Glycodelin-A and interleukin-10 regulate phenotype and functions of human macrophages

Abstract

Decidual macrophage comprises of about 20-30% of total decidual leukocyte population. It helps to establish a successful pregnancy by conferring immune tolerance to the semi-allogenic fetus and ensure proper placenta development. Yet, the factors regulating decidual macrophage differentiation were unclear. IL-10, a cytokine found abundantly in the decidua, along with MCSF induces M2 (anti-inflammatory) phenotype in macrophages. However, this phenotype lacks some of the important decidual macrophage markers (e.g. IDO-1) suggesting that other decidual-derived factors may be involved in this process. Glycodelin-A (GdA) is glycoprotein found abundantly in the decidualized endometrium. It plays an important role in feto-maternal defense by modulating the cellular activity and differentiation of the decidual leukocytes. The glycans of GdA, especially sialic acids, mediate its binding and activities via sialic acid-specific receptors such as L-selectin and Siglec. Siglec-7 is one of the Siglec members that is expressed in monocytes and macrophages. This study hypothesized that GdA and IL-10 drive monocyte differentiation towards a decidual macrophage phenotype and regulate its functions via Siglec-7 receptor. The role of GdA and IL-10 in macrophage differentiation was investigated. Monocytes isolated from human peripheral blood were treated with MCSF (50 ng/mL), GdA (10 μg/mL) and IL-10 (5 ng/mL) for 9 days. Individually, GdA regulates the expression of IDO-1 and CD209, while IL-10 regulates NRP-1 and CD209, but when they come together all the three markers are expressed. The GdA-polarized macrophages exhibited reduced phagocytic capability, higher IDO-1 activity and increased concentration of the cytokine interleukin-13 (IL-13), which might contribute to immune tolerance. Additionally, the conditioned medium-derived from GdA- and GdA/IL-10-polarized macrophages exhibited higher concentration of chemokine C-C motif chemokine 2 (CCL-2), which might enhance chemoattractant feature of monocytes and macrophages. The role of the differentiated macrophages in regulating placental remodeling of the uterine vasculature was investigated. Angiogenesis array and ELISA showed an increase in the expression of insulin-like growth factor binding protein-1 (IGFBP-1) in the conditioned medium of GdA-polarized macrophages. The conditioned medium promoted invasive and integration capacity of trophoblasts and angiogenic capacity of endothelial cells. Moreover, blocking of IGFBP-1 or inhibiting IDO-1 activity reduced the trophoblast invasive capacity induced by the GdA-polarized macrophage conditioned medium. To confirm Siglec-7 is one of the GdA binding receptors on monocytes, co-immunoprecipitation experiments of monocyte membrane extract followed by binding to anti-glycodelin affinity column was performed. It was confirmed that Siglec-7 interacts with GdA in vitro. Flow cytometry analysis showed that blocking of Siglec-7 by blocking antibody prevented GdA binding on monocytes; while blocking of Siglec-7 expression abolished GdA-mediated changes in IDO-1/CD209 expression, kynurenine levels, CCL-2 levels and phagocytic capability. In conclusion, the present findings suggest an important role of GdA and IL-10 in polarizing the macrophage differentiation into a decidual macrophage phenotype that promotes feto-maternal tolerance and placental development. GdA mediates some of its actions via Siglec-7. Results from this study might lay a foundation on the use of GdA as biomarker for predicting the risk of pregnancy-related complications (in early stages) and the possibility of using allogenic monocyte-derived macrophage in alleviating pregnancy/placenta-associated diseases.