RESUMEN
During acute viral infections, innate immune cells invade inflamed tissues and face hypoxic areas. Hypoxia-inducible factors (HIFs) adapt cellular responses towards these conditions. We wanted to investigate the effects of a loss of HIF-2α in macrophages during acute Friend murine leukemia retrovirus (FV) infection in C57BL/6 mice using a Cre/loxP system. Remarkably, mice with floxed Hif-2a (Hif-2afl; Hif-2a is also known as Epas1) did not show any signs of FV infection independent of Cre activity. This prevented a detailed analysis of the role of macrophage HIF-2α for FV infection but allowed us to study a model of unexpected FV resistance. Hif-2afl mice showed a significant decrease in the expression of the Atp6v1e2 gene encoding for the E2 subunit of the vacuolar H+-ATPase, which resulted in a decreased acidification of lysosomes and limited virus entry into the cell. These findings highlight that the insertion of loxP sites is not always without functional consequences and has established a phenotype in the floxed Hif-2a mouse, which is not only unexpected, but unwanted and is of relevance for the use of this mouse strain in (at least virus) experiments.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Virus de la Leucemia Murina de Friend , ATPasas de Translocación de Protón Vacuolares , Animales , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Virus de la Leucemia Murina de Friend/genética , Lisosomas/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , Macrófagos/inmunología , Ratones Endogámicos C57BL , Infecciones por Retroviridae/genética , Infecciones por Retroviridae/metabolismo , Infecciones por Retroviridae/virología , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
Natural killer (NK) cells are forced to cope with different oxygen environments even under resting conditions. The adaptation to low oxygen is regulated by oxygen-sensitive transcription factors, the hypoxia-inducible factors (HIFs). The function of HIFs for NK cell activation and metabolic rewiring remains controversial. Activated NK cells are predominantly glycolytic, but the metabolic programs that ensure the maintenance of resting NK cells are enigmatic. By combining in situ metabolomic and transcriptomic analyses in resting murine NK cells, our study defines HIF-1α as a regulator of tryptophan metabolism and cellular nicotinamide adenine dinucleotide (NAD+ ) levels. The HIF-1α/NAD+ axis prevents ROS production during oxidative phosphorylation (OxPhos) and thereby blocks DNA damage and NK cell apoptosis under steady-state conditions. In contrast, in activated NK cells under hypoxia, HIF-1α is required for glycolysis, and forced HIF-1α expression boosts glycolysis and NK cell performance in vitro and in vivo. Our data highlight two distinct pathways by which HIF-1α interferes with NK cell metabolism. While HIF-1α-driven glycolysis is essential for NK cell activation, resting NK cell homeostasis relies on HIF-1α-dependent tryptophan/NAD+ metabolism.
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NAD , Triptófano , Ratones , Animales , Triptófano/metabolismo , Células Asesinas Naturales , Glucólisis/genética , Hipoxia/metabolismo , Hipoxia de la Célula , Oxígeno/metabolismo , Homeostasis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
Cells of the immune defence, especially leukocytes, often have to perform their function in tissue areas that are characterized by oxygen deficiency, so-called hypoxia. Physiological hypoxia significantly affects leukocyte function and controls the innate and adaptive immune response mainly through transcriptional gene regulation via the hypoxia-inducible factors (HIFs). Multiple pathogens including components of bacteria, such as lipopolysaccharides (LPS) trigger the activation of leukocytes. HIF pathway activation enables immune cells to adapt to both hypoxic environments in physiological and inflammatory settings and modulates immune cell responses through metabolism changes and crosstalk with other immune-relevant signalling pathways. To study the mutual influence of both processes in vivo, we used a human endotoxemia model, challenging participants with an intravenous LPS injection post or prior to a 4-h stay in a hypoxic chamber with normobaric hypoxia of 10.5% oxygen. We analysed changes in gene expression in whole blood cells and determined inflammatory markers to unveil the crosstalk between both processes. Our investigations showed differentially altered gene expression patterns of HIF and target genes upon in vivo treatment with LPS and hypoxia. Further, we found evidence for effects of hypoxic priming upon inflammation in combination with immunomodulatory effects in whole blood cells in vivo. Our work elucidates the complex interplay of hypoxic and inflammatory HIF regulation in human immune cells and offers new perspectives for further clinical research.
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Hipoxia , Inflamación , Lipopolisacáridos , Humanos , Lipopolisacáridos/farmacología , Lipopolisacáridos/toxicidad , Hipoxia/metabolismo , Hipoxia/inmunología , Masculino , Adulto , Inflamación/metabolismo , Endotoxemia/metabolismo , Endotoxemia/inmunología , Femenino , Oxígeno/metabolismoRESUMEN
Tissues often experience hypoxia at sites of inflammation due to malperfusion, massive immune cell recruitment, and increased oxygen consumption. Organisms adapt to these hypoxic conditions through the transcriptional activation of various genes. In fact, there is significant crosstalk between the transcriptional responses to hypoxia and inflammatory processes. This interaction, named inflammatory hypoxia, plays a crucial role in various diseases including malignancies, chronic inflammatory lung diseases and sepsis. To further elucidate the crosstalk between hypoxia and inflammation in vivo and assess its potential for innovative therapies, our study aimed at investigating the impact of acute hypoxic conditions on inflammation-induced immune responses. To this end, we exposed healthy human subjects to hypoxia either before (hypoxia priming) or after a single intravenous (i.v.) injection of 0.4 ng/kg LPS. Our data show that hypoxia exposure prior to LPS injection (hypoxia priming) amplified the pro-inflammatory response. This was reflected by an increase in body temperature, plasma noradrenaline levels and the production of pro-inflammatory cytokines (i.e., IL-6 and TNF-α), compared to LPS control conditions. These effects were not observed when participants were exposed to hypoxia after LPS administration, demonstrating that the interaction between hypoxia and inflammation highly depends on the timing of both stimuli. Our findings suggest that acute hypoxia (i.e., hypoxia priming) modulates transient inflammation, leading to an enhanced pro-inflammatory response in healthy human subjects. This highlights the need for further investigations to understand the pathology of various HIF-associated inflammatory diseases, and to develop suitable, innovative therapies.
RESUMEN
Inflammatory bowel disease such as chronic colitis promotes colorectal cancer, which is a common cause of cancer mortality worldwide. Hypoxia is a characteristic of inflammation as well as of solid tumors and enforces a gene expression response controlled by hypoxia-inducible factors (HIFs). Once established, solid tumors are immunosuppressive to escape their abatement through immune cells. Although HIF activity is known to 1) promote cancer development and 2) drive tumor immune suppression through the secretion of adenosine, both prolyl hydroxylases and an asparaginyl hydroxylase termed factor-inhibiting HIF (FIH) negatively regulate HIF. Thus, FIH may act as a tumor suppressor in colorectal cancer development. In this study, we examined the role of colon epithelial FIH in a mouse model of colitis-induced colorectal cancer. We recapitulated colitis-associated colorectal cancer development in mice using the azoxymethane/dextran sodium sulfate model in Vil1-Cre/FIH+f/+f and wild-type siblings. Colon samples were analyzed regarding RNA and protein expression and histology. Vil1-Cre/FIH+f/+f mice showed a less severe colitis progress compared with FIH+f/+f animals and a lower number of infiltrating macrophages in the inflamed tissue. RNA sequencing analyses of colon tissue revealed a lower expression of genes associated with the immune response in Vil1-Cre/FIH+f/+f mice. However, tumor occurrence did not significantly differ between Vil1-Cre/FIH+f/+f and wild-type mice. Thus, FIH knockout in colon epithelial cells did not modulate colorectal cancer development but reduced the inflammatory response in chronic colitis.
Asunto(s)
Neoplasias Asociadas a Colitis/patología , Colitis/patología , Neoplasias Colorrectales/patología , Mucosa Intestinal/patología , Oxigenasas de Función Mixta/metabolismo , Adenosina/metabolismo , Animales , Azoximetano/toxicidad , Hipoxia de la Célula/fisiología , Colitis/inducido químicamente , Colitis/genética , Neoplasias Asociadas a Colitis/genética , Colon/patología , Neoplasias Colorrectales/genética , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Células Epiteliales/patología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxigenasas de Función Mixta/genética , Prolil Hidroxilasas/metabolismo , Transducción de Señal/fisiología , Escape del Tumor/inmunología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
HIF-2 represents a tissue-specific isoform of the hypoxia-inducible factors (HIFs) which regulate oxygen homeostasis in the cell. In acute oxygen deficiency, HIF transcription factors ensure the timely restoration of adequate oxygen supply. Particularly in medical conditions such as stroke, which have a high mortality risk due to ischaemic brain damage, rapid recovery of oxygen supply is of extraordinary importance. Nevertheless, the endogenous mechanisms are often not sufficient to respond to severe hypoxic stress with restoring oxygenation and fail to protect the tissue. Herein, we analysed murine neurospheres without functioning HIF-2α and found that special importance in the differentiation of neurons can be attributed to HIF-2 in the brain. Other processes, such as cell migration and signal transduction of different signalling pathways, appear to be mediated to some extent via HIF-2 and illustrate the function of HIF-2 in brain remodelling. Without hypoxic stress, HIF-2 in the brain presumably focuses on the fine-tuning of the neural network. However, a therapeutically increase of HIF-2 has the potential to regenerate or replace destroyed brain tissue and help minimize the consequences of an ischaemic stroke.
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Isquemia Encefálica/metabolismo , Regeneración Nerviosa , Neuroprotección , Factores de Transcripción/metabolismo , Animales , Femenino , Hipoxia/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Cultivo Primario de CélulasRESUMEN
Carotid body (CB) type I cells sense the blood Po2 and generate a nervous signal for stimulating ventilation and circulation when blood oxygen levels decline. Three oxygen-sensing enzyme complexes may be used for this purpose: 1) mitochondrial electron transport chain metabolism, 2) heme oxygenase 2 (HO-2)-generating CO, and/or 3) an NAD(P)H oxidase (NOX). We hypothesize that intracellular redox changes are the link between the sensor and nervous signals. To test this hypothesis type I cell autofluorescence of flavoproteins (Fp) and NAD(P)H within the mouse CB ex vivo was recorded as Fp/(Fp+NAD(P)H) redox ratio. CB type I cell redox ratio transiently declined with the onset of hypoxia. Upon reoxygenation, CB type I cells showed a significantly increased redox ratio. As a control organ, the non-oxygen-sensing sympathetic superior cervical ganglion (SCG) showed a continuously reduced redox ratio upon hypoxia. CN-, diphenyleneiodonium, or reactive oxygen species influenced chemoreceptor discharge (CND) with subsequent loss of O2 sensitivity and inhibited hypoxic Fp reduction only in the CB but not in SCG Fp, indicating a specific role of Fp in the oxygen-sensing process. Hypoxia-induced changes in CB type I cell redox ratio affected peptidyl prolyl isomerase Pin1, which is believed to colocalize with the NADPH oxidase subunit p47phox in the cell membrane to trigger the opening of potassium channels. We postulate that hypoxia-induced changes in the Fp-mediated redox ratio of the CB regulate the Pin1/p47phox tandem to alter type I cell potassium channels and therewith CND.
Asunto(s)
Cuerpo Carotídeo/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Oxígeno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Quimiorreceptoras/metabolismo , Flavoproteínas/metabolismo , Hipoxia/metabolismo , Pulmón/metabolismo , Ratones , Mitocondrias/metabolismo , Canales de Potasio/metabolismoRESUMEN
(1) Background: Hypoxia is a common feature of inflammation when hypoxia inducible factors (HIFs) adapt cells to conditions of low oxygen tension and inflammation. We studied the role of HIF-1 and HIF-2 in cells of the myeloid lineage in a mouse model of acute colitis. (2) Methods: Mice with and without a conditional knockout for either Hif-1a or Hif-2a or Hif-1a and Hif-2a in cells of the myeloid lineage were treated with 2.5% dextran sodium sulfate (DSS) for 6 days to induce an acute colitis. We analyzed the course of inflammation with respect to macroscopic (disease activity index) and microscopic (histology score and immunohistochemical staining of immune cells) parameters and quantified the mRNA expression of cytokines and chemokines in the colon and the mesenteric lymph nodes. (3) Results: A conditional knockout of myeloid Hif-1a ameliorated whereas the knockout of Hif-2a aggravated murine DSS colitis by increased recruitment of neutrophils to deeper layers of the colon. This led to higher expression of Il6, Ifng, Cd11c, Cd4, and Cd8 in the colon but also induced anti-inflammatory mediators such as Foxp3 and Il10. A conditional knockout of Hif-1a and Hif-2a did not show any differences compared to wildtype mice. (4) Conclusions: Myeloid HIF-1α and HIF-2α play opposing roles in acute DSS colitis. Thus, not only a cell type specific, but also the isoform specific modulation of HIFs needs to be addressed in attempts to modify HIF for therapeutic purposes.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Enfermedades Inflamatorias del Intestino , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Antígenos CD4/genética , Antígenos CD4/inmunología , Antígenos CD8/genética , Antígenos CD8/inmunología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/fisiopatología , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Ratones , Ratones Noqueados , Células Mieloides/inmunología , Células Mieloides/patologíaRESUMEN
The recent bestowal of the Nobel Prize 2019 in Physiology or Medicine to Gregg L. Semenza, Sir Peter J. Ratcliffe, and William G. Kaelin Jr. celebrates a series of remarkable discoveries that span from the physiological research question on how oxygen deficiency (hypoxia) induces the red blood cell forming hormone erythropoietin (Epo) to the first clinical application of a novel family of Epo-inducing drugs to treat patients suffering from renal anemia. This review looks back at the most important findings made by the three Nobel laureates, highlights current research trends, and sheds an eye on future perspectives of hypoxia research, including emerging and potential clinical applications.
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Oxígeno/metabolismo , Animales , Eritropoyetina/metabolismo , Humanos , Hipoxia/metabolismoRESUMEN
A major challenge in developing new treatments for myocardial infarction (MI) is an improved understanding of the pathophysiology of hypoxic tissue damage and the activation of endogenous adaptive programs to hypoxia. Due to the relevance of oxygen in metabolism, molecular adaptation to hypoxia driven by the hypoxia-inducible factors (HIFs) and the HIF-regulating prolyl hydroxylase domain enzymes (PHDs) is pivotal for the survival of cells and tissue under hypoxia. The heart under ischemic stress will extensively rely on these mechanisms of endogenous cardiac protection until hypoxia becomes too severe. In the past, work from several laboratories has provided evidence that inhibition of HIF-regulating PHDs might improve the outcome in ischemic heart disease (IHD) potentially because the adaptive mechanisms are boosted early and vigorously. Here, we review the role of the HIF hydroxylase pathway in IHD and highlight the potential of PHD inhibitors as a new treatment for MI with special regard to acute ischemia, reperfusion, and regeneration of the heart.
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Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Isquemia Miocárdica/enzimología , Animales , Corazón/fisiología , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Terapia Molecular Dirigida , Reperfusión Miocárdica , RegeneraciónRESUMEN
Nearly 30 years ago hypoxia-inducible factor (HIF) was described as a protein complex bound to regulatory DNA sequences termed hypoxia response elements because HIF binding induced transcription of the erythropoietin gene under hypoxia. However, it soon became clear that HIF is part of a ubiquitous cellular oxygen sensing system, which ensures finely tuned control of HIF abundance and activity in dependence of the cellular oxygen tension. For their discoveries of how cells sense and adapt to oxygen availability Gregg L. Semenza, William G. Kaelin Jr. and Sir Peter J. Ratcliffe received the Nobel Prize in Physiology or Medicine 2019. The Nobel laureates' pioneering work on cellular oxygen sensing has unraveled that HIF has numerous target genes reflecting its multiple functions in cellular metabolism and adaptation to different levels of oxygen. Importantly, HIF is also crucial for the development of the nervous system. HIF has an influence on different neural cell types regarding neurogenesis, maturation and apoptosis. Furthermore, HIF is involved in pathophysiological processes of the brain like stroke and Alzheimer's disease resulting in the development of HIF-related therapeutic approaches.
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Encéfalo/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Oxígeno/metabolismo , Adaptación Fisiológica , Animales , Hipoxia de la Célula , Humanos , Ratones , Neuronas/metabolismoRESUMEN
Hypoxia due to rapid tumor growth with impaired neovascularization and inflammation resulting from immune cell activation are hallmarks of cancer. Hypoxia-inducible factors control transcriptional adaptation in response to low oxygen conditions, both in tumor and immune cells. In addition, sphingolipids become increasingly recognized as important cell mediators in tumor and inflammatory hypoxia. Recent studies have identified acid sphingomyelinase (ASM), a central enzyme in the sphingolipid metabolism, as a regulator of several types of stress stimuli pathways and an important player in the tumor microenvironment. Therefore, this review will address the connection between the hypoxic response and the ASM/ceramide system in the context of inflammatory hypoxia.
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Ceramidas/metabolismo , Hipoxia/metabolismo , Inflamación/metabolismo , Esfingolípidos/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Animales , HumanosRESUMEN
Molecular imaging of the assembly of hypoxia inducible factor (HIF) complexes in living cells may lead to a deeper understanding of cellular oxygen sensing. Sophisticated live cell imaging has extended the toolbox to study the molecular response to changes in oxygen supply. In this respect fluorescence resonance energy transfer (FRET) as a technique to investigate protein-protein interaction in the nanoscale range gets increasing interest. Herein, we review FRET studies related to hypoxia research, emphasizing on recent progress, but also demonstrating how FRET studies are complementary or potentially superior to conventional biochemical as well as histochemical techniques. Technical advances in the application of FRET in living cells will overcome restrictions to end-point analysis on the population rather than single cell level and will thereby provide progress in understanding the cellular hypoxic response by HIF.
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Hipoxia de la Célula/fisiología , Transferencia Resonante de Energía de Fluorescencia , Hipoxia/metabolismo , Imagen Óptica , Oxígeno/metabolismo , Fotoblanqueo , Animales , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Imagen Óptica/métodosAsunto(s)
Arterias/metabolismo , Arterias/fisiología , Cuerpo Carotídeo/fisiología , Complejo IV de Transporte de Electrones/metabolismo , Animales , Cuerpo Carotídeo/metabolismo , Células Quimiorreceptoras/metabolismo , Células Quimiorreceptoras/fisiología , Cristalografía/métodos , Humanos , Ácido Láctico/metabolismo , Oxígeno/metabolismoRESUMEN
Hypoxia is a hallmark of chronically inflamed tissue. Hypoxia develops from vascular dysfunction and increased oxygen consumption by infiltrating leukocytes. With respect to inflammatory bowel disease (IBD), hypoxia is likely to be of particular importance: Impairment of the intestinal barrier during IBD allows anoxia from the lumen of the gut to spread to formerly normoxic tissue. In addition, disturbed perfusion of inflamed tissue and a higher oxygen demand of infiltrating immune cells lead to low oxygen levels in inflamed mucosal tissue. Here, cells become hypoxic and must now adapt to this condition. The hypoxia inducible factor (HIF)-1 complex is a key transcription factor for cellular adaption to low oxygen tension. HIF-1 is a heterodimer formed by two subunits: HIF-α (either HIF-1α or HIF-2α) and HIF-1ß. Under normoxic conditions, hydroxylation of the HIF-α subunit by specific oxygen-dependent prolyl hydroxylases (PHDs) leads to ubiquitin proteasome-dependent degradation. Under hypoxic conditions, however, PHD activity is inhibited; thus, HIF-α can translocate into the nucleus, dimerize with HIF-1ß, and bind to hypoxia-responsive elements of HIF-1 target genes. So far, most studies have addressed the function of HIF-1α in intestinal epithelial cells and the effect of HIF stabilization by PHD inhibitors in murine models of colitis. Furthermore, the role of HIF-1α in immune cells becomes more and more important as T cells or dendritic cells for which HIF-1 is of critical importance are highly involved in the pathogenesis of IBD. This review will summarize the function of HIF-1α and the therapeutic prospects for targeting the HIF pathway in intestinal mucosal inflammation.
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Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Oxígeno/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Mucosa Intestinal/citología , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
UNLABELLED: Angiogenesis is a key feature of liver fibrosis. Although sinusoidal remodeling is believed to contribute to fibrogenesis, the impact of sinusoidal angiogenesis on the resolution of liver fibrosis remains undefined. Myeloid cells, particularly macrophages, constantly infiltrate the fibrotic liver and can profoundly contribute to remodeling of liver sinusoids. We observe that the development of fibrosis is associated with decreased hepatic vascular endothelial growth factor (VEGF) expression as well as sinusoidal rarefication of the fibrotic scar. In contrast, the resolution of fibrosis is characterized by a rise in hepatic VEGF levels and revascularization of the fibrotic tissue. Genetic ablation of VEGF in myeloid cells or pharmacological inhibition of VEGF receptor 2 signaling prevents this angiogenic response and the resolution of liver fibrosis. We observe increased expression of matrix metalloproteases as well as decreased expression of tissue inhibitor of metalloproteases confined to sinusoidal endothelial cells in response to myeloid cell VEGF. Remarkably, reintroduction of myeloid cell-derived VEGF upon recovery restores collagenolytic acitivity and the resolution of fibrosis. CONCLUSION: We identify myeloid cell-derived VEGF as a critical regulator of extracellular matrix degradation by liver endothelial cells, thereby unmasking an unanticipated link between angiogenesis and the resolution of fibrosis.
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Cirrosis Hepática , Hígado/fisiología , Células Mieloides/fisiología , Neovascularización Fisiológica , Animales , Células Endoteliales/enzimología , Matriz Extracelular/metabolismo , Femenino , Fibrosis , Humanos , Hígado/irrigación sanguínea , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Critically ill patients are at high risk to suffer from sepsis, even in the absence of an initial infectious source, but the molecular mechanisms for their increased sepsis susceptibility, including a suppressed immune system, remain unclear. Although microbes and pathogen-associated molecular pattern are accepted inducers of sepsis and septic immunosuppression, the role of endogenous Toll-like receptor (TLR) ligands, such as mitochondrial DNA (mtDNA), in altering the immune response is unknown. METHODS: Mitochondrial DNA serum concentrations of the mitochondrial genes D-Loop and adenosine triphosphatase 6 were determined (quantitative polymerase chain reaction) in 165 septic patients and 50 healthy volunteers. Furthermore, cytotoxic T-cell activity was analyzed in wild-type and TLR9 knockout mice, with/without previous mtDNA administration, followed by injection of an ovalbumin-expressing adenoviral vector. RESULTS: Mitochondrial DNA serum concentrations were increased in septic patients (adenosine triphosphatase 6, 123-fold; D-Loop, 76-fold, P < 0.0001) compared with volunteers. Furthermore, a single mtDNA injection caused profound, TLR9-dependent immunosuppression of adaptive T-cell cytotoxicity in wild-type but not in TLR9 knockout mice and evoked various immunosuppressive mechanisms including the destruction of the splenic microstructure, deletion of cross-presenting dendritic cells, and up-regulation of programmed cell death ligand 1 and indoleamine 2,3-dioxygenase. Several of these findings in mice were mirrored in septic patients, and mtDNA concentrations were associated with an increased 30-day mortality. CONCLUSIONS: The findings of this study imply that mtDNA, an endogenous danger associated molecular pattern, is a hitherto unknown inducer of septic immunoparalysis and one possible link between initial inflammation and subsequent immunosuppression in critically ill patients.
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ADN Mitocondrial/sangre , ADN Mitocondrial/inmunología , Inflamación/sangre , Inflamación/inmunología , Sepsis/sangre , Sepsis/inmunología , Adulto , Anciano , Animales , Enfermedad Crítica , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Humanos , Inmunidad/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Estudios ProspectivosRESUMEN
Hypoxia is a common phenomenon that occurs in a variety of diseases such as cardiovascular ischemia, anemia, and cancer. Cellular oxygen sensors measure changes in tissue oxygenation and induce responses aimed at restoring sufficient supply with oxygen. Genetic adaptation to hypoxia is under control of hypoxia-inducible factors (HIFs), of which two highly homologous subunits HIF-1α and HIF-2α are regulated by oxygen tension. Together with HIF-1ß (=ARNT; aryl hydrocarbon receptor nuclear translocator) they form transcriptionally active complexes under hypoxia which drive the expression of hypoxia inducible genes. The meaning of different HIF complexes, i.e., HIF-1α/ARNT versus HIF-2α/ARNT with respect to target gene or tissue specificity has not been fully resolved. We applied modern microscopic methods like fluorescence resonance energy transfer (FRET) to elucidate protein-protein interactions and fluorescence recovery after photo-bleaching (FRAP) to study mobility of HIF proteins inside the nuclei of living cells. We found differences both in nuclear mobility and the assembly of HIF-1 versus HIF-2 which might help to better understand the assembly of HIF complexes.