ABSTRACT
As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. We identify beta-2-microglobulin (ß2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed ß2m promotes ß2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1ß (IL-1ß) and IL-18. This process depends on activation of the NLRP3 inflammasome after ß2m accumulation, as macrophages from NLRP3-deficient mice lack efficient ß2m-induced IL-1ß production. Moreover, depletion or silencing of ß2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by ß2m-induced inflammasome signaling. Our results provide mechanistic evidence for ß2m's role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM.
Subject(s)
Amyloid/metabolism , Multiple Myeloma/pathology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Tumor-Associated Macrophages/metabolism , beta 2-Microglobulin/metabolism , Animals , Cells, Cultured , Humans , Inflammation/immunology , Interleukin-18/metabolism , Interleukin-1beta/metabolism , Lysosomes/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Phagocytosis/immunology , Signal Transduction/immunology , Tumor Microenvironment/immunology , Tumor-Associated Macrophages/immunology , beta 2-Microglobulin/geneticsABSTRACT
The bone marrow niche has a pivotal role in progression, survival, and drug resistance of multiple myeloma cells. Therefore, it is important to develop means for targeting the multiple myeloma bone marrow microenvironment. Myeloma-associated macrophages (MAM) in the bone marrow niche are M2 like. They provide nurturing signals to multiple myeloma cells and promote immune escape. Reprogramming M2-like macrophages toward a tumoricidal M1 phenotype represents an intriguing therapeutic strategy. This is especially interesting in view of the successful use of mAbs against multiple myeloma cells, as these therapies hold the potential to trigger macrophage-mediated phagocytosis and cytotoxicity. In this study, we observed that MAMs derived from patients treated with the immunomodulatory drug (IMiD) lenalidomide skewed phenotypically and functionally toward an M1 phenotype. Lenalidomide is known to exert its beneficial effects by modulating the CRBN-CRL4 E3 ligase to ubiquitinate and degrade the transcription factor IKAROS family zinc finger 1 (IKZF1). In M2-like MAMs, we observed enhanced IKZF1 levels that vanished through treatment with lenalidomide, yielding MAMs with a bioenergetic profile, T-cell stimulatory properties, and loss of tumor-promoting capabilities that resemble M1 cells. We also provide evidence that IMiDs interfere epigenetically, via degradation of IKZF1, with IFN regulatory factors 4 and 5, which in turn alters the balance of M1/M2 polarization. We validated our observations in vivo using the CrbnI391V mouse model that recapitulates the IMiD-triggered IKZF1 degradation. These data show a role for IKZF1 in macrophage polarization and can provide explanations for the clinical benefits observed when combining IMiDs with therapeutic antibodies.See related Spotlight on p. 254.
Subject(s)
Ikaros Transcription Factor/metabolism , Lenalidomide/pharmacology , Multiple Myeloma/immunology , Tumor Microenvironment/immunology , Tumor-Associated Macrophages/immunology , Adult , Aged , Aged, 80 and over , Animals , Bone Marrow/pathology , Cell Line, Tumor , Disease Models, Animal , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/immunology , Gene Knock-In Techniques , Humans , Ikaros Transcription Factor/antagonists & inhibitors , Interferon Regulatory Factors/metabolism , Lenalidomide/therapeutic use , Male , Mice , Mice, Transgenic , Middle Aged , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Primary Cell Culture , Proteolysis/drug effects , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunology , Tumor Escape/drug effects , Tumor Escape/immunology , Tumor Microenvironment/drug effects , Tumor-Associated Macrophages/drug effects , Tumor-Associated Macrophages/metabolism , Ubiquitination/drug effects , Young AdultABSTRACT
PURPOSE: To evaluate the effect of several culture variables on clonal growth and differentiation of limbal stem cells ex vivo and provide an improved culture technique that supports preferential expansion and preservation of stem cells for therapeutic applications. METHODS: Corneal epithelial stem cells were isolated from human limbal specimens and clonally expanded on a 3T3 feeder layer, followed by subcultivation of holoclones on fibrin gels. The effect of different limbal regions, enzymatic dissociation methods, and culture media supplemented with different calcium, serum, and growth factor concentrations on colony-forming efficiency, colony size, and colony density was compared. A panel of putative stem cell and differentiation markers was used to analyze the epithelial phenotype by morphologic and immunohistochemical methods. RESULTS: Limbal cells obtained from the superior limbus, isolated by a two-step enzymatic dissociation method (dispase II/trypsin-EDTA), and cultured in low to medium (0.03-0.4 mM) calcium concentrations with proper serum levels (10% FCS) and growth factor combinations (EGF, NGF) yielded the highest clonal growth capacity and an undifferentiated cellular phenotype. Subcultivation of holoclones supported the preservation of stem and progenitor cells in the basal layer of the fibrin-based epithelial sheets, as demonstrated by multiple molecular stem cell markers (p63alpha, Bmi-1, K15, and ABCG2), whereas increased calcium concentrations and air-lifting induced terminal differentiation and gradual loss of stem cells. CONCLUSIONS: The proposed culture system supports enrichment and survival of limbal stem and progenitor cells during the entire cultivation process and may be essential for long-term restoration of the damaged ocular surface.