Henrike J. Fischer

Type 1 Diabetes in BioBreeding Rats Is Critically Linked to an Imbalance between Th17 and Regulatory T Cells and an Altered TCR Repertoire

Diabetes-prone BioBreeding (DP-BB) rats spontaneously develop type 1 diabetes mellitus (T1DM) on grounds of their MHC haplotype RT1u and a point mutation in the Gimap5 gene. In this study, we report that DP-BB rats exhibit an increasingly severe imbalance, in particular between Th17 and regulatory T (Treg) cells, within the first months of age. This can be assigned to an excess in effector T cells because neither the percentage nor the function of the Treg cells is compromised. Flow cytometric analysis of Vβ segment usage and CDR3 spectratyping further suggest that the disturbed repertoire of peripheral T cells may also contribute to the development of T1DM in DP-BB rats. Importantly, expansion of Treg cells in vivo by means of a CD28 superagonistic Ab as well as adoptive transfer of Treg cells efficiently interferes with the development of T1DM in DP-BB rats, whereas treatment with conventional Th cells does not afford protection. Using a newly generated strain of enhanced GFP transgenic rats, we could further demonstrate that the transferred Treg cells persist in the recipient rats for several months and partially correct the imbalance between Th17 and Treg cells. Thus, our data support the hypothesis that unchecked effector T cell action and a disturbed T cell repertoire contribute to the development of T1DM in DP-BB rats, which may also have implications for a better understanding of the human disease.

Chemokine-mediated redirection of T cells constitutes a critical mechanism of glucocorticoid therapy in autoimmune CNS responses

Glucocorticoids (GCs) are the standard therapy for treating multiple sclerosis (MS) patients suffering from an acute relapse. One of the main mechanisms of GC action is held to be the induction of T cell apoptosis leading to reduced lymphocyte infiltration into the CNS, yet our analysis of experimental autoimmune encephalomyelitis (EAE) in three different strains of genetically manipulated mice has revealed that the induction of T cell apoptosis is not essential for the therapeutic efficacy of GCs. Instead, we identified the redirection of T cell migration in response to chemokines as a new therapeutic principle of GC action. GCs inhibited the migration of T cells towards CCL19 while they enhanced their responsiveness towards CXCL12. Importantly, blocking CXCR4 signaling in vivo by applying Plerixafor® strongly impaired the capacity of GCs to interfere with EAE, as revealed by an aggravated disease course, more pronounced CNS infiltration and a more dispersed distribution of the infiltrating T cells throughout the parenchyma. Our observation that T cells lacking the GC receptor were refractory to CXCL12 further underscores the importance of this pathway for the treatment of EAE by GCs. Importantly, methylprednisolone pulse therapy strongly increased the capacity of peripheral blood T cells from MS patients of different subtypes to migrate towards CXCL12. This indicates that modulation of T cell migration is an important mechanistic principle responsible for the efficacy of high-dose GC therapy not only of EAE but also of MS.

The potential role of T cell migration and chemotaxis as targets of glucocorticoids in multiple sclerosis and experimental autoimmune encephalomyelitis.

Glucocorticoids (GCs) are the most commonly prescribed drugs for the treatment of acute disease bouts in multiple sclerosis (MS) patients. While T lymphocytes were shown to be essential targets of GC therapy, at least in animal models of MS, the mechanisms by which GCs modulate T cell function are less clear. Until now, apoptosis induction and repression of pro-inflammatory cytokines in T cells have been considered the most critical mechanisms in ameliorating disease symptoms. However, this notion is being challenged by increasing evidence that the control of T cell migration and chemotaxis by GCs might be even more important for the treatment of neuroinflammatory diseases. In this review we aim to provide an overview of how GCs impact the morphological alterations that T cells undergo during activation and migration as well as the influences that GCs have on the directed movement of T cells under the influence of chemokines. A deeper understanding of these processes should not only help to advance our understanding of how GCs exert their beneficial effects in MS therapy but may reveal future strategies to intervene in the pathogenesis of neuroinflammatory diseases.

Glucocorticoids Induce Effector T Cell Depolarization via ERM Proteins, Thereby Impeding Migration and APC Conjugation

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.

The Insulin Receptor Plays a Critical Role in T Cell Function and Adaptive Immunity

T cell activation is an energy-demanding process fueled by increased glucose consumption and accompanied by upregulation of the insulin receptor (INSR). In this article, we report that silencing the INSR in inducible knockdown rats impairs selective T cell functions but not thymocyte development. Glucose transport and glycolysis in activated CD4+ T cells were compromised in the absence of the INSR, which was associated with alterations in intracellular signaling pathways. The observed metabolic defects coincided with reduced cytokine production, proliferation, and migration, as well as increased apoptosis of CD4+ T cells. The cytotoxicity of CD8+ T cells in response to alloantigens was also diminished under these conditions, whereas the frequency and suppressive capacity of regulatory T cells were unaffected. The observed impairments proved to be decisive in vivo because silencing of the INSR attenuated clinical symptoms in animal models of acute graft-versus-host disease and multiple sclerosis. Taken together, our results suggest that upregulation of the INSR on T cells following activation is required for efficient adaptive immunity.

Erythropoietin Responsive Cardiomyogenic Cells Contribute to Heart Repair Post Myocardial Infarction

The role of erythropoietin (Epo) in myocardial repair after infarction remains inconclusive. We observed high Epo receptor (EPOR) expression in cardiac progenitor cells (CPCs). Therefore, we aimed to characterize these cells and elucidate their contribution to myocardial regeneration on Epo stimulation. High EPOR expression was detected during murine embryonic heart development followed by a marked decrease until adulthood. EPOR‐positive cells in the adult heart were identified in a CPC‐enriched cell population and showed coexpression of stem, mesenchymal, endothelial, and cardiomyogenic cell markers. We focused on the population coexpressing early (TBX5, NKX2.5) and definitive (myosin heavy chain [MHC], cardiac Troponin T [cTNT]) cardiomyocyte markers. Epo increased their proliferation and thus were designated as Epo‐responsive MHC expressing cells (EMCs). In vitro, EMCs proliferated and partially differentiated toward cardiomyocyte‐like cells. Repetitive Epo administration in mice with myocardial infarction (cumulative dose 4 IU/g) resulted in an increase in cardiac EMCs and cTNT‐positive cells in the infarcted area. This was further accompanied by a significant preservation of cardiac function when compared with control mice. Our study characterized an EPO‐responsive MHC‐expressing cell population in the adult heart. Repetitive, moderate‐dose Epo treatment enhanced the proliferation of EMCs resulting in preservation of post‐ischemic cardiac function. Stem Cells 2014;32:2480–2491

CD8+ T cell help is required for efficient induction of EAE in Lewis rats

The role of CD8⁺ T cells in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is still unclear. We describe here significantly reduced disease activity of EAE both in Lewis rats depleted of CD8⁺ T cells by monoclonal antibodies and CD8 knockout rats, which was accompanied by reduced leukocyte infiltration into the spinal cord. We detected myelin basic protein (MBP)-specific CD8⁺ T cells in peripheral lymphoid organs of CD8-depleted animals which, however, failed to differentiate into interferon-γ-producing effector cells. Our results indicate that CD8⁺ T cells interact with myelin-specific CD8⁺ T cells early in EAE enabling them to differentiate into pathogenic effector cells.

Deletion of the Mineralocorticoid Receptor in Myeloid Cells Attenuates Central Nervous System Autoimmunity

Myeloid cells play an important role in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Monocytes, macrophages, and microglia can adopt two distinct phenotypes, with M1-polarized cells being more related to inflammation and autoimmunity while M2-polarized cells contribute to tissue repair and anti-inflammatory processes. Here, we show that deletion of the mineralocorticoid receptor (MR) in bone marrow-derived macrophages and peritoneal macrophages caused their polarization toward the M2 phenotype with its distinct gene expression, altered phagocytic and migratory properties, and dampened NO production. After induction of EAE, mice that are selectively devoid of the MR in their myeloid cells (MRlysM mice) showed diminished clinical symptoms and ameliorated histological hallmarks of neuroinflammation. T cells in peripheral lymphoid organs of these mice produced less pro-inflammatory cytokines while their proliferation and the abundance of regulatory T cells were unaltered. The numbers of inflammatory monocytes and reactive microglia in the central nervous system (CNS) in MRlysM mice were significantly lower and they adopted an M2-polarized phenotype based on their gene expression profile, presumably explaining the ameliorated neuroinflammation. Our results indicate that the MR in myeloid cells plays a critical role for CNS autoimmunity, providing a rational to interfere with diseases such as MS by pharmacologically targeting this receptor.

Inducible Knock-Down of the Mineralocorticoid Receptor in Mice Disturbs Regulation of the Renin-Angiotensin-Aldosterone System and Attenuates Heart Failure Induced by Pressure Overload

Mineralocorticoid receptor (MR) inactivation in mice results in early postnatal lethality. Therefore we generated mice in which MR expression can be silenced during adulthood by administration of doxycycline (Dox). Using a lentiviral approach, we obtained two lines of transgenic mice harboring a construct that allows for regulatable MR inactivation by RNAi and concomitant expression of eGFP. MR mRNA levels in heart and kidney of inducible MR knock-down mice were unaltered in the absence of Dox, confirming the tightness of the system. In contrast, two weeks after Dox administration MR expression was significantly diminished in a variety of tissues. In the kidney, this resulted in lower mRNA levels of selected target genes, which was accompanied by strongly increased serum aldosterone and plasma renin levels as well as by elevated sodium excretion. In the healthy heart, gene expression and the amount of collagen were unchanged despite MR levels being significantly reduced. After transverse aortic constriction, however, cardiac hypertrophy and progressive heart failure were attenuated by MR silencing, fibrosis was unaffected and mRNA levels of a subset of genes reduced. Taken together, we believe that this mouse model is a useful tool to investigate the role of the MR in pathophysiological processes.

Targeted delivery of glucocorticoids to macrophages in a mouse model of multiple sclerosis using inorganic-organic hybrid nanoparticles

&NA; Glucocorticoids (GC) are widely used to treat acute relapses in multiple sclerosis (MS) patients, but their application is accompanied by side effects due to their broad spectrum of action. Here, we report on the therapeutic option to apply GC via inorganic‐organic hybrid nanoparticles (IOH‐NP) with the composition [ZrO]2+[(BMP)0.9(FMN)0.1]2− (designated BMP‐NP with BMP: betamethasone phosphate; FMN: flavinmononucleotide). We found that these BMP‐NP have an increased cell type‐specificity compared to free GC while retaining full therapeutic efficacy in a mouse model of MS. BMP‐NP were preferentially taken up by phagocytic cells and modulated macrophages in vivo more efficiently than T cells. When GC were applied in the form of BMP‐NP, treatment of neuroinflammatory disease in mice exclusively depended on the control of macrophage function whereas effects on T cells and brain endothelial cells were dispensable for therapeutic efficacy. Importantly, BMP‐NP were not only active in mice but also showed strong activity towards monocytes isolated from healthy human volunteers. We conclude that application of GC via IOH‐NP has the potential to improve MS therapy in the future. Graphical abstract Figure. No caption available.