Microbiota-dependent activation of the myeloid calcineurin-NFAT pathway inhibits B7H3- and B7H4-dependent anti-tumor immunity in colorectal cancer.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8+ T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8+ T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8+ T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8+ T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC.

Cross-reactivity of a pathogenic autoantibody to a tumor antigen in GABAA receptor encephalitis.

Encephalitis associated with antibodies against the neuronal gamma-aminobutyric acid A receptor (GABAA-R) is a rare form of autoimmune encephalitis. The pathogenesis is still unknown but autoimmune mechanisms were surmised. Here we identified a strongly expanded B cell clone in the cerebrospinal fluid of a patient with GABAA-R encephalitis. We expressed the antibody produced by it and showed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry that it recognizes the GABAA-R. Patch-clamp recordings revealed that it tones down inhibitory synaptic transmission and causes increased excitability of hippocampal CA1 pyramidal neurons. Thus, the antibody likely contributed to clinical disease symptoms. Hybridization to a protein array revealed the cross-reactive protein LIM-domain-only protein 5 (LMO5), which is related to cell-cycle regulation and tumor growth. We confirmed LMO5 recognition by immunoprecipitation and ELISA and showed that cerebrospinal fluid samples from two other patients with GABAA-R encephalitis also recognized LMO5. This suggests that cross-reactivity between GABAA-R and LMO5 is frequent in GABAA-R encephalitis and supports the hypothesis of a paraneoplastic etiology.

Validation of TREK1 ion channel activators as an immunomodulatory and neuroprotective strategy in neuroinflammation.

Modulation of two-pore domain potassium (K2P) channels has emerged as a novel field of therapeutic strategies as they may regulate immune cell activation and metabolism, inflammatory signals, or barrier integrity. One of these ion channels is the TWIK-related potassium channel 1 (TREK1). In the current study, we report the identification and validation of new TREK1 activators. Firstly, we used a modified potassium ion channel assay to perform high-throughput-screening of new TREK1 activators. Dose-response studies helped to identify compounds with a high separation between effectiveness and toxicity. Inside-out patch-clamp measurements of Xenopus laevis oocytes expressing TREK1 were used for further validation of these activators regarding specificity and activity. These approaches yielded three substances, E1, B3 and A2 that robustly activate TREK1. Functionally, we demonstrated that these compounds reduce levels of adhesion molecules on primary human brain and muscle endothelial cells without affecting cell viability. Finally, we studied compound A2 via voltage-clamp recordings as this activator displayed the strongest effect on adhesion molecules. Interestingly, A2 lacked TREK1 activation in the tested neuronal cell type. Taken together, this study provides data on novel TREK1 activators that might be employed to pharmacologically modulate TREK1 activity.

Activation of non-classical NMDA receptors by glycine impairs barrier function of brain endothelial cells.

Blood-brain barrier (BBB) integrity is necessary to maintain homeostasis of the central nervous system (CNS). NMDA receptor (NMDAR) function and expression have been implicated in BBB integrity. However, as evidenced in neuroinflammatory conditions, BBB disruption contributes to immune cell infiltration and propagation of inflammatory pathways. Currently, our understanding of the pathophysiological role of NMDAR signaling on endothelial cells remains incomplete. Thus, we investigated NMDAR function on primary mouse brain microvascular endothelial cells (MBMECs). We detected glycine-responsive NMDAR channels, composed of functional GluN1, GluN2A and GluN3A subunits. Importantly, application of glycine alone, but not glutamate, was sufficient to induce NMDAR-mediated currents and an increase in intracellular Ca2+ concentrations. Functionally, glycine-mediated NMDAR activation leads to loss of BBB integrity and changes in actin distribution. Treatment of oocytes that express NMDARs composed of different subunits, with GluN1 and GluN3A binding site inhibitors, resulted in abrogation of NMDAR signaling as measured by two-electrode voltage clamp (TEVC). This effect was only detected in the presence of the GluN2A subunits, suggesting the latter as prerequisite for pharmacological modulation of NMDARs on brain endothelial cells. Taken together, our findings argue for a novel role of glycine as NMDAR ligand on endothelial cells shaping BBB integrity.

Spillover and crossover effects of exposure to work-related aggression and adversities: A dyadic diary study.

The past two decades have produced extensive evidence on the manifold and severe outcomes for victims of aggression exposure in the workplace. However, due to the dominating individual-centered approach, most findings miss a social network perspective. Consequently, knowledge of negative spillover to different life-domains or crossover to uninvolved individuals alongside a detailed understanding of the involved transmission processes remains scarce. By integrating social aggression theorizing, the present study investigated transmission routes (emphatic, behavioral) of experienced adversities and aggression at work toward perpetration of aggressive behavior and potential spillover and crossover effects into the private life domain in a diary study of 72 mixed dyads. Analyses of mediation based upon the Actor-Partner Interdependence Model revealed an association between the frequency of perpetrating aggressive behavior in the work context and a spillover into the private life domain via aggression-promoting internal states (emotions, cognitions, arousal). Based on the different patterns of mediation, it appears that adversities follow a mental transmission process, whereby experienced aggression displayed behavioral assimilation. In contrast, no crossover effects of exposure to adversities or aggression at work to a study partner at home could be detected. Practical and theoretical implications as well as limitations and ideas for future work are discussed.

Cladribine treatment improves cortical network functionality in a mouse model of autoimmune encephalomyelitis.

BACKGROUND: Cladribine is a synthetic purine analogue that interferes with DNA synthesis and repair next to disrupting cellular proliferation in actively dividing lymphocytes. The compound is approved for the treatment of multiple sclerosis (MS). Cladribine can cross the blood-brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. Here, we explored compartment-specific immunosuppressive as well as potential direct neuroprotective effects of oral cladribine treatment in experimental autoimmune encephalomyelitis (EAE) mice. METHODS: In the current study, we compare immune cell frequencies and phenotypes in the periphery and CNS of EAE mice with distinct grey and white matter lesions (combined active and focal EAE) either orally treated with cladribine or vehicle, using flow cytometry. To evaluate potential direct neuroprotective effects, we assessed the integrity of the primary auditory cortex neuronal network by studying neuronal activity and spontaneous synaptic activity with electrophysiological techniques ex vivo. RESULTS: Oral cladribine treatment significantly attenuated clinical deficits in EAE mice. Ex vivo flow cytometry showed that cladribine administration led to peripheral immune cell depletion in a compartment-specific manner and reduced immune cell infiltration into the CNS. Histological evaluations revealed no significant differences for inflammatory lesion load following cladribine treatment compared to vehicle control. Single cell electrophysiology in acute brain slices was performed and showed an impact of cladribine treatment on intrinsic cellular firing patterns and spontaneous synaptic transmission in neurons of the primary auditory cortex. Here, cladribine administration in vivo partially restored cortical neuronal network function, reducing action potential firing. Both, the effect on immune cells and neuronal activity were transient. CONCLUSIONS: Our results indicate that cladribine exerts a neuroprotective effect after crossing the blood-brain barrier independently of its peripheral immunosuppressant action.

Dimethyl fumarate treatment restrains the antioxidative capacity of T cells to control autoimmunity.

Dimethyl fumarate, an approved treatment for relapsing-remitting multiple sclerosis, exerts pleiotropic effects on immune cells as well as CNS resident cells. Here, we show that dimethyl fumarate exerts a profound alteration of the metabolic profile of human CD4+ as well as CD8+ T cells and restricts their antioxidative capacities by decreasing intracellular levels of the reactive oxygen species scavenger glutathione. This causes an increase in mitochondrial reactive oxygen species levels accompanied by an enhanced mitochondrial stress response, ultimately leading to impaired mitochondrial function. Enhanced mitochondrial reactive oxygen species levels not only result in enhanced T-cell apoptosis in vitro as well as in dimethyl fumarate-treated patients, but are key for the well-known immunomodulatory effects of dimethyl fumarate both in vitro and in an animal model of multiple sclerosis, i.e. experimental autoimmune encephalomyelitis. Indeed, dimethyl fumarate immune-modulatory effects on T cells were completely abrogated by pharmacological interference of mitochondrial reactive oxygen species production. These data shed new light on dimethyl fumarate as bona fide immune-metabolic drug that targets the intracellular stress response in activated T cells, thereby restricting mitochondrial function and energetic capacity, providing novel insight into the role of oxidative stress in modulating cellular immune responses and T cell-mediated autoimmunity.

Plasma kallikrein modulates immune cell trafficking during neuroinflammation via PAR2 and bradykinin release.

Blood-brain barrier (BBB) disruption and transendothelial trafficking of immune cells into the central nervous system (CNS) are pathophysiological hallmarks of neuroinflammatory disorders like multiple sclerosis (MS). Recent evidence suggests that the kallikrein-kinin and coagulation system might participate in this process. Here, we identify plasma kallikrein (KK) as a specific direct modulator of BBB integrity. Levels of plasma prekallikrein (PK), the precursor of KK, were markedly enhanced in active CNS lesions of MS patients. Deficiency or pharmacologic blockade of PK renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by a remarkable reduction of BBB disruption and CNS inflammation. In vitro analysis revealed that KK modulates endothelial cell function in a protease-activated receptor-2-dependent manner, leading to an up-regulation of the cellular adhesion molecules Intercellular Adhesion Molecule 1 and Vascular Cell Adhesion Molecule 1, thereby amplifying leukocyte trafficking. Our study demonstrates that PK is an important direct regulator of BBB integrity as a result of its protease function. Therefore, KK inhibition can decrease BBB damage and cell invasion during neuroinflammation and may offer a strategy for the treatment of MS.

SETDB1 is required for intestinal epithelial differentiation and the prevention of intestinal inflammation.

OBJECTIVE: The intestinal epithelium is a rapidly renewing tissue which plays central roles in nutrient uptake, barrier function and the prevention of intestinal inflammation. Control of epithelial differentiation is essential to these processes and is dependent on cell type-specific activity of transcription factors which bind to accessible chromatin. Here, we studied the role of SET Domain Bifurcated Histone Lysine Methyltransferase 1, also known as ESET (SETDB1), a histone H3K9 methyltransferase, in intestinal epithelial homeostasis and IBD. DESIGN: We investigated mice with constitutive and inducible intestinal epithelial deletion of Setdb1, studied the expression of SETDB1 in patients with IBD and mouse models of IBD, and investigated the abundance of SETDB1 variants in healthy individuals and patients with IBD. RESULTS: Deletion of intestinal epithelial Setdb1 in mice was associated with defects in intestinal epithelial differentiation, barrier disruption, inflammation and mortality. Mechanistic studies showed that loss of SETDB1 leads to de-silencing of endogenous retroviruses, DNA damage and intestinal epithelial cell death. Predicted loss-of-function variants in human SETDB1 were considerably less frequently observed than expected, consistent with a critical role of SETDB1 in human biology. While the vast majority of patients with IBD showed unimpaired mucosal SETDB1 expression, comparison of IBD and non-IBD exomes revealed over-representation of individual rare missense variants in SETDB1 in IBD, some of which are predicted to be associated with loss of function and may contribute to the pathogenesis of intestinal inflammation. CONCLUSION: SETDB1 plays an essential role in intestinal epithelial homeostasis. Future work is required to investigate whether rare variants in SETDB1 contribute to the pathogenesis of IBD.

Loss of hepatic Mboat7 leads to liver fibrosis.

OBJECTIVE: The rs641738C>T variant located near the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) locus is associated with fibrosis in liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease, hepatitis B and C. We aim to understand the mechanism by which the rs641738C>T variant contributes to pathogenesis of NAFLD. DESIGN: Mice with hepatocyte-specific deletion of MBOAT7 (Mboat7Δhep) were generated and livers were characterised by histology, flow cytometry, qPCR, RNA sequencing and lipidomics. We analysed the association of rs641738C>T genotype with liver inflammation and fibrosis in 846 NAFLD patients and obtained genotype-specific liver lipidomes from 280 human biopsies. RESULTS: Allelic imbalance analysis of heterozygous human liver samples pointed to lower expression of the MBOAT7 transcript on the rs641738C>T haplotype. Mboat7Δhep mice showed spontaneous steatosis characterised by increased hepatic cholesterol ester content after 10 weeks. After 6 weeks on a high fat, methionine-low, choline-deficient diet, mice developed increased hepatic fibrosis as measured by picrosirius staining (p<0.05), hydroxyproline content (p<0.05) and transcriptomics, while the inflammatory cell populations and inflammatory mediators were minimally affected. In a human biopsied NAFLD cohort, MBOAT7 rs641738C>T was associated with fibrosis (p=0.004) independent of the presence of histological inflammation. Liver lipidomes of Mboat7Δhep mice and human rs641738TT carriers with fibrosis showed increased total lysophosphatidylinositol levels. The altered lysophosphatidylinositol and phosphatidylinositol subspecies in MBOAT7Δhep livers and human rs641738TT carriers were similar. CONCLUSION: Mboat7 deficiency in mice and human points to an inflammation-independent pathway of liver fibrosis that may be mediated by lipid signalling and a potentially targetable treatment option in NAFLD.

Chemoenzymatic Total Synthesis of Sorbicatechol Structural Analogues and Evaluation of Their Antiviral Potential.

Sorbicillinoids are fungal polyketides characterized by highly complex and diverse molecular structures, with considerable stereochemical intricacy combined with a high degree of oxygenation. Many sorbicillinoids possess promising biological activities. An interesting member of this natural product family is sorbicatechol A, which is reported to have antiviral activity, particularly against influenza A virus (H1N1). Through a straightforward, one-pot chemoenzymatic approach with recently developed oxidoreductase SorbC, the characteristic bicyclo[2.2.2]octane core of sorbicatechol is structurally diversified by variation of its natural 2-methoxyphenol substituent. This facilitates the preparation of a focused library of structural analogues bearing substituted aromatic systems, alkanes, heterocycles, and ethers. Fast access to this structural diversity provides an opportunity to explore the antiviral potential of the sorbicatechol family.

Discovery of the Streptoketides by Direct Cloning and Rapid Heterologous Expression of a Cryptic PKS II Gene Cluster from Streptomyces sp. Tü 6314.

Genome sequencing and bioinformatic analysis have identified numerous cryptic gene clusters that have the potential to produce novel natural products. Within this work, we identified a cryptic type II PKS gene cluster (skt) from Streptomyces sp. Tü 6314. Facilitated by linear plus linear homologous recombination-mediated recombineering (LLHR), we directly cloned the skt gene cluster using the Streptomyces site-specific integration vector pSET152. Direct cloning allowed for rapid heterologous expression in Streptomyces coelicolor, leading to the identification and structural characterization of six polyketides (three known compounds and new streptoketides), four of which exhibit anti-HIV activities. Our study shows that the pSET152 vector can be directly used for LLHR, expanding the Rec/ET direct cloning toolbox and providing the possibility for rapid heterologous expression of gene clusters from Streptomyces.

NOX4-dependent neuronal autotoxicity and BBB breakdown explain the superior sensitivity of the brain to ischemic damage.

Ischemic injury represents the most frequent cause of death and disability, and it remains unclear why, of all body organs, the brain is most sensitive to hypoxia. In many tissues, type 4 NADPH oxidase is induced upon ischemia or hypoxia, converting oxygen to reactive oxygen species. Here, we show in mouse models of ischemia in the heart, brain, and hindlimb that only in the brain does NADPH oxidase 4 (NOX4) lead to ischemic damage. We explain this distinct cellular distribution pattern through cell-specific knockouts. Endothelial NOX4 breaks down the BBB, while neuronal NOX4 leads to neuronal autotoxicity. Vascular smooth muscle NOX4, the common denominator of ischemia within all ischemic organs, played no apparent role. The direct neuroprotective potential of pharmacological NOX4 inhibition was confirmed in an ex vivo model, free of vascular and BBB components. Our results demonstrate that the heightened sensitivity of the brain to ischemic damage is due to an organ-specific role of NOX4 in blood-brain-barrier endothelial cells and neurons. This mechanism is conserved in at least two rodents and humans, making NOX4 a prime target for a first-in-class mechanism-based, cytoprotective therapy in the unmet high medical need indication of ischemic stroke.

Genome-wide association analysis of diverticular disease points towards neuromuscular, connective tissue and epithelial pathomechanisms.

OBJECTIVE: Diverticular disease is a common complex disorder characterised by mucosal outpouchings of the colonic wall that manifests through complications such as diverticulitis, perforation and bleeding. We report the to date largest genome-wide association study (GWAS) to identify genetic risk factors for diverticular disease. DESIGN: Discovery GWAS analysis was performed on UK Biobank imputed genotypes using 31 964 cases and 419 135 controls of European descent. Associations were replicated in a European sample of 3893 cases and 2829 diverticula-free controls and evaluated for risk contribution to diverticulitis and uncomplicated diverticulosis. Transcripts at top 20 replicating loci were analysed by real-time quatitative PCR in preparations of the mucosal, submucosal and muscular layer of colon. The localisation of expressed protein at selected loci was investigated by immunohistochemistry. RESULTS: We discovered 48 risk loci, of which 12 are novel, with genome-wide significance and consistent OR in the replication sample. Nominal replication (p<0.05) was observed for 27 loci, and additional 8 in meta-analysis with a population-based cohort. The most significant novel risk variant rs9960286 is located near CTAGE1 with a p value of 2.3×10-10 and 0.002 (ORallelic=1.14 (95% CI 1.05 to 1.24)) in the replication analysis. Four loci showed stronger effects for diverticulitis, PHGR1 (OR 1.32, 95% CI 1.12 to 1.56), FAM155A-2 (OR 1.21, 95% CI 1.04 to 1.42), CALCB (OR 1.17, 95% CI 1.03 to 1.33) and S100A10 (OR 1.17, 95% CI 1.03 to 1.33). CONCLUSION: In silico analyses point to diverticulosis primarily as a disorder of intestinal neuromuscular function and of impaired connective fibre support, while an additional diverticulitis risk might be conferred by epithelial dysfunction.

Loss of Orai2-Mediated Capacitative Ca2+ Entry Is Neuroprotective in Acute Ischemic Stroke.

Background and Purpose- Ischemic stroke is one of the leading causes of disability and death. The principal goal of acute stroke treatment is the recanalization of the occluded cerebral arteries, which is, however, only effective in a very narrow time window. Therefore, neuroprotective treatments that can be combined with recanalization strategies are needed. Calcium overload is one of the major triggers of neuronal cell death. We have previously shown that capacitative Ca2+ entry, which is triggered by the depletion of intracellular calcium stores, contributes to ischemia-induced calcium influx in neurons, but the responsible Ca2+ channel is not known. Methods- Here, we have generated mice lacking the calcium channel subunit Orai2 and analyzed them in experimental stroke. Results- Orai2-deficient mice were protected from ischemic neuronal death both during acute ischemia under vessel occlusion and during ischemia/reperfusion upon successful recanalization. Calcium signals induced by calcium store depletion or oxygen/glucose deprivation were significantly diminished in Orai2-deficient neurons demonstrating that Orai2 is a central mediator of neuronal capacitative Ca2+ entry and is involved in calcium overload during ischemia. Conclusions- Our experimental data identify Orai2 as an attractive target for pharmaceutical intervention in acute stroke.

Biological evaluation of molecules of the azaBINOL class as antiviral agents: Inhibition of HIV-1 RNase H activity by 7-isopropoxy-8-(naphth-1-yl)quinoline.

Inspired by bioactive biaryl-containing natural products found in plants and the marine environment, a series of synthetic compounds belonging to the azaBINOL chiral ligand family was evaluated for antiviral activity against HIV-1. Testing of 39 unique azaBINOLs and two BINOLs in a single-round infectivity assay resulted in the identification of three promising antiviral compounds, including 7-isopropoxy-8-(naphth-1-yl)quinoline (azaBINOL B#24), which exhibited low-micromolar activity without associated cytotoxicity. The active compounds and several close structural analogues were further tested against three different HIV-1 envelope pseudotyped viruses as well as in a full-virus replication system (EASY-HIT). The in vitro studies indicated that azaBINOL B#24 acts on early stages of viral replication before viral assembly and budding. Next we explored B#24's activity against HIV-1 reverse transcriptase (RT) and individually tested for polymerase and RNase H activity. The azaBINOL B#24 inhibits RNase H activity and binds directly to the HIV-1 RT enzyme. Additionally, we observe additive inhibitory activity against pseudotyped viruses when B#24 is dosed in competition with the clinically used non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz. When tested against a multi-drug resistant HIV-1 isolate with drug resistance associated mutations in regions encoding for HIV-1 RT and protease, B#24 only exhibits a 5.1-fold net decrease in IC50 value, while efavirenz' activity decreases by 7.6-fold. These results indicate that azaBINOL B#24 is a potentially viable, novel lead for the development of new HIV-1 RNase H inhibitors. Furthermore, this study demonstrates that the survey of libraries of synthetic compounds, designed purely with the goal of facilitating chemical synthesis in mind, may yield unexpected and selective drug leads for the development of new antiviral agents.

Comparison of Gene Expression Patterns Between Mouse Models of Nonalcoholic Fatty Liver Disease and Liver Tissues From Patients.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries. Mouse models of NAFLD have been used in studies of pathogenesis and treatment, and have certain features of the human disease. We performed a systematic transcriptome-wide analysis of liver tissues from patients at different stages of NAFLD progression (ranging from healthy obese individuals to those with steatosis), as well as rodent models of NAFLD, to identify those that most closely resemble human disease progression in terms of gene expression patterns. METHODS: We performed a systematic evaluation of genome-wide messenger RNA expression using liver tissues collected from mice fed a standard chow diet (controls) and 9 mouse models of NAFLD: mice on a high-fat diet (with or without fructose), mice on a Western-type diet, mice on a methionine- and choline-deficient diet, mice on a high-fat diet given streptozotocin, and mice with disruption of Pten in hepatocytes. We compared gene expression patterns with those of liver tissues from 25 patients with nonalcoholic steatohepatitis (NASH), 27 patients with NAFLD, 15 healthy obese individuals, and 39 healthy nonobese individuals (controls). Liver samples were obtained from patients undergoing liver biopsy for suspected NAFLD or NASH, or during liver or bariatric surgeries. Data sets were analyzed using the limma R-package. Overlap of functional profiles was analyzed by gene set enrichment analysis profiles. RESULTS: We found differences between human and mouse transcriptomes to be significantly larger than differences between disease stages or models. Of the 65 genes with significantly altered expression in patients with NASH and 177 genes with significantly altered expression in patients with NAFLD, compared with controls, only 1-18 of these genes also differed significantly in expression between mouse models of NAFLD and control mice. However, expression of genes that regulate pathways associated with the development of NAFLD were altered in some mouse models (such as pathways associated with lipid metabolism). On a pathway level, gene expression patterns in livers of mice on the high-fat diet were associated more closely with human fatty liver disease than other models. CONCLUSIONS: In comparing gene expression profiles between liver tissues from different mouse models of NAFLD and patients with different stages of NAFLD, we found very little overlap. Our data set is available for studies of pathways that contribute to the development of NASH and NAFLD and selection of the most applicable mouse models (http://www.nash-profiler.com).

Chemokine CCL17 is expressed by dendritic cells in the CNS during experimental autoimmune encephalomyelitis and promotes pathogenesis of disease.

The CC chemokine ligand 17 (CCL17) and its cognate CC chemokine receptor 4 (CCR4) are known to control leukocyte migration, maintenance of TH17 cells, and regulatory T cell (Treg) expansion in vivo. In this study we characterized the expression and functional role of CCL17 in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). Using a CCL17/EGFP reporter mouse model, we could show that CCL17 expression in the CNS can be found in a subset of classical dendritic cells (DCs) that immigrate into the CNS during the effector phase of MOG-induced EAE. CCL17 deficient (CCL17-/-) mice exhibited an ameliorated disease course upon MOG-immunization, associated with reduced immigration of IL-17 producing CD4+ T cells and peripheral DCs into the CNS. CCL17-/- DCs further showed equivalent MHC class II and costimulatory molecule expression and an equivalent capacity to secrete IL-23 and induce myelin-reactive TH17 cells when compared to wildtype DCs. In contrast, their transmigration in an in vitro model of the blood-brain barrier was markedly impaired. In addition, peripheral Treg cells were enhanced in CCL17-/- mice at peak of disease pointing towards an immunoregulatory function of CCL17 in EAE. Our study identifies CCL17 as a unique modulator of EAE pathogenesis regulating DC trafficking as well as peripheral Treg cell expansion in EAE. Thus, CCL17 operates at distinct levels and on different cell subsets during immune response in EAE, a property harboring therapeutic potential for the treatment of CNS autoimmunity.

The quality of cortical network function recovery depends on localization and degree of axonal demyelination.

Myelin loss is a severe pathological hallmark common to a number of neurodegenerative diseases, including multiple sclerosis (MS). Demyelination in the central nervous system appears in the form of lesions affecting both white and gray matter structures. The functional consequences of demyelination on neuronal network and brain function are not well understood. Current therapeutic strategies for ameliorating the course of such diseases usually focus on promoting remyelination, but the effectiveness of these approaches strongly depends on the timing in relation to the disease state. In this study, we sought to characterize the time course of sensory and behavioral alterations induced by de- and remyelination to establish a rational for the use of remyelination strategies. By taking advantage of animal models of general and focal demyelination, we tested the consequences of myelin loss on the functionality of the auditory thalamocortical system: a well-studied neuronal network consisting of both white and gray matter regions. We found that general demyelination was associated with a permanent loss of the tonotopic cortical organization in vivo, and the inability to induce tone-frequency-dependent conditioned behaviors, a status persisting after remyelination. Targeted, focal lysolecithin-induced lesions in the white matter fiber tract, but not in the gray matter regions of cortex, were fully reversible at the morphological, functional and behavioral level. These findings indicate that remyelination of white and gray matter lesions have a different functional regeneration potential, with the white matter being able to regain full functionality while cortical gray matter lesions suffer from permanently altered network function. Therefore therapeutic interventions aiming for remyelination have to consider both region- and time-dependent strategies.

Metabolic and Homeostatic Changes in Seizures and Acquired Epilepsy-Mitochondria, Calcium Dynamics and Reactive Oxygen Species.

Acquired epilepsies can arise as a consequence of brain injury and result in unprovoked seizures that emerge after a latent period of epileptogenesis. These epilepsies pose a major challenge to clinicians as they are present in the majority of patients seen in a common outpatient epilepsy clinic and are prone to pharmacoresistance, highlighting an unmet need for new treatment strategies. Metabolic and homeostatic changes are closely linked to seizures and epilepsy, although, surprisingly, no potential treatment targets to date have been translated into clinical practice. We summarize here the current knowledge about metabolic and homeostatic changes in seizures and acquired epilepsy, maintaining a particular focus on mitochondria, calcium dynamics, reactive oxygen species and key regulators of cellular metabolism such as the Nrf2 pathway. Finally, we highlight research gaps that will need to be addressed in the future which may help to translate these findings into clinical practice.