ß-Synuclein-reactive T cells induce autoimmune CNS grey matter degeneration.

The grey matter is a central target of pathological processes in neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. The grey matter is often also affected in multiple sclerosis, an autoimmune disease of the central nervous system. The mechanisms that underlie grey matter inflammation and degeneration in multiple sclerosis are not well understood. Here we show that, in Lewis rats, T cells directed against the neuronal protein ß-synuclein specifically invade the grey matter and that this is accompanied by the presentation of multifaceted clinical disease. The expression pattern of ß-synuclein induces the local activation of these T cells and, therefore, determined inflammatory priming of the tissue and targeted recruitment of immune cells. The resulting inflammation led to significant changes in the grey matter, which ranged from gliosis and neuronal destruction to brain atrophy. In humans, ß-synuclein-specific T cells were enriched in patients with chronic-progressive multiple sclerosis. These findings reveal a previously unrecognized role of ß-synuclein in provoking T-cell-mediated pathology of the central nervous system.

Publisher Correction: ß-Synuclein-reactive T cells induce autoimmune CNS grey matter degeneration.

In this Article, owing to an error during the production process, the y-axis label of Fig. 2c should read "Number of Tß-syn cells" rather than "Number of T1ß-syn cells" and the left and right panels of Fig. 4 should be labelled 'a' and 'b', respectively. These errors have been corrected online.

Glucocorticoid Nanoparticles Show Full Therapeutic Efficacy in a Mouse Model of Acute Lung Injury and Concomitantly Reduce Adverse Effects.

Glucocorticoids (GCs) are widely used to treat inflammatory disorders such as acute lung injury (ALI). Here, we explored inorganic-organic hybrid nanoparticles (IOH-NPs) as a new delivery vehicle for GCs in a mouse model of ALI. Betamethasone (BMZ) encapsulated into IOH-NPs (BNPs) ameliorated the massive infiltration of neutrophils into the airways with a similar efficacy as the free drug. This was accompanied by a potent inhibition of pulmonary gene expression and secretion of pro-inflammatory mediators, whereas the alveolar-capillary barrier integrity was only restored by BMZ in its traditional form. Experiments with genetically engineered mice identified myeloid cells and alveolar type II (AT II) cells as essential targets of BNPs in ALI therapy, confirming their high cell-type specificity. Consequently, adverse effects were reduced when using IOH-NPs for GC delivery. BNPs did not alter T and B cell numbers in the blood and also prevented the induction of muscle atrophy after three days of treatment. Collectively, our data suggest that IOH-NPs target GCs to myeloid and AT II cells, resulting in full therapeutic efficacy in the treatment of ALI while being associated with reduced adverse effects.

Glucocorticoids delivered by inorganic-organic hybrid nanoparticles mitigate acute graft-versus-host disease and sustain graft-versus-leukemia activity.

Glucocorticoids (GCs) are widely used to treat acute graft-versus-host disease (aGvHD) due to their immunosuppressive activity, but they also reduce the beneficial graft-versus-leukemia (GvL) effect of the allogeneic T cells contained in the graft. Here, we tested whether aGvHD therapy could be improved by delivering GCs with the help of inorganic-organic hybrid nanoparticles (IOH-NPs) that preferentially target myeloid cells. IOH-NPs containing the GC betamethasone (BMP-NPs) efficiently reduced morbidity, mortality, and tissue damage in a totally MHC mismatched mouse model of aGvHD. Therapeutic activity was lost in mice lacking the GC receptor (GR) in myeloid cells, confirming the cell type specificity of our approach. BMP-NPs had no relevant systemic activity but suppressed cytokine and chemokine gene expression locally in the small intestine, which presumably explains their mode of action. Most importantly, BMP-NPs delayed the development of an adoptively transferred B cell lymphoma better than the free drug, although the overall incidence was unaffected. Our findings thus suggest that employing IOH-NPs could diminish the risk of relapse associated with GC therapy of aGvHD patients while still allowing to efficiently ameliorate the disease.

Glucocorticoid receptor in T cells mediates protection from autoimmunity in pregnancy.

Pregnancy is one of the strongest inducers of immunological tolerance. Disease activity of many autoimmune diseases including multiple sclerosis (MS) is temporarily suppressed by pregnancy, but little is known about the underlying molecular mechanisms. Here, we investigated the endocrine regulation of conventional and regulatory T cells (Tregs) during reproduction. In vitro, we found the pregnancy hormone progesterone to robustly increase Treg frequencies via promiscuous binding to the glucocorticoid receptor (GR) in T cells. In vivo, T-cell-specific GR deletion in pregnant animals undergoing experimental autoimmune encephalomyelitis (EAE), the animal model of MS, resulted in a reduced Treg increase and a selective loss of pregnancy-induced protection, whereas reproductive success was unaffected. Our data imply that steroid hormones can shift the immunological balance in favor of Tregs via differential engagement of the GR in T cells. This newly defined mechanism confers protection from autoimmunity during pregnancy and represents a potential target for future therapy.

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.

Airway Epithelial Cells Are Crucial Targets of Glucocorticoids in a Mouse Model of Allergic Asthma.

Although glucocorticoids (GCs) are a mainstay in the clinical management of asthma, the target cells that mediate their therapeutic effects are unknown. Contrary to our expectation, we found that GC receptor (GR) expression in immune cells was dispensable for successful therapy of allergic airway inflammation (AAI) with dexamethasone. Instead, GC treatment was compromised in mice expressing a defective GR in the nonhematopoietic compartment or selectively lacking the GR in airway epithelial cells. Further, we found that an intact GR dimerization interface was a prerequisite for the suppression of AAI and airway hyperresponsiveness by GCs. Our observation that the ability of dexamethasone to modulate gene expression in airway epithelial cells coincided with its potency to resolve AAI supports a crucial role for transcriptional regulation by the GR in this cell type. Taken together, we identified an unknown mode of GC action in the treatment of allergic asthma that might help to develop more specific therapies in the future.

An alternative for extracorporeal photopheresis: 8-methoxypsoralen and UVA-treated leucocytes from allogeneic donors improve graft-versus-host disease in mice.

BACKGROUND AND OBJECTIVE: Extracorporeal photopheresis (ECP) is an important immune tolerance inducing therapy for graft-versus-host disease (GvHD). However, a sufficient number of ECP cycles cannot be performed in patients with severe GvHD and contraindications for apheresis. Allogeneic sources of leucocytes for use as ECP treatment would be of great benefit. Therefore, this study aimed to test the therapeutic potential of novel sources of leucocytes for ECP. MATERIALS AND METHODS: Graft-versus-host disease mice were treated with ECP using therapeutic cells from different allogeneic sources. Splenocytes were incubated with 8-methoxypsoralen (8-MOP), irradiated with UVA light and injected into GvHD mice as a model for ECP. RESULTS: The therapy with 8-MOP/UVA-treated cells from healthy mice of the bone marrow transplantation (BMT) donor strain reduced the GvHD symptoms, at least in a model of chronic GvHD. In the acute GvHD model, 8-MOP/UVA-treated cells from the BMT donor or recipient strain did not show significant improvements in GvHD symptoms or survival time. Pre-activation of cells by mixed lymphocyte reactions before 8-MOP/UVA treatment also failed to result in significant differences in survival time or GvHD score. In contrast, ECP with third-party 8-MOP/UVA-treated cells from a HLA-mismatched donor resulted in a mean survival time of 37 days compared to 21 days in the control group. CONCLUSION: In our analysis of novel allogeneic leucocyte sources for ECP, we could demonstrate that the source of the 8-MOP/UVA-treated cells is crucial. The underlying immunologic effect of allogeneic 8-MOP/UVA-treated cells needs to be investigated in future studies.

Distinct roles of T-cell lymphopenia and the microbial flora for gastrointestinal and CNS autoimmunity.

T-cell lymphopenia is a major risk factor for autoimmunity. Here we describe congenic Lewis (LEW) rats with a loss-of-function mutation in the Gimap5 gene, leading to a 92% reduction in peripheral T-cell numbers. Gimap5-deficient LEW rats developed eosinophilic autoimmune gastroenteritis accompanied by a 40-fold increase in IgE serum levels. This phenotype was ameliorated by antibiotic treatment, indicating a critical role of the microbial flora in the development of inflammatory bowel disease. Interestingly, Gimap5-deficient LEW rats showed strongly aggravated experimental autoimmune encephalomyelitis (EAE) after immunization with guinea pig myelin basic protein. This phenotype, however, persisted after antibiosis, confirming that the enhanced CNS autoimmune response in T-cell lymphopenic Gimap5-deficient LEW rats was unrelated to the composition of the microbial flora. Rather, it seems that it was caused by the 7-fold increase in the percentage of activated T cells producing IL-17 and IFN-γ, and the skewed T-cell receptor (TCR) repertoire, both of which were the result of T-cell lymphopenia and not affected by antibiosis. This notion was supported by the observation that adoptive T-cell transfer corrected the TCR repertoire and improved EAE. Collectively, our findings confirm a critical albeit differential role of T-cell lymphopenia in the susceptibility to organ-specific autoimmune responses.-Fischer, H. J., Witte, A.-K., Walter, L., Gröne, H.-J., van den Brandt, J., Reichardt, H. M. Distinct roles of T-cell lymphopenia and the microbial flora for gastrointestinal and CNS autoimmunity.

Prediction of graft-versus-host disease: a biomarker panel based on lymphocytes and cytokines.

Graft-versus-host disease (GvHD) still belongs to the major challenges after allogeneic hematopoietic stem cell transplantation (HSCT). Immune-suppressive therapy against GvHD is a double-edged sword due to risk of infections and relapse. The ability to adapt prophylactic treatment according to the probability of severe GvHD would be an essential advantage for the patients. We analyzed different biomarkers for their potential to predict the development of GvHD in 28 patients who underwent allogeneic HSCT. Blood was taken once directly after hematopoietic engraftment. In this study, patients were monitored for 12 months after HSCT for the occurrence of acute GvHD or acute/chronic GvHD overlap syndrome. Soluble IL-2 receptor and CD4/CD8 T cell ratio were independently associated with the occurrence of GvHD in the observation period. However, the largest area under the receiver operating characteristic curve with 0.90 was observed when a 5-parameter biomarker score based on CD4+ T cells, CD8+ T cells, CD19- CD21+ precursor B cells, CD4/CD8 T cell ratio, and soluble IL-2 receptor was used to predict GvHD. In addition, CD8+ T cell levels above 2.3% of all mononuclear cells after engraftment may predict relapse-free survival at least for 12 months. In summary, we found a new biomarker panel for prediction of GvHD which is featured by established laboratory assays and high statistical significance. In order to introduce the biomarker panel into routine clinical protocols, we suggest performing a larger multi-center study.

Novel Drug Delivery Systems Tailored for Improved Administration of Glucocorticoids.

Glucocorticoids (GC) are one of the most popular and versatile classes of drugs available to treat chronic inflammation and cancer, but side effects and resistance constrain their use. To overcome these hurdles, which are often related to the uniform tissue distribution of free GC and their short half-life in biological fluids, new delivery vehicles have been developed including PEGylated liposomes, polymeric micelles, polymer-drug conjugates, inorganic scaffolds, and hybrid nanoparticles. While each of these nanoformulations has individual drawbacks, they are often superior to free GC in many aspects including therapeutic efficacy when tested in cell culture or animal models. Successful application of nanomedicines has been demonstrated in various models of neuroinflammatory diseases, cancer, rheumatoid arthritis, and several other disorders. Moreover, investigations using human cells and first clinical trials raise the hope that the new delivery vehicles may have the potential to make GC therapies more tolerable, specific and efficient in the future.

Thymocyte-derived BDNF influences T-cell maturation at the DN3/DN4 transition stage.

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival, regeneration, and plasticity. Emerging evidence also indicates an essential role for BDNF outside the nervous system, for instance in immune cells. We therefore investigated the impact of BDNF on T cells using BDNF knockout (KO) mice and conditional KO mice lacking BDNF specifically in this lymphoid subset. In both settings, we observed diminished T-cell cellularity in peripheral lymphoid organs and an increase in CD4(+) CD44(+) memory T cells. Analysis of thymocyte development revealed diminished total thymocyte numbers, accompanied by a significant increase in CD4/CD8 double-negative (DN) thymocytes due to a partial block in the transition from the DN3 to the DN4 stage. This was neither due to increased thymocyte apoptosis nor defects in the expression of the TCR-ß chain or the pre-TCR. In contrast, pERK but not pAKT levels were diminished in DN3 BDNF-deficient thymocytes. BDNF deficiency in T cells did not result in gross deficits in peripheral acute immune responses nor in changes of the homeostatic proliferation of peripheral T cells. Taken together, our data reveal a critical autocrine and/or paracrine role of T-cell-derived BDNF in thymocyte maturation involving ERK-mediated TCR signaling pathways.

Glucocorticoids attenuate acute graft-versus-host disease by suppressing the cytotoxic capacity of CD8(+) T cells.

Glucocorticoids (GCs) are released from the adrenal gland during inflammation and help to keep immune responses at bay. Owing to their potent anti-inflammatory activity, GCs also play a key role in controlling acute graft-versus-host disease (aGvHD). Here we demonstrate that mice lacking the glucocorticoid receptor (GR) in T cells develop fulminant disease after allogeneic bone marrow transplantation. In a fully MHC-mismatched model, transfer of GR-deficient T cells resulted in severe aGvHD symptoms and strongly decreased survival times. Histopathological features were aggravated and infiltration of CD8(+) T cells into the jejunum was increased when the GR was not expressed. Furthermore, serum levels of IL-2, IFNγ, and IL-17 were elevated and the cytotoxicity of CD8(+) T cells was enhanced after transfer of GR-deficient T cells. Short-term treatment with dexamethasone reduced cytokine secretion but neither impacted disease severity nor the CTLs' cytolytic capacity. Importantly, in an aGvHD model in which disease development exclusively depends on the presence of CD8(+) T cells in the transplant, transfer of GR-deficient T cells aggravated clinical symptoms and reduced survival times as well. Taken together, our findings highlight that suppression of CD8(+) T-cell function is a crucial mechanism in the control of aGvHD by endogenous GCs.

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

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.

Multifunctional phosphate-based inorganic-organic hybrid nanoparticles.

Phosphate-based inorganic-organic hybrid nanoparticles (IOH-NPs) with the general composition [M](2+)[Rfunction(O)PO3](2-) (M = ZrO, Mg2O; R = functional organic group) show multipurpose and multifunctional properties. If [Rfunction(O)PO3](2-) is a fluorescent dye anion ([RdyeOPO3](2-)), the IOH-NPs show blue, green, red, and near-infrared fluorescence. This is shown for [ZrO](2+)[PUP](2-), [ZrO](2+)[MFP](2-), [ZrO](2+)[RRP](2-), and [ZrO](2+)[DUT](2-) (PUP = phenylumbelliferon phosphate, MFP = methylfluorescein phosphate, RRP = resorufin phosphate, DUT = Dyomics-647 uridine triphosphate). With pharmaceutical agents as functional anions ([RdrugOPO3](2-)), drug transport and release of anti-inflammatory ([ZrO](2+)[BMP](2-)) and antitumor agents ([ZrO](2+)[FdUMP](2-)) with an up to 80% load of active drug is possible (BMP = betamethason phosphate, FdUMP = 5'-fluoro-2'-deoxyuridine 5'-monophosphate). A combination of fluorescent dye and drug anions is possible as well and shown for [ZrO](2+)[BMP](2-)0.996[DUT](2-)0.004. Merging of functional anions, in general, results in [ZrO](2+)([RdrugOPO3]1-x[RdyeOPO3]x)(2-) nanoparticles and is highly relevant for theranostics. Amine-based functional anions in [MgO](2+)[RaminePO3](2-) IOH-NPs, finally, show CO2 sorption (up to 180 mg g(-1)) and can be used for CO2/N2 separation (selectivity up to α = 23). This includes aminomethyl phosphonate [AMP](2-), 1-aminoethyl phosphonate [1AEP](2-), 2-aminoethyl phosphonate [2AEP](2-), aminopropyl phosphonate [APP](2-), and aminobutyl phosphonate [ABP](2-). All [M](2+)[Rfunction(O)PO3](2-) IOH-NPs are prepared via noncomplex synthesis in water, which facilitates practical handling and which is optimal for biomedical application. In sum, all IOH-NPs have very similar chemical compositions but can address a variety of different functions, including fluorescence, drug delivery, and CO2 sorption.

High-grade acute organ toxicity and p16(INK4A) expression as positive prognostic factors in primary radio(chemo)therapy for patients with head and neck squamous cell carcinoma.

BACKGROUND: Superior treatment response and survival for patients with human papilloma virus (HPV)-positive head and neck cancer (HNSCC) are documented in clinical studies. However, the relevance of high-grade acute organ toxicity (HGAOT), which has also been correlated with improved prognosis, has attracted scant attention in HPV-positive HNSCC patients. Hence we tested the hypothesis that both parameters, HPV and HGAOT, are positive prognostic factors in patients with HNSCC treated with definite radiotherapy (RT) or radiochemotherapy (RCT). PATIENTS AND METHODS: Pretreatment tumor tissue and clinical records were available from 233 patients receiving definite RT (62 patients) or RCT (171 patients). HPV infection was analysed by means of HPV DNA detection or p16(INK4A) expression; HGAOT was defined as the occurrence of acute organ toxicity >grade 2 according to the Common Toxicity Criteria. Both variables were correlated with overall survival (OS) using Cox proportional hazards regression. RESULTS: Positivity for HPV DNA (44 samples, 18.9 %) and p16(INK4A) expression (102 samples, 43.8 %) were significantly correlated (p < 0.01), and HGAOT occurred in 77 (33 %) patients. Overall, the 5-year OS was 23 %; stratified for p16(INK4A) expression and HGAOT, OS rates were 47 %, 42 %, 20 % and 10 % for patients with p16(INK4A) expression and HGAOT, patients with HGAOT only, patients with p16(INK4A) expression only, and patients without p16(INK4A) expression or HGAOT, respectively. After multivariate testing p16(INK4A) expression (p = 0.003) and HGAOT (p < 0.001) were significantly associated with OS. CONCLUSION: P16(INK4A) expression and HGAOT are independent prognostic factors for OS of patients with HNSCC, whereas p16(INK4A) expression is particularly important for patients without HGAOT.

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.

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.