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While blood vessels play important roles in bone homeostasis and repair, fundamental aspects of vascular function in the skeletal system remain poorly understood. Here we show that the long bone vasculature generates a peculiar flow pattern, which is important for proper angiogenesis. Intravital imaging reveals that vessel growth in murine long bone involves the extension and anastomotic fusion of endothelial buds. Impaired blood flow leads to defective angiogenesis and osteogenesis, and downregulation of Notch signalling in endothelial cells. In aged mice, skeletal blood flow and endothelial Notch activity are also reduced leading to decreased angiogenesis and osteogenesis, which is reverted by genetic reactivation of Notch. Blood flow and angiogenesis in aged mice are also enhanced on administration of bisphosphonate, a class of drugs frequently used for the treatment of osteoporosis. We propose that blood flow and endothelial Notch signalling are key factors controlling ageing processes in the skeletal system.

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IntroductionCD4 ϩ T cells play a pivotal role in regulation of the immune response through induction of key cytokines and can differentiate into several subsets depending on the stimuli in their environment. These subsets are characterized by different cytokine profiles and distinct functions. Until recently, CD4 ϩ T cells were mostly divided into Th1, Th2, and Treg cells; however, a new subset of IL-17-secreting effector T cells, namely Th17 cells, has recently been characterized. 1,2 IL-17A, IL-17F, and IL-22 produced by Th17 cells play a critical role in inflammation, autoimmunity, and cancer. Their differentiation is critically dependent on the transforming growth factor-␤ (TGF-␤) and IL-6. 3 Both Th17 cells as well as induced regulatory T cells (iTregs) depend on TGF-␤ for their development/induction, whereas TGF-␤ together with retinoic acid (RA) is responsible for induction of Foxp3 Tregs from naive T cells in the periphery in the absence of proinflammatory signals. 4,5 Tregs play an important role in the control of inflammatory responses 6 as well as in the suppression of antitumor immune responses. [7][8][9] Several studies have suggested that the balance of IL-6 and RA in the presence of TGF-␤ determines development of iTregs or Th17 cells by competition between Foxp3 and ROR␥t. [10][11][12] It has been shown that all-trans retinoic acid (atRA) can regulate TGF-␤-dependent immune responses at least partially through inhibiting IL-6 induction of Th17 cells. [13][14][15][16][17] Previous studies have shown that induced regulatory T cells as well as Th17 cells are not at the final stage of their differentiation and have the potential to convert into different subsets of CD4 ϩ T cells depending on the cytokine environment. For example, regulatory T cells stimulated with IL-6 can express IL-17 and down-regulate Foxp3 expression. 18 Similarly, already developed Th17 cells can change into IFN-␥-producing Th1 cells or IL-4-producing Th2 cells when stimulated by Development of iTregs or Th17 cells in mice is shown to be associated with distinct cells of myeloid origin, which express RA and particular cytokines needed for control of these differentiation processes. Several studies have found that mouse intestinal dendritic cells (DCs) induce Th17 cells, whereas macrophages lead to Foxp3 ϩ T-cell generation. [22][23][24] In other studies, the CD103 ϩ DCs were identified as the cells leading to Treg development, whereas the CD103 Ϫ subset induced inflammatory Th17 cells. 14,22-25 However, no human counterpart has been described so far.Myeloid-derived suppressor cells (MDSCs) were originally described as a heterogeneous population of immature cells derived from myeloid progenitors with suppressive capacity in tumorbearing mice. [26][27][28] Human MDSCs are not so well characterized because of the lack of specific markers. We have recently shown a novel population of human MDSCs that are CD14 ϩ HLA-DR Ϫ/low , have arginase activity, and inhibit autologous T cells and NK cells. 29,30 These MDSCs convert naive CD4 ϩ ...

Background and aims Myeloid derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive activity. They accumulate in tumor-bearing mice and humans with different types of cancer, including hepatocellular carcinoma (HCC). The aim of this study was to examine the biology of MDSC in murine HCC models and to identify a model, which mimics the human disease. Methods: The comparative analysis of MDSC was performed in mice, bearing transplantable, diethylnitrosoamine (DEN)-induced and MYC-expressing HCC at different ages. Results: An accumulation of MDSC was found in mice with HCC irrespectively of the model tested. Transplantable tumors rapidly induced systemic recruitment of MDSC, in contrast to slow-growing DEN-induced or MYC-expressing HCC, where MDSC numbers only increased intra-hepatically in mice with advanced tumors. MDSC derived from mice with subcutaneous tumors were more suppressive than those from mice with DEN-induced HCC. Enhanced expression of genes associated with MDSC generation (GM-CSF, VEGF, IL-6, IL-1β) and migration (MCP-1, KC, S100A8, S100A9) was observed in mice with subcutaneous tumors. In contrast, only KC levels increased in mice with DEN-induced HCC. Both KC and GM-CSF over-expression or anti-KC and anti-GM-CSF treatment controlled MDSC frequency in mice with HCC. Finally, the frequency of MDSC decreased upon successful anti-tumor treatment with sorafenib. Conclusions: Our data indicate that MDSC accumulation is a late event during hepatocarcinogenesis and differs significantly depending on the tumor model studied.

A population of monocytes, known as Ly6Clo monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6Chi monocytes into Ly6Clo monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation.

Summary Myeloid‐derived suppressor cells (MDSC) are a heterogeneous population of cells that negatively regulate the immune response during tumour progression, inflammation and infection. Only limited data are available on human MDSC because of the lack of specific markers. We have identified members of the S100 protein family – S100A8, S100A9 and S100A12 – specifically expressed in CD14+ HLA‐DR−/low MDSC. S100A9 staining in combination with anti‐CD14 could be used to identify MDSC in whole blood from patients with colon cancer. An increase in the population of CD14+ S100A9high MDSC was observed in the peripheral blood from colon cancer patients in comparison with healthy controls. Finally, nitric oxide synthase expression, a hallmark of MDSC, was induced in CD14+ S100A9high upon lipopolysaccharide/interferon‐γ stimulation. We propose S100 proteins as useful markers for the analysis and further characterization of human MDSC.

Ischemia causes an inflammatory response that is intended to restore perfusion and homeostasis yet often aggravates damage. Here we show, using conditional genetic deletion strategies together with adoptive cell transfer experiments in a mouse model of hind limb ischemia, that blood vessels control macrophage differentiation and maturation from recruited monocytes via Notch signaling, which in turn promotes arteriogenesis and tissue repair. Macrophage maturation is controlled by Notch ligand Dll1 expressed in vascular endothelial cells of arteries and requires macrophage canonical Notch signaling via Rbpj, which simultaneously suppresses an inflammatory macrophage fate. Conversely, conditional mutant mice lacking Dll1 or Rbpj show proliferation and transient accumulation of inflammatory macrophages, which antagonizes arteriogenesis and tissue repair. Furthermore, the effects of Notch are sufficient to generate mature macrophages from monocytes ex vivo that display a stable anti-inflammatory phenotype when challenged with pro-inflammatory stimuli. Thus, angiocrine Notch signaling fosters macrophage maturation during ischemia.