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The ability of cells to sense and adapt to changes in oxygen is mediated by hypoxia-inducible factor (HIF). Immune cells function in physiologically complex and varying environments whereby oxygen, pH, nutrients, metabolites and cytokines are continuously fluctuating. HIF is well known to play an important role in coordinating the adaptation and function of both innate immune cells and T cells in these complex environments. This review summarises recent discoveries concerning how hypoxia and HIF control B cell behaviour, and regulate antibody quality and decisions concerning tolerance. Hypoxia and HIF activation may provide an important context; coordinating metabolism with variable demands for quiescence, rapid proliferation, and differentiation. Understanding when and how HIF is activated during B cell development and response is important as drugs targeting HIF could influence antibody responses, providing novel therapeutic opportunities for vaccine adjuvants and in treating autoimmunity.

Brexit and the troubled state of the NHS call for re-thinking the UK's approach to health. The EU referendum vote reveals deep social divisions as well as presenting the country with important decisions and negotiations about the future. At the same time, health problems are growing; the NHS faces severe financial constraints and appears to lurch from crisis to crisis, with leaving the European Union likely to exacerbate many problems including staffing issues across the whole sector. However, new scientific developments and digital technology offer societies everywhere massive and unprecedented opportunities for improving health. It is vital for the country that the NHS is able to adopt these discoveries and see them translated into improved patient care and population health, but also that the UK benefits from its capabilities and strengths in these areas.

B-lymphocyte development and selection is central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR)-signaling and occur in bone marrow, an environment with variable hypoxia, but whether Hypoxia-inducible factor (HIF) is involved is unknown. We show high HIF-activity in human bone marrow pro/preB cells that decreased at the immature B cell stage. Stage-specific HIF-suppression was required for B cell development, since constitutive HIF-1α-activation in murine B cells led to immature B cell developmental-arrest, limited BCR-editing and reduced repertoire-diversity. Disruption of HIF-1α-regulation reduced surface BCR and CD19 and increased expression of pro-apoptotic BIM. Transient administration of a clinical HIF-activator reduced immature and transitional B cells, confirming the on-going importance of this axis for the maintenance of the established B cell-compartment. Our work identifies a critical role for HIF-1α in tuning BCR-signal strength and demonstrates that dynamic HIF-regulation is essential for normal B cell development, with significant clinical implications.

Cell-based therapy is a promising strategy in regenerative medicine, but the poor survival rate of the implanted cells remains a major challenge and limits clinical translation. We preconditioned periosteal cells to the hypoxic and ischemic environment of the bone defect site by deleting prolyl hydroxylase domain-containing protein 2 (PHD2), resulting in hypoxia-inducible factor 1 alpha (HIF-1α) stabilization. This strategy increased postimplantation cell survival and improved bone regeneration. The enhanced cell viability was angiogenesis independent but relied on combined changes in glutamine and glycogen metabolism. HIF-1α stabilization stimulated glutaminase-mediated glutathione synthesis, maintaining redox homeostasis at baseline and during oxidative or nutrient stress. Simultaneously, HIF-1α signaling increased glycogen storage, preventing an energy deficit during nutrient or oxygen deprivation. Pharmacological inhibition of PHD2 recapitulated the adaptations in glutamine and glycogen metabolism and, consequently, the beneficial effects on cell survival. Thus, targeting cellular metabolism is an appealing strategy for bone regeneration and cell-based therapy in general. publisher: Elsevier articletitle: HIF-1α Promotes Glutamine-Mediated Redox Homeostasis and Glycogen-Dependent Bioenergetics to Support Postimplantation Bone Cell Survival journaltitle: Cell Metabolism articlelink: http://dx.doi.org/10.1016/j.cmet.2016.01.002 content_type: article copyright: Copyright © 2016 Elsevier Inc. All rights reserved. ispartof: Cell Metabolism vol:23 issue:2 pages:265-79 ispartof: location:United States status: published

Because retinal ischemia is a common cause of vision loss, we sought to determine the effects of ischemia on neuroretinal function and survival in murine oxygen-induced retinopathy (OIR) and to define the role of endogenous erythropoietin (EPO) in this model. OIR is a reproducible model of ischemia-induced retinal neovascularization; it is used commonly to develop antiangiogenic strategies. We investigated the effects of ischemia in murine OIR on retinal function and neurodegeneration by electroretinography and detailed morphology. OIR was associated with significant neuroretinal dysfunction, with reduced photopic and scotopic ERG responses and reduced b-wave/a-wave ratios consistent with specific inner-retinal dysfunction. OIR resulted in significantly increased apoptosis and atrophy of the inner retina in areas of ischemia. EPO deficiency in heterozygous Epo-Tag transgenic mice was associated with more profound retinal dysfunction after OIR, indicated by a significantly greater suppression of ERG amplitudes, but had no measurable effect on the extent of retinal ischemia, preretinal neovascularization, or neuroretinal degeneration in OIR. Systemic administration of recombinant EPO protected EPO-deficient mice against this additional suppression, but EPO supplementation in wild-type animals with OIR did not rescue neuroretinal dysfunction or degeneration. Murine OIR offers a valuable model of ischemic neuroretinal dysfunction and degeneration in which to investigate adaptive tissue responses and evaluate novel therapeutic approaches. Endogenous EPO can protect neuroretinal function in ischemic retinopathy.

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain enzymes. Its potential value and mechanism of actions in preventing/treating gastrointestinal injury are, however, poorly understood. We, therefore, examined the effect of DMOG on influencing gut injury and repair using a variety of in vitro and in vivo models. We performed in vitro studies utilising pro-migratory (wounded monolayer) and proliferation (using DNA quantitation) assays of human stomach (AGS) and colonic (HT29) carcinoma cells. Time course studies examined changes in hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF) levels, a growth factor known to be regulated via HIF. In vivo studies utilised a rat gastric (indomethacin, 20 mg/kg and 3 h restraint) damage model. DMOG stimulated migration in a dose-dependent manner, increasing migration twofold when added at 25μM (P

Complement factor H-related protein 5 (CFHR5) nephropathy is a familial renal disease endemic in Cyprus. It is characterized by persistent microscopic hematuria, synpharyngitic macroscopic hematuria and progressive renal impairment. Isolated glomerular accumulation of complement component 3 (C3) is typical with variable degrees of glomerular inflammation. Affected individuals have a heterozygous internal duplication in the CFHR5 gene, although the mechanism through which this mutation results in renal disease is not understood. Notably, the risk of progressive renal failure in this condition is higher in males than females. We report the first documented case of recurrence of CFHR5 nephropathy in a renal transplant in a 53-year-old Cypriot male. Strikingly, histological changes of CFHR5 nephropathy were evident in the donor kidney 46 days post-transplantation. This unique case demonstrates that renal-derived CFHR5 protein cannot prevent the development of CFHR5 nephropathy.

Background & Aims: Hypoxia inducible factor (HIF) prolyl hydroxylase inhibitors are protective in mouse models of inflammatory bowel disease (IBD). Here, we investigated the therapeutic target(s) and mechanism(s) involved. Methods: The effect of genetic deletion of individual HIF-prolyl hydroxylase (PHD) enzymes on the development of dextran sulphate sodium (DSS)–induced colitis was examined in mice. Results: PHD1−/−, but not PHD2+/− or PHD3−/−, mice were less susceptible to the development of colitis than wild-type controls as determined by weight loss, disease activity, colon histology, neutrophil infiltration, and cytokine expression. Reduced susceptibility of PHD1−/− mice to colitis was associated with increased density of colonic epithelial cells relative to wild-type controls, which was because of decreased levels of apoptosis that resulted in enhanced epithelial barrier function. Furthermore, with the use of cultured epithelial cells it was confirmed that hydroxylase inhibition reversed DSS-induced apo tosis and barrier dysfunction. Finally, PHD1 levels were increased with disease severity in intestinal tissue from patients with IBD and in colonic tissues from DSS-treated mice. Conclusions: These results imply a role for PHD1 as a positive regulator of intestinal epithelial cell apoptosis in the inflamed colon. Genetic loss of PHD1 is protective against colitis through decreased epithelial cell apoptosis and consequent enhancement of intestinal epithelial barrier function. Thus, targeted PHD1 inhibition may represent a new therapeutic approach in IBD. Deposited by bulk import

Oxygen is an essential nutrient for eukaryotic life and in complex organisms a finely tuned system, based on Hypoxia Inducible Factor (HIF), has evolved to control the cellular, local and organism-wide responses to its availability. Cells of the immune system must function in a variety of local environments and it is now clear that oxygen availability is an important signal for the cells involved in immune defence. This review summarises the mechanisms by which activation of the HIF pathway influences, and is influenced by, both immune activation state and oxygen tension. It is likely that understanding how oxygen tension modulates immune function will provide insights into disease pathogenesis, and may offer new opportunities for treatment.

Hepcidin is a critical inhibitor of iron export from macrophages, enterocytes, and hepatocytes. Given that it is filtered and degraded by the kidney, its elevated levels in renal failure have been suggested to play a role in the disordered iron metabolism of uremia, including erythropoietin resistance. Here, we used a novel radioimmunoassay for hepcidin-25, the active form of the hormone, to measure its levels in renal disease. There was a significant diurnal variation of hepcidin and a strong correlation to ferritin levels in normal volunteers. In 44 patients with mild to moderate kidney disease, hepcidin levels were significantly elevated, positively correlated with ferritin but inversely correlated with the estimated glomerular filtration rate. In 94 stable hemodialysis patients, hepcidin levels were also significantly elevated, but this did not correlate with interleukin-6 levels, suggesting that increased hepcidin was not due to a general inflammatory state. Elevated hepcidin was associated with anemia, but, intriguingly, the erythropoietin dose was negatively correlated with hepcidin, suggesting that erythropoietin suppresses hepcidin levels. This was confirmed in 7 patients when hepcidin levels significantly decreased after initiation of erythropoietin treatment. Our results show that hepcidin is elevated in renal disease and suggest that higher hepcidin levels do not predict increased erythropoietin requirements.

Inactivation of the von Hippel-Lindau (VHL) gene and activation of the hypoxia-inducible factor (HIF) in susceptible cells precedes formation of tumorlets and frank tumor in the epididymis of male VHL patients. We performed detailed histologic and molecular pathologic analysis of tumor-free epididymal tissues from VHL patients to obtain further insight into early epididymal tumorigenesis. Four epididymides from two VHL patients were serially sectioned to allow for three-dimensional visualization of morphologic changes. Areas of interest were genetically analyzed by tissue microdissection, immunohistochemistry for HIF and markers for mesonephric differentiation, and in situ hybridization for HIF downstream target vascular endothelial growth factor. Structural analysis of the epididymides revealed marked deviations from the regular anatomic structure resulting from impaired organogenesis. Selected efferent ductules were represented by disorganized mesonephric cells, and the maldeveloped mesonephric material was VHL-deficient by allelic deletion analysis. Furthermore, we observed maldeveloped mesonephric material near cystic structures, which were also VHL-deficient and were apparent derivatives of maldeveloped material. Finally, a subset of VHL-deficient cells was structurally integrated in regular efferent ductules; proliferation of intraductular VHL-deficient cells manifests itself as papillary growth into the ductular lumen. Furthermore, we clarify that that there is a pathogenetic continuum between microscopic tumorlets and formation of tumor. In multiple locations, three-dimensional reconstruction revealed papillary growth to extend deeply into ductular lumina, indicative of progression into early hamartoma-like neoplasia. We conclude epididymal tumorigenesis in VHL disease to occur in two distinct sequential steps: maldevelopment of VHL-deficient mesonephric cells, followed by neoplastic papillary proliferation.

The hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha are activated in hypoxic tumor regions. However, their role in tumorigenesis remains controversial, as tumor growth promoter and suppressor activities have been ascribed to HIF-1alpha, while the role of HIF-2alpha remains largely unknown. Here, we show that overexpression of HIF-2alpha in rat glioma tumors enhances angiogenesis but reduces growth of these tumors, in part by increasing tumor cell apoptosis. Moreover, siRNA knockdown of HIF-2alpha reduced apoptosis in hypoxic human malignant glioblastoma cells. Furthermore, inhibition of HIF by overexpression of a dominant-negative HIF transgene in glioma cells or HIF-2alpha deficiency in teratomas reduced vascularization but accelerated growth of these tumor types. These findings urge careful consideration of using HIF inhibitors as cancer therapeutic strategies.

A key aspect of vascular function is distributing oxygen reliably. Oxygen deficiency is a logical consequence of reducing blood flow, e.g. by an atheromatous plaque. Recently, a transcriptional system centred on the hypoxia-inducible factor-1 (HIF) and the von Hippel-Lindau (VHL) protein has emerged as a major regulator in oxygen homeostasis. HIF regulation in normoxia depends on VHL-mediated destruction. When the oxygen supply is reduced, the HIF pathway is activated. Together with activation of HIF in normoxia, VHL-deficient cells show a striking defect in extracellular matrix assembly, raising the question as to whether HIF underlies this. If so, altered oxygen tension in pathological settings could have major effects on matrix behaviour.

Mutations in the von Hippel-Lindau (VHL) gene are associated with hereditary and sporadic clear cell renal carcinoma. VHL acts in a ubiquitin ligase complex regulating hypoxia-inducible factor-1 (HIF-1), but the link between this function and cancer development is unclear. Here we show that in the kidneys of patients with VHL disease, HIF activation is an early event occurring in morphologically normal single cells within the renal tubules. In comparison, dysplastic lesions, cystic lesions, and tumors showed evidence of additional mechanisms that amplify HIF activation. Detection of cells with constitutive HIF activation identified a large number of previously unrecognized foci of VHL inactivation. In proximal tubules these were almost entirely unicellular, whereas multicellular foci were almost exclusively seen in the distal nephron.

Deprivation of oxygen (hypoxia) and/or glucose (hypoglycemia) represents a serious stress that affects cellular survival. The hypoxia-inducible transcription factor-1alpha (HIF-1alpha), which has been implicated in the cellular response to hypoxia (Semenza, G. L. (1999) Annu. Rev. Cell Dev. Biol. 15, 551-578), mediates apoptosis during hypoxia (Halterman, M. W., Miller, C. C., and Federoff, H. J. (1999) J. Neurosci. 19, 6818-6824 and Carmeliet, P., Dor, Y., Herbert, J. M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C. J., Ratcliffe, P., Moons, L., Jain, R. K., Collen, D., and Keshet, E. (1998) Nature 394, 485-490), but the function of its homologue HIF-2alpha remains unknown. Therefore, the role of HIF-2alpha in cellular survival was studied by targeted inactivation of the HIF-2alpha gene (HIF-2alpha(-/-)) in murine embryonic stem (ES) cells. In contrast to HIF-1alpha deficiency, loss of HIF-2alpha did not protect ES cells against apoptosis during hypoxia. Both HIF-1alpha(-/-) and HIF-2alpha(-/-) ES cells were, however, resistant to apoptosis in response to hypoglycemia. When co-cultured with wild type ES cells, HIF-2alpha(-/-) ES cells became rapidly and progressively enriched in hypoglycemia but not in hypoxia. Thus, HIF-1alpha and HIF-2alpha may have distinct roles in responses to environmental stress, and despite its name, HIF-2alpha may be more important in the survival response to environmental variables other than the level of oxygen.

Hypoxia-inducible expression has been demonstrated for many groups of mammalian genes, and studies of transcriptional control have revealed the existence of hypoxia-responsive elements (HREs) in the cis-acting sequences of several of these genes. These sequences generally contain one or more binding sites for a heterodimeric DNA binding complex termed hypoxia-inducible factor-1 (HIF-1). To analyze this response further, Chinese hamster ovary cells were stably transfected with plasmids bearing HREs linked to genes encoding immunoselectable cell surface markers, and clones that showed reduced or absent hypoxia-inducible marker expression were selected from a mutagenized culture of cells. Analysis of these cells revealed several clones with transacting defects in HRE activation, and in one the defect was identified as a failure to express the α-subunit of HIF-1. Comparison of hypoxia-inducible gene expression in wild type, HIF-1α-defective, and HIF-1α-complemented cells revealed two types of response. For some genes (e.g. glucose transporter-1), hypoxia-inducible expression was critically dependent on HIF-1α, whereas for other genes (e.g. heme oxygenase-1) hypoxia-inducible expression appeared largely independent of the expression of HIF-1α. These experiments show the utility of mutagenesis and selection of mutant cells in the analysis of mammalian transcriptional responses to hypoxia and demonstrate the operation of HIF-1α-dependent and HIF-1α-independent pathways of hypoxia-inducible gene expression in Chinese hamster ovary cells.

Efficient energy metabolism is a fundamental biochemical requirement which is approached in different ways by different organisms [reviewed in 1]. Many bacteria, for instance, are able to live in diverse chemical environments. In hypoxia they can regulate gene expression to provide enzymatic systems which utilize a variety of alternate electron acceptors such as nitrate, or fumarate instead of the more energetically efficient oxygen. In higher animals alternate electron acceptors are not used in this way and some form of aerobic environment is obligatory. A variety of specialized systems (lungs, heart, vessels and blood) have evolved to provide oxygen in optimal concentrations for cellular respiration. Since oxygen in excess is potentially toxic, the matching of oxygen supply with cellular metabolism must be precise. Comparative anatomists and physiologists have demonstrated many examples of this precision. For instance, among muscles from different species and different parts of the body, there is a striking concordance of measures of capillary and mitochondrial volume density, implying a close coordination of oxygen supply and demand [21. Despite the detail of this type of organization, which is seen in the gross anatomy of species with different athletic specialization, in metabolic zonation within organs such as liver and kidney [3], and in the disposition of organelles within cells, physiological texts dealing with oxygen sensing have tended to stress the importance of chemoreceptors with control over major functions such as ventilatory rate, cardiac output, or red cell production. Relatively little consideration has been given to the implications of the detailed organization of oxygen supply and demand at the microscopic level for cellular oxygen sensing. It now seems likely that recent advances arising from the study of one of the major systemic regulators, erythropoietin production, may also shed light on the transcriptional processes underlying these local responses to oxygenation.

SummaryMutations in VHL, RET, NF1, SDHB, SDHC, and SDHD can give rise to pheochromocytoma/paraganglioma. These different genetic lesions may all act by decreasing the activity of a 2-oxoglutarate-dependent oxygenase, SM-20/EglN3/PHD3, resulting in reduced apoptosis of neural crest cells during development.

As Morelle et al.1 point out, one might expect to see a reduction in plasma hepcidin levels post dialysis—on size considerations alone, perhaps a 20–30% reduction would be anticipated, but in the six patients studied in our report,2 no reduction was observed. These patients were dialyzed for 4 h using 2.1 m2 low-flux acetate membranes with a minimum ultrafiltrate volume of 2 l, delivering a dialysis dose (spKt/V) of over 1.5 in all patients.