title: The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization.
abstract: BACKGROUND
  The nuclear receptors PPAR-γ and LXRs regulate macrophage lipid metabolism and macrophage mediated inflammation. We examined the influence of these molecules on macrophage alternative activation, with particular focus on differentiation of "M2c" anti-inflammatory cells.
  
  
  METHODS
  We cultured human monocytes in M0, M1, M2a or M2c macrophage differentiating conditions, in the presence or absence of PPAR-γ and LXR ligands. Flow cytometry was used to analyze membrane expression of phenotypic markers. Basal and LPS-stimulated production of soluble mediators was measured by ELISA. Efferocytosis assays were performed by coincubating monocytes/macrophages with apoptotic neutrophils.
  
  
  RESULTS
  We found that PPAR-γ inhibition, using the PPAR-γ antagonist GW9662, elicits differentiation of M2c-like (CD206(+) CD163(+) CD16(+)) cells and upregulation of the MerTK/Gas6 axis. Exposure of differentiating macrophages to IFN-γ, GM-CSF or LPS (M1 conditions), however, hampers GW9662 induction of MerTK and Gas6. When macrophages are differentiated with IL-4 (M2a conditions), addition of GW9662 results into an M2a (CD206(+) CD209(+) CD163(-) MerTK(-)) to M2c (CD206(high) CD209(-) CD163(+) MerTK(+)) polarization shift. Conversely, in the presence of dexamethasone (M2c conditions), the PPAR-γ agonist rosiglitazone attenuates CD163 and MerTK upregulation. The LXR agonist T0901317 induces MerTK independently of M2c polarization; indeed, CD206, CD163 and CD16 are downregulated. GW9662-differentiated M2c-like cells secrete high levels of Gas6 and low amounts of TNF-α and IL-10, mimicking dexamethasone effects in vitro. However, unlike conventional M2c cells, GW9662-differentiated cells do not show enhanced efferocytic ability.
  
  
  CONCLUSIONS
  Our results provide new insights into the role of PPAR-γ and LXR receptors in human macrophage activation and reveal the existence of different patterns regulating MerTK expression. Unexpectedly, PPAR-γ appears to negatively control the expansion of a discrete subset of M2c-like anti-inflammatory macrophages.
authors.affiliation: Temple Autoimmunity Center, Temple University, 3500 N. Broad Street, 19140 Philadelphia, PA USA ; Department of Medicine, Section of Rheumatology, Temple University, 3322 N. Broad Street, 19140 Philadelphia, PA USA.
authors.forename: Gaetano
authors.initials: G
authors.lastname: Zizzo
authors.affiliation: Temple Autoimmunity Center, Temple University, 3500 N. Broad Street, 19140 Philadelphia, PA USA ; Department of Medicine, Section of Rheumatology, Temple University, 3322 N. Broad Street, 19140 Philadelphia, PA USA.
authors.forename: Philip L
authors.initials: PL
authors.lastname: Cohen
keywords: CD163; Gas6; Liver X Receptor (LXR); M2; Macrophages; Mer receptor Tyrosine Kinase (MerTK); Peroxisome Proliferator Activated Receptor-gamma (PPAR-γ)