Monoclonal immunoglobulins promote bone loss in multiple myeloma.

Most patients with multiple myeloma develop a severe osteolytic bone disease. The myeloma cells secrete immunoglobulins, and the presence of monoclonal immunoglobulins in the patient's sera is an important diagnostic criterion. Here, we show that immunoglobulins isolated from myeloma patients with bone disease promote osteoclast differentiation when added to human preosteoclasts in vitro, whereas immunoglobulins from patients without bone disease do not. This effect was primarily mediated by immune complexes or aggregates. The function and aggregation behavior of immunoglobulins are partly determined by differential glycosylation of the immunoglobulin-Fc part. Glycosylation analyses revealed that patients with bone disease had significantly less galactose on immunoglobulin G (IgG) compared with patients without bone disease and also less sialic acid on IgG compared with healthy persons. Importantly, we also observed a significant reduction of IgG sialylation in serum of patients upon onset of bone disease. In the 5TGM1 mouse myeloma model, we found decreased numbers of lesions and decreased CTX-1 levels, a marker for osteoclast activity, in mice treated with a sialic acid precursor, N-acetylmannosamine (ManNAc). ManNAc treatment increased IgG-Fc sialylation in the mice. Our data support that deglycosylated immunoglobulins promote bone loss in multiple myeloma and that altering IgG glycosylation may be a therapeutic strategy to reduce bone loss.

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.

TLR8 and complement C5 induce cytokine release and thrombin activation in human whole blood challenged with Gram-positive bacteria.

We recently showed that TLR8 is critical for the detection of Gram-positive bacteria by human monocytes. Here, we hypothesized that TLR8 and complement together regulate antibacterial responses in human blood. Anticoagulated blood was treated with selective inhibitors of TLR8 and/or complement C5, and then challenged with live Streptococcus agalactiae (Group B streptococcus, GBS), Staphylococcus aureus, or Escherichia coli. Cytokine production, plasma membrane permeability, bacterial survival, phagocytosis, and activation of coagulation was examined. GBS and S. aureus, but not E. coli, triggered TLR8-dependent production of IL-12p70, IL-1ß, TNF, and IL-6 in fresh human whole blood. In purified polymorphonuclear neutrophils (PMN), GBS and S. aureus induced IL-8 release in part via TLR8, whereas PMN plasma membrane leakage and extracellular DNA levels increased independently of TLR8. TLR8 was more important than C5 for bacteria-induced production of IL-12p70, IL-1ß, and TNF in blood, whereas IL-8 release was more C5 dependent. Both TLR8 and C5 induced IL-6 release and activation of prothrombin cleavage, and here their combined effects were additive. Blocking of C5 or C5aR1 attenuated phagocytosis and increased the extracellular growth of GBS in blood, whereas TLR8 inhibition neither reduced phagocytosis nor intracellular killing of GBS and S. aureus. In conclusion, TLR8 is more important than C5 for production of IL-12p70, IL-1ß, and TNF upon GBS and S. aureus infection in blood, whereas C5 is central for IL-8 release and phagocytosis. Both TLR8 and C5 mediate IL-6 release and activation of coagulation during challenge with Gram-positive bacteria in blood.

Human Toll-like Receptor 8 (TLR8) Is an Important Sensor of Pyogenic Bacteria, and Is Attenuated by Cell Surface TLR Signaling.

TLR8 is an endosomal sensor of RNA degradation products in human phagocytes, and is involved in the recognition of viral and bacterial pathogens. We previously showed that in human primary monocytes and monocyte derived macrophages, TLR8 senses entire Staphylococcus aureus and Streptococcus agalactiae (group B streptococcus, GBS), resulting in the activation of IRF5 and production of IFNß, IL-12p70, and TNF. However, the quantitative and qualitative impact of TLR8 for the sensing of bacteria have remained unclear because selective inhibitors have been unavailable. Moreover, while we have shown that TLR2 activation attenuates TLR8-IRF5 signaling, the molecular mechanism of this crosstalk is unknown. We here used a recently developed chemical antagonist of TLR8 to determine its role in human primary monocytes challenged with S. aureus, GBS, Streptococcus pneumonia, Pseudomonas aeruginosa, and E. coli. The inhibitor completely blocked cytokine production in monocytes stimulated with TLR8-agonists, but not TLR2-, and TLR4-agonists. Upon challenge with S. aureus, GBS, and S. pneumonia, the TLR8 inhibitor almost eliminated the production of IL-1ß and IL-12p70, and it strongly reduced the release of IL-6, TNF, and IL-10. With P. aeruginosa infection, the TLR8 inhibitor impaired the production of IL-12p70 and IL-1ß, while with E. coli infection the inhibitor had less effect that varied depending on the strain and conditions. Signaling via TLR2, TLR4, or TLR5, but not TLR8, rapidly eliminated IRAK-1 detection by immunoblotting due to IRAK-1 modifications during activation. Silencing of IRAK-1 reduced the induction of IFNß and TNF by TLR8 activation, suggesting that IRAK-1 is required for TLR8-IRF5 signaling. The TLR-induced modifications of IRAK-1 also correlated closely with attenuation of TLR8-IRF5 activation, suggesting that sequestration and/or modification of Myddosome components by cell surface TLRs limit the function of TLR8. Accordingly, inhibition of CD14- and TLR4-activation during E. coli challenge increased the activation of IRF5 and the production of IL-1ß and IL-12p70. We conclude that TLR8 is a dominating sensor of several species of pyogenic bacteria in human monocytes, while some bacteria attenuate TLR8-signaling via cell surface TLR- activation. Taken together, TLR8 appears as a more important sensor in the antibacterial defense system than previously known.

Caspase-8 regulates the expression of pro- and anti-inflammatory cytokines in human bone marrow-derived mesenchymal stromal cells.

INTRODUCTION: Mesenchymal stem cells, also called mesenchymal stromal cells, MSCs, have great potential in stem cell therapy partly due to their immunosuppressive properties. How these cells respond to chronic inflammatory stimuli is therefore of importance. Toll-like receptors (TLR)s are innate immune receptors that mediate inflammatory signals in response to infection, stress, and damage. Caspase-8 is involved in activation of NF-kB downstream of TLRs in immune cells. Here we investigated the role of caspase-8 in regulating TLR-induced cytokine production from human bone marrow-derived mesenchymal stromal cells (hBMSCs). METHODS: Cytokine expression in hBMCs in response to poly(I:C) and LPS was evaluated by PCR, multiplex cytokine assay, and ELISA. TLR3, TRIF, and caspase-8 were silenced using siRNA. Caspase-8 was also inhibited using a caspase-8 inhibitor, z-IEDT. RESULTS: We found that TLR3 agonist poly(I:C) and TLR4 agonist LPS induced secretion of several pro-inflammatory cytokines in a TLR-dependent manner which required the TLR signaling adaptor molecule TRIF. Further, poly(I:C) reduced the expression of anti-inflammatory cytokines HGF and TGFß whereas LPS reduced HGF expression only. Notably, caspase-8 was involved in the induction of IL- IL-1ß, IL-6, CXCL10, and in the inhibition of HGF and TGFß. CONCLUSION: Caspase-8 appears to modulate hBMSCs into gaining a pro-inflammatory phenotype. Therefore, inhibiting caspase-8 in hBMSCs might promote an immunosuppressive phenotype which could be useful in clinical applications to treat inflammatory disorders.

The proportion of CD16(+)CD14(dim) monocytes increases with tumor cell load in bone marrow of patients with multiple myeloma.

Multiple myeloma is an incurable cancer with expansion of malignant plasma cells in the bone marrow. Previous studies have shown that monocytes and macrophages in the bone marrow milieu are important for tumor growth and may play a role in the drug response. We therefore characterized monocytes in bone marrow aspirates by flow cytometry. We found that there was significant correlation between the proportion of CX3CR1 (+), CD16(+)CD14(dim) non classical monocytes, and percent plasma cells (PC) in the bone marrow of myeloma patients. The bone marrow monocytes could be stimulated by TLR ligands to produce cytokines which promote myeloma cell growth. The proportion of the non-classical monocytes increased with the tumor load, particularly in patients with tumor loads in the range of 10-30% bone marrow PC.