IL21R expressing CD14+CD16+ monocytes expand in multiple myeloma patients leading to increased osteoclasts

Abstract

Bone marrow monocytes are primarily committed to osteoclast formation. It is, however, unknown whether potential primary alterations are specifically present in bone marrow monocytes from patients with multiple myeloma, smoldering myeloma or monoclonal gammopathy of undetermined significance. We analyzed the immunophenotypic and transcriptional profiles of bone marrow CD14⁺ monocytes in a cohort of patients with different types of monoclonal gammopathies to identify alterations involved in myeloma-enhanced osteoclastogenesis. The number of bone marrow CD14⁺CD16⁺ cells was higher in patients with active myeloma than in those with smoldering myeloma or monoclonal gammopathy of undetermined significance. Interestingly, sorted bone marrow CD14⁺CD16⁺ cells from myeloma patients were more pro-osteoclastogenic than CD14⁺CD16-cells in cultures ex vivo Moreover, transcriptional analysis demonstrated that bone marrow CD14⁺ cells from patients with multiple myeloma (but neither monoclonal gammopathy of undetermined significance nor smoldering myeloma) significantly upregulated genes involved in osteoclast formation, including IL21RIL21R mRNA over-expression by bone marrow CD14⁺ cells was independent of the presence of interleukin-21. Consistently, interleukin-21 production by T cells as well as levels of interleukin-21 in the bone marrow were not significantly different among monoclonal gammopathies. Thereafter, we showed that IL21R over-expression in CD14⁺ cells increased osteoclast formation. Consistently, interleukin-21 receptor signaling inhibition by Janex 1 suppressed osteoclast differentiation from bone marrow CD14⁺ cells of myeloma patients. Our results indicate that bone marrow monocytes from multiple myeloma patients show distinct features compared to those from patients with indolent monoclonal gammopathies, supporting the role of IL21R over-expression by bone marrow CD14⁺ cells in enhanced osteoclast formation.

Figure 1.

Immunophenotype of bone marrow monocytes in patients with monoclonal gammopathies and their pro-osteoclastogenic ex vivo properties. (A) CD14 and CD16 expression by BM monocytes in patients with monoclonal gammopathies: example of plots of flow cytometry data. (B) Box plots representing the median percentage values of CD14⁺CD16⁺ cells evaluated in BM samples obtained from patients with MGUS, SMM or MM (P calculated using the Mann-Whitney test). (C, D) Box plots representing the median percentage values of CD14⁺CD16⁺ cells evaluated in BM samples obtained (C) from patients with (w) or without (w/o) osteolysis and (D) from patients with high bone disease (HBD) or low bone disease (LBD). (E) Osteoclast assay. CD14⁺ cells were purified from BM samples of patients with mono clonal gammopathies by an immunomagnetic method and then sorted into the two sub-populations CD14⁺CD16⁻ and CD14⁺CD16⁺ by a flow cell sorter as described in the Methods section. CD14⁺CD16⁻ or CD14⁺CD16⁺ cells (200,000 cells/well) were seeded in 96-well plates in αMEM with 10% FBS, rhM-CSF 25 ng/mL and rhRANKL 60 ng/mL. After 28 days of culture, osteoclasts were identified as multinucleated TRAP-positive cells and counted by light microscopy. (E) Bar graph represents the median number of osteoclasts/well of each condition, divided into osteoclasts with ≥5 nuclei or <5 nuclei (P calculated using the Mann-Whitney test, CD14⁺CD16⁻ versus CD14⁺CD16⁺ cells). On the right there is a representative image of the osteoclastogenesis assay stained by TRAP from BM sorted CD14⁺CD16-and CD14⁺CD16⁺ cells (original magnification, 4×).

Figure 2.

Transcriptional fingerprints evaluated by gene expression profiling of purified bone marrow CD14⁺ cells from patients with different monoclonal gammopathies. (A) Heatmap of the trancriptional profiles resulting from the unsupervised analysis of all the MM, SMM and MGUS monocyte samples. (B) Scatterplots showing the correlation between IL21R expression and that of CD40, SLAMF7 and CCR5 by BM monocytes as determined from gene expression analysis. The lines represent the linear regression between each couple of genes. (C) Quantitative real-time polymerase chain reaction of IL21R, CCR5, CD40 and SLAMF7 genes performed on BM monocytes purified from patients with monoclonal gammopathies. Values represent the median of the −ΔCt values of the reactions (*: fold change >1.5). (D) Quantitative real-time polymerase chain reaction of IL21R, CCR5, CD40 and SLAMF7 genes performed on BM monocytes purified from MM patients with (w) or without (w/o) osteolysis. Values represent the mean of the −∆Ct values of the reactions.

Figure 3.

IL-21R over-expression by bone marrow CD14⁺ cells from patients with multiple myeloma compared to those from patients with smoldering multiple myeloma or monoclonal gammopathy of undetermined significance. (A) IL21R mRNA expression was evaluated by real-time polymerase chain reaction (PCR) in purified BM CD14⁺ obtained from patients with monoclonal gammopathies. Box plots show the median −ΔCt levels (P calculated by the Mann-Whitney test). (B) IL21R mRNA expression was evaluated by real-time PCR in purified BM CD14⁺ obtained from MM patients versus SMM plus MGUS patients. Box plots show the median −ΔCt levels (P calculated by the Mann-Whitney test). (C) CD360/IL-21R expression was evaluated by flow cytometry in BM CD14⁺CD16⁻ and CD14⁺CD16⁺ cells. Comparison between CD360 expression by CD14⁺CD16⁻ and CD14⁺CD16⁺ monocyte populations, stained with anti-CD360 or control IgG1, is shown in three representative MM patients (i, ii, and iii) (MFI: median fluorescence intensity). (D) Active STAT3 levels were determined by the STAT family assay kit in nuclear extracts of purified BM CD14⁺ cells obtained from MGUS (n=3), SMM (n=3) and MM (n=3) patients. The bar chart represents the mean±SD level of active STAT3 checked as optical density (OD) at 450 nm with a reference wavelength of 620 nm, after subtracting the blank. (E) BM CD14⁺ cells purified from patients with MM, SMM or MGUS were treated with or without rhIL-21 (30 pg/mL) for 24 h. IL21R mRNA level was evaluated by real-time PCR. The bar chart represents the median −ΔCt of IL21R mRNA of three replicates (Con: untreated control). (F) The monocytic cell line THP-1 was treated for 48 h with or without rhIL-6 (20 ng/mL) or TNF-α (10 ng/mL) or both cytokines. IL21R mRNA levels were evaluated by real-time PCR in three independent experiments (P calculated using the t test). The bar chart represents the mean±SD fold change of mRNA IL21R (Con: untreated control).

Figure 4.

IL21 mRNA expression by the bone marrow microenvironment and levels of interleukin-21 in the bone marrow in patients with multiple myeloma, smoldering multiple myeloma or monoclonal gammopathy of undetermined significance. (A) IL21 mRNA expression by purified CD3⁺ cells from MM (n=5), SMM (n=7) or MGUS (n=7) patients evaluated by real-time polymerase chain reaction. The bar chart shows the mean±SD −ΔCt of IL21 mRNA. (B) BM IL-21 levels were evaluated by enzyme linked immunosorbent assay in a cohort of 76 newly diagnosed MM, 42 SMM and 41 MGUS patients. The scatter dot plot represents BM IL-21 levels in the cohort of patients with the lines representing median levels.

Figure 5.

IL21R over-expression by a lentiviral vector in monocytes increases the differentiation of osteoclasts. (A) IL21R was over-expressed in peripheral blood CD14⁺ cells obtained from three different healthy donors transduced with a specific lentiviral vector for IL21R (CD14⁺ IL21R vector) as compared to those infected with the empty control vector (CD14⁺ empty vector) or not transduced (CD14⁺). IL21R mRNA levels were checked by real-time polymerase chain reaction. Bar graph represents the median −ΔCt levels of three independent experiments. CD14⁺ transduced cells with IL21R or empty lentiviral vectors (200,000 cells/well) were seeded in 96-well plates in αMEM with 10% FBS, rhM-CSF 10 ng/mL and rhRANKL 50 ng/mL in the presence or absence of the IL-21R signaling inhibitor Janex 1 (10 μM) or vehicle (DMSO). After 28 days of culture, osteoclasts were identified as multinucleated (>3 nuclei) TRAP-positive cells and counted by light microscopy. (B) The bar graph shows the mean±SD number of osteoclasts for each well (P calculated using the t test) in three independent experiments with CD14⁺ from three different healthy donors (left panel). The box plot represents the osteoclast area (P calculated by the Mann-Whitney test) in a representative experiment performed at least in triplicate (right panel). (C) Images of one representative experiment of the osteoclastogenesis assay stained by TRAP of CD14⁺ IL21R vector and CD14⁺ empty vector cells performed in the presence or absence of Janex 1 (original magnification, 4×).

Figure 6.

Interleukin-21 receptor signaling inhibition blocks Interleukin-21 driven osteoclastogenesis. BM mononuclear cells, obtained from MM patients, were seeded at the concentration of 4×10⁵ cells/well in 96-well plates in αMEM with 10% FBS, rhM-CSF 25 ng/mL and rhRANKL 20 ng/mL in the presence or absence of rIL-21 (30 pg/mL) and the IL-21R signaling inhibitor Janex 1 (10 μM) or vehicle (DMSO) for 28 days, replacing the medium every 3 days. At the end of the culture period osteoclasts were identified as multinucleated (>3 nuclei) TRAP-positive cells and counted by light microscopy (Con: untreated control). (A) The bar graph represents the mean±SD osteoclast number for each well (P calculated using the t test) (upper panel). The box plot shows the osteoclast (OC) area (P calculated by the Mann-Whitney test) in one representative experiment performed at least in triplicate (lower panel). (B) Representative images of osteoclasts stained with TRAP after 28 days of culture (original magnification, 4×).