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: The RA synovium is a multi‐cellular tissue, and while many cell types interact to promote the inflammatory response, their metabolic requirements differ, and understanding the complex interplay between hypoxia‐induced signalling pathways, metabolic pathways and theinflammatory response will provide better insight into the underlying mechanisms of disease pathogenesis.
Abstract: Rheumatoid arthritis is characterized by synovial proliferation, neovascularization and leucocyte extravasation leading to joint destruction and functional disability. The blood vessels in the inflamed synovium are highly dysregulated, resulting in poor delivery of oxygen; this, along with the increased metabolic demand of infiltrating immune cells and inflamed resident cells, results in the lack of key nutrients at the site of inflammation. In these adverse conditions synovial cells must adapt to generate sufficient energy to support their proliferation and activation status, and thus switch their cell metabolism from a resting regulatory state to a highly metabolically active state. This alters redox-sensitive signalling pathways and also results in the accumulation of metabolic intermediates which, in turn, can act as signalling molecules that further exacerbate the inflammatory response. The RA synovium is a multi-cellular tissue, and while many cell types interact to promote the inflammatory response, their metabolic requirements differ. Thus, understanding the complex interplay between hypoxia-induced signalling pathways, metabolic pathways and the inflammatory response will provide better insight into the underlying mechanisms of disease pathogenesis.
45 citations
TL;DR: It is suggested that HIF-1α is a major player in the establishment of chronic Leishmania infection and is crucial for enhancing immunosuppressive functions and lowering leishmanicidal capacity of myeloid cells.
Abstract: Leishmania donovani is known to induce myelopoiesis and to dramatically increase extramedullary myelopoiesis. This results in splenomegaly, which is then accompanied by disruption of the splenic microarchitecture, a chronic inflammatory environment, and immunosuppression. Chronically inflamed tissues are typically hypoxic. The role of hypoxia on myeloid cell functions during visceral leishmaniasis has not yet been studied. Here we show that L. donovani promotes the output from the bone marrow of monocytes with a regulatory phenotype that function as safe targets for the parasite. We also demonstrate that splenic myeloid cells acquire MDSC-like function in a HIF-1α-dependent manner. HIF-1α is also involved in driving the polarization towards M2-like macrophages and rendering intermediate stage monocytes more susceptible to L. donovani infection. Our results suggest that HIF-1α is a major player in the establishment of chronic Leishmania infection and is crucial for enhancing immunosuppressive functions and lowering leishmanicidal capacity of myeloid cells.
39 citations
TL;DR: By focusing on key pathological conditions, namely obesity and diabetes, rheumatoid arthritis, atherosclerosis and cancer, this work reviews the metabolic targets suitable for therapeutic intervention in macrophages and discusses the major obstacles and challenges related to the development of therapeutic strategies for a pharmacological targeting of macrophage's metabolism.
32 citations
TL;DR: Hypoxia treatment of PrECs, prostate cell lines, and a macrophage cell line (THP-1) increased the levels of NLRP3, AIM2, and pro-IL-1β, which supports the idea that hypoxia in human prostatic tumors contributes to PCI, in part, by priming cells for the activation ofNLRP3 and AIM1 inflammasome.
Abstract: // Ravichandran Panchanathan 1, 2 , Hongzhu Liu 1, 2 , Divaker Choubey 1, 2 1 Cincinnati VA Medical Center, Cincinnati, OH 45220, USA 2 Department of Environmental Health, University of Cincinnati, Cincinnati, OH 45267, USA Correspondence to: Divaker Choubey, email: Divaker.choubey@uc.edu Keywords: hypoxia, prostate, inflammasome, inflammation, cancer Received: December 15, 2015 Accepted: March 28, 2016 Published: April 5, 2016 ABSTRACT The molecular mechanisms by which hypoxia contributes to prostatic chronic inflammation (PCI) remain largely unknown. Because hypoxia stimulates the transcriptional activity of NF-κB, which “primes” cells for inflammasome activation by inducing the expression of NLRP3 or AIM2 receptor and pro-IL-1β, we investigated whether hypoxia could activate the NLRP3 and AIM2 inflammasome in human normal prostate epithelial cells (PrECs) and cancer cell lines. Here we report that hypoxia (1% O 2 ) treatment of PrECs, prostate cell lines, and a macrophage cell line (THP-1) increased the levels of NLRP3, AIM2, and pro-IL-1β. Further, hypoxia in cells potentiated activation of the NLRP3 and AIM2 inflammasome activity. Notably, hypoxia “primed” cells for NLRP3 and AIM2 inflammasome activation through stimulation of the NF-κB activity. Our observations support the idea that hypoxia in human prostatic tumors contributes to PCI, in part, by priming cells for the activation of NLRP3 and AIM2 inflammasome.
31 citations
Additional excerpts
...Because PMA-treatment of THP-1 monocytic cells induces cell differentiation and increases the levels of HIF-1α and stimulates the expression of HIF-1α target genes [40], we used undifferentiated THP-1 monocytic cells to investigate whether hypoxia could “prime” activation of the NLRP3 or AIM2 inflammasome....
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TL;DR: More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses, as well as the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings.
Abstract: Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses.
27 citations
References
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TL;DR: It is demonstrated that Hif-1 DNA binding activity is also induced by hypoxia in a variety of mammalian cell lines in which the EPO gene is not transcribed, providing evidence that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to Hypoxia.
Abstract: Transcription of the human erythropoietin (EPO) gene is activated in Hep3B cells exposed to hypoxia. Hypoxia-inducible factor 1 (HIF-1) is a nuclear factor whose DNA binding activity is induced by hypoxia in Hep3B cells, and HIF-1 binds at a site in the EPO gene enhancer that is required for hypoxic activation of transcription. In this paper, we demonstrate that HIF-1 DNA binding activity is also induced by hypoxia in a variety of mammalian cell lines in which the EPO gene is not transcribed. The composition of the HIF-1 DNA binding complex and its isolated DNA binding subunit and the mechanism of HIF-1 activation appear to be similar or identical in EPO-producing and non-EPO-producing cells. Transcription of reporter genes containing the EPO gene enhancer is induced by hypoxia in non-EPO-producing cells and mutations that eliminate HIF-1 binding eliminate inducibility. These results provide evidence that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to hypoxia.
1,405 citations
"Human monocytes and macrophages dif..." refers background in this paper
...In other cells (for example, T-cells), it is known that the transcription factor HIF-1 under hypoxic conditions is translocated into the nucleus and binds to promoter regions of target genes to enable the necessary adaptation and maintenance of basic functions like motion, activation and effector cell function [12,13]....
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TL;DR: It is demonstrated that induction of both Hif-1 and erythropoietin RNA is inhibited by the protein kinase inhibitor 2-aminopurine, consistent with the proposed function of HIF-1 as a physiologic regulator of gene expression that responds to changes in cellular oxygen tension.
875 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: It is described that oxygen availability is a determinant parameter in the setting of chemotactic responsiveness to stromal-derived factor 1 (CXCL12), and the Hyp–Hyp-inducible factor 1 α–CXCR4 pathway may regulate trafficking in and out of hypoxic tissue microenvironments.
Abstract: Cell adaptation to hypoxia (Hyp) requires activation of transcriptional programs that coordinate expression of genes involved in oxygen delivery (via angiogenesis) and metabolic adaptation (via glycolysis). Here, we describe that oxygen availability is a determinant parameter in the setting of chemotactic responsiveness to stromal-derived factor 1 (CXCL12). Low oxygen concentration induces high expression of the CXCL12 receptor, CXC receptor 4 (CXCR4), in different cell types (monocytes, monocyte-derived macrophages, tumor-associated macrophages, endothelial cells, and cancer cells), which is paralleled by increased chemotactic responsiveness to its specific ligand. CXCR4 induction by Hyp is dependent on both activation of the Hyp-inducible factor 1 alpha and transcript stabilization. In a relay multistep navigation process, the Hyp-Hyp-inducible factor 1 alpha-CXCR4 pathway may regulate trafficking in and out of hypoxic tissue microenvironments.
834 citations
"Human monocytes and macrophages dif..." refers background in this paper
...CXCR4 transcript levels have been shown to increase in monocytes facing hypoxia, which suggests HIF is crucially involved in regulating the trafficking [15]....
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...showed that in human monocytes and human MDMs, hypoxia induced expression of CXCR4 at the protein level [15]....
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TL;DR: The intracellular signaling mechanism that leads to induction of COx-2 by hypoxia includes binding of p65 to the relatively 3′ NF-κB consensus element in the COX-2 upstream promoter region in human vascular endothelial cells.
683 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: Functional studies in a mutant cell line expressing neither HIF-1alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPas-1 transactivation of the VEGF promoter than the LDH-A promoter.
661 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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