AMT) reportedly improves herpetic stromal keratitis (HSK). Here we studied the role of the amniotic membrane (AM) on macrophages.
Methods:
BALB/c mice with necrotizing HSK received an AMT or tarsorrhaphy (TAR) as control. Apoptosis of F4/80+ cells was determined using the annexinV/7-AAD system. Macrophage invasion was determined using a cornea invasion assay. Cytokine secretion was quantified by ELISA. Arginase activity was measured by bioassay. Expression of nuclear factor (NF)-κB or peroxisome proliferator-activated receptor (PPAR)-γ related proteins was detected by Western blot analysis, and the expression of costimulatory surface molecules or PPAR-γ by flow cytometry. Lipid accumulation was observed by Oil red O and Sudan B staining.
Results:
After AMT apoptotic features of corneal macrophages, but also macrophage invasion increased. IL-6, IL-10, IL-12, TNF-α, and NF-κB content in HSK corneas had decreased with AMT. AMT increased expression of PPAR-γ, arginase 1 and 2, and arginase activity in AM-treated HSK corneas. In vitro, NF-κB, cytokine production, costimulatory molecules (CD80, CD86, CD40), phagocytic capacity, proliferation, viability, and accessory function to herpes simplex virus (HSV)-1 specific draining lymph node (DLN) cells were reduced in bone marrow derived macrophages (BM) cocultured with AM, while CD206, CD204, CD163, and CD68, lipid accumulation in the cytoplasm, PPAR-γ expression, and arginase activity was increased. An increase in viability and proliferation was observed in the presence of AM combined with apoptotic cells, compared with AM alone.
Conclusions:
Based on these results it can be concluded that the action mechanism of AM is associated with modulation of classically activated macrophages into alternatively activated macrophages or macrophage cell death, probably by engaging lipid metabolism and activating the PPAR-γ pathway, consequently curtailing effector T cell functions. Apoptotic cells induced in the environment with AM support the presence and survival of such macrophages.
Insights
Amniotic membrane transplantation (AMT) modulates macrophages in herpetic stromal keratitis (HSK) by promoting cell death and alternative activation, likely via lipid metabolism and PPAR-γ activation. This reduces inflammation and curbs effector T cell responses.
Area of Science:
- Ophthalmology
- Immunology
- Cell Biology
Background:
- Herpetic stromal keratitis (HSK) is an inflammatory eye disease often treated with amniotic membrane transplantation (AMT).
- The precise immunomodulatory mechanisms of AMT, particularly its effects on macrophages, remain incompletely understood.
Purpose of the Study:
- To investigate the role of amniotic membrane (AM) in modulating macrophage behavior during herpetic stromal keratitis (HSK).
- To elucidate the cellular and molecular pathways involved in AMT's therapeutic effects on HSK.
Main Methods:
- BALB/c mice with HSK underwent AMT or tarsorrhaphy (TAR).
- Macrophage apoptosis, invasion, cytokine secretion, arginase activity, and protein expression (NF-κB, PPAR-γ) were analyzed.
- In vitro studies involved co-culturing bone marrow-derived macrophages (BMDM) with AM.
Main Results:
- AMT increased macrophage apoptosis and invasion in HSK corneas but decreased pro-inflammatory cytokines (IL-6, TNF-α) and NF-κB.
- AMT upregulated PPAR-γ, arginase 1 & 2, and arginase activity in HSK corneas.
- In vitro, AM reduced BMDM pro-inflammatory markers and increased alternatively activated macrophage markers (CD206, CD204, CD163, CD68), lipid accumulation, PPAR-γ, and arginase activity.
Conclusions:
- AMT likely functions by shifting classically activated macrophages towards an alternatively activated phenotype or inducing cell death.
- This modulation is probably mediated by lipid metabolism and PPAR-γ pathway activation, suppressing effector T cell responses.
- The presence of apoptotic cells in an AM-treated environment supports the survival of these regulatory macrophages.
