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Diet‐induced obesity is associated with enhanced systemic inflammation that limits bone regeneration. HDAC inhibitors are currently being explored as anti‐inflammatory agents. Prior reports show that myeloid progenitor‐directed Hdac3 ablation enhances intramembranous bone healing in female mice. In this study, we determined if Hdac3 ablation increased intramembranous bone regeneration in mice fed a high‐fat/high‐sugar (HFD) diet. Micro‐CT analyses demonstrated that HFD‐feeding enhanced the formation of periosteal reaction tissue of control littermates, reflective of suboptimal bone healing. We confirmed enhanced bone volume within the defect of Hdac3‐ablated females and showed that Hdac3 ablation reduced the amount of periosteal reaction tissue following HFD feeding. Osteoblasts cultured in a conditioned medium derived from Hdac3‐ablated cells exhibited a four‐fold increase in mineralization and enhanced osteogenic gene expression. We found that Hdac3 ablation elevated the secretion of several chemokines, including CCL2. We then confirmed that Hdac3 deficiency increased the expression of Ccl2. Lastly, we show that the proportion of CCL2‐positve cells within bone defects was significantly higher in Hdac3‐deficient mice and was further enhanced by HFD. Overall, our studies demonstrate that Hdac3 deletion enhances intramembranous bone healing in a setting of diet‐induced obesity, possibly through increased production of CCL2 by macrophages within the defect. [ABSTRACT FROM AUTHOR]

Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3+ MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the antiinflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-ß signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-ß neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-ß-dependent mechanisms. [ABSTRACT FROM AUTHOR]

Circadian regulation of gene expression is prevalent and plays critical roles in cell differentiation. However, its roles in the reprogramming of differentiated cells remain largely unknown. Here, we found that one of the master circadian regulators PER1 promoted virus-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to induced neurons (iNs) and induced pluripotent stem cells (iPSCs). Unexpectedly, PER1 achieved this by repressing inflammatory activation of contaminating macrophages in the MEF culture, rather than by directly modulating the reprogrammability of MEFs. More specifically, we found that transduced viruses activated inflammatory genes in macrophages, such as Tnf encoding TNFα, one of the central inflammatory regulators and an autocrine activator of macrophages. TNFα inhibited iN reprogramming, whereas a TNFα inhibitor promoted iN reprogramming, connecting the inflammatory responses to iN reprogramming. In addition, macrophages were induced to proliferate and mature by non-macrophage cells serving as feeders, which also supported up-regulation of TNFα in macrophages without virus transduction. Furthermore, the 2 inflammatory responses were repressed by the circadian regulator PER1 in macrophages, making reprogrammability dependent on time-of-day of virus transduction. Similar results were obtained with iPSC reprogramming, suggesting a wide occurrence of macrophage-mediated inhibition of cell reprogramming. This study uncovers mechanistic links between cell reprogramming, bystander inflammatory macrophages, and circadian rhythms, which are particularly relevant to in vivo reprogramming and organoid formation incorporating immune cells. Circadian regulation of gene expression plays critical roles in cell differentiation, but what about reprogramming of differentiated cells? This study shows that contaminating macrophages in fibroblast culture play unsuspected inhibitory roles during reprogramming to neurons and pluripotent stem cells, controlled by the circadian rhythm gene Per1. [ABSTRACT FROM AUTHOR]

Disruptions in the bone remodeling cycle that occur with increasing age lead to degeneration of the skeleton and increased risk of fragility fractures. Our understanding of how bone remodeling within cortical bone is controlled and altered with age in males and females is limited. Here, we generated bone marrow chimeric mice to understand the impacts of age and sex on bone remodeling. We demonstrate that transplantation of aged male or female bone marrow into young, lethally irradiated male hosts unexpectedly enhances cortical bone mass without impacting cancellous bone. Our single cell RNA-sequencing data show that mice reconstituted with aged bone marrow exhibited subsets of cells marked by CD11B/CD36 expression that demonstrate enhanced production of anabolic cytokines as compared to young counterparts, and that these myeloid subsets exist under conditions of normal physiology in aged mice. Importantly, CD11B+CD36+ cells do not differentiate into osteoclasts in vitro, and CD36 does not mark TRAP+ cells in vivo. Instead, CD36+ cells localize to resorption sites, including within cortical bone defects, suggesting their involvement in cortical bone remodeling and healing. CD11B+CD36+ cells also express elevated levels of bone anabolic WNT ligands, especially Wnt6. In functional assays, we demonstrate that soluble factors produced by CD11B+CD36+ cells enhance osteoblast progenitor commitment, mineralization, and activation of WNT signaling in vitro. Moreover, CD11B/CD36 exquisitely mark a subset of anabolic myeloid cells within human bone marrow. In conclusion, our studies identified a novel population of aged macrophages that limit cortical bone loss.