IL-4 shapes microglia-dependent pruning of the cerebellum during postnatal development.

Interleukin-4 (IL-4) is a type 2 cytokine with pleiotropic functions in adaptive immunity, allergies, and cognitive processes. Here, we show that low levels of IL-4 in the early postnatal stage delineate a critical period in which microglia extensively prune cerebellar neurons. Elevating the levels of this cytokine via peripheral injection, or using a mouse model of allergic asthma, leads to defective pruning, permanent increase in cerebellar granule cells, and circuit alterations. These animals also show a hyperkinetic and impulsive-like phenotype, reminiscent of attention-deficit hyperactivity disorder (ADHD). These alterations are blocked in Il4rαfl/fl::Cx3cr1-CreER mice, which are deficient in IL-4 receptor signaling in microglia. These findings demonstrate a previously unknown role for IL-4 during a neuroimmune critical period of cerebellar maturation and provide a first putative mechanism for the comorbidity between allergic disease and ADHD observed in humans.

Interleukin-4 as a therapeutic target.

Interleukin-4 (IL-4) is a pleiotropic cytokine mainly known for its role in type 2 immunity. Therapies antagonizing or blocking IL-4 activity have been developed to counteract diseases such as atopic dermatitis and asthma. In contrast, other disorders experimentally benefit from IL-4-related effects and IL-4 recently demonstrated beneficial activity in experimental stroke, spinal cord injury and the animal model of multiple sclerosis. To exploit IL-4-related activity for therapeutic concepts, current experimental efforts include modifying the pathway without inducing type 2 immune response and targeting of the cytokine to specific tissues. Here, we review different activities of IL-4 as well as therapeutic strategies.

Interleukin-4 receptor signaling modulates neuronal network activity.

Evidence is emerging that immune responses not only play a part in the central nervous system (CNS) in diseases but may also be relevant for healthy conditions. We discovered a major role for the interleukin-4 (IL-4)/IL-4 receptor alpha (IL-4Rα) signaling pathway in synaptic processes, as indicated by transcriptome analysis in IL-4Rα-deficient mice and human neurons with/without IL-4 treatment. Moreover, IL-4Rα is expressed presynaptically, and locally available IL-4 regulates synaptic transmission. We found reduced synaptic vesicle pools, altered postsynaptic currents, and a higher excitatory drive in cortical networks of IL-4Rα-deficient neurons. Acute effects of IL-4 treatment on postsynaptic currents in wild-type neurons were mediated via PKCγ signaling release and led to increased inhibitory activity supporting the findings in IL-4Rα-deficient neurons. In fact, the deficiency of IL-4Rα resulted in increased network activity in vivo, accompanied by altered exploration and anxiety-related learning behavior; general learning and memory was unchanged. In conclusion, neuronal IL-4Rα and its presynaptic prevalence appear relevant for maintaining homeostasis of CNS synaptic function.

CNS-localized myeloid cells capture living invading T cells during neuroinflammation.

To study the role of myeloid cells in the central nervous system (CNS) in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), we used intravital microscopy, assessing local cellular interactions in vivo in EAE animals and ex vivo in organotypic hippocampal slice cultures. We discovered that myeloid cells actively engulf invading living Th17 lymphocytes, a process mediated by expression of activation-dependent lectin and its T cell-binding partner, N-acetyl-D-glucosamine (GlcNAc). Stable engulfment resulted in the death of the engulfed cells, and, remarkably, enhancement of GlcNAc exposure on T cells in the CNS ameliorated clinical EAE symptoms. These findings demonstrate the ability of myeloid cells to directly react to pathogenic T cell infiltration by engulfing living T cells. Amelioration of EAE via GlcNAc treatment suggests a novel first-defense pathway of myeloid cells as an initial response to CNS invasion and demonstrates that T cell engulfment by myeloid cells can be therapeutically exploited in vivo.

Targeting CD52 does not affect murine neuron and microglia function.

The humanized anti-CD52 antibody alemtuzumab is successfully used in the treatment of multiple sclerosis (MS) and is thought to exert most of its therapeutic action by depletion and repopulation of mainly B and T lymphocytes. Although neuroprotective effects of alemtuzumab have been suggested, direct effects of anti-CD52 treatment on glial cells and neurons within the CNS itself have not been investigated so far. Here, we show CD52 expression in murine neurons, astrocytes and microglia, both in vitro and in vivo. As expected, anti CD52-treatment caused profound lymphopenia and improved disease symptoms in mice subjected to experimental autoimmune encephalomyelitis (EAE). CD52 blockade also had a significant effect on microglial morphology in organotypic hippocampal slice cultures but did not affect microglial functions. Furthermore, anti-CD52 neither changed baseline neuronal calcium, nor did it act neuroprotective in excitotoxicity models. Altogether, our findings argue against a functionally significant role of CD52 blockade on CNS neurons and microglia. The beneficial effects of alemtuzumab in MS may be exclusively mediated by peripheral immune mechanisms.

The Cerebral Thrombin System Is Activated after Intracerebral Hemorrhage and Contributes to Secondary Lesion Growth and Poor Neurological Outcome in C57Bl/6 Mice.

With increasing evidence for the existence of a cerebral thrombin system, coagulation factor IIa (thrombin) is suspected to influence the pathogenesis of secondary injury progression after intracerebral hemorrhage (ICH). We hypothesized that mechanisms associated with local volume expansion after ICH, rather than blood constituents, activate the cerebral thrombin system and are responsible for detrimental neurological outcome. To test this hypothesis, we examine the local thrombin expression after ICH in a C57BL/6N mouse model in the presence and absence of blood constituents. ICH was established using stereotaxic orthotopic injection of utologous blood (n = 10) or silicone oil as inert volume substance (n = 10) into the striatum. Intracranial pressure (ICP), cerebral blood flow (CBF), and mean arterial blood pressure (MAP) were monitored during and 30 min after the procedure. No significant differences between ICP, CBF, and MAP were found between both groups. Prothrombin messenger RNA expression was upregulated early after ICH. Immunohistochemistry showed an increase of perilesional thrombin in both groups (blood, 4.24-fold; silicone, 3.10-fold), whereas prothrombin fragment (F1.2) was elevated only in the absence of whole blood. Thrombin expression is colocalized with neuronal antigen expression. After 24 h, lesion size and neuronal loss were similar. Perihematomal thrombin correlated with increased neuronal loss and detrimental neurological outcome in vivo. In our study, we demonstrate, for the first time, that the local cerebral thrombin system is activated after ICH and that this activation is independent of the presence of whole-blood constituents. In our study, neuronal damage is driven by local thrombin expression and leads to an adverse clinical outcome.

Publisher Correction: Maladaptive cortical hyperactivity upon recovery from experimental autoimmune encephalomyelitis.

In the version of this article initially published, Inigo Ruiz de Azua's name was miscategorized. His given name is Inigo and his surname is Ruiz de Azua. This has been corrected in the HTML coding.

Maladaptive cortical hyperactivity upon recovery from experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) patients exhibit neuropsychological symptoms in early disease despite the immune attack occurring predominantly in white matter and spinal cord. It is unclear why neurodegeneration may start early in the disease and is prominent in later stages. We assessed cortical microcircuit activity by employing spiking-specific two-photon Ca2+ imaging in proteolipid protein-immunized relapsing-remitting SJL/J mice in vivo. We identified the emergence of hyperactive cortical neurons in remission only, independent of direct immune-mediated damage and paralleled by elevated anxiety. High levels of neuronal activity were accompanied by increased caspase-3 expression. Cortical TNFα expression was mainly increased by excitatory neurons in remission; blockade with intraventricular infliximab restored AMPA spontaneous excitatory postsynaptic current frequencies, completely recovered normal neuronal network activity patterns and alleviated elevated anxiety. This suggests a dysregulation of cortical networks attempting to achieve functional compensation by synaptic plasticity mechanisms, indicating a link between immune attack and early start of neurodegeneration.

Pharmacological Suppression of CNS Scarring by Deferoxamine Reduces Lesion Volume and Increases Regeneration in an In Vitro Model for Astroglial-Fibrotic Scarring and in Rat Spinal Cord Injury In Vivo.

Lesion-induced scarring is a major impediment for regeneration of injured axons in the central nervous system (CNS). The collagen-rich glial-fibrous scar contains numerous axon growth inhibitory factors forming a regeneration-barrier for axons. We demonstrated previously that the combination of the iron chelator 2,2'-bipyridine-5,5'-decarboxylic acid (BPY-DCA) and 8-Br-cyclic AMP (cAMP) inhibits scar formation and collagen deposition, leading to enhanced axon regeneration and partial functional recovery after spinal cord injury. While BPY-DCA is not a clinical drug, the clinically approved iron chelator deferoxamine mesylate (DFO) may be a suitable alternative for anti-scarring treatment (AST). In order to prove the scar-suppressing efficacy of DFO we modified a recently published in vitro model for CNS scarring. The model comprises a co-culture system of cerebral astrocytes and meningeal fibroblasts, which form scar-like clusters when stimulated with transforming growth factor-ß (TGF-ß). We studied the mechanisms of TGF-ß-induced CNS scarring and compared the efficiency of different putative pharmacological scar-reducing treatments, including BPY-DCA, DFO and cAMP as well as combinations thereof. We observed modulation of TGF-ß-induced scarring at the level of fibroblast proliferation and contraction as well as specific changes in the expression of extracellular matrix molecules and axon growth inhibitory proteins. The individual and combinatorial pharmacological treatments had distinct effects on the cellular and molecular aspects of in vitro scarring. DFO could be identified as a putative anti-scarring treatment for CNS trauma. We subsequently validated this by local application of DFO to a dorsal hemisection in the rat thoracic spinal cord. DFO treatment led to significant reduction of scarring, slightly increased regeneration of corticospinal tract as well as ascending CGRP-positive axons and moderately improved locomotion. We conclude that the in vitro model for CNS scarring is suitable for efficient pre-screening and identification of putative scar-suppressing agents prior to in vivo application and validation, thus saving costs, time and laboratory animals.

High glucose enhances thrombin responses via protease-activated receptor-4 in human vascular smooth muscle cells.

OBJECTIVE: Diabetes is associated with vascular remodeling and increased thrombin generation. Thrombin promotes vascular smooth muscle cell (SMC) mitogenesis and migration via protease-activated receptors (PAR)-1, PAR-3, and PAR-4. We investigated the effect of high glucose on expression and function of vascular thrombin receptors. METHODS AND RESULTS: In human vascular SMCs, high glucose (25 versus 5.5 mmol/L) induced a rapid and sustained increase in PAR-4 mRNA, protein, and cell surface expression. PAR-1 and PAR-3 expression were not changed. High glucose pretreatment (48 hours) enhanced thrombin or PAR-4-activating peptide but not PAR-1-activating peptide evoked intracellular calcium mobilization, migration, and tumor necrosis factor α gene expression. This enhancement of thrombin-stimulated migration and gene expression by high glucose was abolished by endogenous PAR-4 knockdown. PAR-4 regulation was prevented by inhibition of protein kinase (PK)C-ß and -δ isoforms or nuclear factor (NF)κB. Nuclear translocation of NFκB in high glucose-stimulated SMCs led to PKC-dependent NFκB binding to the PAR-4 promoter in a chromatin immunoprecipitation assay. Furthermore, in situ hybridization and immunohistochemistry confirmed high abundance of PAR-4 in human diabetic vessels as compared with nondiabetic vessels. CONCLUSIONS: High glucose enhances SMC responsiveness to thrombin through transcriptional upregulation of PAR-4, mediated via PKC-ß, -δ, and NFκB. This may play an important role in the vascular complications of diabetes.