Over-expression of pro-osteoclastogenic cytokine receptors and chemokines by bone marrow CD14+ monocytes of Multiple Myeloma (MM) patients as compared to Smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS): role of IL-21R/IL-21 axis in MM-induced osteoclastogenesis.

Multiple myeloma (MM) is characterized by the uncoupled increase in bone marrow (BM) of osteoclast formation and activation, which lead to bone destruction, as compared to patients with smoldering MM (SMM) and monoclonal gammopathy of uncertain significance (MGUS). Although the molecular analysis of clonal plasma cells (PCs) identified several genes whose overexpression is associated with the occurrence of bone lesions, a clear transcriptional fingerprint able to distinguish the different PC dyscrasias is lacking. As the close relationship between PCs and BM microenvironment plays a pivotal role in MM pathogenesis, ongoing studies are focusing on the presence of potential molecular alterations in the microenvironment. Among the different cell types of BM microenvironment, monocytes are known to be primarily involved in osteoclastogenesis, angiogenesis and immune function. The aim of this study was to analyze the transcriptional and proteomic profiles of the BM CD14+ cells across the different types of monoclonal gammopathies and to identify alterations potentially involved in the pathogenesis of the increased osteoclastogenesis. The expression profiles of CD14+ samples were evaluated by Affymetrix GeneChip HG-U133Plus 2.0 arrays in 25 MM, 11 SMM and 8 MGUS patients. The proteomic analysis of CD14+ cells of 5 MM, 5 SMM and 5 MGUS was run on Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific) and the data analyzed with Proteome Discoverer 1.4 software. A multiclass analysis identified 18 differentially expressed genes in MGUS, SMM and MM. The comparison of MM with both SMM and MGUS samples identified 61 genes differentially expressed in CD14+ cells (37 up-regulated and 24 down-regulated). Interestingly, we found specific cytokine receptors (IL21R and IL-15R) and pro-osteoclastogenic chemokines (CXCL10) that were over-expressed in CD14+ of MM patients, as compared to SMM and MGUS. Similarly, the proteomic analysis reinforced that different CD14+ monocyte protein profiles were found comparing MM patients with MGUS and SMM ones. Interestingly, MM monocytes over-expressed proteins involved in cell adhesion and inflammation and down-regulated molecules implicated in antimicrobial functions. Because recent data indicate that IL-21 is a growth factor for MM cells and may promote osteoclastogenesis in some pathological conditions such as rheumatoid arthritis, we further investigate the potential role of IL-21R over-expression by MM CD14+ cells in osteoclastogenesis. Firstly, we confirmed the IL-21R up-regulation by CD14+ of MM patients at both mRNA and protein level as compared to both SMM and MGUS. On the other hand any significant difference was not observed in the BM plasma IL-21 levels between MM, SMM and MGUS. The treatment with rhIL-21, at the concentration observed in the BM plasma, increased the size of osteoclasts in CD14+-derived in vitro osteoclastogenesis assays, in MM patients but not in SMM and MGUS; this suggested higher sensitivity to the IL-21-dependent pro-osteoclastogenic differentiation effect in MM patients. Finally the overexpression of IL-21R by lentiviral vectors, as well as the use of an IL-21R signaling inhibitor, was analyzed in our in vitro model to delineate a new anti-osteoclastogenic strategy. In conclusion our results indicate that different expression fingerprints characterize BM CD14+ monocytes of patients with MM as compared to those with SMM and MGUS, including over-expression of IL-21R, putatively involved in MM-induced osteoclast formation and activation through an increased sensitivity to IL-21.