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In order to recover from infection, organisms must balance robust immune responses to pathogens with the tolerance of immune-mediated pathology. This balance is particularly critical within the central nervous system, whose complex architecture, essential function, and limited capacity for self-renewal render it susceptible to both pathogen-and immune-mediated pathology. Here, we identify the alarmin IL-33 and its receptor ST2 as critical for host survival to neuroinvasive flavivirus infection. We identify oligodendrocytes as the critical source of IL-33, and microglia as the key cellular responders. Notably, we find that the IL-33/ST2 axis does not impact viral control or adaptive immune responses; rather, it is required to promote the activation and survival of microglia. In the absence of intact IL-33/ST2 signaling in the brain, neuroinvasive flavivirus infection triggered aberrant recruitment of monocyte-derived peripheral immune cells, increased neuronal stress, and neuronal cell death, effects that compromised organismal survival. These findings identify IL-33 as a critical mediator of CNS tolerance to pathogen-initiated immunity and inflammation.Author SummaryThe central nervous system (CNS) is an extraordinarily complex organ system with limited capacity for repair and renewal. When infectious pathogens invade the CNS, resulting immune responses must clear the pathogen while limiting immune-mediated tissue pathology. IL-33 is an “alarmin” cytokine, whose release from dying cells can promote a variety of immune responses. IL-33 is expressed at high levels in the brain, and previous work has implicated signaling by IL-33 in CNS development as well as in the response to parasitic infection of the CNS. Here, we identify IL-33 as a key mediator of disease tolerance in the context of neuroinvasive infection with the flaviviruses West Nile virus and Zika virus. Notably, we find that IL-33 signaling is not involved in the clearance of virus from the brain; rather, IL-33 is required to promote the survival and reprogramming brain-resident immune cells (microglia). When IL-33 signaling is disrupted in mice, flavivirus infection results in defective microglial activation, increased death of both microglia and neurons, increased invasion of the brain by peripheral immune cells, and increased host death. This work suggests that IL-33 is a key mediator of host tolerance upon neuroinvasive flavivirus infection.

Dysfunctional meningeal immunity is associated with cognitive decline, impaired glymphatics, and enhanced Alzheimer’s pathology., Aging leads to a progressive deterioration of meningeal lymphatics and peripheral immunity, which may accelerate cognitive decline. We hypothesized that an age-related reduction in C-C chemokine receptor type 7 (CCR7)–dependent egress of immune cells through the lymphatic vasculature mediates some aspects of brain aging and potentially exacerbates cognitive decline and Alzheimer’s disease–like brain β-amyloid (Aβ) pathology. We report a reduction in CCR7 expression by meningeal T cells in old mice that is linked to increased effector and regulatory T cells. Hematopoietic CCR7 deficiency mimicked the aging-associated changes in meningeal T cells and led to reduced glymphatic influx and cognitive impairment. Deletion of CCR7 in 5xFAD transgenic mice resulted in deleterious neurovascular and microglial activation, along with increased Aβ deposition in the brain. Treating old mice with anti-CD25 antibodies alleviated the exacerbated meningeal regulatory T cell response and improved cognitive function, highlighting the therapeutic potential of modulating meningeal immunity to fine-tune brain function in aging and in neurodegenerative diseases.

In recent years, there has been great progress in the field of neuroimmunology, implicating both parenchymal macrophages (microglia) and meningeal immunity in homeostatic brain function. In this review, Norris and Kipnis discuss these two immune compartments and how they are critically segregated to preserve homoeostasis., Recent advances have directed our knowledge of the immune system from a narrative of “self” versus “nonself” to one in which immune function is critical for homeostasis of organs throughout the body. This is also the case with respect to the central nervous system (CNS). CNS immunity exists in a segregated state, with a marked partition occurring between the brain parenchyma and meningeal spaces. While the brain parenchyma is patrolled by perivascular macrophages and microglia, the meningeal spaces are supplied with a diverse immune repertoire. In this review, we posit that such partition allows for neuro–immune crosstalk to be properly tuned. Convention may imply that meningeal immunity is an ominous threat to brain function; however, recent studies have shown that its presence may instead be a steady hand directing the CNS to optimal performance.

Norris et al. show that microglia are the key phagocytes in removal of synaptic debris in the dorsal lateral geniculate nucleus after optic nerve injury. This microglial function is dependent on recognition of neurodegeneration and is mediated by the complement system., Phagocytosis of synaptic material by microglia is critical for central nervous system development. Less well understood is this microglial function in the injured adult brain. Assay of microglial phagocytosis is challenging, because peripheral myeloid cells engraft the site of injury, which could obscure interpretation of microglial roles. The model used here, optic nerve crush injury, results in degeneration of synapses in the dorsal lateral geniculate nucleus (dLGN), which stimulates rapid activation and engulfment of synaptic material by resident microglia without myeloid cell engraftment. Pharmacological depletion of microglia causes postinjury accumulation of synaptic debris, suggesting that microglia are the dominant postinjury phagocytes. Genetic or pharmacological manipulations revealed that neuronal activity does not trigger microglia phagocytosis after injury. RNA sequencing reveals C1q and CD11b/CR3 involvement in clearance of debris by dLGN-resident microglia. Indeed, C1qa−/− and Itgam−/− mice exhibit impaired postinjury debris clearance. Our results show how neurodegenerative debris is cleared by microglia and offers a model for studying its mechanisms and physiological roles.

A key process in central sensory circuit development involves activity-dependent “pruning” of early exuberant terminals. Here, we studied gustatory terminal field maturation in the postnatal mouse nucleus of the solitary tract (NST) during normal development and in mice where their mothers were fed a low NaCl diet for a limited period during early embryonic development. “Pruning” of terminal fields in controls involved the complement system and appears driven by NaCl-elicited taste activity. In contrast, offspring of mothers with the early dietary manipulation failed to “prune” gustatory terminal fields even though peripheral taste activity developed normally. The ability to “prune” in these mice was rescued by activating myeloid cells postnatally, and conversely, “pruning” was arrested in controls with the loss of myeloid cell function. The altered “pruning” and myeloid cell function appears programmed before the peripheral gustatory system is assembled and corresponds to the embryonic period when microglia progenitors migrate into the brain.

SummaryMutations in MECP2, encoding the epigenetic regulator methyl-CpG-binding protein 2, are the predominant cause of Rett syndrome, a disease characterized by both neurological symptoms and systemic abnormalities. Microglial dysfunction is thought to contribute to disease pathogenesis, and here we found microglia become activated and subsequently lost with disease progression in Mecp2-null mice. Mecp2 was found to be expressed in peripheral macrophage and monocyte populations, several of which also became depleted in Mecp2-null mice. RNA-seq revealed increased expression of glucocorticoid- and hypoxia-induced transcripts in Mecp2-deficient microglia and peritoneal macrophages. Furthermore, Mecp2 was found to regulate inflammatory gene transcription in response to TNF stimulation. Postnatal re-expression of Mecp2 using Cx3cr1creER increased the lifespan of otherwise Mecp2-null mice. These data suggest that Mecp2 regulates microglia and macrophage responsiveness to environmental stimuli to promote homeostasis. Dysfunction of tissue-resident macrophages might contribute to the systemic pathologies observed in Rett syndrome.

IL-4 has been extensively studied in the context of its role in immunity. Accumulating evidence indicates, however, that it also plays a critical role in higher functions of the normal brain, such as memory and learning. In this review, we summarize current knowledge of the basic immunology of IL-4, describe how and where this cytokine appears to operate in normal brain function, and propose a hypothesis concerning its potential role in neurological pathologies.

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC–Neuroblastoma-related (MYCN)– amplified ( P < 0.0001) and high-risk ( P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC 50 values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC 50 values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity ( P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.

As long-lived post-mitotic cells, neurons employ unique strategies to resist pathogen infection while preserving cellular function. Here, using a murine model of Zika virus (ZIKV) infection, we identified an innate immune pathway that restricts ZIKV replication in neurons and is required for survival upon ZIKV infection of the central nervous system (CNS). We found that neuronal ZIKV infection activated the nucleotide sensor ZBP1 and the kinases RIPK1 and RIPK3, core components of virus-induced necroptotic cell death signaling. However, activation of this pathway in ZIKV-infected neurons did not induce cell death. Rather, RIPK signaling restricted viral replication by altering cellular metabolism via upregulation of the enzyme IRG1 and production of the metabolite itaconatemechanism of viral restriction during neur. Itaconate inhibited the activity of succinate dehydrogenase, generating a metabolic state in neurons that suppresses replication of viral genomes. These findings demonstrate an immunometabolic mechanism of viral restriction during neuroinvasive infection.

The mechanisms underlying genetic susceptibility at loci discovered by genome-wide association study (GWAS) approaches in human cancer remain largely undefined. In this study, we characterized the high-risk neuroblastoma association at the BRCA1-related locus, BARD1, showing that disease-associated variations correlate with increased expression of the oncogenically activated isoform, BARD1β. In neuroblastoma cells, silencing of BARD1β showed genotype-specific cytotoxic effects, including decreased substrate-adherence, anchorage-independence, and foci growth. In established murine fibroblasts, overexpression of BARD1β was sufficient for neoplastic transformation. BARD1β stabilized the Aurora family of kinases in neuroblastoma cells, suggesting both a mechanism for the observed effect and a potential therapeutic strategy. Together, our findings identify BARD1β as an oncogenic driver of high-risk neuroblastoma tumorigenesis, and more generally, they illustrate how robust GWAS signals offer genomic landmarks to identify molecular mechanisms involved in both tumor initiation and malignant progression. The interaction of BARD1β with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma. Cancer Res; 72(8); 2068–78. ©2012 AACR.

Genome-wide association studies have proven to be a powerful tool to identify susceptibility loci in cancer, however the molecular basis of a majority of these association signals remain undefined. We have previously reported nine common single nucleotide polymorphism (SNP) alleles at chromosome 2q35, within and 5′ upstream of the BRCA1-associated RING domain (BARD1) gene, to be significantly associated with high-risk neuroblastoma (Capasso, M et al., Nature Genetics 2009). Here, we focus on understanding the biological underpinnings of this genetic association by investigating how common variation at this locus correlates with changes in BARD1 expression in both the germline and in neuroblastoma samples, and further how these BARD1 alterations are involved in driving neuroblastoma tumorigenesis. We first showed that the neuroblastoma associated SNP variation at 2q35 is in linkage disequilibrium (LD) with multiple genomic areas dense with regulatory domains, including many putative binding sites for the neuronal splicing regulator, FOX2. We next demonstrated that in peripheral blood mononuclear cells, these SNPs correlate both with a trend towards increased overall BARD1 expression and with differential BARD1 exon expression. Specifically, a majority of the neuroblastoma associated SNPs were significantly associated with decreased expression of the RING domain forming BARD1 exons 2 and 3 (P = 0.0008-0.034). We have identified 16 unique BARD1 isoforms in the neuroblastoma tissue of origin, sympathetic fetal ganglia, with the β-BARD1 isoform specifically splicing out exons 2 and 3. β-BARD1 was found to be ubiquitously expressed in a panel of fetal sympathetic ganglia, neuroblastoma cell lines, and high-risk primary tumors, with its expression being significantly increased in cell lines harboring a homozygous BARD1 risk allele genotype at the most significantly associated 2q35 SNP, rs6435862 (N = 22; P = 0.04). Transient transfection of a specific siRNA targeting the alternatively spliced boundary of β-BARD1 in a panel of neuroblastoma cell lines (N = 12) showed that neuroblastoma cells harboring at least one risk allele at SNP rs6435862 and the highest β-BARD1 expression showed a significant inhibition of cell proliferation upon β-BARD1 knockdown (% growth inhibition = 45-81%, P = 0.0004-0.034), while six of seven cell lines with a homozygous major allele genotype at rs6435862 and lower β-BARD1 expression did not show significant growth inhibition. These data are consistent with β-BARD1 acting as an oncogene in neuroblastoma and we hypothesize that common genomic variation drives formation of this isoform, underlying neuroblastoma predisposition on 2q35. Ongoing work will focus on further defining the role of common variation in BARD1's RNA splicing patterns and identifying the molecular basis of β-BARD1's oncogenicity in neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3867.