Nathalie Dehne

HIF-1 in the inflammatory microenvironment

Hypoxia-inducible factor (HIF) is a transcriptional activator that coordinates adaptive responses to hypoxia. An increased activity is recognized in the majority of clinical relevant hypoxic/ischemic episodes and human cancers. However, studies with HIF-1alpha knockout mice revealed an important role of HIF-1 for physiology such as embryogenesis or glycolytic energy production. The discovery that HIF-1 activity is not only restricted to pathological conditions of reduced oxygen availability but also is needed for the normal O2-homeostasis by regulating O2-delivery and consumption opens a diverse spectrum of so far unappreciated HIF-1 functions in several organs, including the immune system. Innate immune responses are orchestrated by macrophages. These cells respond to environmental input signals and in turn generate appropriate answers to initiate resolution of inflammation. It appears that multiple pathways in the inflammatory microenvironment are used to adjust HIF-1alpha levels to affect macrophage biology. This review summarizes mechanisms of HIF activation in mammalian immune cells, especially in macrophages and neutrophils, and outlines how HIF moderates inflammation.

Redox Control of Inflammation in Macrophages

Macrophages are present throughout the human body, constitute important immune effector cells, and have variable roles in a great number of pathological, but also physiological, settings. It is apparent that macrophages need to adjust their activation profile toward a steadily changing environment that requires altering their phenotype, a process known as macrophage polarization. Formation of reactive oxygen species (ROS), derived from NADPH-oxidases, mitochondria, or NO-producing enzymes, are not necessarily toxic, but rather compose a network signaling system, known as redox regulation. Formation of redox signals in classically versus alternatively activated macrophages, their action and interaction at the level of key targets, and the resulting physiology still are insufficiently understood. We review the identity, source, and biological activities of ROS produced during macrophage activation, and discuss how they shape the key transcriptional responses evoked by hypoxia-inducible transcription factors, nuclear-erythroid 2-p45-related factor 2 (Nrf2), and peroxisome proliferator-activated receptor-γ. We summarize the mechanisms how redox signals add to the process of macrophage polarization and reprogramming, how this is controlled by the interaction of macrophages with their environment, and addresses the outcome of the polarization process in health and disease. Future studies need to tackle the option whether we can use the knowledge of redox biology in macrophages to shape their mediator profile in pathophysiology, to accelerate healing in injured tissue, to fight the invading pathogens, or to eliminate settings of altered self in tumors.

Knockout of HIF-1α in tumor-associated macrophages enhances M2 polarization and attenuates their pro-angiogenic responses

Tumor-associated macrophages (TAMs) constitute major infiltrates of solid tumors and express a marker profile that characterizes alternatively activated macrophages (MФs). TAMs accumulate in hypoxic tumor regions, express high amounts of hypoxia-inducible factor-1 (HIF-1) and contribute to tumor angiogenesis and invasiveness. However, the precise role of HIF-1 on MФ infiltration and phenotype alterations remains poorly defined. Therefore, we cocultured wild type (wt) versus HIF-1α(-/-) MФs with tumor spheroids. Both, wt and HIF-1α(-/-) MФs, infiltrated hypoxic regions of tumor spheroids at equal rates and got alternatively activated. Interestingly, significantly higher amounts of HIF-1α(-/-) MФs expressed the TAM markers CD206 and stabilin-1 compared with wt phagocytes. Stimulation of infiltrated TAMs with lipopolysaccharide (LPS)/interferon-γ revealed a reduced expression of the pro-inflammatory markers interleukin (IL)-6, tumor necrosis factor-α and inducible nitric oxide synthase in HIF-1α(-/-) MФs. Furthermore, HIF-1α(-/-) MФs were less cytotoxic toward tumor cells. Although infiltration of MФs increased the invasive potential of tumor spheroids independently of HIF-1, the ability to stimulate differentiation of stem cells toward CD31-positive cells was triggered by wt but not by HIF-1α(-/-) MФs. Our data suggest that HIF-1α-deficient MФs develop a more prominent TAM marker profile accompanied by reduced cytotoxicity, whereas HIF-1 seems indispensable for the angiogenesis-promoting properties of TAMs.

Hypoxia causes epigenetic gene regulation in macrophages by attenuating Jumonji histone demethylase activity

Epigenetic processes elicit changes in gene expression by modifying DNA bases or histone side chains without altering DNA sequences. Recently discovered Jumonji histone demethylases (JHDMs) affect gene expression by demethylating lysine residues of histone tails. JHDMs belong to a family of dioxygenases and share similarities with prolyl hydroxylases (PHDs). Therefore, we investigated the influence of hypoxia in macrophages on histone methylation. All JHDM family members JMJD1A-C and JMJD2A-D are expressed in macrophages. Thus, we analyzed the methylation status of histone H3 residues not only under hypoxia but also after treatment with the dioxygenase-inhibitors DMOG, NO and ROS. Western analysis revealed increased methylations in H3K9me2/me3 and H3K36me3 at pO₂ below 3%, DMOG, NO and ROS treatment. Chromatin immunoprecipitation (ChIP) assays demonstrated increased repressive marks H3K9me2 and H3K9me3 in specific promoter regions of the chemokine Ccl2 and the chemokine receptors Ccr1 and Ccr5, which correlated with a downregulation of their mRNA expression under hypoxic conditions. In contrasts, the hypoxia-inducible factor (HIF) target gene adrenomedullin (ADM) mRNA was upregulated and no increase in its histone modification was observed. We suggest that hypoxia and a concomitant loss of JHDM activity increases H3K9 methylation and decreases chemokine expression.

Roles of hypoxia‐inducible factor‐1α (HIF‐1α) versus HIF‐2α in the survival of hepatocellular tumor spheroids

Hypoxia‐inducible factors (HIFs) provoke adaptation to hypoxic stress occurring in rapidly growing tumor tissues. Therefore, overexpression of HIF‐1 or HIF‐2 is a common feature in hepatocellular carcinoma but their specific function is still controversially discussed. To analyze HIF function in hypoxia‐induced cell death we created a stable knockdown of HIF‐1α and HIF‐2α in HepG2 cells and generated tumor spheroids as an in vitro hepatocellular carcinoma model. Knockdown of HIF‐1α enhanced expression of HIF‐2α and vice versa. Unexpectedly, knockdown of HIF‐1α or HIF‐2α increased cell viability as well as spheroid size and decreased caspase‐3 activity. Antiapoptotic Bcl‐XL expression increased in both knockdown spheroids, whereas proapoptotic Bax was only reduced in HIF‐1α‐knockdown cells. Furthermore, an HIF‐2α‐knockdown significantly increased Bcl‐2/adenovirus E1B 19 kDa‐interacting protein 3 (BNIP3) expression in an HIF‐1α‐dependent manner. Concomitantly, electron microscopy revealed a substantial increase in autophagosomal structures in HIF‐2α‐knockdown spheroids and mito‐/lysotracker costaining confirmed lysosomal activity of these autophagosomes. Blocking autophagosome maturation using 3‐methyladenine restored cell death in HIF‐2α‐knockdown clones comparable to wildtype cells. Conclusion: An HIF‐1α‐knockdown increases HIF‐2α expression and shifts the balance of Bcl‐2 family members toward survival. The knockdown of HIF‐2α raises autophagic activity and attenuates apoptosis by enhancing HIF‐1α expression. Our data indicate that enhanced expression of one HIF‐isoform causes a survival advantage in hepatocellular carcinoma development. HEPATOLOGY 2010

Role of sestrin2 in peroxide signaling in macrophages.

Reactive oxygen species not only serve as signaling molecules, they also contribute to oxidative stress and cell damage. The thioredoxin and glutaredoxin systems form along with peroxiredoxins a precisely regulated defense system to maintain the cellular redox homeostasis. There is evidence that nitric oxide (NO) protects cells from oxidative stress by preventing inactivation of peroxiredoxins by sulfinylation. Here we demonstrate that NO and hypoxia upregulate Sestrin2 by HIF‐1‐dependent and additional mechanisms and that Sestrin2 contributes to preventing peroxiredoxins from sulfinylation. We conclude that Sestrin2 plays a role in peroxide defense as a reductase for peroxiredoxins.

Vitamin D Promotes Vascular Regeneration

Background— Vitamin D deficiency in humans is frequent and has been associated with inflammation. The role of the active hormone 1,25-dihydroxycholecalciferol (1,25-dihydroxy-vitamin D3; 1,25-VitD3) in the cardiovascular system is controversial. High doses induce vascular calcification; vitamin D3 deficiency, however, has been linked to cardiovascular disease because the hormone has anti-inflammatory properties. We therefore hypothesized that 1,25-VitD3 promotes regeneration after vascular injury. Methods and Results— In healthy volunteers, supplementation of vitamin D3 (4000 IU cholecalciferol per day) increased the number of circulating CD45-CD117+Sca1+Flk1+ angiogenic myeloid cells, which are thought to promote vascular regeneration. Similarly, in mice, 1,25-VitD3 (100 ng/kg per day) increased the number of angiogenic myeloid cells and promoted reendothelialization in the carotid artery injury model. In streptozotocin-induced diabetic mice, 1,25-VitD3 also promoted reendothelialization and restored the impaired angiogenesis in the femoral artery ligation model. Angiogenic myeloid cells home through the stromal cell–derived factor 1 (SDF1) receptor CXCR4. Inhibition of CXCR4 blocked 1,25-VitD3–stimulated healing, pointing to a role of SDF1. The combination of injury and 1,25-VitD3 increased SDF1 in vessels. Conditioned medium from injured, 1,25-VitD3–treated arteries elicited a chemotactic effect on angiogenic myeloid cells, which was blocked by SDF1-neutralizing antibodies. Conditional knockout of the vitamin D receptor in myeloid cells but not the endothelium or smooth muscle cells blocked the effects of 1,25-VitD3 on healing and prevented SDF1 formation. Mechanistically, 1,25-VitD3 increased hypoxia-inducible factor 1-&agr; through binding to its promoter. Increased hypoxia-inducible factor signaling subsequently promoted SDF1 expression, as revealed by reporter assays and knockout and inhibitory strategies of hypoxia-inducible factor 1-&agr;. Conclusions— By inducing SDF1, vitamin D3 is a novel approach to promote vascular repair.

Nitric oxide causes macrophage migration via the HIF-1-stimulated small GTPases Cdc42 and Rac1.

Hypoxia-inducible factor 1 (HIF-1) is a key regulator of tumor development. Recently, the tumor microenvironment, with the presence of tumor-associated macrophages (TAMs), has gained considerable interest. The mechanisms of macrophage/TAM migration as well as the role of HIF-1 in macrophages for tumor progression are still under debate. We present evidence that under normoxic conditions, nitric oxide (NO) promotes macrophage migration. The response was impaired in macrophages from leukocyte conditional HIF-1 alpha(-/-) mice. NO production and cell migration in response to cytokines were attenuated in macrophages from iNOS(-/-) mice, suggesting that iNOS-derived NO transmits cytokine signaling toward cell migration. We further identified the small GTPases Cdc42 and Rac1 as effectors of the NO-HIF axis to drive macrophage migration by modulating the actin cytoskeleton. Our observations strengthen the role of HIF-1 in macrophages as a target of NO in facilitating functional responses such as migration.

Sensors, Transmitters, and Targets in Mitochondrial Oxygen Shortage—A Hypoxia-Inducible Factor Relay Story

SIGNIFICANCE Cells sense and respond to a shortage of oxygen by activating the hypoxia-inducible transcription factors HIF-1 and HIF-2 and evoking adaptive responses. RECENT ADVANCES Mitochondria are at the center of a hypoxia sensing and responding relay system. CRITICAL ISSUES Under normoxia, reactive oxygen species (ROS) and nitric oxide (NO) are HIF activators. As their individual flux rates determine their diffusion-controlled interaction, predictions how these radicals affect HIF appear context-dependent. Considering that the oxygen requirement for NO formation limits its role in activating HIF to conditions of ambient oxygen tension. Given the central role of mitochondrial complex IV as a NO target, especially under hypoxia, allows inhibition of mitochondrial respiration by NO to spare oxygen thus, raising the threshold for HIF activation. HIF targets seem to configure a feedback-signaling circuit aimed at gradually adjusting mitochondrial function. In hypoxic cancer cells, mitochondria redirect Krebs cycle intermediates to preserve their biosynthetic ability. Persistent HIF activation lowers the entry of electron-delivering compounds into mitochondria to reduce Krebs cycle fueling and β-oxidation, attenuates the expression of electron transport chain components, limits mitochondria biosynthesis, and provokes their removal by autophagy. FUTURE DIRECTIONS Mitochondria can be placed central in a hypoxia sensing-hypoxia responding circuit. We need to determine to which extent and how mitochondria contribute to sense hypoxia, explore whether modulating their oxygen-consuming capacity redirects hypoxic responses in in vivo relevant disease conditions, and elucidate how the multiple HIF targets in mitochondria shape conditions of acute versus chronic hypoxia.

The supernatant of apoptotic cells causes transcriptional activation of hypoxia-inducible factor-1alpha in macrophages via sphingosine-1-phosphate and transforming growth factor-beta.

Macrophages infiltrating solid tumors exhibit a tumor-supporting phenotype and are critical for tumor development. Little is known which tumor-derived signal provokes this phenotype shift and how these signals are interpreted in macrophages to support tumor growth. We used the supernatant of apoptotic cells and noticed transcriptional, nuclear factor of activated T cells-dependent up-regulation of hypoxia-inducible factor (HIF)-1alpha mRNA, subsequent protein expression, and HIF-1 activity. Blocking calcineurin with cyclosporine A attenuated nuclear factor of activated T cells binding during electrophoretic mobility shift assay analysis and circumvented the HIF-1alpha mRNA increase. Knockdown experiments, receptor analysis, and antibody neutralization pointed to sphingosine-1-phosphate and transforming growth factor-beta as the initiators of the HIF-1 response. The use of macrophages from conditional HIF-1alpha knockout mice revealed that macrophages, under the impact of apoptotic cell supernatants, use HIF-1 to produce factors that induce CD31 expression in murine embryonic stem cells. Our study supports the notion that soluble factors produced from apoptotic tumor cells activate the HIF-1 system under normoxia in macrophages to enhance their tumor-promoting capacity by, for example, releasing vascular endothelial growth factor. This shows the importance of HIF-1-elicited responses in regulatory macrophages under normoxia.