Amniotic Membrane Induces Peroxisome Proliferator-Activated Receptor-γ Positive Alternatively Activated Macrophages

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)-kappa B or peroxisome proliferator-activated receptor (PPAR)-gamma related proteins was detected by Western blot analysis, and the expression of costimulatory surface molecules or PPAR-gamma 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-alpha, and NF-kappa B content in HSK corneas had decreased with AMT. AMT increased expression of PPAR-gamma, arginase 1 and 2, and arginase activity in AM-treated HSK corneas. In vitro, NF-kappa B, 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-gamma 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-gamma pathway, consequently curtailing effector T cell functions. Apoptotic cells induced in the environment with AM support the presence and survival of such macrophages. (Invest Ophthalmol Vis Sci. 2012; 53: 799-810) DOI: 10.1167/iovs.11-7617