Versikine, a novel DAMP with immunoregulatory roles in the myeloma microenvironment. (A-B) Freshly explanted MAM from the human myeloma marrow cases indicated were exposed to 1 μM versikine (Vkine) for 12 hours. Relative expression of IL-1β and IL-6 transcripts is shown. Black bars, vehicle; gray bars, versikine-treated; NS, not significant; veh, vehicle. (C-D) Wild-type (WT) and Tpl2−/− BMDMs were treated with 1 μM versikine, and cytokine concentrations were measured in the culture supernatant at 12 hours postexposure. (C) Il-1β (left), Il-6 (right); before Il-1β assay, cells were treated with 5 mM ATP for 20 minutes. (D) Il-10 (left); Il-12p40 (right). (E) Versikine modulates macrophage polarization. BMDMs were exposed to vehicle, versikine alone, or versikine + OVA/anti-OVA immune complexes (IC), as previously described.17 Versikine exposure resulted in M1-like phenotype (Il-12hi, Il-10lo) in the absence of concurrent Fcγ ligation. Versikine + IC promoted macrophage polarization toward an M2b-like, immunoregulatory phenotype (Il-12lo, Il-10hi). (F) WT and Tlr2−/− BMDM were stimulated by versikine for 12 hours and Il-6 protein was measured in the supernatant. (G) Signaling mediators induced by versikine stimulation of WT and Tpl2−/− BMDMs. BMDMs were collected following stimulation with versikine at designed time-points (each number reflects minutes) and subjected to immunoblot analysis with the antibodies shown. (H) RNA-seq analysis of MM1.S myeloma cells exposed to versikine-transduced macrophages for 48 hours. Only 23 genes were differentially expressed and all were upregulated. Thirteen of 23 upregulated genes were ISGs (highlighted in gray). VCAN gene transcription changes are underlined. (I-J) Myeloma cell-macrophage cocultures were exposed to 0.5 μM purified versikine for 4, 18, or 48 hours. Representative ISG transcription is shown for THP-1 (I) and MM1.S cells (J). Relative mRNA transcription is normalized to vehicle-only control at each time point. *P < .05, **P < .01, ***P < .001, ****P < .0001.
