Multiple Myeloma Cell-Derived Interleukin-32gamma Increases the Immunosuppressive Function of Macrophages By Promoting Indoleamine 2,3-Dioxygenase (IDO) Expression

Background: The interaction of multiple myeloma (MM) cells with macrophages (MΦs) in the bone marrow microenvironment contributes to the pathophysiology of MM. In addition to promoting angiogenesis through vasculogenic mimicry, MM-associated MΦs (mMΦs) protect MM cells from spontaneous and chemotherapy-induced apoptosis. mMΦs therefore represent a potential target for myeloma treatment and it is essential to explore the mechanisms underlying normal MΦ polarization to mMΦs. We previously showed that IL-32 is overexpressed in MM patients and is mainly derived from MM cells. The present study was designed to explore the clinical significance of IL-32 in MM and to further elucidate the molecular mechanisms underlying the IL-32-mediated immune function of MΦs.

Methods: We examined the expression of IL-32 in bone marrow biopsy samples using immunohistochemistry. Quantitative real-time PCR, western blot analysis and immunofluorescence were applied to measure the expression of IL-32, IDO and proteinase 3 (PR3). We obtained the global transcriptional profile of the IL-32γ-treated MΦs by RNA sequencing (RNA-Seq). Immunoprecipitation (IP) and GST pulldown experiments was applied to confirm the binding affinity of PR3 for IL-32. We created IL-32-knockdown MM cells by transfection of IL-32 shRNA and silenced PR3 expression in MΦs using siRNA targeting PR3. CD4⁺ T cell proliferation and IL-2, IFN-γ and TNF-α production were measured by flow cytometry.

Results: We found that high IL-32 expression in MM patients was associated with advanced clinical stage and high serum β2-microglobulin levels. Several isoforms of IL-32 were detected in MM cells and IL-32γ was the most active subtype. RNA sequencing revealed that IL-32γ significantly induced the production of the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO) in MΦs and this effect was verified at the protein level. Furthermore, IL-32-knockdown MM cells showed less ability than control MM cells to promote IDO expression. As a binding protein for IL-32, PR3 was universally expressed on the surface of MΦs and knockdown of PR3 or inhibition of the STAT3 and nuclear factor κB (NF-κB) pathways hindered the IL-32γ-mediated stimulation of IDO expression. Finally, IDO-positive IL-32γ-educated MΦs inhibited CD4⁺ T cell proliferation and IL-2, IFN-γ and TNF-α production in response to activation.

Conclusion: Our study showed that MM cell-derived IL-32γ induced IDO production in MΦs through PR3 and the downstream STAT3 and NF-κB pathways, resulting in the suppression of the proliferation and effector function of CD4⁺ T cells. High IL-32 expression in MM may contribute to an immunosuppressive microenvironment by upregulating IDO production in MΦs and promote MM progression.