Publications by authors named "Denise Tischner"

23 Publications

  • Page 1 of 1

Glucocorticoid Receptor-Deficient Foxp3 Regulatory T Cells Fail to Control Experimental Inflammatory Bowel Disease.

Front Immunol 2019 18;10:472. Epub 2019 Mar 18.

Division of Developmental Immunology, Biocenter, Medical University Innsbruck, Innsbruck, Austria.

Activation of the immune system increases systemic adrenal-derived glucocorticoid (GC) levels which downregulate the immune response as part of a negative feedback loop. While CD4 T cells are essential target cells affected by GC, it is not known whether these hormones exert their major effects on CD4 helper T cells, CD4Foxp3 regulatory T cells (Treg cells), or both. Here, we generated mice with a specific deletion of the glucocorticoid receptor (GR) in Foxp3 Treg cells. Remarkably, while basal Treg cell characteristics and suppression capacity were unchanged, Treg cells lacking the GR did not prevent the induction of inflammatory bowel disease in an mouse model. Under inflammatory conditions, GR-deficient Treg cells acquired Th1-like characteristics and expressed IFN-gamma, but not IL-17, and failed to inhibit pro-inflammatory CD4 T cell expansion . These findings reveal that the GR is critical for Foxp3 Treg cell function and suggest that endogenous GC prevent Treg cell plasticity toward a Th1-like Treg cell phenotype in experimental colitis. When equally active in humans, a rationale is provided to develop GC-mimicking therapeutic strategies which specifically target Foxp3 Treg cells for the treatment of inflammatory bowel disease.
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http://dx.doi.org/10.3389/fimmu.2019.00472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431616PMC
September 2020

Single-cell profiling reveals GPCR heterogeneity and functional patterning during neuroinflammation.

JCI Insight 2017 Aug 3;2(15). Epub 2017 Aug 3.

Department of Pharmacology.

GPCR expression was intensively studied in bulk cDNA of leukocyte populations, but limited data are available with respect to expression in individual cells. Here, we show a microfluidic-based single-cell GPCR expression analysis in primary T cells, myeloid cells, and endothelial cells under naive conditions and during experimental autoimmune encephalomyelitis, the mouse model of multiple sclerosis. We found that neuroinflammation induces characteristic changes in GPCR heterogeneity and patterning, and we identify various functionally relevant subgroups with specific GPCR profiles among spinal cord-infiltrating CD4 T cells, macrophages, microglia, or endothelial cells. Using GPCRs CXCR4, S1P1, and LPHN2 as examples, we show how this information can be used to develop new strategies for the functional modulation of Th17 cells and activated endothelial cells. Taken together, single-cell GPCR expression analysis identifies functionally relevant subpopulations with specific GPCR repertoires and provides a basis for the development of new therapeutic strategies in immune disorders.
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http://dx.doi.org/10.1172/jci.insight.95063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543912PMC
August 2017

EBI2 Is Highly Expressed in Multiple Sclerosis Lesions and Promotes Early CNS Migration of Encephalitogenic CD4 T Cells.

Cell Rep 2017 01;18(5):1270-1284

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany. Electronic address:

Arrival of encephalitogenic T cells at inflammatory foci represents a critical step in development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. EBI2 and its ligand, 7α,25-OHC, direct immune cell localization in secondary lymphoid organs. CH25H and CYP7B1 hydroxylate cholesterol to 7α,25-OHC. During EAE, we found increased expression of CH25H by microglia and CYP7B1 by CNS-infiltrating immune cells elevating the ligand concentration in the CNS. Two critical pro-inflammatory cytokines, interleukin-23 (IL-23) and interleukin-1 beta (IL-1β), maintained expression of EBI2 in differentiating Th17 cells. In line with this, EBI2 enhanced early migration of encephalitogenic T cells into the CNS in a transfer EAE model. Nonetheless, EBI2 was dispensable in active EAE. Human Th17 cells do also express EBI2, and EBI2 expressing cells are abundant within multiple sclerosis (MS) white matter lesions. These findings implicate EBI2 as a mediator of CNS autoimmunity and describe mechanistically its contribution to the migration of autoreactive T cells into inflamed organs.
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http://dx.doi.org/10.1016/j.celrep.2017.01.020DOI Listing
January 2017

S1P2/G12/13 Signaling Negatively Regulates Macrophage Activation and Indirectly Shapes the Atheroprotective B1-Cell Population.

Arterioscler Thromb Vasc Biol 2016 Jan 24;36(1):37-48. Epub 2015 Nov 24.

From the Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany (M.G., D.T., K.T., J.A.J., K.K.S., N.W.); Pharmazentrum Frankfurt/ZAFES, Clinical Pharmacology (N.F.B., G.G.) and Centre for Molecular Medicine, Medical Faculty (N.W.), J.W. Goethe University Frankfurt, Frankfurt, Germany; and Department of Laboratory Medicine, Medical University of Vienna and Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria (C.J.B.).

Objectives: Monocyte/macrophage recruitment and activation at vascular predilection sites plays a central role in the pathogenesis of atherosclerosis. Heterotrimeric G proteins of the G12/13 family have been implicated in the control of migration and inflammatory gene expression, but their function in myeloid cells, especially during atherogenesis, is unknown.

Approach And Results: Mice with myeloid-specific deficiency for G12/13 show reduced atherosclerosis with a clear shift to anti-inflammatory gene expression in aortal macrophages. These changes are because of neither altered monocyte/macrophage migration nor reduced activation of inflammatory gene expression; on the contrary, G12/13-deficient macrophages show an increased nuclear factor-κB-dependent gene expression in the resting state. Chronically increased inflammatory gene expression in resident peritoneal macrophages results in myeloid-specific G12/13-deficient mice in an altered peritoneal micromilieu with secondary expansion of peritoneal B1 cells. Titers of B1-derived atheroprotective antibodies are increased, and adoptive transfer of peritoneal cells from mutant mice conveys atheroprotection to wild-type mice. With respect to the mechanism of G12/13-mediated transcriptional control, we identify an autocrine feedback loop that suppresses nuclear factor-κB-dependent gene expression through a signaling cascade involving sphingosine 1-phosphate receptor subtype 2, G12/13, and RhoA.

Conclusions: Together, these data show that selective inhibition of G12/13 signaling in macrophages can augment atheroprotective B-cell populations and ameliorate atherosclerosis.
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http://dx.doi.org/10.1161/ATVBAHA.115.306066DOI Listing
January 2016

Endothelial Gαq/11 is required for VEGF-induced vascular permeability and angiogenesis.

Cardiovasc Res 2015 Oct 13;108(1):171-80. Epub 2015 Aug 13.

Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany J.W. Goethe University Frankfurt, 60590 Frankfurt, Germany

Aims: VEGF A (VEGF-A) is a central regulator of pre- and postnatal vascular development. In vitro studies suggested that heterotrimeric G-proteins of the Gq/11 family contribute to VEGF receptor 2 (VEGFR2) signalling, but the mechanism and physiological relevance of this finding is unknown. The aim of this study is to understand the role of endothelial Gαq/11 in VEGF-dependent regulation of vascular permeability and angiogenesis.

Methods And Results: We show here that VEGF-A-induced signalling events, such as VEGFR2 autophosphorylation, calcium mobilization, or phosphorylation of Src and Cdh5, were reduced in Gαq/11-deficient endothelial cells (ECs), resulting in impaired VEGF-dependent barrier opening, tube formation, and proliferation. Agonists at Gq/11-coupled receptors facilitated VEGF-A-induced VEGFR2 autophosphorylation in a Gαq/11-dependent manner, thereby enhancing downstream VEGFR2 signalling. In vivo, EC-specific Gαq/11- and Gαq-deficient mice showed reduced VEGF-induced fluid extravasation, and retinal angiogenesis was significantly impaired. Gαq-deficient ECs showed reduced proliferation, Cdh5 phosphorylation, and fluid extravasation, whereas apoptosis was increased.

Conclusion: Gαq/11 critically contributes to VEGF-A-dependent permeability control and angiogenic behaviour in vitro and in vivo.
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http://dx.doi.org/10.1093/cvr/cvv216DOI Listing
October 2015

Tyrphostin AG126 exerts neuroprotection in CNS inflammation by a dual mechanism.

Glia 2015 Jun 2;63(6):1083-99. Epub 2015 Mar 2.

Institute of Neuropathology, University of Göttingen, Germany.

The putative protein tyrosine kinase (PTK) inhibitor tyrphostin AG126 has proven beneficial in various models of inflammatory disease. Yet molecular targets and cellular mechanisms remained enigmatic. We demonstrate here that AG126 treatment has beneficial effects in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. AG126 alleviates the clinical symptoms, diminishes encephalitogenic Th17 differentiation, reduces inflammatory CNS infiltration as well as microglia activation and attenuates myelin damage. We show that AG126 directly inhibits Bruton's tyrosine kinase (BTK), a PTK associated with B cell receptor and Toll-like receptor (TLR) signaling. However, BTK inhibition cannot account for the entire activity spectrum. Effects on TLR-induced proinflammatory cytokine expression in microglia involve AG126 hydrolysis and conversion of its dinitrile side chain to malononitrile (MN). Notably, while liberated MN can subsequently mediate critical AG126 features, full protection in EAE still requires delivery of intact AG126. Its anti-inflammatory potential and especially interference with TLR signaling thus rely on a dual mechanism encompassing BTK and a novel MN-sensitive target. Both principles bear great potential for the therapeutic management of disturbed innate and adaptive immune functions.
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http://dx.doi.org/10.1002/glia.22803DOI Listing
June 2015

Glucocorticoids induce effector T cell depolarization via ERM proteins, thereby impeding migration and APC conjugation.

J Immunol 2013 Apr 8;190(8):4360-70. Epub 2013 Mar 8.

Institute for Virology and Immunobiology, University of Würzburg, 97078 Würzburg, Germany.

Glucocorticoids (GCs) repress lymphocyte function by controlling gene expression. In this study, we investigated Ag-specific effector T cells and provide evidence that GCs also modulate these cells' cytoskeletal architecture by nongenomic mechanisms. Following GC treatment, effector T cells rapidly lose their polarized morphology, which impedes both their migratory capacity and their interaction with APCs. The cytoskeleton rearrangements are preceded by an activation of ezrin-radixin-moesin proteins, which transiently increases the cellular rigidity but seems to occur independently of altered tyrosine phosphorylation. Phospholipase C activity is critically involved in mediating these nongenomic effects, because its inhibition prevents both T cell depolarization and ezrin-radixin-moesin phosphorylation after GC exposure. GC administration in vivo induced similar morphological changes in effector T cells as observed in vitro, suggesting that the above process plays a role in modulating inflammatory diseases. Taken together, our findings identify a novel mechanism through which GCs rapidly repress T cell function independently of gene transcription.
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http://dx.doi.org/10.4049/jimmunol.1201520DOI Listing
April 2013

Haematopoietic stem cell survival and transplantation efficacy is limited by the BH3-only proteins Bim and Bmf.

EMBO Mol Med 2013 01 24;5(1):122-36. Epub 2012 Nov 24.

Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria.

Anti-apoptotic Bcl-2 family members are critical for the regulation of haematopoietic stem and progenitor cell (HSPC) survival. Little is known about the role of their pro-apoptotic antagonists, i.e. 'BH3-only' proteins, in this cell compartment. Based on the analysis of cytokine deprivation-induced changes in mRNA expression levels of Bcl-2 family proteins, we determined the consequences of BH3-only protein depletion on HSPC survival in culture and, for selected candidates, on engraftment in vivo. Thereby, we revealed a critical role for Bim and Bmf as regulators of HSPC dynamics both during early engraftment and long-term reconstitution. HSPCs derived from wild-type donors were readily displaced by Bim- or Bmf-deficient or Bcl-2-overexpressing HSPCs as early as 10 days after engraftment. Moreover, in the absence of Bim, significantly lower numbers of transplanted HSPCs were able to fully engraft radio-depleted recipients. Finally, we provide proof of principle that RNAi-based reduction of BIM or BMF, or overexpression of BCL-2 in human CD34(+) cord blood cells may be an attractive therapeutic option to increase stem cell survival and transplantation efficacy.
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http://dx.doi.org/10.1002/emmm.201201235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3569658PMC
January 2013

Necrosis-like death can engage multiple pro-apoptotic Bcl-2 protein family members.

Apoptosis 2012 Nov;17(11):1197-1209

Division of Developmental Immunology, Biocenter, Medical University Innsbruck, 6020 Innsbruck, Austria.

Necroptosis is a physiologically relevant mode of cell death with some well-described initiating events, but largely unknown executioners. Here we investigated necrostatin-1 (Nec-1) sensitive death elicited by different necroptosis stimuli in L929 mouse fibrosarcoma cells, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages. We found that TNFα- or zVAD-induced necroptosis occurs independently of the recently implicated executioners Bmf or PARP-2, but can involve the Bcl-2 family proteins Bid and Bak. Furthermore, this type of necroptosis is associated with mitochondrial cytochrome c release and partly sensitive to cyclosporine A inhibition, suggesting a cross talk with the mitochondrial permeability transition pore. Necroptosis triggered by cadmium (Cd) exposure caused fully Nec-1-sensitive and caspase-independent death in L929 cells that was associated with autocrine TNFα-mediated feed-forward signalling. In MEF Cd-exposure elicited a mixed mode of cell death that was to some extent Nec-1-sensitive but also displayed features of apoptosis. It was partly dependent on Bmf and Bax/Bak, proteins typically considered to act pro-apoptotic, but ultimately insensitive to caspase inhibition. Overall, our study indicates that inducers of "extrinsic" and "intrinsic" necroptosis can both trigger TNF-receptor signalling. Further, necroptosis may depend on mitochondrial changes engaging proteins considered critical for MOMP during apoptosis that ultimately contribute to caspase-independent necrotic cell death.
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http://dx.doi.org/10.1007/s10495-012-0756-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918797PMC
November 2012

PINCH-1 promotes Bcl-2-dependent survival signalling and inhibits JNK-mediated apoptosis in the primitive endoderm.

J Cell Sci 2012 Nov 3;125(Pt 21):5233-40. Epub 2012 Sep 3.

Max Planck Institute of Biochemistry, Department for Molecular Medicine, 82152 Martinsried, Germany.

The focal adhesion (FA) protein PINCH-1 is required for the survival of primitive endoderm (PrE) cells. How PINCH-1 regulates this fundamental process is not known. Here, we use embryoid bodies (EBs) and isolated EB-derived PrE cells to investigate the mechanisms by which PINCH-1 promotes PrE survival. We report that loss of PINCH-1 in PrE cells leads to a sustained activity of JNK and the pro-apoptotic factor Bax. Mechanistically, the sustained JNK activation was due to diminished levels of the JNK inhibitory factor Ras suppressor protein-1 (RSU-1), whose stability was severely reduced upon loss of PINCH-1. Chemical inhibition of JNK attenuated apoptosis of PrE cells but failed to reduce Bax activity. The increased Bax activity was associated with reduced integrin signalling and diminished Bcl-2 levels, which were shown to inhibit Bax. Altogether our findings show that PINCH-1 is a pro-survival factor that prevents apoptosis of PrE cells by modulating two independent signalling pathways; PINCH-1 inhibits JNK-mediated apoptosis by stabilising the PINCH-1 binding protein RSU-1 and promotes Bcl-2-dependent pro-survival signalling downstream of integrins.
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http://dx.doi.org/10.1242/jcs.112029DOI Listing
November 2012

Targeting antiapoptotic A1/Bfl-1 by in vivo RNAi reveals multiple roles in leukocyte development in mice.

Blood 2012 Jun 11;119(25):6032-42. Epub 2012 May 11.

Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innrain 80-82, Innsbruck, Austria.

Gene-targeting studies in mice have identified the essential roles of most prosurvival Bcl-2 family members in normal physiology and under conditions of stress. The function of one member, Bcl2a1/Bfl-1/A1, is only poorly understood because of quadruplication of its gene locus in mice, hindering conventional knockout studies. To overcome this problem, we generated mouse models allowing traceable constitutive or reversible ablation of A1 in the hematopoietic system by RNA interference. Knockdown of A1 impaired early stages of T-cell differentiation, B-cell homeostasis, and sensitized transitional as well as follicular B cells to apoptosis induced by ligation of the B-cell receptor. As a consequence, B-cell proliferation in response to mitogens was severely impaired, whereas that of T cells appeared unaffected. Furthermore, depending on the extent of A1 knockdown, granulocytes showed increased spontaneous death in culture or failed to accumulate in significant numbers in vivo. These models highlight the critical role of A1 in leukocyte development and homeostasis, constituting valuable tools for investigating presumed roles of this Bcl-2 family member in immunity, tumorigenesis, and drug resistance.
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http://dx.doi.org/10.1182/blood-2011-12-399089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418769PMC
June 2012

A1/Bfl-1 in leukocyte development and cell death.

Exp Cell Res 2012 Jul 4;318(11):1291-303. Epub 2012 Feb 4.

Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria.

The function of the anti-apoptotic Bcl-2 family member Bcl2a1/Bfl-1/A1 is poorly understood due to the lack of appropriate loss-of-function mouse models and redundant effects with other Bcl-2 pro-survival proteins upon overexpression. Expression analysis of A1 suggests predominant roles in leukocyte development, their survival upon viral or bacterial infection, as well as during allergic reactions. In addition, A1 has been implicated in autoimmunity and the pathology and therapy resistance of hematological as well as solid tumors that may aberrantly express this protein. In this review, we aim to summarize current knowledge on A1 biology, focusing on its role in the immune system and compare it to that of other pro-survival Bcl-2 proteins.
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http://dx.doi.org/10.1016/j.yexcr.2012.01.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405526PMC
July 2012

Defective cell death signalling along the Bcl-2 regulated apoptosis pathway compromises Treg cell development and limits their functionality in mice.

J Autoimmun 2012 Feb 17;38(1):59-69. Epub 2012 Jan 17.

Biocenter, Division of Developmental Immunology, Innsbruck Medical University, A-6020 Innsbruck, Austria.

The Bcl-2 regulated apoptosis pathway is critical for the elimination of autoreactive lymphocytes, thereby precluding autoimmunity. T cells escaping this process can be kept in check by regulatory T (Treg) cells expressing the transcription and lineage commitment factor Foxp3. Despite the well-established role of Bcl-2 family proteins in shaping the immune system and their frequent deregulation in autoimmune pathologies, it is poorly understood how these proteins affect Treg cell development and function. Here we compared the relative expression of a panel of 40 apoptosis-associated genes in Treg vs. conventional CD4(+) T cells. Physiological significance of key-changes was validated using gene-modified mice lacking or overexpressing pro- or anti-apoptotic Bcl-2 family members. We define a key role for the Bim/Bcl-2 axis in Treg cell development, homeostasis and function but exclude a role for apoptosis induction in responder T cells as relevant suppression mechanism. Notably, only lack of the pro-apoptotic BH3-only protein Bim or Bcl-2 overexpression led to accumulation of Treg cells while loss of pro-apoptotic Bad, Bmf, Puma or Noxa had no effect. Remarkably, apoptosis resistant Treg cells showed reduced suppressive capacity in a model of T cell-driven colitis, posing a caveat for the use of such long-lived cells in possible therapeutic settings.
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http://dx.doi.org/10.1016/j.jaut.2011.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314992PMC
February 2012

Acid sphingomyelinase is required for protection of effector memory T cells against glucocorticoid-induced cell death.

J Immunol 2011 Nov 23;187(9):4509-16. Epub 2011 Sep 23.

Department of Cellular and Molecular Immunology, University of Göttingen Medical School, Göttingen 37073, Germany.

The activity of acid sphingomyelinase (aSMase) was previously reported to be involved in glucocorticoid-induced cell death (GICD) of T lymphocytes. This mechanism in turn is believed to contribute to the therapeutic efficacy of glucocorticoids (GCs) in the treatment of inflammatory diseases. In this study, we reassessed the role of aSMase in GICD by using aSMase knockout mice. The absence of aSMase largely abolished the partial protection that effector memory CD4(+) T cells in wild-type mice possess against GICD. Reduced IL-2 secretion by aSMase-deficient CD4(+) T cells suggested that a lack of this important survival factor might be the cause of these cells' enhanced susceptibility to GICD. Indeed, addition of IL-2 restored the protection against GICD, whereas neutralization of IL-2 abrogated the otherwise protective effect seen in wild-type effector memory CD4(+) T cells. The therapeutic implications of the altered sensitivity of aSMase-deficient T cells to GICD were assessed in models of inflammatory disorders; namely, experimental autoimmune encephalomyelitis and acute graft-versus-host disease. Surprisingly, GC treatment was equally efficient in both models in terms of ameliorating the diseases, regardless of the genotype of the T cells. Thus, our data reveal a hitherto unrecognized contribution of aSMase to the sensitivity of effector memory CD4(+) T cells to GICD and call into question the traditionally attributed importance of GICD of T cells to the treatment of inflammatory diseases by GCs.
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http://dx.doi.org/10.4049/jimmunol.1100911DOI Listing
November 2011

Shaping the T-cell repertoire: a matter of life and death.

Immunol Cell Biol 2011 Jan 9;89(1):33-9. Epub 2010 Nov 9.

Division of Developmental Immunology, Biocenter, Medical University Innsbruck, Innsbruck, Austria.

Thymocyte selection aims to shape a T-cell repertoire that, on the one hand, is able to recognize and respond to foreign peptides and, on the other hand, tolerizes the presence of self-peptides in the periphery. Deletion of T cells or their precursors that fail to fulfill these criteria is mainly mediated by the Bcl-2-regulated apoptosis pathway. Absence of T-cell receptor (TCR)-mediated signals or hyperactivation of the TCR by high-affinity self-peptide-major histocompatibility complexes can both trigger apoptotic cell death in developing thymocytes. Notably, TCR-signaling strength also defines survival and outgrowth of the fittest antigen-specific T-cell clones in the periphery. TCR threshold activity leading to such drastically opposing signaling outcomes (life or death) is modulated in part by cytokines and other factors, such as glucocorticoids, that fine-tune the Bcl-2 rheostat, thereby impacting on cell survival. This review aims to highlight the role of Bcl-2-regulated cell death for clonal T-cell selection.
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http://dx.doi.org/10.1038/icb.2010.127DOI Listing
January 2011

Therapeutic and adverse effects of a non-steroidal glucocorticoid receptor ligand in a mouse model of multiple sclerosis.

PLoS One 2009 Dec 7;4(12):e8202. Epub 2009 Dec 7.

Institute for Multiple Sclerosis Research, University of Göttingen and Gemeinnützige Hertie-Stiftung, Göttingen, Germany.

Background: Dissociating glucocorticoid receptor (GR) ligands hold great promise for treating inflammatory disorders since it is assumed that they exert beneficial activities mediated by transrepression but avoid adverse effects of GR action requiring transactivation. Here we challenged this paradigm by investigating 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (CpdA), a dissociating non-steroidal GR ligand, in the context of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).

Methodology/principal Findings: CpdA inhibited pro-inflammatory mediators in myelin-specific T cells and fibroblasts in a GR-dependent manner while gene activation was abolished. However, it also induced massive apoptosis in various cell types even in the absence of the GR by engaging a Bcl-2- and caspase-dependent pathway. (1)H NMR spectroscopy corroborated these findings by revealing that CpdA dissolved in buffered solutions rapidly decomposes into aziridine intermediates known to act as alkylating pro-apoptotic agents. Importantly, the dichotomy of CpdA action also became evident in vivo. Administration of high-dose CpdA to mice was lethal while treatment of EAE with low to intermediate amounts of CpdA dissolved in water significantly ameliorated the disease. The beneficial effect of CpdA required expression of the GR in T cells and was achieved by down regulating LFA-1 and CD44 on peripheral Th cells and by repressing IL-17 production.

Conclusions/significance: CpdA has significant therapeutic potential although adverse effects severely compromise its application in vivo. Hence, non-steroidal GR ligands require careful analysis prior to their translation into new therapeutic concepts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008202PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781169PMC
December 2009

Stable silencing of the glucocorticoid receptor in myelin-specific T effector cells by retroviral delivery of shRNA: insight into neuroinflammatory disease.

Eur J Immunol 2009 Sep;39(9):2361-70

Department of Cellular and Molecular Immunology, University of Göttingen Medical School, Göttingen, Germany.

Autoimmune responses in the CNS can be induced by adoptive transfer of CD4(+) T effector cells after antigen-restimulation and expansion of clonal cell lines in vitro. However, pathogenic factors remain partially elusive due to the lack of appropriate methods to achieve gene inactivation. Here we describe a protocol for stable gene silencing in differentiated rat T cells by retroviral transfer of small hairpin RNAs. Through the combination of an expression cassette containing the green fluorescent protein with a puromycin selection cassette this allows for the generation of pure knockdown cell lines suitable for tracking in animals. Exemplified for the glucocorticoid receptor, we demonstrate that gene silencing renders T effector cells unresponsive to ligand-induced apoptosis and gene regulation without affecting their ability to induce EAE in rats. Interestingly, glucocorticoid administration remains effective in the treatment of EAE despite strongly diminished glucocorticoid receptor expression in antigen-specific T cells. This highlights an important role of other cell types and bystander T cells as targets of glucocorticoid therapy. Collectively, our approach provides a simple tool for stable and efficient gene silencing in T effector cells, which should help to better understand brain autoimmune pathophysiology.
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http://dx.doi.org/10.1002/eji.200939490DOI Listing
September 2009

Peripheral T cells are the therapeutic targets of glucocorticoids in experimental autoimmune encephalomyelitis.

J Immunol 2008 Jun;180(12):8434-43

Institute for Multiple Sclerosis Research, University of Göttingen and Gemeinnützige Hertie-Stiftung, Göttingen, Germany.

High-dose glucocorticoid (GC) therapy is widely used to treat multiple sclerosis (MS), but the underlying mechanisms remain debatable. In this study, we investigated the impact of GC administration on experimental autoimmune encephalomyelitis using different GC receptor (GR)-deficient mutants. Heterozygous GR knockout mice were less sensitive to dexamethasone therapy, indicating that the expression level of the receptor determines therapeutic efficacy. Mice reconstituted with homozygous GR knockout fetal liver cells showed an earlier onset of the disease and were largely refractory to GC treatment, indicating that the GR in hematopoietic cells is essential for the beneficial effects of endogenous GCs and dexamethasone. Using cell-type specific GR-deficient mice, we could demonstrate that GCs mainly act on T cells, while modulation of macrophage function was largely dispensable in this context. The therapeutic effects were achieved through induction of apoptosis and down-regulation of cell adhesion molecules in peripheral T(H)17 and bystander T cells, while similar effects were not observed within the spinal cord. In addition, dexamethasone inhibited T cell migration into the CNS, confirming that peripheral but not CNS-residing T lymphocytes are the essential targets of GCs. Collectively, our findings reveal a highly selective mechanism of GC action in experimental autoimmune encephalomyelitis and presumably multiple sclerosis.
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http://dx.doi.org/10.4049/jimmunol.180.12.8434DOI Listing
June 2008

A CD28 superagonistic antibody elicits 2 functionally distinct waves of T cell activation in rats.

J Clin Invest 2008 Apr;118(4):1405-16

Institute for Virology and Immunobiology, University of Wuerzburg, Wuerzburg, Germany.

Administration of the CD28 superagonistic antibody JJ316 is an efficient means to treat autoimmune diseases in rats, but the humanized antibody TGN1412 caused devastating side effects in healthy volunteers during a clinical trial. Here we show that JJ316 treatment of rats induced a dramatic redistribution of T lymphocytes from the periphery to the secondary lymphoid organs, resulting in severe T lymphopenia. Live imaging of secondary lymphoid organs revealed that JJ316 administration almost instantaneously (<2 minutes) arrested T cells in situ. This reduction in T cell motility was accompanied by profound cytoskeletal rearrangements and increased cell size. In addition, surface expression of lymphocyte function-associated antigen-1 was enhanced, endothelial differentiation sphingolipid G protein-coupled receptor 1 and L selectin levels were downregulated, and the cells lost their responsiveness to sphingosine 1-phosphate-directed migration. These proadhesive alterations were accompanied by signs of strong activation, including upregulation of CD25, CD69, CD134, and proinflammatory mediators. However, this did not lead to a cytokine storm similar to the clinical trial. While most of the early changes disappeared within 48 hours, we observed that CD4+CD25+FoxP3+ regulatory T cells experienced a second phase of activation, which resulted in massive cell enlargement, extensive polarization, and increased motility. These data suggest that CD28 superagonists elicit 2 qualitatively distinct waves of activation.
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http://dx.doi.org/10.1172/JCI32698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2269726PMC
April 2008

Glucocorticoids in the control of neuroinflammation.

Mol Cell Endocrinol 2007 Sep 4;275(1-2):62-70. Epub 2007 May 4.

University of Göttingen, Medical School, Department of Cellular and Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany.

Glucocorticoids are a class of steroid hormones that are endowed with profound anti-inflammatory and immunosuppressive activities. Endogenous glucocorticoids are key players in the modulation of the immune system and establish an endocrine basis of many inflammatory diseases. In addition, synthetic glucocorticoids are amongst the most commonly prescribed drugs worldwide for the treatment of autoimmune disorders. In this review we summarize our present knowledge on the mechanisms by which glucocorticoids impact on multiple sclerosis (MS), a highly prevalent neuroinflammatory disease, and its animal model experimental autoimmune encephalomyelitis (EAE). In spite of the new methodologies that have become available during recent years, we are still far from a comprehensive picture of the mechanism by which glucocorticoids control neuroinflammation.
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http://dx.doi.org/10.1016/j.mce.2007.03.007DOI Listing
September 2007

Enhanced glucocorticoid receptor signaling in T cells impacts thymocyte apoptosis and adaptive immune responses.

Am J Pathol 2007 Mar;170(3):1041-53

Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany.

To study the effect of enhanced glucocorticoid signaling on T cells, we generated transgenic rats overexpressing a mutant glucocorticoid receptor with increased ligand affinity in the thymus. We found that this caused massive thymocyte apoptosis at physiological hormone levels, which could be reversed by adrenalectomy. Due to homeostatic proliferation, a considerable number of mature T lymphocytes accumulated in the periphery, responding normally to costimulation but exhibiting a perturbed T-cell repertoire. Furthermore, the transgenic rats showed increased resistance to experimental autoimmune encephalomyelitis, which manifests in a delayed onset and milder disease course, impaired leukocyte infiltration into the central nervous system and a distinct cytokine profile. In contrast, the ability of the transgenic rats to mount an allergic airway response to ovalbumin was not compromised, although isotype switching of antigen-specific immunoglobulins was altered. Collectively, our findings suggest that endogenous glucocorticoids impact T-cell development and favor the selection of Th2- over Th1-dominated adaptive immune responses.
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http://dx.doi.org/10.2353/ajpath.2007.060804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1864890PMC
March 2007

Antigen therapy of experimental autoimmune encephalomyelitis selectively induces apoptosis of pathogenic T cells.

J Neuroimmunol 2007 Feb 2;183(1-2):146-50. Epub 2007 Jan 2.

Institute for Virology and Immunobiology, University of Würzburg, Versbacher Strasse 7, 97078 Würzburg, and Department of Neurology, St. Josef-Hospital, University of Bochum, Germany.

Administration of high-dose myelin antigen induces massive T cell apoptosis in experimental autoimmune encephalomyelitis (EAE) but the nature of the target cells remains elusive. Here we have used a cell line established in eGFP-transgenic Lewis rats to distinguish between pathogenic and bystander T cells in adoptive transfer EAE. Intravenous application of gpMBP strongly reduced the amount of encephalitogenic cells in spinal cord and spleen while the number of the other T cells remained constant. This could be attributed to their differential sensitivity to apoptosis. Thus, antigen therapy selectively targets pathogenic T cells and should therefore limit potential adverse effects.
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http://dx.doi.org/10.1016/j.jneuroim.2006.11.031DOI Listing
February 2007

Polyclonal expansion of regulatory T cells interferes with effector cell migration in a model of multiple sclerosis.

Brain 2006 Oct 18;129(Pt 10):2635-47. Epub 2006 Aug 18.

Institute for Virology and Immunobiology, University of Würzburg, Germany.

Recruitment of naturally occurring CD4+ CD25+ regulatory T (T(reg)) cells is a highly promising approach for the treatment of experimental autoimmune encephalomyelitis (EAE), a widely used model of multiple sclerosis. Here, we studied the in vivo interaction of T(reg) cells, induced by the monoclonal anti-CD28 antibody JJ316, with encephalitogenic T cell lines established from eGFP-transgenic rats. By tracking these fluorescent cells using flow cytometry and confocal microscopy, we found that the activation and expansion of T(reg) cells inhibited infiltration of the CNS by pathogenic T cells. Interference with effector cell migration occured within the secondary lymphoid organs, since the early therapeutic effects were achieved despite the absence of T(reg) cells in the spinal cord. However, the delayed homing to the CNS seen after prophylactic JJ316 administration indicates that T(reg) cells may play an additional role within the target tissue. In addition, the blood-brain barrier remained largely intact after JJ316 treatment, the secretion of T(H)2 cytokines was augmented and the encephalitogenic T cells exhibited a reduced secretion of IFN-gamma. This in turn resulted in a reduced expression of the chemokine receptor CXCR-3 on effector T cells which may interfere with their capacity to infiltrate the CNS. Importantly, these effects were not achieved by direct action of JJ316 on the encephalitogenic cells. Our data rather suggest that polyclonal activation of T(reg) cells in the secondary lymphoid organs is instrumental in preventing the pathological transmigration of encephalitogenic T cells into the CNS. We anticipate that these results may help to better understand the role of T(reg) cells in controlling autoimmunity in the CNS.
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http://dx.doi.org/10.1093/brain/awl213DOI Listing
October 2006