C.L. Tran

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.

Pathophysiological hypoxia affects the redox state and IL‐2 signalling of human CD4+ T cells and concomitantly impairs survival and proliferation

Inflamed areas are characterized by infiltration of immune cells, local hypoxia and alterations of cellular redox states. We investigated the impact of hypoxia on survival, proliferation, cytokine secretion, intracellular energy and redox state of human CD4+ T cells. We found that pathophysiological hypoxia (<2% O2) significantly decreased CD4+ T‐cell survival after mitogenic stimulation. This effect was not due to an increased caspase‐3/7‐mediated apoptosis or adenosine‐5′‐triphosphate (ATP) consumption/depletion. However, the ability of stimulated T cells to proliferate was reduced under hypoxic conditions, despite increased expression of CD25. Pathophysiological hypoxia was also found to modify intracellular ROS (iROS) levels in stimulated T cells over time as compared with levels found in normoxia. Physiological hypoxia (5% O2) did not decrease CD4+ T‐cell survival and proliferation or modify iROS levels as compared with normoxia. We conclude that pathophysiological hypoxia affects T‐cell proliferation and viability via disturbed IL‐2R signalling downstream of STAT5a phosphorylation, but not as a result of impaired cellular energy homeostasis. We suggest iROS links early events in T‐cell stimulation to the inhibition of the lymphoproliferative response under pathophysiological hypoxic conditions. The level of iROS may therefore act as a mediator of immune functions leading to down‐regulation of long‐term T‐cell activity in inflamed tissues.

Immunologically restricted patients exhibit a pronounced inflammation and inadequate response to hypoxia in fracture hematomas

For patients who are known to have an impaired immune system, bone healing is often impaired. Therefore, it has been suggested that an effectively functioning immune system will have an influence on the quality of bone healing. Here, we demonstrate that cells within the fracture hematoma of immunologically restricted patients (1) exhibit a disturbed osteogenic differentiation (normal SPP1 but diminished RUNX2 expression), (2) show a strong inflammatory reaction (high IL8 and CXCR4), and (3) react on local hypoxia (high expression of HIF1A) but with inadequate target gene responses (diminished LDHA and PGK1 expression). Thus, it is already within the early inflammatory phase of fracture healing that the local gene expression in fracture hematomas of immunologically restricted patients points toward a critical regeneration.

Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo

In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory.

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

CTLA-4 Mediates Inhibitory Function of Mesenchymal Stem/Stromal Cells

Mesenchymal stem/stromal cells (MSCs) are stem cells of the connective tissue, possess a plastic phenotype, and are able to differentiate into various tissues. Besides their role in tissue regeneration, MSCs perform additional functions as a modulator or inhibitor of immune responses. Due to their pleiotropic function, MSCs have also gained therapeutic importance for the treatment of autoimmune diseases and for improving fracture healing and cartilage regeneration. However, the therapeutic/immunomodulatory mode of action of MSCs is largely unknown. Here, we describe that MSCs express the inhibitory receptor CTLA-4 (cytotoxic T lymphocyte antigen 4). We show that depending on the environmental conditions, MSCs express different isoforms of CTLA-4 with the secreted isoform (sCTLA-4) being the most abundant under hypoxic conditions. Furthermore, we demonstrate that the immunosuppressive function of MSCs is mediated mainly by the secretion of CTLA-4. These findings open new ways for treatment when tissue regeneration/fracture healing is difficult.

A7.19 Systemic inhibition of MIR-148A by antagomirs reduces CD4+ T helper cell numbers and alleviates inflammation in a preclinical model of transfer colitis

Background and objectives T helper type 1 (Th1) cells are involved in rheumatic diseases such as Crohn´s disease or rheumatoid arthritis and have a history of chronic autoantigenic stimulation. We have shown that Th1 cells adapt to chronic inflammation by upregulating the expression of the microRNA (miR)-148a. MiR-148a promotes the survival of chronically activated Th1 cells by regulating the expression of the proapoptotic protein Bim. Thus, we tested the suitability of miR-148a as a therapeutic target for the selective elimination of proinflammatory Th1 cells in a preclinical model of colitis. Methods Chronically activated Th1 cells were transferred into Rag1-deficient mice to induce colitis. Then, mice were intravenously injected with antagomirs that specifically target the microRNA-148a (antagomir-148a) or with control antagomirs (antagomir-scr). For assessing the function of T cell-intrinsic expression of miR-148a in colitis, we transduced Th1 cells with inducible microRNA sponges prior to transfer into Rag1-deficient mice. Colitic inflammation was determined by the weight-to-length ratios of colons. Colonic Th cells were sorted by FACS to measure miR-148a expression by qRT-PCR. The expression of Bim and Bcl-2 as well as numbers of viable Th1 cells, were determined by flow cytometry. Results Systemic inhibition of miR-148a by antagomirs alleviated colitis in mice as measured by reduced weight-to-length ratios of their colons. The numbers of viable Th1 cells were reduced up to 50% in mice that were treated with antagomir-148a when compared to mice that were injected with antagomir-scr. Antagomir-148a injections were efficient and resulted in a 30% reduction of miR-148a in colonic Th1 cells. The expression of the proapoptotic protein Bim was increased up to 30% in Th cells from antagomir-148a treated mice, while the anti-apoptotic protein Bcl-2 was unchanged. Th cell specific inhibition of miR-148a by miR-sponges during colitis led to a 30% reduction of Th cells in the colons of colitic mice and reduced the weight-to-length ratio. Conclusions We suggest that miR-148a controls the survival of proinflammatory Th1 cells in chronic inflammation by inhibiting Bim expression in a Th cell intrinsic fashion. Thus, miR-148a might represent a suitable target for the selective depletion of proinflammatory Th1 cells in chronic inflammation.

AB0038 Modulation of the Survival of Proinflammatory TH1 Lymphocytes by Stable Expression of MIR-148A Sponges in a Murine Model of Transfer Colitis

Background Proinflammatory T helper (Th) cells are critically involved in the initiation and perpetuation of chronic inflammatory diseases (CID). In these diseases, proinflammatory Th cells persist in inflamed tissues and are refractory to current immunosuppressive therapies. The molecular factors regulating the survival of proinflammatory Th cells are not fully understood. Recently, we have shown that proinflammatory Th1 cells adapt to chronic inflammation by upregulating the expression of the microRNA (miRNA, miR)-148a1. MiR-148a promotes the survival of proinflammatory Th1 cells by regulating the expression of the pro-apoptotic protein Bim. Memory Th cells isolated from inflamed synovia of patients with rheumatoid arthritis induce the expression of miR-148a, presumably enhancing their survival. Therefore we hypothesize, that miR-148a could represent a therapeutic target for the selective elimination of proinflammatory Th1 cells in CID. Objectives To test the suitability of miR-148a as a therapeutic target in a preclinical murine model of transfer colitis. Methods We have generated a retroviral based inducible miR-148a inhibitor, containing complementary binding sites for miR-148a, termed “miR-148a sponge”. We transduced transgenic Th cells with the miR-148a sponge or a scrambled (scr) control sponge. Induction of sponge expression was initiated by tamoxifen treatment. Expression of miRNA sponges was detected by GFP reporter gene expression and the functionality of miR148a inhibition was determined by real-time PCR for the miR-148a target Bim. MiR-148a sponge-transduced CD4+ Th1 cells were FACS-sorted and transferred into Rag1-/- mice. After manifestation of colitis, mice were sacrificed and T cells were re-isolated from the spleens and colons for determining their phenotype and number by FACS analysis. Results Expression of miR-148a sponges resulted in a 2 fold increase of Bim expression in activated Th1 cells in vitro. In addition, we found reduced numbers of miR-148a-sponge expressing Th cells as compared to scr-sponge expressing Th cells in the spleens and colons of colitic mice. Finally, miR-148a sponge-expressing Th cells from spleens and colons of colitic mice showed higher levels of Bim expression per cell than scr-sponge expressing cells. Conclusions Our data suggest that miR-148a controls the survival of pro-inflammatory Th1 cells in chronic inflammation by inhibiting Bim expression. Thus, therapeutic targeting of miR-148a probably is suitable for the selective depletion of proinflammatory Th1 cells in chronic inflammation. References Haftmann, C. et al. miR-148a is upregulated by Twist1 and T-bet and promotes Th1-cell survival by regulating the proapoptotic gene Bim. European journal of immunology, doi:10.1002/eji.201444633 (2014). Acknowledgements Patrick Maschmeyer is supported by EUTRAIN, a FP7 Marie Curie Initial Training Network for Early Stage Researchers funded by the European Union. Disclosure of Interest None declared

AB0026 Pathophysiological hypoxia affects both redox state and il-2 signaling of human cd4+ t cells and concomitantly impairs survival and proliferation

Background Rheumatoid arthritis (RA) is a chronic inflammatory disease of diarthrodial joints, characterized by the infiltration of immune cells, alterations of cellular redox state and pathophysiological local tissue hypoxia (<1% O2). Objectives We investigated the impact of different hypoxia levels on survival, proliferation, cytokine secretion, intracellular energy and redox state of quiescent and mitogen stimulated human CD4+ T cells. Methods Isolated human CD4+ T cells were exposed to normoxia (18% O2), physiological hypoxia (5% O2) and pathophysiological hypoxia (<1% O2), respectively and cultured with or without PHA stimulation at defined periods of time (6h, 24, 48, and 72h). Cells were then analyzed by measuring IL-2R signaling (CD25 expression, phosphoSTAT5a), proliferation and the production of intracellular ROS (iROS) using flow cytometry. In addition, caspase-3/7 activity and ATP levels were determined by luminometric assays. Cellular oxidative damage and cytokine release were analyzed by ELISA based quantification of protein carbonylation (ROS damage) and IL2 production. Results We found pathophysiological hypoxia (<1% O2) to decrease significantly CD4+ T cell survival after mitogenic stimulation. This effect was neither due to induced caspase-3/7-mediated apoptosis nor to increased ATP consumption or increased ATP depletion. However, the ability of stimulated T cells to proliferate was both delayed and suppressed under these hypoxic conditions, despite increased expression of CD25 and unchanged secreted IL-2 amounts. Hypoxia was also found to modify iROS levels in stimulated T cells over time, but without producing measurable effects on oxidative protein damage as compared to normoxia. In contrast, both normoxia (18% O2) and physiological hypoxia (5% O2) did not decrease CD4+ T cell survival and proliferation after mitogenic stimulation. Conclusions We conclude that pathophysiological hypoxia (<1% O2) but not physiological hypoxia (5% O2) affects T cell proliferation and viability via disturbed IL-2R signaling downstream of STAT5a phosphorylation. However, the decreased T cell proliferation was not linked to an impaired cellular energy homeostasis. We suggest iROS links early events in T cell stimulation to the inhibition of the lymphoproliferative response under hypoxic conditions. The level of iROS may therefore act as a mediator of immune functions leading to down-regulation of long-term T cell activity in inflamed tissues. Disclosure of Interest None Declared

OP0271 Differential regulatory functions of HIF-1α and HIF-2α during angiogenesis of human microvascular endothelial cells (HMECS)

Background Hypoxia and angiogenesis are features of inflamed and injured tissues. The transcription factors Hypoxia inducible factor (HIF)-1α and (HIF)-2α control cellular metabolic response to decreased oxygen tension thereby promoting angiogenesis and having implications on the pathogenesis of RA. Objectives We focused on the effects of HIF-1α and HIF-2α on angiogenesis and developed a human microvascular endothelial cells (HMEC) lentiviral based knockdown system for both transcription factors allowing us to analyze angiogenesis of HMECs under hypoxia in the absence of HIF-1α or HIF-2α, respectively. Methods Specific knockdown of HIF-1α or HIF-2α was achieved using lentiviral-based shRNA technology. The reduction of HIF-1α or HIF-2α was confirmed on transcriptional and translational level by realtime RT-PCR and Western blot. Angiogenesis of transduced HMECs in comparison to scrambled cells was studied by investigating both tubuli and node formation under hypoxia (<1% O2). Expression of hypoxia driven genes HIF1A, HIF2A, VEGFA and IL8 was quantified by realtime RT-PCR. Multiplex suspension array technology was used to measure the concentrations of secreted VEGF and IL8. Results The successful knockdown of HIF-1α and HIF-2α was confirmed by demonstrating considerably reduced gene expression levels of HIF1A and HIF2A by up to 71% (p=0.0222) under normoxic and hypoxic conditions. As a consequence, strongly reduced HIF-1α and HIF-2α protein levels were detected by Western blot. Targeting of HIF-1α led to a significantly decreased node formation (1.6-fold change under hypoxia, p=0.0067) with similar effects by trend on tubuli formation. The HIF-2α knockdown also led to a significantly decreased tubuli formation (1.7-fold change, p=0.0444) with similar effects by trend on node formation. Furthermore, HIF-1α targeted cells did not show any significant decrease in the gene expressions of VEGFA and IL8 but surprisingly raised cytokine levels of IL8 under hypoxia (1.4-fold change, p=0.021) compared to the control. In contrast, targeting of HIF-2α gave rise to reduced levels of secreted VEGF and IL8 with a significant suppression of IL8 gene expression under normoxia (2.1-fold change, p=0.0101). Conclusions Our findings show essential and overlapping functions of HIF-1α and HIF-2α with regard to their regulatory potential of angiogenesis in HMECs. Both transcription factors have the same impact on VEGF but differ in their effects on IL8 expression. These findings provide new insights into basic principles of angiogenesis in inflamed tissues and are therefore considered to be of clinical importance. Disclosure of Interest None Declared