Macrophages activated by the gram negative bacterial product lipopolysaccharide (LPS) switch their core metabolism from oxidative phosphorylation to glycolysis1. Inhibition of glycolysis with 2-deoxyglucose (2DG) suppressed LPS-induced Interleukin-1 beta (IL-1β) but not Tumour necrosis factor alpha (TNFα) in macrophages. A comprehensive metabolic map of LPS-activated macrophages revealed up-regulation of glycolytic and down-regulation of mitochondrial genes, which correlated directly with the expression profiles of altered metabolites. LPS strongly increased the TCA cycle intermediate succinate. Glutamine-dependent anerplerosis was the major source of succinate with the ‘Gamma-Aminobutyric Acid (GABA)-shunt’ pathway also playing a role. LPS-induced succinate stabilized Hypoxia-inducible factor 1α (HIF-1α), an effect inhibited by 2DG, with IL-1β as an important target. LPS also increases succinylation of several proteins. Succinate is therefore identified as a metabolite in innate immune signalling which leads to enhanced IL-1β production during inflammation.

In multiple myeloma (MM) osteolytic lesions rarely heal because of persistent suppressed osteoblast differentiation resulting in a high fracture risk. Herein, chromatin immunoprecipitation analyses reveal that MM cells induce repressive epigenetic histone changes at the Runx2 locus that prevent osteoblast differentiation. The most pronounced MM-induced changes were at the Runx2-P1 promoter, converting it from a poised bivalent state to a repressed state. Previously it was observed that MM induce the transcription repressor GFI1 in osteoblast precursors, which correlates with decreased Runx2 expression. Thus, prompting detailed characterization of the MM and TNFα-dependent GFI1-response element within the Runx2-P1 promoter. Further analyses reveal that MM-induced GFI1 binding to Runx2 in osteoblast precursors and recruitment of the histone modifiers HDAC1, LSD1, and EZH2 is required to establish and maintain Runx2 repression in osteogenic conditions. These GFI1-mediated repressive chromatin changes persist even after removal of MM. Ectopic GFI1 is sufficient to bind to Runx2, recruit HDAC1 and EZH2, increase H3K27me3 on the gene, and prevent osteogenic induction of endogenous Runx2 expression. Gfi1 knockdown in MC4 cells blocked MM-induced recruitment of HDAC1 and EZH2 to Runx2, acquisition of repressive chromatin architecture, and suppression of OB differentiation. Importantly, inhibition of EZH2 or HDAC1 activity in pre-osteoblasts after MM exposure in vitro or in osteoblast precursors from MM patients reversed the repressive chromatin architecture at Runx2 and rescued osteoblast differentiation. Implications This study suggests that therapeutically targeting EZH2 or HDAC1 activity may reverse the profound MM-induced osteoblast suppression and allow repair of the lytic lesions.

Interleukin-1β and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPβ and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators.

Extracellular vesicles (EVs) are characterized by complex cargo composition and carry a wide array of signalling cargo, including growth factors (GFs). Beyond surface‐associated GFs, it is unclear if EV intralumenal growth factors are biologically active. Here, bone morphogenetic protein‐2 (BMP2), loaded directly into the lumen of EVs designated engineered BMP2‐EVs (eBMP2‐EVs), was comprehensively characterized including its regulation of osteoblastogenesis. eBMP2‐EVs and non‐EV ‘free’ BMP2 were observed to similarly regulate osteoblastogenesis. Furthermore, cell trafficking experiments suggest rapid BMP2 recycling and its extracellular release as ‘free’ BMP2 and natural occurring BMP2‐EVs (nBMP2‐EVs), with both being osteogenic. Interestingly, BMP2 occurs on the EV surface of nBMP2‐EVs and is susceptible to proteolysis, inhibition by noggin and complete dissociation from nBMP2‐EVs over 3 days. Whereas, within the eBMP2‐EVs, BMP2 is protected from proteolysis, inhibition by noggin and is retained in EV lumen at 100% for the first 24 h and ∼80% after 10 days. Similar to ‘free’ BMP2, bioprinted eBMP2‐EV microenvironments induced osteogenesis in vitro and in vivo in spatial registration to the printed patterns. Taken together, BMP2 signalling involves dynamic BMP2 cell trafficking in and out of the cell involving EVs, with distinct differences between these nBMP2‐EVs and eBMP2‐EVs attributable to the BMP2 cargo location with EVs. Lastly, eBMP2‐EVs appear to deliver BMP2 directly into the cytoplasm, initiating BMP2 signalling within the cell, bypassing its cell surface receptors.

Multiple myeloma (MM) is the most frequent cancer to involve the skeleton with patients developing osteolytic bone lesions due to hyperactivation of osteoclasts and suppression of BMSCs differentiation into functional osteoblasts. Although new therapies for MM have greatly improved survival, MM remains incurable for most patients. Despite the major advances in current anti-MM and anti-resorptive treatments that can significantly improve osteolytic bone lysis, many bone lesions can persist even after therapeutic remission of active disease. Bone marrow mesenchymal stem cells (BMSCs) from MM patients are phenotypically distinct from their healthy counterparts and the mechanisms associated with the long-term osteogenic suppression are largely unknown. In this review we will highlight recent results of transcriptomic profiling studies that provide new insights into the establishment and maintenance of the persistent pathological alterations in MM-BMSCs that occur in MM. We will we discuss the role of genomic instabilities and senescence in propagating the chronically suppressed state and pro-inflammatory phenotype associated with MM-BMSCs. Lastly we describe the role of epigenetic-based mechanisms in regulating osteogenic gene expression to establish and maintain the pro-longed suppression of MM-BMSC differentiation into functional OBs.