Microvesicles carrying LRP5 induce macrophage polarization to an anti-inflammatory phenotype.

Microvesicles (MV) contribute to cell-to-cell communication through their transported proteins and nucleic acids. MV, released into the extracellular space, exert paracrine regulation by modulating cellular responses after interaction with near and far target cells. MV are released at high concentrations by activated inflammatory cells. Different subtypes of human macrophages have been characterized based on surface epitopes being CD16+ macrophages associated with anti-inflammatory phenotypes. We have previously shown that low-density lipoprotein receptor-related protein 5 (LRP5), a member of the LDLR family that participates in lipid homeostasis, is expressed in macrophage CD16+ with repair and survival functions. The goal of our study was to characterize the cargo and tentative function of macrophage-derived MV, whether LRP5 is delivered into MV and whether these MV are able to induce inflammatory cell differentiation to a specific CD16- or CD16+ phenotype. We show, for the first time, that lipid-loaded macrophages release MV containing LRP5. LDL loading induces increased expression of macrophage pro-inflammatory markers and increased release of MV containing pro-inflammatory markers. Conditioning of fresh macrophages with MV released by Lrp5-silenced macrophages induced the transcription of inflammatory genes and reduced the transcription of anti-inflammatory genes. Thus, MV containing LRP5 induce anti-inflammatory phenotypes in macrophages.

Heterozygous deletion of LRP5 gene in mice alters profile of immune cells and modulates differentiation of osteoblasts.

Skeletal homeostasis is dynamically influenced by the immune system. Low density lipoprotein receptor-related protein-5 (LRP5) is a co-receptor of the Wnt signaling pathway, which modulates bone metabolism in humans and mice. Immune disorders can lead to abnormal bone metabolism. It is unclear whether and how LRP5 alters the balance of the immune system to modulate bone homeostasis. In this study, we used primary osteoblast to detect the differentiation of osteoblasts in vitro, the immune cells of spleen and bone marrow of 6-month old LRP5 heterozygote (HZ) and wild-type (WT) mice were analyzed by Flow cytometry. We found that LRP5+/- could influence the differentiation of osteoblasts by decreasing the mRNA level of Osterix, and increasing the mRNA level of Runx2 and the ratio of receptor activator for nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG). In the LRP5+/- mice, percentages of NK cells, CD3e+ cells, and CD8a+ T cells were increased in both spleen and bone marrow, and percentages of CD106+ cells and CD11c+ cells were increased in spleen while decreased in bone marrow, conversely, CD62L+ cells were decreased in spleen while increased in bone marrow compared to the WT mice. Percentages of CD4+ cells, CD14+ cells, and CD254+ cells were increased in the spleen, and CTLA4+ cells were increased in the bone marrow of the LRP5+/- mice. The mRNA level of Wnt signaling molecules such as ß-catenin, and c-myc were decreased and APC was increased in spleen lymphocytes and bone marrow lymphocytes, and the mRNA level of Wnt3a was decreased in spleen lymphocytes while no change in bone marrow lymphocytes was seen with silencing LRP5 by specific small interfering RNA. In conclusion, heterozygous deletion of the LRP5 gene in mice could alter the profile of the immune cells, influence the balance of immune environment, and modulate bone homeostasis, which might present a potential mechanism to explore the Wnt signaling pathway in the modulation of the immune system.

LRP5 associates with specific subsets of macrophages: Molecular and functional effects.

Innate and acquired immunity is involved in the progression of atherosclerosis. The molecular mechanisms ruling monocyte to macrophage (Mø) differentiation are not yet fully understood. Different subtypes of plaque macrophages that have differentiated from monocytes recruited from circulating blood, have been characterized based on surface epitopes. We have recently shown that LRP5, a member of the LDL receptor superfamily supporting Wnt signalling, has an important role in monocyte to macrophage differentiation. The aim of this study was to investigate whether the CD16- and CD16+ macrophage subsets found in human atherosclerotic plaques have a differential LRP5 expression/function and Wnt signalling potential. We show for the first time that LRP5 expression is significantly higher in human CD16+Mø derived from CD14(+)CD16(+) monocytes than in CD16-Mø macrophages derived from CD14(+)CD16(-) monocytes. LRP5 is not found in human healthy vessel or arterial intimal thickening but is found in advanced human atherosclerotic lesions co-localizing only with the CD16+Mø macrophage subset. LRP5 expressing macrophages infiltrate the deep layers of atherosclerotic plaques towards the intima-media boundaries showing increased migratory activity and higher phagocytic activity. The equivalent for human patrolling CD14(+)CD16(+) monocytes in mice, CD115(+)GR1(low) monocytes, also show an increased expression of LRP5. In summary, classical CD14(+)CD16(-)monocytes that differentiate into CD16-Mø do not express LRP5. Instead, human monocytes expressing LRP5 differentiate into CD16+Mø antiinflammatory macrophages. These antiinflammatory macrophages are found in advanced atherosclerotic human plaques. Thus LRP5 is a signature of the anti-inflammatory defensive phenotype of macrophages.