Although proinflammatory cytokines are key mediators of tissue damage during graft-versus-host disease (GVHD), IFN␥ has previously been attributed with both protective and pathogenic effects. We have resolved this paradox by using wildtype (wt), IFN␥ ؊/؊ , and IFN␥R ؊/؊ mice as donors or recipients in well-described models of allogeneic stem cell transplantation (SCT). We show that donor-derived IFN␥ augments acute GVHD via direct effects on (1) the donor T cell to promote T helper 1 (Th1) differentiation and (2) the gastrointestinal (GI) tract to augment inflammatory cytokine generation. However, these detrimental effects are overwhelmed by a protective role of IFN␥ in preventing the development of idiopathic pneumonia syndrome (IPS). This is the result of direct effects on pulmonary parenchyma to prevent donor cell migration and expansion within the lung. Thus, IFN␥ is the key cytokine differentially controlling the development of IPS and gastrointestinal GVHD after allogeneic SCT. IntroductionAllogeneic bone marrow transplantation (BMT) is a definitive curative therapy for most hematologic malignancies and severe immunodeficiencies. The major complication of allogeneic BMT remains graft-versus-host disease (GVHD) in which the skin, gastrointestinal (GI) tract, liver, and lung are preferentially damaged by the transplanted donor immune system. 1 GVHD occurs in most (50%-70%) recipients and is largely responsible for the high mortality associated with allogeneic BMT. Idiopathic pneumonia syndrome (IPS) is an acute noninfectious lung injury that typically occurs 3 to 4 weeks after BMT, responds poorly to therapy, and is associated with a high mortality. 2 There is thus a pressing need for new treatment approaches to both prevent and treat the full spectrum of GVHD, based on a logical understanding of the underlying disease pathophysiology.Current paradigms suggest that GVHD occurs via a complex cellular network initiated by the interaction of antigen-presenting cells (APCs) and naive donor T cells. [3][4][5] Subsequent T helper 1 (Th1) differentiation leads to the generation of donor cytotoxic T lymphocytes (CTLs) and large amounts of inflammatory cytokines that damage host tissue by both major histocompatibility complex (MHC)-dependent and -independent pathways. 6 Of the Th1 cytokines, IFN␥ is perhaps the most immunologically dominant, influencing a plethora of cell subsets during allograft rejection. 7 However the effects of this cytokine on GVHD are unclear, with a number of contradictory studies [8][9][10][11] suggesting that a clearer understanding of the mechanisms involved are needed. We have re-examined this issue using both IFN␥ Ϫ/Ϫ and IFN␥R Ϫ/Ϫ stem cell transplantation (SCT) donors or recipients following myeloablative conditioning. We demonstrate that donor-derived IFN␥ indeed has both positive and negative effects on GVHD due to differential effects on donor and host tissue, and individual target organs. First, IFN␥ augments acute GVHD via direct affects on the donor T cell to promote Th1 differen...

Despite the essential role of the lymphatic vasculature in tissue homeostasis and disease, knowledge of the organ-specific origins of lymphatic endothelial progenitor cells remains limited. The assumption that most murine embryonic lymphatic endothelial cells (LECs) are venous derived has recently been challenged. Here, we show that the embryonic dermal blood capillary plexus constitutes an additional, local source of LECs that contributes to the formation of the dermal lymphatic vascular network. We describe a novel mechanism whereby rare PROX1-positive endothelial cells exit the capillary plexus in a -dependent manner to establish discrete LEC clusters. As development proceeds, these clusters expand and further contribute to the growing lymphatic system. Lineage tracing and analyses of-deficient mice confirmed that these clusters are endothelial in origin. Furthermore, ectopic expression of in the vasculature increased the number of PROX1-positive progenitors within the capillary bed. Our work reveals a novel source of lymphatic endothelial progenitors employed during construction of the dermal lymphatic vasculature and demonstrates that the blood vasculature is likely to remain an ongoing source of LECs during organogenesis, raising the question of whether a similar mechanism operates during pathological lymphangiogenesis.

Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by well-studied signaling and transcriptional mechanisms. Less well understood is the ongoing elaboration of vessels to form a network. This involves cell polarisation, coordinated migration, adhesion, mixing, regression and cell shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. We found a mutation in polycystic kidney disease 1a to be responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial sprouting of lymphatic precursors is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice also has no effect on sprouting of precursors but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development.

During embryogenesis, lymphatic endothelial progenitor cells first arise from a subset of blood vascular endothelial cells in the dorsolateral aspects of the cardinal veins. The molecular cues responsible for defining the regionalisation of such a discrete pool of progenitors remain uncharacterised. Here we identify a novel function for CYP26B1, an enzyme known to play a role in tissue morphogenesis by fine-tuning retinoic acid (RA) concentration, in regulating lymphangiogenesis. Cyp26b1-null mice, in which RA levels are elevated, exhibited an increased number of lymphatic endothelial progenitor cells in the cardinal veins, together with hyperplastic, blood filled lymph sacs and hyperplastic dermal lymphatic vessels. Conversely, mice over-expressing Cyp26b1 had hypoplastic lymph sacs and lymphatic vessels. Our data suggest that RA clearance by CYP26B1 in the vicinity of lymphatic endothelial progenitor cells is important for determining the position and size of the progenitor pool specified. Our studies identify a genetic pathway that underpins the architecture of the developing lymphatics and define CYP26B1 as a novel modulator of lymphatic vascular patterning.

Key Points• Haploinsufficiency of Sox18 reveals an important role for VEGFD in regulating blood vascular development in vivo in vertebrates.• VEGFD acts through mitogenactivated protein kinase kinaseextracellular signal-regulated kinase to modulate the activity and nuclear concentration of endothelial-specific transcription factor SOX18.Vascular endothelial growth factor-D (VEGFD) is a potent pro-lymphangiogenic molecule during tumor growth and is considered a key therapeutic target to modulate metastasis. Despite roles in pathological neo-lymphangiogenesis, the characterization of an endogenous role for VEGFD in vascular development has remained elusive. Here, we used zebrafish to assay for genetic interactions between the Vegf/Vegf-receptor pathway and SoxF transcription factors and identified a specific interaction between Vegfd and Sox18. Double knockdown zebrafish embryos for Sox18/Vegfd and Sox7/Vegfd exhibit defects in arteriovenous differentiation. Supporting this observation, we found that Sox18/Vegfd double but not single knockout mice displayed dramatic vascular development defects. We find that VEGFD-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase signaling modulates SOX18-mediated transcription, functioning at least in part by enhancing nuclear concentration and transcriptional activity in vascular endothelial cells. This work suggests that VEGFD-mediated pathologies include or involve an underlying dysregulation of SOXF-mediated transcriptional networks. (Blood. 2014;123(7):1102-1112)