Overexpression 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 Interleukin(IL)-21 Receptor/IL-21 Axis in MM-Induced Osteoclastogenesis
Authors: Bolzoni Marina , Ronchetti Domenica , Storti Paola , Guasco Daniela , Marchica Valentina , Agnelli Luca , Dalla Palma Benedetta , Accardi Fabrizio , Costa Federica , Toscani Denise , Bonomini Sabrina , Thomas Benjamin , Neri Antonino , Aversa Franco , Giuliani Nicola 
Published: November 2019

Abstract
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 type 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 GeneChip HG-U133Plus 2.0 arrays (Affymetrix®) 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 and CXCL11) 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. Consistently we found that the conditioned media of human MM cell lines up-regulated IL21R mRNA expression by CD14+ cells. On the other hand any significant difference was not observed in the BM plasma IL-21 levels between MM, SMM and MGUS in a larger cohort of 160 patients. The treatment with rhIL-21, at the concentration observed in the BM plasma increased the osteoclast differentiation of the monocytic cell lines THP-1 and stimulated CD14+-derived in vitro osteoclastogenesis increasing the number and the size of osteoclasts, 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 or the knockout of IL-21R by lentivirus vectors, as well as the use of an IL-21R signaling inhibitor, was analyzed in the monocytic cell lines THP-1 to delineate a new anti-osteoclastogenic in vitro 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.

Disclosures
Giuliani: Celgene Italy: Research Funding.