Human monocytes and macrophages differ in their mechanisms of adaptation to hypoxia
TL;DR: It is demonstrated that during differentiation of monocytes into macrophages, crucial cellular adaptation mechanisms are decisively changed, apparently as an adaptation to a low oxygen environment.
Abstract: Inflammatory arthritis is a progressive disease with chronic inflammation of joints, which is mainly characterized by the infiltration of immune cells and synovial hyperproliferation. Monocytes migrate towards inflamed areas and differentiate into macrophages. In inflamed tissues, much lower oxygen levels (hypoxia) are present in comparison to the peripheral blood. Hence, a metabolic adaptation process must take place. Other studies suggest that Hypoxia Inducible Factor 1-alpha (HIF-1α) may regulate this process, but the mechanism involved for human monocytes is not yet clear. To address this issue, we analyzed the expression and function of HIF-1α in monocytes and macrophages, but also considered alternative pathways involving nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB). Isolated human CD14+ monocytes were incubated under normoxia and hypoxia conditions with or without phorbol 12-myristate 13-acetate (PMA) stimulation, respectively. Nuclear and cytosolic fractions were prepared in order to detect HIF-1α and NFκB by immunoblot. For the experiments with macrophages, primary human monocytes were differentiated into human monocyte derived macrophages (hMDM) using human macrophage colony-stimulating factor (hM-CSF). The effects of normoxia and hypoxia on gene expression were compared between monocytes and hMDMs using quantitative PCR (quantitative polymerase chain reaction). We demonstrate, using primary human monocytes and hMDM, that the localization of transcription factor HIF-1α during the differentiation process is shifted from the cytosol (in monocytes) into the nucleus (in macrophages), apparently as an adaptation to a low oxygen environment. For this localization change, protein kinase C alpha/beta 1 (PKC-α/β1 ) plays an important role. In monocytes, it is NFκB1, and not HIF-1α, which is of central importance for the expression of hypoxia-adjusted genes. These data demonstrate that during differentiation of monocytes into macrophages, crucial cellular adaptation mechanisms are decisively changed.
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TL;DR: Understanding the complex interplay between hypoxia-induced signaling pathways, oxidative stress and mitochondrial function will provide better insight into the underlying mechanisms of disease pathogenesis.
278 citations
TL;DR: The crosstalk between HIF and NF‐κB in the control of the immune response in different immune cell types including macrophages, neutrophils and B and T cells is reviewed.
Abstract: Hypoxia and inflammation have been associated with a number of pathological conditions, in particular inflammatory diseases. While hypoxia is mainly associated with the activation of hypoxia-inducible factors (HIFs), inflammation activates the family of transcription factor called nuclear factor-kappa B (NF-κB). An extensive crosstalk between these two main molecular players involved in hypoxia and inflammation has been demonstrated. This crosstalk includes common activating stimuli, shared regulators and targets. In this review, we discuss the current understanding of the role of NF-κB and HIF in the context of the immune response. We review the crosstalk between HIF and NF-κB in the control of the immune response in different immune cell types including macrophages, neutrophils and B and T cells. Furthermore the importance of the molecular crosstalk between HIFs and NF-κB for a variety of medical conditions will be discussed.
228 citations
TL;DR: The reprogramming of hypoxia-mediated pathways in synovial cells, such as fibroblasts, dendritic cells, macrophages and T cells, is implicated in the pathogenesis of rheumatoid arthritis and other inflammatory conditions, and might therefore provide an opportunity for therapeutic intervention.
Abstract: Synovial proliferation, neovascularization and leukocyte extravasation transform the normally acellular synovium into an invasive tumour-like 'pannus'. The highly dysregulated architecture of the microvasculature creates a poor oxygen supply to the synovium, which, along with the increased metabolic turnover of the expanding synovial pannus, creates a hypoxic microenvironment. Abnormal cellular metabolism and mitochondrial dysfunction thus ensue and, in turn, through the increased production of reactive oxygen species, actively induce inflammation. When exposed to hypoxia in the inflamed joint, immune-inflammatory cells show adaptive survival reactions by activating key proinflammatory signalling pathways, including those mediated by hypoxia-inducible factor-1α (HIF-1α), nuclear factor κB (NF-κB), Janus kinase-signal transducer and activator of transcription (JAK-STAT) and Notch, which contribute to synovial invasiveness. The reprogramming of hypoxia-mediated pathways in synovial cells, such as fibroblasts, dendritic cells, macrophages and T cells, is implicated in the pathogenesis of rheumatoid arthritis and other inflammatory conditions, and might therefore provide an opportunity for therapeutic intervention.
222 citations
TL;DR: This review gives a comprehensive overview on the taxonomy of the currently known macrophage subtypes, with a lack on a good summary on the current taxonomy, functions and phenotypes of macrophages in my recent projects on the biocompatibility of biomaterials in the mouse model.
214 citations
TL;DR: Findings indicate that whenever possible the original haematoma formed upon injury should be conserved during clinical fracture treatment to benefit from the inherent healing potential.
Abstract: Fracture treatment is an old endeavour intended to promote bone healing and to also enable early loading and regain of function in the injured limb. However, in today’s clinical routine the healing potential of the initial fracture haematoma is still not fully recognized. The Arbeitsgemeinschaft fur Osteosynthesefragen (AO) formed in Switzerland in 1956 formulated four AO principles of fracture treatment which are still valid today. Fracture treatment strategies have continued to evolve further, as for example the relatively new concept of minimally invasive plate osteosynthesis (MIPO). This MIPO treatment strategy harbours the benefit of an undisturbed original fracture haematoma that supports the healing process. The extent of the supportive effect of this haematoma for the bone healing process has not been considered in clinical practice so far. The rising importance of osteoimmunological aspects in bone healing supports the essential role of the initial haematoma as a source for inflammatory cells that release the cytokine pattern that directs cell recruitment towards the injured tissue. In reviewing the potential benefits of the fracture haematoma, the early development of angiogenic and osteogenic potentials within the haematoma are striking. Removing the haematoma during surgery could negatively influence the fracture healing process. In an ovine open tibial fracture model the haematoma was removed 4 or 7 days after injury and the bone that formed during the first two weeks of healing was significantly reduced in comparison with an undisturbed control. These findings indicate that whenever possible the original haematoma formed upon injury should be conserved during clinical fracture treatment to benefit from the inherent healing potential.
124 citations
Cites background from "Human monocytes and macrophages dif..."
...Among them the M1 macorphages which instantly switch to an anaerobic energy supply and remain active (Fangradt et al. 2012)....
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TL;DR: The relationship between bioenergetics and immunity is described and current therapeutic approaches for targeting crucial processes of energy metabolism in immune cells are discussed.
276 citations
"Human monocytes and macrophages dif..." refers background in this paper
...Mature monocytes migrate towards sites of inflammation and infection where they differentiate into inflammatory macrophages or into dendritic cells (DCs) [1,4,5]....
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...Monocytes and macrophages require energy in the form of adenosine triphosphate (ATP) in order to facilitate motion (migration to achieve phagocytosis), activation (signal cascade, initiation, maintenance, cytokine production) and effector functions (phagocytosis, antigen presentation, regulatory functions) [4]....
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TL;DR: The induction of detoxicating enzyme expression in HT29 cells exposed to hypoxia results from the induction of both transactivating factors that bind to the AP-1 element and of redox proteins that enhance their affinity for this element.
Abstract: Many solid tumors contain substantial fractions of hypoxic cells which are relatively resistant to both radiation therapy and certain cytotoxic drugs. We have previously shown that exposure of human HT29 cells to hypoxic conditions results in the overexpression of certain enzymes involved in the detoxication of xenobiotics, including NAD(P)H:(quinone acceptor) oxidoreductase (DT)-diaphorase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione synthesis. This hypoxic effect on DT-diaphorase was shown to involve both transcriptional induction and altered message stability. We have investigated the effects of hypoxia on elements in the promoter region of DT-diaphorase. Electrophoretic mobility shift assays demonstrate the induction of a binding activity to the AP-1 response element of DT-diaphorase. Supershift assays suggest that this binding is due to AP-1 nuclear factors and that members of the jun family are induced to a greater degree than fos by hypoxia. Analysis of the kinetics of transcription factor expression indicates that the expression of c-jun and junD is induced during hypoxic exposure; mRNA levels fall during reoxygenation. Induction of fos on the other hand is not as florid during hypoxia (5-fold) and is most pronounced (17-fold) 24 h after the restoration of an oxic environment. Thus, the hypoxic response of DT-diaphorase expression is mediated in part through AP-1, initially by a jun-related mechanism and then by the involvement of fos. The affinity of transcription factors for the AP-1 binding site depends on the redox state of a cysteine residue located close to the DNA-binding region of both Fos and Jun. A nuclear protein, Ref-1, maintains the reduced state of Fos and Jun and promotes binding to AP-1. Nuclear extracts of HT29 cells exposed to hypoxia show markedly increased Ref-1 protein content. Elevation of ref-1 steady-state mRNA levels occurs as an early event following induction of hypoxia and persists when cells are restored to a normally oxygenated environment. Nuclear run-on analysis demonstrates that induction of transcription is the mechanism of ref-1 mRNA elevation. Electrophoretic mobility shift assays and immunodepletion assays were used to further define the interaction of Ref-1 with specific AP-1-binding proteins under hypoxic conditions. These data demonstrate that the induction of detoxicating enzyme expression in HT29 cells exposed to hypoxia results from the induction of both transactivating factors that bind to the AP-1 element and of redox proteins that enhance their affinity for this element.
238 citations
"Human monocytes and macrophages dif..." refers background in this paper
...In the literature, it has been reported that adaptive responses to hypoxia are regulated by several transcription factors, including HIF-1, HIF-2, ETS-1, cAMP response element binding protein, activator protein-1 and nuclear factor- B [26-33]....
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TL;DR: This is the first study to show a direct in vivo correlation between synovial tPO2, inflammation and cell migration, thus it is proposed that hypoxia is a possible primary driver of inflammatory processes in the arthritic joint.
Abstract: Introduction Hypoxia is a microenvironmental feature in the inflamed joint, which promotes survival advantage for cells. The aim of this study was to examine the relationship of partial oxygen pressure in the synovial tissue (tPO 2 ) in patients with inflammatory arthritis with macroscopic/microscopic inflammation and local levels of proinflammatory mediators. Methods Patients with inflammatory arthritis underwent full clinical assessment and video arthroscopy to quantify macroscopic synovitis and measure synovial tPO 2 under direct visualisation. Cell specific markers (CD3 (T cells), CD68 (macrophages), Ki67 (cell proliferation) and terminal deoxynucleotidyl transferase dUTP nick end labelling (cell apoptosis)) were quantified by immunohistology. In vitro migration was assessed in primary and normal synoviocytes (synovial fibroblast cells (SFCs)) using a wound repair scratch assay. Levels of tumour necrosis factor α (TNFα), interleukin 1β (IL1β), interferon γ (IFNγ), IL6, macrophage inflammatory protein 3α (MIP3α) and IL8 were quantified, in matched serum and synovial fluid, by multiplex cytokine assay and ELISA. Results The tPO 2 was 22.5 (range 3.2–54.1) mm Hg and correlated inversely with macroscopic synovitis (r=−0.421, p=0.02), sublining CD3 cells (−0.611, p 2 in vivo) induced cell migration. This was coupled with significantly higher levels of synovial fluid tumour necrosis factor α (TNFα), IL1β, IFNγ and MIP3α in patients with tPO 2 Conclusions This is the first study to show a direct in vivo correlation between synovial tPO 2 , inflammation and cell migration, thus it is proposed that hypoxia is a possible primary driver of inflammatory processes in the arthritic joint.
205 citations
"Human monocytes and macrophages dif..." refers background in this paper
...Hypoxia occurs in joint inflammation such as during the pathogenesis of rheumatoid arthritis [1,5], fracture hematomas [8], and malignant tumors [9]....
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...Inflammatory arthritis is characterized by infiltration of immune cells and local tissue hypoxia [1-3]....
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...Mature monocytes migrate towards sites of inflammation and infection where they differentiate into inflammatory macrophages or into dendritic cells (DCs) [1,4,5]....
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TL;DR: This study represents the first transcriptome analysis of hypoxic primary human monocytes, which provides novel insights into monocyte functional behavior within ischemic/hypoxic tissues.
Abstract: Peripheral blood monocytes migrate to and accumulate in hypoxic areas of inflammatory and tumor lesions. To characterize the molecular bases underlying monocyte functions within a hypoxic microenvironment, we investigated the transcriptional profile induced by hypoxia in primary human monocytes using high-density oligonucleotide microarrays. Profound changes in the gene expression pattern were detected following 16 h exposure to 1% O 2 , with 536 and 677 sequences showing at least a 1.5-fold increase and decrease, respectively. Validation of this analysis was provided by quantitative RT-PCR confirmation of expression differences of selected genes. Among modulated genes, 74 were known hypoxia-responsive genes, whereas the majority were new genes whose responsiveness to hypoxia had not been previously described. The hypoxic transcriptome was characterized by the modulation of a significant cluster of genes with immunological relevance. These included scavenger receptors ( CD163 , STAB1 , C1qR1 , MSR1 , MARCO , TLR7 ), immunoregulatory, costimulatory, and adhesion molecules ( CD32 , CD64 , CD69 , CD89 , CMRF-35H , ITGB5 , LAIR1 , LIR9 ), chemokines/cytokines and receptors ( CCL23 , CCL15 , CCL8 , CCR1 , CCR2 , RDC1 , IL-23A , IL-6ST ). Furthermore, we provided conclusive evidence of hypoxic induction of CCL20 , a chemoattractant for immature dendritic cells, activated/memory T lymphocytes, and naive B cells. CCL20 mRNA up-regulation was paralleled by increased protein expression and secretion. This study represents the first transcriptome analysis of hypoxic primary human monocytes, which provides novel insights into monocyte functional behavior within ischemic/hypoxic tissues. CCL20 up-regulation by hypoxia may constitute an important mechanism to promote recruitment of specific leukocyte subsets at pathological sites and may have implications for the pathogenesis of chronic inflammatory diseases.
198 citations
"Human monocytes and macrophages dif..." refers background in this paper
...terisation of monocytes incubated under hypoxia [40]....
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...[40], we showed that hypoxia strengthens the genetic expression of the glycolysis enzyme LDHA....
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TL;DR: It is reported that the DNA-binding and transcriptional activity of transcription factor AP-1 is very strongly induced in a biphasic response when HeLa cells are exposed to reduced oxygen pressure, and data suggest that the genomic response to hypoxia is primarily mediated byAP-1 while the inflammatory response to reoxygenation is mediated by NF-kappa B.
Abstract: Hypoxia and reoxygenation are important pathophysiological conditions that occur during injury, ischemia, reperfusion and stroke. In tumors, hypoxia and oxidative stress are regarded as triggers for enhanced proliferation and metastasis. Hypoxia and reoxygenation exert part of their biological effects by inducing the expression of novel genes but very little is known about the transcription factors involved. Here, we have compared the behaviour of two redox-controlled factors, AP-1 and NF-kappa B, during hypoxia and reoxygenation. We report that the DNA-binding and transcriptional activity of transcription factor AP-1 is very strongly induced in a biphasic response when HeLa cells are exposed to reduced oxygen pressure. This induction required new AP-1 protein synthesis. Different members of the Jun/Fos family of transcription factors were found in the first and second maxima of activation. The pathogen-responsive, pre-existing transcription factor NF-kappa B was not activated under hypoxic conditions. However, a p50-p65 heterodimer of NF-kappa B was rapidly and strongly activated when HeLa cells were re-exposed to normal oxygen pressure. This explains the induction of NF-kappa B-controlled inflammatory cytokine genes during reperfusion of ischemic tissue. Our data suggest that the genomic response to hypoxia is primarily mediated by AP-1 while the inflammatory response to reoxygenation is mediated by NF-kappa B.
183 citations
"Human monocytes and macrophages dif..." refers background in this paper
...In the literature, it has been reported that adaptive responses to hypoxia are regulated by several transcription factors, including HIF-1, HIF-2, ETS-1, cAMP response element binding protein, activator protein-1 and nuclear factor- B [26-33]....
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