The Impact of VEGF-C-Induced Dural Lymphatic Vessel Growth on Ischemic Stroke Pathology.

Timely relief of edema and clearance of waste products, as well as promotion of anti-inflammatory immune responses, reduce ischemic stroke pathology, and attenuate harmful long-term effects post-stroke. The discovery of an extensive and functional lymphatic vessel system in the outermost meningeal layer, dura mater, has opened up new possibilities to facilitate post-stroke recovery by inducing dural lymphatic vessel (dLV) growth via a single injection of a vector encoding vascular endothelial growth factor C (VEGF-C). In the present study, we aimed to improve post-stroke outcomes by inducing dLV growth in mice. We injected mice with a single intracerebroventricular dose of adeno-associated viral particles encoding VEGF-C before subjecting them to transient middle cerebral artery occlusion (tMCAo). Behavioral testing, Gadolinium (Gd) contrast agent-enhanced magnetic resonance imaging (MRI), and immunohistochemical analysis were performed to define the impact of VEGF-C on the post-stroke outcome. VEGF-C improved stroke-induced behavioral deficits, such as gait disturbances and neurological deficits, ameliorated post-stroke inflammation, and enhanced an alternative glial immune response. Importantly, VEGF-C treatment increased the drainage of brain interstitial fluid (ISF) and cerebrospinal fluid (CSF), as shown by Gd-enhanced MRI. These outcomes were closely associated with an increase in the growth of dLVs around the region where we observed increased vefgc mRNA expression within the brain, including the olfactory bulb, cortex, and cerebellum. Strikingly, VEGF-C-treated ischemic mice exhibited a faster and stronger Gd-signal accumulation in ischemic core area and an enhanced fluid outflow via the cribriform plate. In conclusion, the VEGF-C-induced dLV growth improved the overall outcome post-stroke, indicating that VEGF-C has potential to be included in the treatment strategies of post-ischemic stroke. However, to maximize the therapeutic potential of VEGF-C treatment, further studies on the impact of an enhanced dural lymphatic system at clinically relevant time points are essential.

Cytokines impact natural killer cell phenotype and functionality against glioblastoma in vitro.

Objective: Natural killer (NK) cells are a part of the innate immune system and first-line defense against cancer. Since they possess natural mechanisms to recognize and kill tumor cells, NK cells are considered as a potential option for an off-the-shelf allogeneic cell-based immunotherapy. Here, our objective was to identify the optimal cytokine-based, feeder-free, activation and expansion protocol for cytotoxic NK cells against glioblastoma in vitro. Methods: NK cells were enriched from human peripheral blood and expanded for 16 days with different activation and cytokine combinations. The expansion conditions were evaluated based on NK cell viability, functionality, expansion rate and purity. The cytotoxicity and degranulation of the expanded NK cells were measured in vitro from co­cultures with the glioma cell lines U­87 MG, U­87 MG EGFR vIII, LN-229, U-118 and DK-MG. The best expansion protocols were selected from ultimately 39 different conditions: three magnetic cell­selection steps (Depletion of CD3+ cells, enrichment of CD56+ cells, and depletion of CD3+ cells followed by enrichment of CD56+ cells); four activation protocols (continuous, pre-activation, re-activation, and boost); and four cytokine combinations (IL-2/15, IL­21/15, IL­27/18/15 and IL-12/18/15). Results: The expansion rates varied between 2-50-fold, depending on the donor and the expansion conditions. The best expansion rate and purity were gained with sequential selection (Depletion of CD3+ cells and enrichment of CD56+ cells) from the starting material and pre-activation with IL­12/18/15 cytokines, which are known to produce cytokine-induced memory-like NK cells. The cytotoxicity of these memory-like NK cells was enhanced with re-activation, diminishing the donor variation. The most cytotoxic NK cells were produced when cells were boosted at the end of the expansion with IL-12/18/15 or IL-21/15. Conclusion: According to our findings the ex vivo proliferation capacity and functionality of NK cells is affected by multiple factors, such as the donor, composition of starting material, cytokine combination and the activation protocol. The cytokines modified the NK cells' phenotype and functionality, which was evident in their reactivity against the glioma cell lines. To our knowledge, this is the first comprehensive comparative study performed to this extent, and these findings could be used for upscaling clinical NK cell manufacturing.

An arylthiazyne derivative is a potent inhibitor of lipid peroxidation and ferroptosis providing neuroprotection in vitro and in vivo.

Lipid peroxidation-initiated ferroptosis is an iron-dependent mechanism of programmed cell death taking place in neurological diseases. Here we show that a condensed benzo[b]thiazine derivative small molecule with an arylthiazine backbone (ADA-409-052) inhibits tert-Butyl hydroperoxide (TBHP)-induced lipid peroxidation (LP) and protects against ferroptotic cell death triggered by glutathione (GSH) depletion or glutathione peroxidase 4 (GPx4) inhibition in neuronal cell lines. In addition, ADA-409-052 suppresses pro-inflammatory activation of BV2 microglia and protects N2a neuronal cells from cell death induced by pro-inflammatory RAW 264.7 macrophages. Moreover, ADA-409-052 efficiently reduces infarct volume, edema and expression of pro-inflammatory genes in a mouse model of thromboembolic stroke. Targeting ferroptosis may be a promising therapeutic strategy in neurological diseases involving severe neuronal death and neuroinflammation.

Metabolic alterations in Parkinson's disease astrocytes.

In Parkinson`s disease (PD), the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta is associated with Lewy bodies arising from the accumulation of alpha-synuclein protein which leads ultimately to movement impairment. While PD has been considered a disease of the DA neurons, a glial contribution, in particular that of astrocytes, in PD pathogenesis is starting to be uncovered. Here, we report findings from astrocytes derived from induced pluripotent stem cells of LRRK2 G2019S mutant patients, with one patient also carrying a GBA N370S mutation, as well as healthy individuals. The PD patient astrocytes manifest the hallmarks of the disease pathology including increased expression of alpha-synuclein. This has detrimental consequences, resulting in altered metabolism, disturbed Ca2+ homeostasis and increased release of cytokines upon inflammatory stimulation. Furthermore, PD astroglial cells manifest increased levels of polyamines and polyamine precursors while lysophosphatidylethanolamine levels are decreased, both of these changes have been reported also in PD brain. Collectively, these data reveal an important role for astrocytes in PD pathology and highlight the potential of iPSC-derived cells in disease modeling and drug discovery.

Developmental Dysfunction of the Central Nervous System Lymphatics Modulates the Adaptive Neuro-Immune Response in the Perilesional Cortex in a Mouse Model of Traumatic Brain Injury.

Rationale: The recently discovered meningeal lymphatic vessels (mLVs) have been proposed to be the missing link between the immune and the central nervous system. The role of mLVs in modulating the neuro-immune response following a traumatic brain injury (TBI), however, has not been analyzed. Parenchymal T lymphocyte infiltration has been previously reported as part of secondary events after TBI, suggestive of an adaptive neuro-immune response. The phenotype of these cells has remained mostly uncharacterized. In this study, we identified subpopulations of T cells infiltrating the perilesional areas 30 days post-injury (an early-chronic time point). Furthermore, we analyzed how the lack of mLVs affects the magnitude and the type of T cell response in the brain after TBI. Methods: TBI was induced in K14-VEGFR3-Ig transgenic (TG) mice or in their littermate controls (WT; wild type), applying a controlled cortical impact (CCI). One month after TBI, T cells were isolated from cortical areas ipsilateral or contralateral to the trauma and from the spleen, then characterized by flow cytometry. Lesion size in each animal was evaluated by MRI. Results: In both WT and TG-CCI mice, we found a prominent T cell infiltration in the brain confined to the perilesional cortex and hippocampus. The majority of infiltrating T cells were cytotoxic CD8+ expressing a CD44hiCD69+ phenotype, suggesting that these are effector resident memory T cells. K14-VEGFR3-Ig mice showed a significant reduction of infiltrating CD4+ T lymphocytes, suggesting that mLVs could be involved in establishing a proper neuro-immune response. Extension of the lesion (measured as lesion volume from MRI) did not differ between the genotypes. Finally, TBI did not relate to alterations in peripheral circulating T cells, as assessed one month after injury. Conclusions: Our results are consistent with the hypothesis that mLVs are involved in the neuro-immune response after TBI. We also defined the resident memory CD8+ T cells as one of the main population activated within the brain after a traumatic injury.

NF-E2-related factor 2 activation boosts antioxidant defenses and ameliorates inflammatory and amyloid properties in human Presenilin-1 mutated Alzheimer's disease astrocytes.

Alzheimer's disease (AD) is a common dementia affecting a vast number of individuals and significantly impairing quality of life. Despite extensive research in animal models and numerous promising treatment trials, there is still no curative treatment for AD. Astrocytes, the most common cell type of the central nervous system, have been shown to play a role in the major AD pathologies, including accumulation of amyloid plaques, neuroinflammation, and oxidative stress. Here, we show that inflammatory stimulation leads to metabolic activation of human astrocytes and reduces amyloid secretion. On the other hand, the activation of oxidative metabolism leads to increased reactive oxygen species production especially in AD astrocytes. While healthy astrocytes increase glutathione (GSH) release to protect the cells, Presenilin-1-mutated AD patient astrocytes do not. Thus, chronic inflammation is likely to induce oxidative damage in AD astrocytes. Activation of NRF2, the major regulator of cellular antioxidant defenses, encoded by the NFE2L2 gene, poses several beneficial effects on AD astrocytes. We report here that the activation of NRF2 pathway reduces amyloid secretion, normalizes cytokine release, and increases GSH secretion in AD astrocytes. NRF2 induction also activates the metabolism of astrocytes and increases the utilization of glycolysis. Taken together, targeting NRF2 in astrocytes could be a potent therapeutic strategy in AD.

PSEN1ΔE9, APPswe, and APOE4 Confer Disparate Phenotypes in Human iPSC-Derived Microglia.

Here we elucidate the effect of Alzheimer disease (AD)-predisposing genetic backgrounds, APOE4, PSEN1ΔE9, and APPswe, on functionality of human microglia-like cells (iMGLs). We present a physiologically relevant high-yield protocol for producing iMGLs from induced pluripotent stem cells. Differentiation is directed with small molecules through primitive erythromyeloid progenitors to re-create microglial ontogeny from yolk sac. The iMGLs express microglial signature genes and respond to ADP with intracellular Ca2+ release distinguishing them from macrophages. Using 16 iPSC lines from healthy donors, AD patients and isogenic controls, we reveal that the APOE4 genotype has a profound impact on several aspects of microglial functionality, whereas PSEN1ΔE9 and APPswe mutations trigger minor alterations. The APOE4 genotype impairs phagocytosis, migration, and metabolic activity of iMGLs but exacerbates their cytokine secretion. This indicates that APOE4 iMGLs are fundamentally unable to mount normal microglial functionality in AD.

Peripheral Administration of IL-13 Induces Anti-inflammatory Microglial/Macrophage Responses and Provides Neuroprotection in Ischemic Stroke.

Neuroinflammation is strongly induced by cerebral ischemia. The early phase after the onset of ischemic stroke is characterized by acute neuronal injury, microglial activation, and subsequent infiltration of blood-derived inflammatory cells, including macrophages. Therefore, modulation of the microglial/macrophage responses has increasingly gained interest as a potential therapeutic approach for the ischemic stroke. In our study, we investigated the effects of peripherally administered interleukin 13 (IL-13) in a mouse model of permanent middle cerebral artery occlusion (pMCAo). Systemic administration of IL-13 immediately after the ischemic insult significantly reduced the lesion volume, alleviated the infiltration of CD45+ leukocytes, and promoted the microglia/macrophage alternative activation within the ischemic region, as determined by arginase 1 (Arg1) immunoreactivity at 3 days post-ischemia (dpi). Moreover, IL-13 enhanced the expression of M2a alternative activation markers Arg1 and Ym1 in the peri-ischemic (PI) area, as well as increased plasma IL-6 and IL-10 levels at 3 dpi. Furthermore, IL-13 treatment ameliorated gait disturbances at day 7 and 14 and sensorimotor deficits at day 14 post-ischemia, as analyzed by the CatWalk gait analysis system and adhesive removal test, respectively. Finally, IL-13 treatment decreased neuronal cell death in a coculture model of neuroinflammation with RAW 264.7 macrophages. Taken together, delivery of IL-13 enhances microglial/macrophage anti-inflammatory responses in vivo and in vitro, decreases ischemia-induced brain cell death, and improves sensory and motor functions in the pMCAo mouse model of cerebral ischemia.

Dysfunction of Cellular Proteostasis in Parkinson's Disease.

Despite decades of research, current therapeutic interventions for Parkinson's disease (PD) are insufficient as they fail to modify disease progression by ameliorating the underlying pathology. Cellular proteostasis (protein homeostasis) is an essential factor in maintaining a persistent environment for neuronal activity. Proteostasis is ensured by mechanisms including regulation of protein translation, chaperone-assisted protein folding and protein degradation pathways. It is generally accepted that deficits in proteostasis are linked to various neurodegenerative diseases including PD. While the proteasome fails to degrade large protein aggregates, particularly alpha-synuclein (α-SYN) in PD, drug-induced activation of autophagy can efficiently remove aggregates and prevent degeneration of dopaminergic (DA) neurons. Therefore, maintenance of these mechanisms is essential to preserve all cellular functions relying on a correctly folded proteome. The correlations between endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) that aims to restore proteostasis within the secretory pathway are well-established. However, while mild insults increase the activity of chaperones, prolonged cell stress, or insufficient adaptive response causes cell death. Modulating the activity of molecular chaperones, such as protein disulfide isomerase which assists refolding and contributes to the removal of unfolded proteins, and their associated pathways may offer a new approach for disease-modifying treatment. Here, we summarize some of the key concepts and emerging ideas on the relation of protein aggregation and imbalanced proteostasis with an emphasis on PD as our area of main expertise. Furthermore, we discuss recent insights into the strategies for reducing the toxic effects of protein unfolding in PD by targeting the ER UPR pathway.

Activation of P2X7 Receptors in Peritoneal and Meningeal Mast Cells Detected by Uptake of Organic Dyes: Possible Purinergic Triggers of Neuroinflammation in Meninges.

Extracellular ATP activates inflammasome and triggers the release of multiple cytokines in various immune cells, a process primarily mediated by P2X7 receptors. However, the expression and functional properties of P2X7 receptors in native mast cells in tissues such as meninges where migraine pain originates from have not been explored. Here we report a novel model of murine cultured meningeal mast cells and using these, as well as easily accessible peritoneal mast cells, studied the mechanisms of ATP-mediated mast cell activation. We show that ATP induced a time and dose-dependent activation of peritoneal mast cells as analyzed by the uptake of organic dye YO-PRO1 as well as 4,6-diamidino-2-phenylindole (DAPI). Both YO-PRO1 and DAPI uptake in mast cells was mediated by the P2X7 subtype of ATP receptors as demonstrated by the inhibitory effect of P2X7 antagonist A839977. Consistent with this, significant YO-PRO1 uptake was promoted by the P2X7 agonist 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP). Extracellular ATP-induced degranulation of native and cultured meningeal mast cells was shown with Toluidine Blue staining. Taken together, these data demonstrate the important contribution of P2X7 receptors to ATP-driven activation of mast cells, suggesting these purinergic mechanisms as potential triggers of neuroinflammation and pain sensitization in migraine.

Long-term interleukin-33 treatment delays disease onset and alleviates astrocytic activation in a transgenic mouse model of amyotrophic lateral sclerosis.

Inflammation is a prominent feature of the neuropathology of amyotrophic lateral sclerosis (ALS). Emerging evidence suggests that inflammatory cascades contributing to the disease progression are not restricted to the central nervous system (CNS) but also occur peripherally. Indeed, alterations in T cell responses and their secreted cytokines have been detected in ALS patients and in animal models of ALS. One key cytokine responsible for the shift in T cell responses is interleukin-33 (IL-33), which stimulates innate type 2 immune cells to produce a large amount of Th2 cytokines that are possibly beneficial in the recovery processes of CNS injuries. Since the levels of IL-33 have been shown to be decreased in patients affected with ALS, we sought to determine whether a long-term recombinant IL-33 treatment of a transgenic mouse model of ALS expressing G93A-superoxide dismutase 1 (SOD1-G93A) alters the disease progression and ameliorates the ALS-like disease pathology. SOD1-G93A mice were treated with intraperitoneal injections of IL-33 and effects on disease onset and inflammatory status were determined. Spinal cord (SC) neurons, astrocytes and T-cells were exposed to IL-33 to evaluate the cell specific responses to IL-33. Treatment of SOD1-G93A mice with IL-33 delayed the disease onset in female mice, decreased the proportion of CD4+ and CD8 + T cell populations in the spleen and lymph nodes, and alleviated astrocytic activation in the ventral horn of the lumbar SC. Male SOD1-G93A mice were unresponsive to the treatment. In vitro studies showed that IL-33 is most likely not acting directly on neurons and astrocytes, but rather conveying its effects through peripheral T-cells. Our results suggest that strategies directed to the peripheral immune system may have therapeutic potential in ALS. The effect of gender dimorphisms to the treatment efficacy needs to be taken into consideration when designing new therapeutic strategies for CNS diseases.

ADAMTS-4 in central nervous system pathologies.

ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs type 4) is a metalloproteinase specialized in the degradation of chondroitin sulfate proteoglycans, contributing to cartilage breakdown during arthritis. In this review, we first focus on the modifications of ADAMTS-4 expression during CNS physiological and pathological conditions, including chronic diseases and injuries. Then, we discuss the contributions of ADAMTS-4 to mechanisms mediating neuroplasticity, neuroinflammation and neurodegeneration during spinal cord injury, ischemic stroke, amyotrophic lateral sclerosis and Alzheimer's disease. Here, we provide an overview of ADAMTS-4 functions and effects in the CNS, and we discuss directions for future studies and treatments. Overall, this review highlights that ADAMTS-4 is a unique multifaceted metalloproteinase which influences various CNS disease pathophysiologies. © 2017 Wiley Periodicals, Inc.

The Copper bis(thiosemicarbazone) Complex CuII(atsm) Is Protective Against Cerebral Ischemia Through Modulation of the Inflammatory Milieu.

Developing new therapies for stroke is urgently needed, as this disease is the leading cause of death and disability worldwide, and the existing treatment is only available for a small subset of patients. The interruption of blood flow to the brain during ischemic stroke launches multiple immune responses, characterized by infiltration of peripheral immune cells, the activation of brain microglial cells, and the accumulation of immune mediators. Copper is an essential trace element that is required for many critical processes in the brain. Copper homeostasis is disturbed in chronic neurodegenerative diseases and altered in stroke patients, and targeted copper delivery has been shown to be protective against chronic neurodegeneration. This study was undertaken to assess whether the copper bis(thiosemicarbazone) complex, CuII(atsm), is beneficial in acute brain injury, in preclinical mouse models of ischemic stroke. We demonstrate that the copper complex CuII(atsm) protects neurons from excitotoxicity and N2a cells from OGD in vitro, and is protective in permanent and transient ischemia models in mice as measured by functional outcome and lesion size. Copper delivery in the ischemic brains modulates the inflammatory response, specifically affecting the myeloid cells. It reduces CD45 and Iba1 immunoreactivity, and alters the morphology of Iba1 positive cells in the ischemic brain. CuII(atsm) also protects endogenous microglia against ischemic insult and reduces the proportion of invading monocytes. These results demonstrate that the copper complex CuII(atsm) is an inflammation-modulating compound with high therapeutic potential in stroke and is a strong candidate for the development of therapies for acute brain injury.

Aß and Inflammatory Stimulus Activate Diverse Signaling Pathways in Monocytic Cells: Implications in Retaining Phagocytosis in Aß-Laden Environment.

Background: Accumulation of amyloid ß (Aß) is one of the main hallmarks of Alzheimer's disease (AD). The enhancement of Aß clearance may provide therapeutic means to restrict AD pathology. The cellular responses to different forms of Aß in monocytic cells are poorly known. We aimed to study whether different forms of Aß induce inflammatory responses in monocytic phagocytes and how Aß may affect monocytic cell survival and function to retain phagocytosis in Aß-laden environment. Methods: Monocytic cells were differentiated from bone marrow hematopoietic stem cells (HSC) in the presence of macrophage-colony stimulating factor. Monocytic cells were stimulated with synthetic Aß42 and intracellular calcium responses were recorded with calcium imaging. The formation of reactive oxygen species (ROS), secretion of cytokines and cell viability were also assessed. Finally, monocytic cells were introduced to native Aß deposits ex vivo and the cellular responses in terms of cell viability, pro-inflammatory activation and phagocytosis were determined. The ability of monocytic cells to phagocytose Aß plaques was determined after intrahippocampal transplantation in vivo. Results: Freshly solubilized Aß induced calcium oscillations, which persisted after removal of the stimulus. After few hours of aggregation, Aß was not able to induce oscillations in monocytic cells. Instead, lipopolysaccharide (LPS) induced calcium responses divergent from Aß-induced response. Furthermore, while LPS induced massive production of pro-inflammatory cytokines, neither synthetic Aß species nor native Aß deposits were able to induce pro-inflammatory activation of monocytic cells, contrary to primary microglia. Finally, monocytic cells retained their viability in the presence of Aß and exhibited phagocytic activity towards native fibrillar Aß deposits and congophilic Aß plaques. Conclusion: Monocytic cells carry diverse cellular responses to Aß and inflammatory stimulus LPS. Even though Aß species cause specific responses in calcium signaling, they completely lack the ability to induce pro-inflammatory phenotype of monocytic cells. Monocytes retain their viability and function in Aß-laden brain.

Bexarotene targets autophagy and is protective against thromboembolic stroke in aged mice with tauopathy.

Stroke is a highly debilitating, often fatal disorder for which current therapies are suitable for only a minor fraction of patients. Discovery of novel, effective therapies is hampered by the fact that advanced age, primary age-related tauopathy or comorbidities typical to several types of dementing diseases are usually not taken into account in preclinical studies, which predominantly use young, healthy rodents. Here we investigated for the first time the neuroprotective potential of bexarotene, an FDA-approved agent, in a co-morbidity model of stroke that combines high age and tauopathy with thromboembolic cerebral ischemia. Following thromboembolic stroke bexarotene enhanced autophagy in the ischemic brain concomitantly with a reduction in lesion volume and amelioration of behavioral deficits in aged transgenic mice expressing the human P301L-Tau mutation. In in vitro studies bexarotene increased the expression of autophagy markers and reduced autophagic flux in neuronal cells expressing P301L-Tau. Bexarotene also restored mitochondrial respiration deficits in P301L-Tau neurons. These newly described actions of bexarotene add to the growing amount of compelling data showing that bexarotene is a potent neuroprotective agent, and identify a novel autophagy-modulating effect of bexarotene.

UDP-sugar substrates of HAS3 regulate its O-GlcNAcylation, intracellular traffic, extracellular shedding and correlate with melanoma progression.

Hyaluronan content is a powerful prognostic factor in many cancer types, but the molecular basis of its synthesis in cancer still remains unclear. Hyaluronan synthesis requires the transport of hyaluronan synthases (HAS1-3) from Golgi to plasma membrane (PM), where the enzymes are activated. For the very first time, the present study demonstrated a rapid recycling of HAS3 between PM and endosomes, controlled by the cytosolic levels of the HAS substrates UDP-GlcUA and UDP-GlcNAc. Depletion of UDP-GlcNAc or UDP-GlcUA shifted the balance towards HAS3 endocytosis, and inhibition of hyaluronan synthesis. In contrast, UDP-GlcNAc surplus suppressed endocytosis and lysosomal decay of HAS3, favoring its retention in PM, stimulating hyaluronan synthesis, and HAS3 shedding in extracellular vesicles. The concentration of UDP-GlcNAc also controlled the level of O-GlcNAc modification of HAS3. Increasing O-GlcNAcylation reproduced the effects of UDP-GlcNAc surplus on HAS3 trafficking, while its suppression showed the opposite effects, indicating that O-GlcNAc signaling is associated to UDP-GlcNAc supply. Importantly, a similar correlation existed between the expression of GFAT1 (the rate limiting enzyme in UDP-GlcNAc synthesis) and hyaluronan content in early and deep human melanomas, suggesting the association of UDP-sugar metabolism in initiation of melanomagenesis. In general, changes in glucose metabolism, realized through UDP-sugar contents and O-GlcNAc signaling, are important in HAS3 trafficking, hyaluronan synthesis, and correlates with melanoma progression.

Immunomodulation by interleukin-33 is protective in stroke through modulation of inflammation.

Cerebral stroke induces massive Th1-shifted inflammation both in the brain and the periphery, contributing to the outcome of stroke. A Th1-type response is neurotoxic whereas a Th2-type response is accompanied by secretion of anti-inflammatory cytokines, such as interleukin-4 (IL-4). Interleukin-33 (IL-33) is a cytokine known to induce a shift towards the Th2-type immune response, polarize macrophages/microglia towards the M2-type, and induce production of anti-inflammatory cytokines. We found that the plasma levels of the inhibitory IL-33 receptor, sST2, are increased in human stroke and correlate with a worsened stroke outcome, suggesting an insufficient IL-33-driven Th2-type response. In mouse, peripheral administration of IL-33 reduced stroke-induced cell death and improved the sensitivity of the contralateral front paw at 5days post injury. The IL-33-treated mice had increased levels of IL-4 in the spleen and in the peri-ischemic area of the cortex. Neutralization of IL-4 by administration of an IL-4 antibody partially prevented the IL-33-mediated protection. IL-33 treatment also reduced astrocytic activation in the peri-ischemic area and increased the number of Arginase-1 immunopositive microglia/macrophages at the lesion site. In human T-cells, IL-33 treatment induced IL-4 secretion, and the conditioned media from IL-33-exposed T-cells reduced astrocytic activation. This study demonstrates that IL-33 is protective against ischemic insult by induction of IL-4 secretion and may represent a novel therapeutic approach for the treatment of stroke.

Interleukin-33 treatment reduces secondary injury and improves functional recovery after contusion spinal cord injury.

Interleukin-33 (IL-33) is a member of the interleukin-1 cytokine family and highly expressed in the naïve mouse brain and spinal cord. Despite the fact that IL-33 is known to be inducible by various inflammatory stimuli, its cellular localization in the central nervous system and role in pathological conditions is controversial. Administration of recombinant IL-33 has been shown to attenuate experimental autoimmune encephalomyelitis progression in one study, yet contradictory reports also exist. Here we investigated for the first time the pattern of IL-33 expression in the contused mouse spinal cord and demonstrated that after spinal cord injury (SCI) IL-33 was up-regulated and exhibited a nuclear localization predominantly in astrocytes. Importantly, we found that treatment with recombinant IL-33 alleviated secondary damage by significantly decreasing tissue loss, demyelination and astrogliosis in the contused mouse spinal cord, resulting in dramatically improved functional recovery. We identified both central and peripheral mechanisms of IL-33 action. In spinal cord, IL-33 treatment reduced the expression of pro-inflammatory tumor necrosis factor-alpha and promoted the activation of anti-inflammatory arginase-1 positive M2 microglia/macrophages, which chronically persisted in the injured spinal cord for up to at least 42 days after the treatment. In addition, IL-33 treatment showed a tendency towards reduced T-cell infiltration into the spinal cord. In the periphery, IL-33 treatment induced a shift towards the Th2 type cytokine profile and reduced the percentage and absolute number of cytotoxic, tumor necrosis factor-alpha expressing CD4+ cells in the spleen. Additionally, IL-33 treatment increased expression of T-regulatory cell marker FoxP3 and reduced expression of M1 marker iNOS in the spleen. Taken together, these results provide the first evidence that IL-33 administration is beneficial after CNS trauma. Treatment with IL33 may offer a novel therapeutic strategy for patients with acute contusion SCI.

Enhanced polyamine catabolism disturbs hematopoietic lineage commitment and leads to a myeloproliferative disease in mice overexpressing spermidine/spermine N¹-acetyltransferase.

Spermidine/spermine N(1)-acetyltransferase (SSAT) regulates intracellular polyamine levels by catabolizing spermidine and spermine which are essential for cell proliferation and differentiation. Hematological characterization of SSAT overexpressing mice (SSAT mice) revealed enhanced myelopoiesis and thrombocytopoiesis leading to increased amounts of myeloid cells in bone marrow, peripheral blood, and spleen compared to wild-type animals. The level of SSAT activity in the bone marrow cells was associated with the bone marrow cellularity and spleen weight which both were significantly increased in SSAT mice. The result of bone marrow transplantations indicated that both the intrinsic SSAT overexpression of bone marrow cells and bone marrow microenvironment had an impact on the observed hematopoietic phenotype. The Lineage-negative Sca-1(+) c-Kit(+) hematopoietic stem cell (HSC) compartment in SSAT mice, showed enhanced proliferation, increased proportion of long-term HSCs and affected expression of transcription factors associated with lineage priming and myeloid differentiation. The proportions of common myeloid and megakaryocytic/erythroid progenitors were decreased and the proportion of granulocyte-macrophage progenitors was increased in SSAT bone marrow. The data suggest that SSAT overexpression and the concomitantly accelerated polyamine metabolism in hematopoietic cells and bone marrow microenvironment affect lineage commitment and lead to the development of a mouse myeloproliferative disease in SSAT mice.