IL-32 and multiple myeloma bone disease

Multiple myeloma is a malignancy of plasma cells characterized by multiple tumors in the bone marrow and progressive osteolytic bone disease. The bone lesions are caused by increased bone resorption by osteoclasts and decreased bone formation by osteoblasts. Recently, we found that myeloma cells obtained from bone marrow plasma had high copy number of IL-32, a novel interleukin that has not previously been related to multiple myeloma. Further investigation revealed that IL-32 was secreted by the myeloma cells bound to exosomes, indicating that the interleukin could influence the bone marrow microenvironment. Preliminary results from the group showed that rhIL-32 and even more potently, IL-32 in myeloma-derived exosomes, increased osteoclastogenesis, both in vitro and in vivo. Here, these findings were validated by treating primary monocytes with exosomes derived from IL-32 expressing- and IL-32 KO myeloma cells. Strikingly, number of osteoclasts in cultures was increased by treatment with IL-32-containing exosomes, while IL32 depleted exosomes did not increase osteoclastogenesis, which support that IL-32 may act as an inducer of osteoclast differentiation. The influence of IL-32 on osteoblasts was not previously studied, and therefore addressed in this thesis. We evaluated the effects of IL-32 on osteoblast differentiation from primary mesenchymal stromal/stem cells (MSCs). Our results indicate that IL-32 alone has no effect on osteoblast differentiation or proliferation. IL32 has previously been shown to upregulate CYP27B11, the enzyme that catalyze conversion of pro- to active vitamin D. We therefore hypothesized that IL-32 could influence osteoblasts by increasing CYP27B1 and thereby activate vitamin D-dependent signaling pathways in the cells. However, we found no clear evidence for effects of IL-32 in the presence of the pro form of vitamin D (25(OH)D3), evaluated by extent of various parameters of osteogenic differentiation. There was a tendency for increased biosynthesis of the active form of vitamin D, 1,25(OH)2D3 in response to IL-32, but an optimized protocol is needed to conclude on this matter. Furthermore, CYP27B1 mRNA was not upregulated in response to IL-32, but the mRNA detection levels were low, suggesting that CYP27B1 expression should be evaluated by other means. Finally, as our preliminary results showed that IL-32 is upregulated in myeloma cells in response to hypoxic conditions, we investigated the effects of hypoxia on MSCs. Interestingly, these cells were found to express IL-32, and the expression is increased by hypoxia and by stimulation with IL-32 itself, the latter suggesting an positive feedback loop between myeloma cells and MSCs in the bone marrow. Taken together, these findings contribute to an increased understanding of the role of IL-32 in multiple myeloma bone disease. However, more research is required to conclude on the role of IL-32 in osteoblasts and MSCs, as well as determining the cellular mechanisms behind IL-32 promoted osteoclastogenesis

sMETASeq: combined profiling of microbiota and host small RNAs

Understanding microbial communities' roles in human health and disease requires methods that accurately characterize the microbial composition and their activity and effects within human biological samples. We present sMETASeq (small RNA Metagenomics by Sequencing), a novel method that uses sequencing of small RNAs to jointly measure host small RNA expression and create metagenomic profiles and detect small bacterial RNAs. We evaluated the performance of sMETASeq on a mock bacterial community and demonstrated its use on different human samples, including colon cancer, oral leukoplakia, cervix cancer, and a panel of human biofluids. In all datasets, the detected microbes reflected the biology of the different sample types

Diet-induced obesity reduces bone marrow T and B cells and promotes tumor progression in a transplantable Vk*MYC model of multiple myeloma

Abstract Obesity is associated with an increased risk of developing multiple myeloma (MM). The molecular mechanisms causing this association is complex and incompletely understood. Whether obesity affects bone marrow immune cell composition in multiple myeloma is not characterized. Here, we examined the effect of diet-induced obesity on bone marrow immune cell composition and tumor growth in a Vk*MYC (Vk12653) transplant model of multiple myeloma. We find that diet-induced obesity promoted tumor growth in the bone marrow and spleen and reduced the relative number of T and B cells in the bone marrow. Our results suggest that obesity may reduce MM immune surveillance and thus may contribute to increased risk of developing MM

Intracellular IL-32 regulates mitochondrial metabolism, proliferation, and differentiation of malignant plasma cells

Interleukin-32 (IL-32) is a nonclassical cytokine expressed in cancers, inflammatory diseases, and infections. Its expression is regulated by two different oxygen sensing systems; HIF1α and cysteamine dioxygenase (ADO), indicating that IL-32 may be involved in the response to hypoxia. We here demonstrate that endogenously expressed, intracellular IL-32 interacts with components of the mitochondrial respiratory chain and promotes oxidative phosphorylation. Knocking out IL-32 in three myeloma cell lines reduced cell survival and proliferation in vitro and in vivo. High-throughput transcriptomic and MS-metabolomic profiling of IL-32 KO cells revealed that cells depleted of IL-32 had perturbations in metabolic pathways, with accumulation of lipids, pyruvate precursors, and citrate. IL-32 was expressed in a subgroup of myeloma patients with inferior survival, and primary myeloma cells expressing IL-32 had a gene signature associated with immaturity, proliferation, and oxidative phosphorylation. In conclusion, we demonstrate a previously unrecognized role of IL-32 in the regulation of plasma cell metabolism

Intracellular IL-32 regulates mitochondrial metabolism, proliferation, and differentiation of malignant plasma cells

Interleukin-32 (IL-32) is a nonclassical cytokine expressed in cancers, inflammatory diseases, and infections. Its expression is regulated by two different oxygen sensing systems; HIF1α and cysteamine dioxygenase (ADO), indicating that IL-32 may be involved in the response to hypoxia. We here demonstrate that endogenously expressed, intracellular IL-32 interacts with components of the mitochondrial respiratory chain and promotes oxidative phosphorylation. Knocking out IL-32 in three myeloma cell lines reduced cell survival and proliferation in vitro and in vivo. High-throughput transcriptomic and MS-metabolomic profiling of IL-32 KO cells revealed that cells depleted of IL-32 had perturbations in metabolic pathways, with accumulation of lipids, pyruvate precursors, and citrate. IL-32 was expressed in a subgroup of myeloma patients with inferior survival, and primary myeloma cells expressing IL-32 had a gene signature associated with immaturity, proliferation, and oxidative phosphorylation. In conclusion, we demonstrate a previously unrecognized role of IL-32 in the regulation of plasma cell metabolism

Hypoxia promotes IL-32 expression in myeloma cells, and high expression is associated with poor survival and bone loss

Multiple myeloma (MM) is a hematologic cancer characterized by expansion of malignant plasma cells in the bone marrow. Most patients develop an osteolytic bone disease, largely caused by increased osteoclastogenesis. The myeloma bone marrow is hypoxic, and hypoxia may contribute to MM disease progression, including bone loss. Here we identified interleukin-32 (IL-32) as a novel inflammatory cytokine expressed by a subset of primary MM cells and MM cell lines. We found that high IL-32 gene expression in plasma cells correlated with inferior survival in MM and that IL-32 gene expression was higher in patients with bone disease compared with those without. IL-32 was secreted from MM cells in extracellular vesicles (EVs), and those EVs, as well as recombinant human IL-32, promoted osteoclast differentiation both in vitro and in vivo. The osteoclast-promoting activity of the EVs was IL-32 dependent. Hypoxia increased plasma-cell IL-32 messenger RNA and protein levels in a hypoxia-inducible factor 1α–dependent manner, and high expression of IL-32 was associated with a hypoxic signature in patient samples, suggesting that hypoxia may promote expression of IL-32 in MM cells. Taken together, our results indicate that targeting IL-32 might be beneficial in the treatment of MM bone disease in a subset of patients