CNS Microenvironment Research Articles

Single cell mapping of human brain tumors reveals tumor-specific education of tissue-invading leukocytes.

2509 Background: Brain tumors can be both of intracranial origin (e.g. gliomas) or spread from another location in the body (metastases). Tumor growth in the CNS is commonly favored by a highly immunosuppressive tumor microenvironment (TME). Whether the TME is predominantly shaped by the CNS microenvironment or by the nature of the malignancy is unknown, as is the origin and function of CNS tumor-associated macrophages (TAM). Methods: We have mapped the leukocyte landscape of brain tumors using high-dimensional profiling with mass cytometry (CyTOF). We designed two CyTOF panels (one lymphoid and one myeloid focused) measuring 74 parameters at the single-cell level and analyzed samples from 47 individuals with the most common tumor entities being glioblastoma and metastases from melanoma and lung cancer as well as control tissue from epilepsy surgery. Results: The heterogeneous composition of tissue-resident and invading immune cells within the TME alone permitted a clear distinction between glioblastoma and metastases. Tissue-invading leukocytes accumulate in metastases, whereas gliomas predominantly contain tissue-resident reactive microglia. In gliomas, isocitrate dehydrogenase 1 (IDH1) mutations are associated with minimal host responses whereas IDH1-wildtype gliomas comprise monocyte-derived macrophages (MDM) similar to metastases. These MDM show a distinctive signature trajectory indicative of tumor-driven education. Conclusions: The immune cell composition of the brain tumor TME is tumor entity-specific, rather than dictated by the CNS tissue. Defining the distinct immunological signature of brain tumors can facilitate the rational design of targeted immunotherapy strategies.

A dynamic perfusion based blood-brain barrier model for cytotoxicity testing and drug permeation

The blood-brain barrier (BBB) serves to protect and regulate the CNS microenvironment. The development of an in-vitro mimic of the BBB requires recapitulating the correct phenotype of the in-vivo BBB, particularly for drug permeation studies. However the majority of widely used BBB models demonstrate low transendothelial electrical resistance (TEER) and poor BBB phenotype. The application of shear stress is known to enhance tight junction formation and hence improve the barrier function. We utilised a high TEER primary porcine brain microvascular endothelial cell (PBMEC) culture to assess the impact of shear stress on barrier formation using the Kirkstall QuasiVivo 600 (QV600) multi-chamber perfusion system. The application of shear stress resulted in a reorientation and enhancement of tight junction formation on both coverslip and permeable inserts, in addition to enhancing and maintaining TEER for longer, when compared to static conditions. Furthermore, the functional consequences of this was demonstrated with the reduction in flux of mitoxantrone across PBMEC monolayers. The QV600 perfusion system may service as a viable tool to enhance and maintain the high TEER PBMEC system for use in in-vitro BBB models.

LS2 METASTATIC BRAIN TUMORS / MENINGIOMAS: CURRENT CONCEPTS AND THERAPEUTIC PERSPECTIVES

Non-glial brain tumors are the most common neoplasms affecting the central nervous system. Brain metastases are a heterogenous complication of systemic cancers. Recent research has provided insight into some important aspects of brain metastasis development, interaction of brain-metastatic tumor cells with resident cells of the CNS microenvironment and growth patterns within the brain parenchyma.Treatment choices have to consider clinical presentation, number, size and localisation of brain metastases, status of extracranial tumor burden, prior therapies, co-morbidities and histological and molecular tumor characteristics. Recent advances show that targeted therapies and immunotherapies have activity against established brain metastases and some targeted therapies are able to prevent brain metastasis development. Meningiomas are common and can be cured in 70–80% of cases by surgical resection. However, the rest of cases cannot be resected completey due to surgically inaccessibility (e.g. skull base) or show non-benign histopathological features that are associated with tumor recurrence. Meningiomas have been shown to be molecularly variable and carry distinct and reccurent genetic and epigenetic alterations that seem to enable targeted therapy and refined prognostication. Overall, significant advances in the biologcial understanding of brain metastases and meningiomas drive clinical trial design and improvement of clinical management strategies for these common tumors.

P: 67 Hippocampal and Cerebellar Astrocytes Morphological Alterations in a Rat Model of Chronic Hepatic Encephalopathy

BACKGROUND: Chronic hepatic encephalopathy (CHE) is a spectrum of neuropsychiatric abnormalities in patients with chronic liver disease. The hippocampus and cerebellum are key regions implicated in the cognitive and fine motor deficits of CHE.1 Astrocytes can sense neuronal activity through neurotransmitter-receptors and ion-channels, modulate the neural circuits and control energy homeostasis through morphological plasticity. They adjust their volume by releasing osmolytes (inositol, taurine, creatine) and can remodel their processes.2–4 They initiate synaptic development and regulate synaptic plasticity in both the healthy and injured brain.5 Astrocytes convert the neurotoxin ammonia into glutamine, regulate cerebral hemodynamics and cytokine responses to inflammation-related signaling pathways.3,6 Histological studies of BDL rat brains are controversial to date, some reporting no change, increased or decreased of anti-glial-fibrillary-acidic-protein (GFAP).Our aim was to investigate whether and how astrocytes react in the hippocampus and cerebellum of bile-duct-ligated (BDL) rat. METHODS: BDL-rats and sham-rats at 4 and 8-weeks post-BDL (n = 3/group/2-time-points) were anesthetized with 4% isoflurane and Temgesic (ESSEX) 0.1mg/kg before transcardial PBS perfusion. Brains were fixed in 4%-formaldehyde and cryopreserved in 30%-sucrose, embedded in a Tissue-Tek®OCT. Immunohistochemistry: On 16 micron sagittal-sections, GFAP7 and DAPI (nucleus) were used. For each rat (n = 3 at 4-weeks and n = 3 at 8-weeks post-BDL, n = 3 Shams), seven slides/rat were analyzed (distance between sections ∼250 microns). Morphometric measurements were performed using Sholl-analysis8 (∼1000 processes/group/region) (Figure 1c). RESULTS: Astrocytes activation is represented by the significant increase in GFAP+ cells at week4 post-BDL in the hippocampus (+47.5%) and cerebellum (+48.7%) vs SHAM (Figure 1a,b). Decrease in the processes intersection was observed already at week4 post-BDL (Figure 1e). Although at week8 post-BDL a significant reduction of astrocytes number was observed (∼20%) (Figure 1b) the astrocytes were altered morphologically, showing shortening (hippocampus: week 4 −13.3%, week 8 −32.4%; cerebellum: week 4: −17.3%, week 8: −35.5%) and decreased number of processes (hippocampus: week 4:−5.8%, week 8:−18.77%; cerebellum: week 4:−32.2%, week 8:−41.5%) and processes intersections at week 8 post−BDL (hippocampus: ring1: −14.5%, ring 2: −39%, ring 3: −72.5%; cerebellum: ring1: −17.6%, ring 2: −27.4%, ring 3: −58.7%) as well as minor processes thickening (Figure 1d). The reported % changes are relative to SHAM. CONCLUSIONS: To our knowledge, this is the first report showing significant alterations in astrocytes count and important morphological changes already 4-weeks post-BDL in the hippocampus and cerebellum. Increase in GFAP+ cells may be related to the stimulation of mature astrocytes and reentering into proliferation cycle which is similar to proliferating neonatal astrocytes and is a common situation in various neuropathological disorders.9,10 The proliferation of activated glial-cells can induce additional inflammatory reactions and creation of glial-scar, something to be investigated in future studies. Finally, the astrocytosis and astrocyte morphology changes may alter the CNS microenvironment that usually ensures neuronal health and may contribute to the cognitive impairment of BDL rats.

A T Cell Suppressive Circuitry Mediated by CD39 and Regulated by ShcC/Rai Is Induced in Astrocytes by Encephalitogenic T Cells.

Multiple sclerosis is an autoimmune disease caused by autoreactive immune cell infiltration into the central nervous system leading to inflammation, demyelination, and neuronal loss. While myelin-reactive Th1 and Th17 are centrally implicated in multiple sclerosis pathogenesis, the local CNS microenvironment, which is shaped by both infiltrated immune cells and central nervous system resident cells, has emerged a key player in disease onset and progression. We have recently demonstrated that ShcC/Rai is as a novel astrocytic adaptor whose loss in mice protects from experimental autoimmune encephalomyelitis. Here, we have explored the mechanisms that underlie the ability of Rai−/− astrocytes to antagonize T cell-dependent neuroinflammation. We show that Rai deficiency enhances the ability of astrocytes to upregulate the expression and activity of the ectonucleotidase CD39, which catalyzes the conversion of extracellular ATP to the immunosuppressive metabolite adenosine, through both contact-dependent and–independent mechanisms. As a result, Rai-deficient astrocytes acquire an enhanced ability to suppress T-cell proliferation, which involves suppression of T cell receptor signaling and upregulation of the inhibitory receptor CTLA-4. Additionally, Rai-deficient astrocytes preferentially polarize to the neuroprotective A2 phenotype. These results identify a new mechanism, to which Rai contributes to a major extent, by which astrocytes modulate the pathogenic potential of autoreactive T cells.

TLR signals license CD8 Tcells to destroy oligodendrocytes expressing an antigen shared with a Listeria pathogen.

Increasing evidence suggests a role of CD8 Tcells in autoimmune demyelinating CNS disease, which, however, is still controversially discussed. Mice, which express ovalbumin (OVA) as cytosolic self-antigen in oligodendrocytes (ODC-OVA mice), respond to CNS infection induced by OVA-expressing attenuated Listeria with CD8 Tcell-mediated inflammatory demyelination. This model is suitable to decipher the contribution of CD8 Tcells and the pathogen in autoimmune CNS disease. Here, we show that both antigen and pathogen are required in the CNS for disease induction, though not in a physically linked fashion. Intracerebral challenge with combined toll like receptor (TLR) TLR2 and TLR9 as well as TLR7 and TLR9 agonists substituted for the bacterial stimulus, but not with individual TLR agonists (TLR2, TLR3,TLR5,TLR7, TLR9). Furthermore, MyD88 inactivation rendered ODC-OVA mice resistant to disease induction. Collectively, CD8 Tcell-mediated destruction of oligodendrocytes is activated if (i) an antigen shared with an infectious agent is provided in the CNS microenvironment and (ii) innate immune signals inform the CNS microenvironment that pathogen removal warrants an immune attack by CD8 Tcells, even at the expense of locally restricted demyelination.

Competitive repopulation of an empty microglial niche yields functionally distinct subsets of microglia-like cells

Circulating monocytes can compete for virtually any tissue macrophage niche and become long-lived replacements that are phenotypically indistinguishable from their embryonic counterparts. As the factors regulating this process are incompletely understood, we studied niche competition in the brain by depleting microglia with >95% efficiency using Cx3cr1CreER/+R26DTA/+ mice and monitored long-term repopulation. Here we show that the microglial niche is repopulated within weeks by a combination of local proliferation of CX3CR1+F4/80lowClec12a– microglia and infiltration of CX3CR1+F4/80hiClec12a+ macrophages that arise directly from Ly6Chi monocytes. This colonization is independent of blood brain barrier breakdown, paralleled by vascular activation, and regulated by type I interferon. Ly6Chi monocytes upregulate microglia gene expression and adopt microglia DNA methylation signatures, but retain a distinct gene signature from proliferating microglia, displaying altered surface marker expression, phagocytic capacity and cytokine production. Our results demonstrate that monocytes are imprinted by the CNS microenvironment but remain transcriptionally, epigenetically and functionally distinct.

Repulsive Environment Attenuation during Adult Mouse Optic Nerve Regeneration.

The regenerative capacity of CNS tracts has ever been a great hurdle to regenerative medicine. Although recent studies have described strategies to stimulate retinal ganglion cells (RGCs) to regenerate axons through the optic nerve, it still remains to be elucidated how these therapies modulate the inhibitory environment of CNS. Thus, the present work investigated the environmental content of the repulsive axon guidance cues, such as Sema3D and its receptors, myelin debris, and astrogliosis, within the regenerating optic nerve of mice submitted to intraocular inflammation + cAMP combined to conditional deletion of PTEN in RGC after optic nerve crush. We show here that treatment was able to promote axonal regeneration through the optic nerve and reach visual targets at twelve weeks after injury. The Regenerating group presented reduced MBP levels, increased microglia/macrophage number, and reduced astrocyte reactivity and CSPG content following optic nerve injury. In addition, Sema3D content and its receptors are reduced in the Regenerating group. Together, our results provide, for the first time, evidence that several regenerative repulsive signals are reduced in regenerating optic nerve fibers following a combined therapy. Therefore, the treatment used made the CNS microenvironment more permissive to regeneration.

Treatment-Resistant Human Extraparenchymal Neurocysticercosis: An Immune-Inflammatory Approach to Cysticidal Treatment Outcome

Objective: The aim of this study is to analyze the immune-endocrine profile in neurocysticercosis (NC) patients resistant to cysticidal treatment. Methods: The inflammatory and regulatory responses of 8 resistant NC patients with extraparenchymal parasites and 5 healthy controls were evaluated through flow cytometry. Serum interleukin levels were measured by ELISA and catecholamines levels by high performance liquid chromatography. Results: Higher percentages of Tr1, CD4+CD25+FOXP3+CD127– and CD4+CD45RO+FOXP3^HI were found in NC patients compared with healthy controls, but no difference was found in catecholamine levels. Antigen-specific proliferative immune response was observed in NC patients. Neither anti-inflammatory nor pro-inflammatory cytokines showed differences between patients and controls, but IL-6 levels were lower in treatment-resistant NC patients. In addition, TGFβ showed a significant negative correlation with dopamine. Conclusions: Altogether, these results may point to a modulation of the neuroinflammation in these patients that could indirectly favor cysticercal survival in CNS microenvironment.

Galectin-3 in M2 Macrophages Plays a Protective Role in Resolution of Neuropathology in Brain Parasitic Infection by Regulating Neutrophil Turnover.

Macrophages/microglia with M2-activation phenotype are thought to play important anti-inflammatory and tissue reparative functions in the brain, yet the molecular bases of their functions in the CNS remain to be clearly defined. In a preclinical model of neurocysticercosis using brain infection with a parasite Mesocestoides corti, we previously reported the presence of large numbers of M2 cells in the CNS. In this study using female mice, we report that M2 macrophages in the parasite-infected brain display abundant galectin-3 expression. Disease severity was increased in Galectin-3-/- mice correlating with increased neurological defects, augmented cell death and, importantly, massive accumulation of neutrophils and M2 macrophages in the CNS of these mice. Because neutrophil clearance by efferocytosis is an important function of M2 macrophages, we investigated a possible role of galectin-3 in this process. Indeed, galectin-3-deficient M2 macrophages exhibited a defect in efferocytic clearance of neutrophils in vitro Furthermore, adoptive transfer of M2 macrophages from galectin-3-sufficient WT mice reduced neutrophilia in the CNS and ameliorated disease severity in parasite-infected Galectin-3-/- mice. Together, these results demonstrate, for the first time, a novel role of galectin-3 in M2 macrophage function in neutrophil turnover and resolution of inflammatory pathology in the CNS. This likely will have implications in neurocysticercosis and neuroinflammatory diseases.SIGNIFICANCE STATEMENT Macrophages/microglia with M1-activation phenotype are thought to promote CNS pathology, whereas M2-anti-inflammatory phenotype promote CNS repair. However, the mechanisms regulating M2 cell-protective functions in the CNS microenvironment are undefined. The current study reports that helminth infection of the brain induces an increased expression of galectin-3 in M2 macrophages accumulated in the CNS. Using multiple experimental models in vivo and in vitro, they show that galectin-3 in M2 macrophages functions to clear neutrophils accumulated in the CNS. Importantly, galectin-3 in M2 macrophages plays a central role in the containment of neuropathology and disease severity. These results provide a direct mechanistic evidence of the protective function of M2 macrophages in the CNS.

Uloga glija ćelija, krvno-moždane barijere i citokina u nastanku konvulzija - implikacije za terapeutske modalitete

Epilepsy is a chronic, common, neurological disorder marked by transient, paroxysmal and hypersynchronous activity of the brain neurons, behaviorally manifested as seizures. It is developed through the process of epileptogenesis which alters neuronal excitability, establishes critical interconnections and develop neuronal hyperexcitability and degeneration, as well as the neuronal network reorganization as its main mechanisms. There are a number of different mechanisms of epileptogenesis, including neuroinflammation as a recently highlighted important novel mechanism. In this review paper, our focus will be to light up the latest findings about neuroinflammation as a pathogenic factor in epileptogenesis. Neuroinflammation is characterized by the structural and functional alteration of the CNS glial cells and peripherally derived immune cells with the presence of blood-brain barrier (BBB) dysfunction as main mechanisms. Disequilibrium in the CNS microenvironment is often followed by increased synthesis of proinflammatory cytokines (IL-6, IL-1β, TNF-α, IFN-γ) and chemokines. The interplay between glial alteration, BBB dysfunction, cytokines and chemokines establish a positive feedback cascade for further epileptogenesis. It is still unclear if neuroinflammation is causing epileptogenesis or whether in a consequence of that, but, there are clear findings about positive feedback between these two processes. This interconnection could be a helpful key to better target therapeutic treatment of neuroinflammation for providing beneficial effects for patients with epilepsy.

Stress disinhibits microglia via down-regulation of CD200R: A mechanism of neuroinflammatory priming

Exposure to stressors primes the neuroinflammatory and microglial proinflammatory response to subsequent immune challenges, suggesting that stress might attenuate immunoregulatory mechanisms in the CNS microenvironment. CD200:CD200R is a key immunoregulatory signaling dyad that constrains microglial activation, and disruption of CD200:CD200R signaling primes microglia to subsequent immune challenges. Therefore, the present study examined the mediating role of CD200:CD200R signaling in stress-induced microglial priming. Here, we found that exposure to an acute stressor reduced CD200R expression across sub-regions of the hippocampus, amygdala as well as in isolated hippocampal microglia. A transcriptional suppressor of CD200R, CAAT/Enhancer Binding Proteinβ, was induced by stress and inversely associated with CD200R expression. To examine whether disrupted CD200:CD200R signaling plays a mediating role in stress-induced microglial priming, a soluble fragment of CD200 (mCD200Fc) was administered intra-cisterna magna prior to stressor exposure and stress-induced microglia priming assessed ex vivo 24 h later. Treatment with mCD200Fc blocked the stress-induced priming of the microglial pro-inflammatory response. Further, treatment with mCD200R1Fc recapitulated the effects of stress on microglial priming. We previously found that stress increases the alarmin high mobility group box-1 (HMGB1) in hippocampus, and that HMGB1 mediates stress-induced priming of microglia. Thus, we examined whether stress-induced increases in hippocampal HMGB1 are a consequence of disrupted CD200:CD200R signaling. Indeed, treatment with mCD200Fc prior to stress exposure blocked the stress-induced increase in hippocampal HMGB1. The present study suggests that stress exposure disrupts immunoregulatory mechanisms in the brain, which typically constrain the immune response of CNS innate immune cells. This attenuation of immunoregulatory mechanisms may thus permit a primed activation state of microglia to manifest.

Systemic Neutrophil Depletion Modulates the Migration and Fate of Transplanted Human Neural Stem Cells to Rescue Functional Repair.

The interaction of transplanted stem cells with local cellular and molecular cues in the host CNS microenvironment may affect the potential for repair by therapeutic cell populations. In this regard, spinal cord injury (SCI), Alzheimer's disease, and other neurological injuries and diseases all exhibit dramatic and dynamic changes to the host microenvironment over time. Previously, we reported that delayed transplantation of human CNS-derived neural stem cells (hCNS-SCns) at 9 or 30 d post-SCI (dpi) resulted in extensive donor cell migration, predominantly neuronal and oligodendrocytic donor cell differentiation, and functional locomotor improvements. Here, we report that acute transplantation of hCNS-SCns at 0 dpi resulted in localized astroglial differentiation of donor cells near the lesion epicenter and failure to produce functional improvement in an all-female immunodeficient mouse model. Critically, specific immunodepletion of neutrophils (polymorphonuclear leukocytes) blocked hCNS-SCns astroglial differentiation near the lesion epicenter and rescued the capacity of these cells to restore function. These data represent novel evidence that a host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population, and support targeting the inflammatory microenvironment in combination with cell transplantation after SCI.SIGNIFICANCE STATEMENT The interaction of transplanted cells with local cellular and molecular cues in the host microenvironment is a key variable that may shape the translation of neurotransplantation research to the clinical spinal cord injury (SCI) human population, and few studies have investigated these events. We show that the specific immunodepletion of polymorphonuclear leukocyte neutrophils using anti-Ly6G inhibits donor cell astrogliosis and rescues the capacity of a donor cell population to promote locomotor improvement after SCI. Critically, our data demonstrate novel evidence that a specific host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population.

Association of CNS involvement in childhood acute lymphoblastic leukaemia with cholesterol biosynthesis upregulation

BackgroundAcute lymphoblastic leukaemia is the most common childhood cancer. Despite advances in treatment, meningeal-infiltrating CNS disease (found in up to 90% of children if untreated) remains a challenge. Every child with acute lymphoblastic leukaemia receives intensive and toxic treatment targeting CNS disease, yet CNS involvement at relapse is common. We tested our hypothesis that leukaemic cells must adapt metabolically to survive in the nutritionally poor CNS microenvironment. MethodsHuman acute lymphoblastic leukaemia cell lines (SEM and REH) were xenografted into immunodeficient (NSG) mice. Leukaemic cells purified from the CNS and spleen underwent RNA-sequencing (NextSeq 500, Illumina, SanDiego, CA, USA). Clinical validity of findings was explored in publicly available microarray datasets (GSE11877/TARGET and GSE60926) with gene set enrichment and pathway analyses. The effect of simvastatin on SEM cells in vitro, and the development of CNS leukaemia in vivo, was investigated. FindingsRNA-sequencing analysis showed upregulation of the cholesterol biosynthesis pathway in CNS compared with that in systemic leukaemia. GSE60926 microarray data confirmed this finding in leukaemic cells from cerebrospinal fluid in children with acute lymphoblastic leukaemia at CNS relapse versus cells from bone marrow at diagnosis and relapse. Analysis of GSE11877/TARGET data showed that upregulation of two or more cholesterol synthesis genes (z-score ≥2) in the bone marrow blasts at diagnosis was associated with an increased rate of CNS relapse (10 [30·3%] of 33 patients with upregulation vs 15 [8·6%] of 174 patients without; log-rank [Mantel-Cox] p<0·0001). In-vitro study showed that disruption of cholesterol synthesis via hydroxymethylglutaryl-CoA reductase inhibition with simvastatin decreased leukaemic cell proliferation and induced apoptosis. Interestingly, oral simvastatin treatment increased CNS leukaemia in male though not female NSG mice (three per group, Student's t test p=0·0004). InterpretationOur findings suggest that cholesterol metabolism could be important in CNS acute lymphoblastic leukaemia. Cholesterol biosynthesis is upregulated in CNS leukaemia, and a cholesterol gene signature in diagnostic bone marrow predicts CNS relapse. Disruption of cholesterol synthesis is toxic in vitro but not effective in vivo. Therefore, reduced circulating cholesterol might induce a CNS-like phenotype in systemic acute lymphoblastic leukaemia cells, increasing cholesterol synthesis and adaptation for the CNS niche. FundingWilliam and Elizabeth Davies Foundation, Chief Scientist Office (ETM/374).

ATP-induced IL-1β secretion is selectively impaired in microglia as compared to hematopoietic macrophages.

Under stressful conditions nucleotides are released from dying cells into the extracellular space, where they can bind to purinergic P2X and P2Y receptors. High concentrations of extracellular ATP in particular induce P2X7-mediated signaling, which leads to inflammasome activation. This in turn leads to the processing and secretion of pro-inflammatory cytokines, like interleukin (IL)-1β. During neurodegenerative diseases, innate immune responses are shaped by microglia and we have previously identified microglia-specific features of inflammasome-mediated responses. Here, we compared ATP-induced IL-1β secretion in primary rhesus macaque microglia and bone marrow-derived macrophages (BMDM). We assessed the full expression profile of P2 receptors and characterized the induction and modulation of IL-1β secretion by extracellular nucleotides. Microglia secreted significantly lower levels of IL-1β in response to ATP when compared to BMDM. We demonstrate that this is not due to differences in sensitivity, kinetics or expression of ATP-processing enzymes, but rather to differences in purinergic receptor expression levels and usage. Using a combined approach of purinergic receptor agonists and antagonists, we demonstrate that ATP-induced IL-1β secretion in BMDM was fully dependent on P2X7 signaling, whereas in microglia multiple purinergic receptors were involved, including P2X7 and P2X4. These cell type-specific features of conserved innate immune responses may reflect adaptations to the vulnerable CNS microenvironment. GLIA 2016;64:2231-2246.

Enhanced antagonism of BST-2 by a neurovirulent SIV envelope.

Current antiretroviral therapy (ART) is not sufficient to completely suppress disease progression in the CNS, as indicated by the rising incidence of HIV-1-associated neurocognitive disorders (HAND) among infected individuals on ART. It is not clear why some HIV-1-infected patients develop HAND, despite effective repression of viral replication in the circulation. SIV-infected nonhuman primate models are widely used to dissect the mechanisms of viral pathogenesis in the CNS. Here, we identified 4 amino acid substitutions in the cytoplasmic tail of viral envelope glycoprotein gp41 of the neurovirulent virus SIVsm804E that enhance replication in macrophages and associate with enhanced antagonism of the host restriction factor BM stromal cell antigen 2 (BST-2). Rhesus macaques were inoculated with a variant of the parental virus SIVsmE543-3 that had been engineered to contain the 4 amino acid substitutions present in gp41 of SIVsm804E. Compared with WT virus-infected controls, animals infected with mutant virus exhibited higher viral load in cerebrospinal fluid. Together, these results are consistent with a potential role for BST-2 in the CNS microenvironment and suggest that BST-2 antagonists may serve as a possible target for countermeasures against HAND.

Neuropathologic, phenotypic and functional analyses of Mucosal Associated Invariant T cells in Multiple Sclerosis

BackgroundThe involvement of Mucosal Associated Invariant T (MAIT) cells, which are anti-microbial semi-invariant T cells, remains elusive in Multiple Sclerosis (MS). ObjectiveDeciphering the potential involvement of MAIT cells in the MS inflammatory process. MethodsBy flow cytometry, blood MAIT cells from similar cohorts of MS patients and healthy volunteers (HV) were compared for frequency, phenotype, activation potential after in vitro TCR engagement by bacterial ligands and transmigration abilities through an in vitro model of blood-brain barrier. MS CNS samples were also studied by immunofluorescent staining and quantitative PCR. Results and conclusionBlood MAIT cells from relapsing-remitting MS patients and HV presented similar frequency, ex vivo effector phenotype and activation abilities. MAIT cells represented 0.5% of the total infiltrating T cells on 39 MS CNS lesions. This is low as compared to blood frequency (p<0.001), but consistent with their low transmigration rate. Finally, transcriptional over-expression of MR1 – which presents cognate antigens to MAIT cells – and of the activating cytokines IL-18 and IL-23 was evidenced in MS lesions, suggesting that the CNS microenvironment is suited to activate the few infiltrating MAIT cells. Taken together, these data place MAIT cells from MS patients as minor components of the inflammatory pathological process.

Genetic risk factors for spontaneous intracerebral haemorrhage

Intracerebral haemorrhage (ICH) is associated with the greatest morbidity and mortality of all stroke subtypes. Established risk factors for ICH include hypertension, alcohol use, current cigarette smoking, and use of oral anticoagulants and/or antiplatelet agents. Familial aggregation of ICH has been observed, and the heritability of ICH risk has been estimated at 44%. Few genes have been found to be associated with ICH at the population level, and much of the evidence for genetic risk factors for ICH comes from single studies conducted in relatively small and homogenous populations. In this Review, we summarize the current knowledge of genetic variants associated with primary spontaneous ICH. Two variants of the gene encoding apolipoprotein E (APOE) - which also contributes to the pathogenesis of cerebral amyloid angiopathy - are the most likely candidates for variants that increase the risk of ICH. Other promising candidates for risk alleles in ICH include variants of the genes ACE, PMF1/SLC25A44, COL4A2, and MTHFR. Other genetic variants, related to haemostasis, lipid metabolism, inflammation, and the CNS microenvironment, have been linked to ICH in single candidate gene studies. Although evidence for genetic contributions to the risk of ICH exists, we do not yet fully understand how and to what extent this information can be utilized to prevent and treat ICH.

The inflammatory microenvironment in brain metastases: potential treatment target?

Brain metastases (BM) are frequent in cancer patients and are associated with high morbidity and mortality. The incidence of BM is increasing and only limited treatment strategies are available. Immunomodulatory agents are emerging as effective cancer therapeutics and a deeper understanding of the inflammatory microenvironment in BM might reveal new treatment possibilities. Although the brain has long been considered an 'immune-privileged' organ with limited capacity for inflammatory response, BM do contain tumor infiltrating lymphocytes (TILs). Moreover, dense infiltration with TIL showed an association with favorable patients' overall survival times. Microglia/macrophages were shown to be involved in the pivotal steps of BM formation by providing a "pre-metastatic niche" attracting BM initiating cells and to promote growth and survival of tumor cells in the CNS microenvironment. The anti-cytotoxic T lymphocyte antigen 4 (CTLA4) immune checkpoint inhibitor ipilimumab showed activity against melanoma BM in a clinical trial, thus providing proof of concept for effective therapeutic immunomodulation in patients with CNS metastases. Collectively, the available data show that BM harbors an active inflammatory microenvironment that may be exploited as treatment target.

A novel size-based sorting mechanism of pinocytic luminal cargoes in microglia.

Microglia are the resident immune cells in the CNS and play diverse roles in the maintenance of CNS homeostasis. Recent studies have shown that microglia continually survey the CNS microenvironment and scavenge cell debris and aberrant proteins by phagocytosis and pinocytosis, and that reactive microglia are capable to present antigens to T cells and initiate immune responses. However, how microglia process the endocytosed contents and evoke an immune response remain unclear. Here we report that a size-dependent selective transport of small soluble contents from the pinosomal lumen into lysosomes is critical for the antigen processing in microglia. Using fluorescent probes and water-soluble magnetic nanobeads of defined sizes, we showed in cultured rodent microglia, and in a cell-free reconstructed system that pinocytosed proteins become degraded immediately following pinocytosis and the resulting peptides are selectively delivered to major histocompatibility complex class II (MHC-II) containing lysosomes, whereas undegraded proteins are retained in the pinosomal lumen. This early size-based sorting of pinosomal contents relied on the formation of transient tunnel between pinosomes and lysosomes in a Rab7- and dynamin II-dependent manner, which allowed the small contents to pass through but restricted large ones. Inhibition of the size-based sorting markedly reduced proliferation and cytokine release of cocultured CD4(+) T cells, indicating that the size-based sorting is required for efficient antigen presentation by microglial cells. Together, these findings reveal a novel early sorting mechanism for pinosomal luminal contents in microglial cells, which may explain how microglia efficiently process protein antigens and evoke an immune response.