Mechanisms of intermittent hypoxia-mediated macrophage activation - potential therapeutic targets for obstructive sleep apnoea.

Intermittent hypoxia (IH) plays a key role in the pathogenesis of insulin resistance (IR) in obstructive sleep apnoea (OSA). IH induces a pro-inflammatory phenotype of the adipose tissue with M1 macrophage polarisation, subsequently impeding adipocyte insulin signalling, and these changes are in striking similarity to those seen in obesity. However, the detailed molecular mechanisms of IH-induced macrophage polarisation are unknown and identification of same should lead to the identification of novel therapeutic targets. In the present study, we tested the hypothesis that IH acts through similar mechanisms as obesity, activating Toll-like-receptor (TLR)4/nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) signalling pathways leading to the upregulation and secretion of the key cytokines interleukin (IL)-1ß and IL-6. Bone-marrow derived macrophages (BMDMs) from lean and obese C57BL/6 male mice were exposed to a state-of-the-art in vitro model of IH. Independent of obesity, IH led to a pro-inflammatory M1 phenotype characterised by increased inducible nitric oxide synthase and IL-6 mRNA expression, robust increase in NF-κB DNA-binding activity and IL-6 secretion. Furthermore, IH significantly increased pro-IL-1ß mRNA and protein expression and mature IL-1ß secretion compared to control treatment. Providing mechanistic insight, pre-treatment with the TLR4 specific inhibitor, TAK-242, prevented IH-induced M1 polarisation and upregulation of IL-1ß mRNA and pro-IL-1ß protein expression. Moreover, IH-induced increase in IL-1ß secretion was prevented in BMDMs isolated from NLRP3 knockout mice. Thus, targeting TLR4/NF-κB and NLRP3 signalling pathways may provide novel therapeutic options for metabolic complications in OSA.

2-Hydroxyglutarate Metabolism Is Altered in an in vivo Model of LPS Induced Endotoxemia.

The metabolic response to endotoxemia closely mimics those seen in sepsis. Here, we show that the urinary excretion of the metabolite 2-hydroxyglutarate (2HG) is dramatically suppressed following lipopolysaccharide (LPS) administration in vivo, and in human septic patients. We further show that enhanced activation of the enzymes responsible for 2-HG degradation, D- and L-2-HGDH, underlie this effect. To determine the role of supplementation with 2HG, we carried out co-administration of LPS and 2HG. This co-administration in mice modulates a number of aspects of physiological responses to LPS, and in particular, protects against LPS-induced hypothermia. Our results identify a novel role for 2HG in endotoxemia pathophysiology, and suggest that this metabolite may be a critical diagnostic and therapeutic target for sepsis.

Immunometabolism and Sepsis: A Role for HIF?

Metabolic reprogramming of innate immune cells occurs during both the hyperinflammatory and immunotolerant phases of sepsis. The hypoxia inducible factor (HIF) signaling pathway plays a vital role in regulating these metabolic changes. This review initially summarizes the HIF-driven changes in metabolic dynamics of innate immune cells in response to sepsis. The hyperinflammatory phase of sepsis is accompanied by a metabolic switch from oxidative phosphorylation to HIF-1α mediated glycolysis. Furthermore, HIF driven alterations in arginine metabolism also occur during this phase. This promotes sepsis pathophysiology and the development of clinical symptoms. These early metabolic changes are followed by a late immunotolerant phase, in which suppressed HIF signaling promotes a switch from aerobic glycolysis to fatty acid oxidation, with a subsequent anti-inflammatory response developing. Recently the molecular mechanisms controlling HIF activation during these early and late phases have begun to be elucidated. In the final part of this review the contribution of toll-like receptors, transcription factors, metabolic intermediates, kinases and reactive oxygen species, in governing the HIF-induced metabolic reprogramming of innate immune cells will be discussed. Importantly, understanding these regulatory mechanisms can lead to the development of novel diagnostic and therapeutic strategies targeting the HIF-dependent metabolic state of innate immune cells.

Adipose tissue as a key player in obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is a major health concern worldwide and adversely affects multiple organs and systems. OSA is associated with obesity in >60% of cases and is independently linked with the development of numerous comorbidities including hypertension, arrhythmia, stroke, coronary heart disease and metabolic dysfunction. The complex interaction between these conditions has a significant impact on patient care and mortality. The pathophysiology of cardiometabolic complications in OSA is still incompletely understood; however, the particular form of intermittent hypoxia (IH) observed in OSA, with repetitive short cycles of desaturation and re-oxygenation, probably plays a pivotal role. There is fast growing evidence that IH mediates some of its detrimental effects through adipose tissue inflammation and dysfunction. This article aims to summarise the effects of IH on adipose tissue in experimental models in a comprehensive way. Data from well-designed controlled trials are also reported with the final goal of proposing new avenues for improving phenotyping and personalised care in OSA.

Hydroxylase Inhibition Selectively Induces Cell Death in Monocytes.

Hypoxia is a common and prominent feature of the microenvironment at sites of bacteria-associated inflammation in inflammatory bowel disease. The prolyl-hydroxylases (PHD1/2/3) and the asparaginyl-hydroxylase factor-inhibiting HIF are oxygen-sensing enzymes that regulate adaptive responses to hypoxia through controlling the activity of HIF and NF-κB-dependent transcriptional pathways. Previous studies have demonstrated that the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) is effective in the alleviation of inflammation in preclinical models of inflammatory bowel disease, at least in part, through suppression of IL-1ß-induced NF-κB activity. TLR-dependent signaling in immune cells, such as monocytes, which is important in bacteria-driven inflammation, shares a signaling pathway with IL-1ß. In studies into the effect of pharmacologic hydroxylase inhibition on TLR-induced inflammation in monocytes, we found that DMOG selectively triggers cell death in cultured THP-1 cells and primary human monocytes at concentrations well tolerated in other cell types. DMOG-induced apoptosis was independent of increased caspase-3/7 activity but was accompanied by reduced expression of the inhibitor of apoptosis protein 1 (cIAP1). Based on these data, we hypothesize that pharmacologic inhibition of the HIF-hydroxylases selectively targets monocytes for cell death and that this may contribute to the anti-inflammatory activity of HIF-hydroxylase inhibitors.

Glycolytic Response to Inflammation Over Time: Role of Myeloid HIF-1alpha.

The in vivo response to lipopolysaccharide (LPS) occurs rapidly and has profound physiological and metabolic effects. The hypoxia inducible (HIF) transcription factor is an intrinsic and essential part of inflammation, and is induced by LPS. To determine the importance of the HIF response in regulating metabolism following an LPS response, glucose uptake was quantified in a time dependent manner in mice lacking HIF-1α in myeloid cells. We found that deletion of HIF-1α has an acute protective effect on LPS-induced hypoglycemia. Furthermore, reduced glucose uptake was observed in the heart and brown fat, in a time dependent manner, following loss of HIF-1α. To determine the physiological significance of these findings, cardiovascular, body temperature, and blood pressure changes were subsequently quantified in real time using radiotelemetry measurements. These studies reveal the temporal aspects of HIF-1α as a regulator of the metabolic response to acute LPS-induced inflammation.

Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-κB-dependent manner.

Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1(-/-) hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease.

A dynamic model of the hypoxia-inducible factor 1α (HIF-1α) network.

Activation of the hypoxia-inducible factor (HIF) pathway is a critical step in the transcriptional response to hypoxia. Although many of the key proteins involved have been characterised, the dynamics of their interactions in generating this response remain unclear. In the present study, we have generated a comprehensive mathematical model of the HIF-1α pathway based on core validated components and dynamic experimental data, and confirm the previously described connections within the predicted network topology. Our model confirms previous work demonstrating that the steps leading to optimal HIF-1α transcriptional activity require sequential inhibition of both prolyl- and asparaginyl-hydroxylases. We predict from our model (and confirm experimentally) that there is residual activity of the asparaginyl-hydroxylase FIH (factor inhibiting HIF) at low oxygen tension. Furthermore, silencing FIH under conditions where prolyl-hydroxylases are inhibited results in increased HIF-1α transcriptional activity, but paradoxically decreases HIF-1α stability. Using a core module of the HIF network and mathematical proof supported by experimental data, we propose that asparaginyl hydroxylation confers a degree of resistance upon HIF-1α to proteosomal degradation. Thus, through in vitro experimental data and in silico predictions, we provide a comprehensive model of the dynamic regulation of HIF-1α transcriptional activity by hydroxylases and use its predictive and adaptive properties to explain counter-intuitive biological observations.

Gremlin plays a key role in the pathogenesis of pulmonary hypertension.

BACKGROUND: Pulmonary hypertension occurs in chronic hypoxic lung diseases, significantly worsening morbidity and mortality. The important role of altered bone morphogenetic protein (BMP) signaling in pulmonary hypertension was first suspected after the identification of heterozygous BMP receptor mutations as the underlying defect in the rare heritable form of pulmonary arterial hypertension. Subsequently, it was demonstrated that BMP signaling was also reduced in common forms of pulmonary hypertension, including hypoxic pulmonary hypertension; however, the mechanism of this reduction has not previously been elucidated. METHODS AND RESULTS: Expression of 2 BMP antagonists, gremlin 1 and gremlin 2, was higher in the lung than in other organs, and gremlin 1 was further increased in the walls of small intrapulmonary vessels of mice during the development of hypoxic pulmonary hypertension. Hypoxia stimulated gremlin secretion from human pulmonary microvascular endothelial cells in vitro, which inhibited endothelial BMP signaling and BMP-stimulated endothelial repair. Haplodeficiency of gremlin 1 augmented BMP signaling in the hypoxic mouse lung and reduced pulmonary vascular resistance by attenuating vascular remodeling. Furthermore, gremlin was increased in the walls of small intrapulmonary vessels in idiopathic pulmonary arterial hypertension and the rare heritable form of pulmonary arterial hypertension in a distribution suggesting endothelial localization. CONCLUSIONS: These findings demonstrate a central role for increased gremlin in hypoxia-induced pulmonary vascular remodeling and the increased pulmonary vascular resistance in hypoxic pulmonary hypertension. High levels of basal gremlin expression in the lung may account for the unique vulnerability of the pulmonary circulation to heterozygous mutations of BMP type 2 receptor in pulmonary arterial hypertension.

NFκB and HIF display synergistic behaviour during hypoxic inflammation.

The oxygen-sensitive transcription factor hypoxia inducible factor (HIF) is a key regulator of gene expression during adaptation to hypoxia. Crucially, inflamed tissue often displays regions of prominent hypoxia. Recent studies have shown HIF signalling is intricately linked to that of the pro-inflammatory transcription factor nuclear factor kappa B (NFκB) during hypoxic inflammation. We describe the relative temporal contributions of each to hypoxia-induced inflammatory gene expression and investigate the level of crosstalk between the two pathways using a novel Gaussia princeps luciferase (Gluc) reporter system. Under the control of an active promoter, Gluc is expressed and secreted into the cell culture media, where it can be sampled and measured over time. Thus, Gluc constructs under the control of either HIF or NFκB were used to resolve their temporal transcriptional dynamics in response to hypoxia and to cytokine stimuli, respectively. We also investigated the interactions between HIF and NFκB activities using a construct containing the sequence from the promoter of the inflammatory gene cyclooxygenase 2 (COX-2), which includes functionally active binding sites for both HIF and NFκB. Finally, based on our experimental data, we constructed a mathematical model of the binding affinities of HIF and NFκB to their respective response elements to analyse transcriptional crosstalk. Taken together, these data reveal distinct temporal HIF and NFκB transcriptional activities in response to hypoxic inflammation. Furthermore, we demonstrate synergistic activity between these two transcription factors on the regulation of the COX-2 promoter, implicating a co-ordinated role for both HIF and NFκB in the expression of COX-2 in hypoxic inflammation.

MicroRNA-155 promotes resolution of hypoxia-inducible factor 1alpha activity during prolonged hypoxia.

The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1α (but not HIF-2α) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxia-inducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1α mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1α in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1α mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1α stabilization and activity. Based on these data and a mathematical model of HIF-1α suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1α activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1α-dependent transcription.

An intact canonical NF-κB pathway is required for inflammatory gene expression in response to hypoxia.

Hypoxia is a feature of the microenvironment in a number of chronic inflammatory conditions due to increased metabolic activity and disrupted perfusion at the inflamed site. Hypoxia contributes to inflammation through the regulation of gene expression via key oxygen-sensitive transcriptional regulators including the hypoxia-inducible factor (HIF) and NF-κB. Recent studies have revealed a high degree of interdependence between HIF and NF-κB signaling; however, the relative contribution of each to hypoxia-induced inflammatory gene expression remains unclear. In this study, we use transgenic mice expressing luciferase under the control of NF-κB to demonstrate that hypoxia activates NF-κB in the heart and lungs of mice in vivo. Using small interfering RNA targeted to the p65 subunit of NF-κB, we confirm a unidirectional dependence of hypoxic HIF-1α accumulation upon an intact canonical NF-κB pathway in cultured cells. Cyclooxygenase-2 and other key proinflammatory genes are transcriptionally induced by hypoxia in a manner that is both HIF-1 and NF-κB dependent, and in mouse embryonic fibroblasts lacking an intact canonical NF-κB pathway, there is a loss of hypoxia-induced inflammatory gene expression. Finally, under conditions of hypoxia, HIF-1α and the p65 subunit of NF-κB directly bind to the cyclooxygenase-2 promoter. These results implicate an essential role for NF-κB signaling in inflammatory gene expression in response to hypoxia both through the regulation of HIF-1 and through direct effects upon target gene expression.

NF-κB links CO2 sensing to innate immunity and inflammation in mammalian cells.

Molecular O(2) and CO(2) are the primary substrate and product of aerobic metabolism, respectively. Levels of these physiologic gases in the cell microenvironment vary dramatically both in health and in diseases, such as chronic inflammation, ischemia, and cancer, in which metabolism is significantly altered. The identification of the hypoxia-inducible factor led to the discovery of an ancient and direct link between tissue O(2) and gene transcription. In this study, we demonstrate that mammalian cells (mouse embryonic fibroblasts and others) also sense changes in local CO(2) levels, leading to altered gene expression via the NF-κB pathway. IKKα, a central regulatory component of NF-κB, rapidly and reversibly translocates to the nucleus in response to elevated CO(2). This response is independent of hypoxia-inducible factor hydroxylases, extracellular and intracellular pH, and pathways that mediate acute CO(2)-sensing in nematodes and flies and leads to attenuation of bacterial LPS-induced gene expression. These results suggest the existence of a molecular CO(2) sensor in mammalian cells that is linked to the regulation of genes involved in innate immunity and inflammation.