Monique Fangradt

Hypoxia Promotes Osteogenesis but Suppresses Adipogenesis of Human Mesenchymal Stromal Cells in a Hypoxia-Inducible Factor-1 Dependent Manner

Background Bone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages. Methodology/Principal Findings Bone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O2) or hypoxic (less than 2% O2) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis. Conclusions/Significance Hypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches.

Origin and Functional Activity of the Membrane-Bound Glucocorticoid Receptor

OBJECTIVE Glucocorticoids (GCs) exert their antiinflammatory and immunosuppressive effects in humans primarily via the cytosolic GC receptor (cGR) but also via rapid, nongenomic mechanisms. Most likely, membrane-bound GRs (mGR) are involved in nongenomic GC signaling. The aim of this study was to investigate the origin and functional activity of mGR. METHODS We analyzed the origin of mGR using mGR-expressing HEK 293T cells, by transient and stable RNA interference-mediated GR reduction. GR messenger RNA (mRNA) and cGR and mGR protein levels were analyzed by real-time quantitative polymerase chain reaction, immunoblotting, and high-sensitivity immunofluorescence staining. Furthermore, we analyzed the functional activity of mGR, using membrane-impermeable bovine serum albumin (BSA)-bound dexamethasone (DEX-BSA) in human monocytes. Membrane-bound GR-expressing monocytes were treated with DEX, DEX-BSA, or BSA. Cell lysates were analyzed using PepChip arrays in order to identify kinases triggered by DEX-BSA, with validation using Bio-Plex assays and immunoblotting. RESULTS Our data showed that transient reduction of GR mRNA in HEK 293T cells decreased cGR protein levels but not mGR protein levels. However, stably transfected cells showed reduced cGR protein expression and significantly reduced mGR protein expression. Furthermore, 51 kinase substrates were identified for which phosphorylation was either reduced or increased. We observed p38 MAP kinase (MAPK) as one possible upstream kinase. Validation of these data by Bio-Plex phosphoprotein assay and immunoblotting showed increased phosphorylation of p38 MAPK after treatment with DEX-BSA. CONCLUSION Our data demonstrate that the human GR gene encodes for both cGR and mGR. Membrane-bound GR retains functional activity, as indicated by induced phosphorylation of p38 MAPK due to DEX-BSA treatment. Membrane-bound GR-mediated cellular signaling needs to be investigated further in order to clarify its therapeutic potential.

Macrophage Migration Inhibitory Factor Counterregulates Dexamethasone-Mediated Suppression of Hypoxia-Inducible Factor-1α Function and Differentially Influences Human CD4+ T Cell Proliferation under Hypoxia

Hypoxia, a feature of inflammation and tumors, is a potent inducer of the proinflammatory cytokine macrophage migration inhibitory factor (MIF). In transformed cells, MIF was shown to modulate and to be modulated via the oxygen-sensitive transcription factor hypoxia-inducible factor (HIF)-1. Furthermore, anti-inflammatory glucocorticoids (GCs) were described to regulate MIF action. However, in-depth studies of the interaction between MIF and HIF-1 and GC action in nontransformed primary human CD4+ T cells under hypoxia are missing. Therefore, we investigated the functional relationship between MIF and HIF and the impact of the GC dexamethasone (DEX) on these key players of inflammation in human CD4+ T cells. In this article, we show that hypoxia, and specifically HIF-1, is a potent and rapid inducer of MIF expression in primary human CD4+ T cells, as well as in Jurkat T cells. MIF signaling via CD74, in turn, is essential for hypoxia-mediated HIF-1α expression and HIF-1 target gene induction involving ERK/mammalian target of rapamycin activity complemented by PI3K activation upon mitogen stimulation. Furthermore, MIF signaling enhances T cell proliferation under normoxia but not hypoxia. MIF also counterregulates DEX-mediated suppression of MIF and HIF-1α expression. Based on these data, we suggest that hypoxia significantly affects the expression of HIF-1α in a MIF-dependent manner leading to a positive-feedback loop in primary human CD4+ T cells, thus influencing the lymphoproliferative response and DEX action via the GC receptor. Therefore, we suggest that HIF and/or MIF could be useful targets to optimize GC therapy when treating inflammation.

Effects of hypoxia and/or lack of glucose on cellular energy metabolism and cytokine production in stimulated human CD4+ T lymphocytes.

Oxidative phosphorylation and/or glycolysis provide energy, mainly in the form of ATP, which ensures proper functioning of immune cells such as CD4(+) T lymphocytes. However, the main substrates, namely oxygen and glucose, are known to remain for a relatively short time in the inflamed tissue and in other clinical situations where immune cells need to function properly. Therefore, we examined the effect of hypoxia and/or lack of glucose on cellular energy metabolism and on cytokine secretion in stimulated human CD4(+) T lymphocytes. Human CD4(+) T cells were MACS-isolated using peripheral blood obtained from healthy donors. Stimulated cells were incubated in medium with or without glucose for 6h in a sealed chamber which led to cumulative hypoxia. During this incubation period, (i) oxygen saturation was measured continuously using a Clark-type electrode, and (ii) samples were taken at different time points in order to quantify for each the viability of cells, intracellular reactive oxygen species (iROS), ATP levels, glycolytic enzyme activity, mRNA expression of hexokinase-1 and superoxide dismutase-1, and concentrations of several different cytokines. Stimulated CD4(+) T cells which were incubated under normoxic conditions served as controls. Under hypoxic conditions, lack of glucose exerted a biphasic effect on cellular oxygen consumption: initially higher but later lower respiration rates were measured when compared to conditions where glucose was available. Lack of glucose strongly increased the number of dead cells and the formation of iROS under normoxia but not under hypoxia. Under both normoxic and hypoxic conditions, intracellular ATP levels remained almost unchanged during the incubation period if glucose was present, but decreased significantly in the absence of glucose, despite the enhanced glycolytic enzyme activity. Measurements of stimulated cytokine production demonstrated (i) that cumulative hypoxia stimulates especially the secretion of IL-1beta, IL-10 and IL-8, and (ii) that lack of glucose results in lower cytokine concentrations. We demonstrate that CD4(+) T cells are highly adaptive in bioenergetic terms which ensure their proper function under extreme conditions of glucose and/or oxygen availability as found under physiological and pathophysiological conditions. Hypoxia seems to facilitate inflammatory reactions and angiogenesis.

Adaptation of Human CD4+ T Cells to Pathophysiological Hypoxia: A Transcriptome Analysis

Objective. Inflamed tissues are usually characterized by low oxygen levels. We investigated whether pathophysiological hypoxia (pO2 < 1%) as found in the rheumatoid synovium modulates the transcriptome of human CD4+ T cells. Methods. We analyzed the extent to which hypoxia influences the transcriptome in the rheumatoid synovium according to a gene cluster reflecting adaptation to low oxygen levels. Hypoxia-inducible factor-1α (HIF-1α) was detected in the rheumatoid synovium using immunohistochemistry. Isolated human CD4+ T cells were exposed to hypoxia and analyzed using microarray analysis, quantitative polymerase chain reaction, and immunoblot detection. Results. In rheumatoid arthritis (RA) synovial tissue samples, hypoxia modulates the transcription profile. This profile is similar, but not identical, to that found in isolated CD4+ T cells incubated under hypoxic conditions. We show that HIF-1α is expressed in synovial tissue samples and in hypoxic CD4+ cells; and that hypoxia directly affects differential gene expression in human T cells with up to 4.8% modulation of the transcriptome. Functional genome analysis revealed substantial effects of hypoxia on immune response, transcriptional regulation, protein modification, cell growth and proliferation, and cell metabolism. Conclusion. Severe hypoxia, a feature of joint inflammation, considerably modulates the transcriptome of cells found in the rheumatoid synovium. Human CD4+ T cells adapt to hypoxic conditions mainly by HIF-1-driven effects on the transcriptome reflecting a profound influence on immune functions. Thus, hypoxia must be taken into account when therapeutically targeting inflammation.

Hypoxia: how does the monocyte-macrophage system respond to changes in oxygen availability?

Hypoxia is an important feature of inflamed tissue, such as the RA joint. Activated monocytes/macrophages and endothelial cells play a pivotal role in the pathogenesis of RA, implicated in the mechanism of inflammation and erosion. During development, myeloid progenitor cells sequentially give rise to monoblasts, promonocytes, and monocytes that are released from the bone marrow into the bloodstream. After extravasation, monocytes differentiate into long‐lived, tissue‐specific macrophages or DCs. The effect of different oxygen concentrations experienced by these cells during maturation represents a novel aspect of this developmental process. In inflamed joint tissue, the microvascular architecture is highly dysregulated; thus, efficiency of oxygen supply to the synovium is poor. Therefore, invading cells must adapt instantaneously to changes in the oxygen level of the microenvironment. Angiogenesis is an early event in the inflammatory joint, which is important in enabling activated monocytes to enter via endothelial cells by active recruitment to expand the synovium into a “pannus”, resulting in cartilage degradation and bone destruction. The increased metabolic turnover of the expanding synovial pannus outpaces the dysfunctional vascular supply, resulting in hypoxia. The abnormal bioenergetics of the microenvironment further promotes synovial cell invasiveness. In RA, joint hypoxia represents a potential threat to cell function and survival. Notably, oxygen availability is a crucial parameter in the cellular energy metabolism, itself an important factor in determining the function of immune cells.

Human monocytes and macrophages differ in their mechanisms of adaptation to hypoxia

IntroductionInflammatory 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).MethodsIsolated 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).ResultsWe 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.ConclusionsThese data demonstrate that during differentiation of monocytes into macrophages, crucial cellular adaptation mechanisms are decisively changed.

Preoperative irradiation for the prevention of heterotopic ossification induces local inflammation in humans

Radiation of the hip is an established method to prevent heterotopic ossification (HO) following total hip arthroplasty (THA) but the precise mechanism is unclear. As inflammatory processes are suggested to be involved in the pathogenesis of HO, we hypothesized that the preoperative irradiation impacts local immune components. Therefore, we quantified immune cell populations and cytokines in hematomas resulting from the transection of the femur in two groups of patients receiving THA: patients irradiated preoperatively (THA-X-hematoma: THA-X-H group) in the hip region (7 Gy) in order to prevent HO and patients who were not irradiated (THA-H group) but were postoperatively treated with non-steroidal anti-inflammatory drugs (NSAIDs). Radiation resulted in significantly increased frequencies of T cells, cytotoxic T cells, NKT cells and CD25+CD127- Treg cells, whereas the number of naive CD45RA-expressing cytotoxic T cells was reduced. These results indicate differential immune cell activation, corroborated by our findings of significantly higher concentrations of pro-inflammatory cytokines (e.g., IL-6, IFNγ) and chemokines (e.g., MCP-1, RANTES) in the THA-X-H group as compared to THA-H group. In contrast, the concentration of the angiogenic VEGF was significantly suppressed in the THA-X-H group. We conclude that preoperative irradiation results in significant changes in immune cell composition and cytokine secretion in THA-hematomas, establishing a specific - rather proinflammatory - milieu. This increase of inflammatory activity together with the observed suppression in VEGF secretion may contribute to the prevention of HO.

OP0086 Alterations of immune cellular circadian rhythms in rheumatoid arthritis

Background The circadian variation of clinical symptoms and the underlying variation of cytokine and hormone levels in rheumatoid arthritis (RA) are well described and have already led to the successful application of chronotherapy with prednisone (Buttgereit et al., Lancet, 2008). Much less is known about the circadian rhythms of different immune cell populations in RA. Objectives In this pilot study we investigated molecular, cellular and humoral circadian parameters in postmenopausal female RA patients in comparison to healthy control subjects. Methods Blood samples from postmenopausal female patients with active RA (DAS 28 ≥4.2) (n=5) and postmenopausal female healthy controls (n=5) were collected every 2 hours for 24 hours and analysed by flow cytometry and multiplex suspension array of 28 cytokines. Clock gene expression of isolated CD14+ monocytes was analysed by quantitative RT-PCR. Endogenous circadian rhythm dynamics of macrophages were determined by means of a Bmal1-promotor driven luciferase reporter construct. COSINOR analysis was used for statistical analysis of the groups. Results Expression of the clock gene RevErbα in CD14+ monocytes showed a significant circadian expression pattern in both RA patients and healthy controls subjects, whereas the clock genes Per2 and Per3 were not expressed in a circadian manner in RA patients but in healthy controls only. The amplitude of the endogenous circadian rhythm of macrophages tended to be lower in RA patients than in healthy controls, whereas period length was not altered. In flow cytometric analysis of surface marker expression of blood cells we found a significant circadian rhythm in RA patients and healthy subjects for the frequency of CD3-CD56+ natural killer (NK) cells, Interleukin-8 Receptor (IL-8R) expressing CD4+ T helper and CD8+ cytotoxic T cells, and CXCR4 expressing CD4+ T helper and CD8+ cytotoxic cells. A significant circadian rhythm was not detectable in RA patients but in healthy controls only for CD3+CD56+ NK T cells. In contrast, a significant circadian expression of IL-8R+ monocytes was found in RA patients only but not in healthy subjects. Of note, CCR7 did not at all show a circadian expression. A significant circadian cytokine expression was detected only for MCP-1 in healthy controls. Conclusions This is the first indication of alterations of clock gene expression and endogenous circadian rhythms in immune cells of RA patients. Traffic of peripheral blood cells shows circadian variation in RA patients and healthy controls with characteristic peak phases, especially in NK cells and chemokine receptor expressing cells. NKT and other cells may lose their normal circadian rhythm in RA, whereas IL-8R expression on monocytes may be established as new “inflammatory” circadian rhythm in RA patients. These findings provide new aspects of RA chronobiology and may have therapeutic implications. Disclosure of Interest C. Spies: None Declared, T. Gaber: None Declared, P. Hoff: None Declared, J. Mazuch: None Declared, B. Maier: None Declared, M. Hahne: None Declared, C. Strehl: None Declared, C. Tran: None Declared, N. Soboleva: None Declared, A. Stoehr: None Declared, M. Wagegg: None Declared, M. Fangradt: None Declared, M. Jakstadt: None Declared, D. Huscher: None Declared, G.-R. Burmester: None Declared, J. Detert: None Declared, A. Kramer: None Declared, F. Buttgereit Grant/Research support from: This study was supported by Horizon Pharma AG, Reinach, Switzerland and Merck KGaA, Darmstadt, Germany. Dr. Buttgereit reports receiving consultancy fees, honoraria and travel expenses from Merck Serono, Horizon Pharma (formerly Nitec Pharma) Mundipharma Int Ltd and grant support from Merck Serono and Horizon Pharma., Consultant for: see above, Speakers Bureau: see above

OP0105 Accumulation of CD34+ hematopoietic stem cells in the initial inflammatory human fracture hematoma is driven by rantes and eotaxin

Background We have previously shown the early phase of human fracture healing to be characterized by hypoxia which promotes inflammation and chemoattraction. Hypoxia is also known to promote proliferation, survival and migration of different stem/progenitor cells like mesenchymal stem cells, endothelial progenitor cells or hematopoietic stem cells (HSC). However, the clinical relevance of hypoxia and inflammation in the early phase of fracture healing for HSC remains unclear. Objectives To investigate immunological events in fracture healing, we quantified (i) CD34+ hematopoietic stem cells and (ii) inflammatory chemokines present in the early (<72h) human fracture hematoma (FH) at the fracture gap. To investigate the chronologic development, we also analyzed hematomas which resulted from the transection of the femur in patients receiving a total hip arthroplasty (THA). The THA-hematomas (THA-H) were defined as a model for fracture hematomas at time point 0h. Methods The proportion of HSC in the fracture hematoma from healthy patients (n=42) and patients receiving a THA (n=20) was analyzed by flow cytometry. Secreted factors were quantified by multiplex suspension array. Results A fracture destroys bone architecture and vascular network leading to bioenergetically restricted conditions such as hypoxia within the fracture hematoma. Although the cells present have to face those conditions, we were able to find a higher proportion of CD34+ hematopoietic stem cells in the FH as compared to THA-H (7.6±1.5 vs. 3.8±0.5% of mononuclear cells) indicating proliferation and/or immigration of HSC in the FH. As CD34+ hematopoietic stem cells express CCR3, we investigated the concentrations of its ligands RANTES and Eotaxin. Indeed, both chemokines were present at significantly higher concentrations in the FH as compared to THA-H (RANTES: 16867±1632 vs. 9830±1397 pg/ml, p<0.01; Eotaxin: 327±76 vs. 125±15 pg/ml, p<0.001). We also identified the macrophage migration inhibitory factor (MIF) to be significantly increased in the FH (179431±28538 vs. 21751±2973 pg/ml, p<0.001). Conclusions Hypoxia and other bioenergetically adverse conditions in a FH contribute to the induction of inflammation, including the secretion of RANTES, Eotaxin and MIF. We suppose the high concentrations of RANTES and Eotaxin to facilitate the immigration of CD34+ HSC. The initial hypoxic conditions also mediate the secretion of the proinflammatory MIF which has been already shown to be important for successful fracture healing. Thus, the inflammatory microenvironment in the FH is among the crucial factors determining fracture healing. Disclosure of Interest None Declared