Radiotherapy induces persistent innate immune reprogramming of microglia into a primed state.

Over half of patients with brain tumors experience debilitating and often progressive cognitive decline after radiotherapy treatment. Microglia, the resident macrophages in the brain, have been implicated in this decline. In response to various insults, microglia can develop innate immune memory (IIM), which can either enhance (priming or training) or repress (tolerance) the response to subsequent inflammatory challenges. Here, we investigate whether radiation affects the IIM of microglia by irradiating the brains of rats and later exposing them to a secondary inflammatory stimulus. Comparative transcriptomic profiling and protein validation of microglia isolated from irradiated rats show a stronger immune response to a secondary inflammatory insult, demonstrating that radiation can lead to long-lasting molecular reprogramming of microglia. Transcriptomic analysis of postmortem normal-appearing non-tumor brain tissue of patients with glioblastoma indicates that radiation-induced microglial priming is likely conserved in humans. Targeting microglial priming or avoiding further inflammatory insults could decrease radiotherapy-induced neurotoxicity.

Glioblastoma and its treatment are associated with extensive accelerated brain aging.

Progressive neurocognitive dysfunction is the leading cause of a reduced quality of life in patients with primary brain tumors. Understanding how the human brain responds to cancer and its treatment is essential to improve the associated cognitive sequelae. In this study, we performed integrated transcriptomic and tissue analysis on postmortem normal-appearing non-tumor brain tissue from glioblastoma (GBM) patients that had received cancer treatments, region-matched brain tissue from unaffected control individuals and Alzheimer's disease (AD) patients. We show that normal-appearing non-tumor brain regions of patients with GBM display hallmarks of accelerated aging, in particular mitochondrial dysfunction, inflammation, and proteostasis deregulation. The extent and spatial pattern of this response decreased with distance from the tumor. Gene set enrichment analyses and a direct comparative analysis with an independent cohort of brain tissue samples from AD patients revealed a significant overlap in differentially expressed genes and a similar biological aging trajectory. Additionally, these responses were validated at the protein level showing the presence of increased lysosomal lipofuscin, phosphorylated microtubule-associated protein Tau, and oxidative DNA damage in normal-appearing brain areas of GBM patients. Overall, our data show that the brain of GBM patients undergoes accelerated aging and shared AD-like features, providing the basis for novel or repurposed therapeutic targets for managing brain tumor-related side effects.

Cold Mechanical Isolation of Placental Macrophages as a Method to Limit Procedure-Induced Activation of Macrophages.

Isolation of placental macrophages using enzymatic digestion at warm temperatures is widely used for in vitro studies. However, studies in brain and kidney tissue show that this method activates immune cells, immediate early genes, and heat shock proteins. Isolating placental macrophages while preserving their tissue-specific characteristics as much as possible is pivotal to reliably studying their functions. We therefore developed a mechanical dissociation protocol at low temperatures and compared this to enzymatic digestion at high temperatures. Decidual and villous macrophages were isolated from term human placentas. A cell suspension was generated by mechanical dissociation using a gentleMACS. For warm enzymatic digestion, Accutase was added, followed by incubation at 37°C. Macrophages were isolated after Ficoll density gradient centrifugation. Cell types were analyzed with flow cytometry (CD45, CD14, CD80, CD86, CD163, and CD206) and their activation status with real-time PCR (FOS, JUN, HSP27, HSP70, IL1ß, TNFα, IL10, and TGFß) after cell sorting. A higher proportion of leukocytes and macrophages was obtained from the villi with cold mechanical dissociation (p < 0.05). Compared to warm enzymatic digestion, cold mechanical dissociation resulted in a higher expression of CD163 in villous and decidual macrophages (p < 0.05). Warm enzymatic digestion showed higher levels of TNFα, IL1ß, and IL10 in decidual and villous macrophages, and HSP70 in villous macrophages. Our data show that mechanical dissociation of placental tissue at low temperatures is associated with less activation of placental macrophages. This suggests that cold mechanical dissociation is a preferred method, resulting in macrophages that more closely resemble their in-tissue state.

Prevention of microgliosis halts early memory loss in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline, the neuropathological formation of amyloid-beta (Aß) plaques and neurofibrillary tangles. The best cellular correlates of the early cognitive deficits in AD patients are synapse loss and gliosis. In particular, it is unclear whether the activation of microglia (microgliosis) has a neuroprotective or pathological role early in AD. Here we report that microgliosis is an early mediator of synaptic dysfunction and cognitive impairment in APP/PS1 mice, a mouse model of increased amyloidosis. We found that the appearance of microgliosis, synaptic dysfunction and behavioral impairment coincided with increased soluble Aß42 levels, and occurred well before the presence of Aß plaques. Inhibition of microglial activity by treatment with minocycline (MC) reduced gliosis, synaptic deficits and cognitive impairments at early pathological stages and was most effective when provided preventive, i.e., before the onset of microgliosis. Interestingly, soluble Aß levels or Aß plaques deposition were not affected by preventive MC treatment at an early pathological stage (4 months) whereas these were reduced upon treatment at a later stage (6 months). In conclusion, this study demonstrates the importance of early-stage prevention of microgliosis on the development of cognitive impairment in APP/PS1 mice, which might be clinically relevant in preventing memory loss and delaying AD pathogenesis.

Early-life stress lastingly impacts microglial transcriptome and function under basal and immune-challenged conditions.

Early-life stress (ELS) leads to increased vulnerability to psychiatric disorders including depression later in life. Neuroinflammatory processes have been implicated in ELS-induced negative health outcomes, but how ELS impacts microglia, the main tissue-resident macrophages of the central nervous system, is unknown. Here, we determined the effects of ELS-induced by limited bedding and nesting material during the first week of life (postnatal days [P]2-9) on microglial (i) morphology; (ii) hippocampal gene expression; and (iii) synaptosome phagocytic capacity in male pups (P9) and adult (P200) mice. The hippocampus of ELS-exposed adult mice displayed altered proportions of morphological subtypes of microglia, as well as microglial transcriptomic changes related to the tumor necrosis factor response and protein ubiquitination. ELS exposure leads to distinct gene expression profiles during microglial development from P9 to P200 and in response to an LPS challenge at P200. Functionally, synaptosomes from ELS-exposed mice were phagocytosed less by age-matched microglia. At P200, but not P9, ELS microglia showed reduced synaptosome phagocytic capacity when compared to control microglia. Lastly, we confirmed the ELS-induced increased expression of the phagocytosis-related gene GAS6 that we observed in mice, in the dentate gyrus of individuals with a history of child abuse using in situ hybridization. These findings reveal persistent effects of ELS on microglial function and suggest that altered microglial phagocytic capacity is a key contributor to ELS-induced phenotypes.

Brain macrophages acquire distinct transcriptomes in multiple sclerosis lesions and normal appearing white matter.

Multiple sclerosis (MS) is a disease of the central nervous system that is characterized by inflammation and focal areas of demyelination, ultimately resulting in axonal degradation and neuronal loss. Several lines of evidence point towards a role for microglia and other brain macrophages in disease initiation and progression, but exactly how lesion formation is triggered is currently unknown. Here, we characterized early changes in MS brain tissue through transcriptomic analysis of normal appearing white matter (NAWM). We found that NAWM was characterized by enriched expression of genes associated with inflammation and cellular stress derived from brain macrophages. Single cell RNA sequencing confirmed a stress response in brain macrophages in NAWM and identified specific microglia and macrophage subsets at different stages of demyelinating lesions. We identified both phagocytic/activated microglia and CAM clusters that were associated with various MS lesion types. These overall changes in microglia and macrophages associated with lesion development in MS brain tissue may provide therapeutic targets to limit lesion progression and demyelination.

Identification of distinct and age-dependent p16High microglia subtypes.

Cells expressing high levels of the cyclin-dependent kinase (CDK)4/6 inhibitor p16 (p16High ) accumulate in aging tissues and promote multiple age-related pathologies, including neurodegeneration. Here, we show that the number of p16High cells is significantly increased in the central nervous system (CNS) of 2-year-old mice. Bulk RNAseq indicated that genes expressed by p16High cells were associated with inflammation and phagocytosis. Single-cell RNAseq of brain cells indicated p16High cells were primarily microglia, and their accumulation was confirmed in brains of aged humans. Interestingly, we identified two distinct subpopulations of p16High microglia in the mouse brain, with one being age-associated and one present in young animals. Both p16High clusters significantly differed from previously described disease-associated microglia and expressed only a partial senescence signature. Taken together, our study provides evidence for the existence of two p16-expressing microglia populations, one accumulating with age and another already present in youth that could positively and negatively contribute to brain homeostasis, function, and disease.

The effects of postmortem delay on mouse and human microglia gene expression.

Microglia are specialized macrophages of the central nervous system (CNS) and first to react to pathogens or injury. Over the last decade, transcriptional profiling of microglia significantly contributed to our understanding of their functions. In the case of human CNS samples, either potential CNS pathology in the case of surgery samples, or a postmortem delay (PMD) due to the time needed for tissue access and collection, are potential factors that affect gene expression profiles. To determine the effect of PMD on the microglia transcriptome, we first analyzed mouse microglia, where genotype, antemortem conditions and PMD can be controlled. Microglia were isolated from mice after different PMDs (0, 4, 6, 12, and 24 hr) using fluorescence-activated cell sorting (FACS). The number of viable microglia significantly decreased with increasing PMD, but even after a 12 hr PMD, high-quality RNA could be obtained. PMD had very limited effect on mouse microglia gene expression, only 50 genes were differentially expressed between different PMDs. These genes were related to mitochondrial, ribosomal, and protein binding functions. In human microglia transcriptomes we previously generated, 31 of the 50 PMD-associated mouse genes had human homologs, and their relative expression was also affected by PMD. This study provides a set of genes that shows relative expression changes in relation to PMD, both in mouse and human microglia. Although the gene expression changes detected are subtle, these genes need to be accounted for when analyzing microglia transcriptomes generated from samples with variable PMDs.

Regionally diverse astrocyte subtypes and their heterogeneous response to EAE.

Astrocytes fulfil many functions in the central nervous system (CNS), including contribution to the blood brain barrier, synapse formation, and trophic support. In addition, they can mount an inflammatory response and are heterogeneous in morphology and function. To extensively characterize astrocyte subtypes, we FACS-isolated and gene expression profiled distinct astrocyte subtypes from three central nervous system regions; forebrain, hindbrain and spinal cord. Astrocyte subpopulations were separated based on GLAST/SLC1A3 and ACSA-2/ATP1B2 cell surface expression. The local brain environment proved key in establishing different transcriptional programs in astrocyte subtypes. Transcriptional differences between subtypes were also apparent in experimental autoimmune encephalomyelitis (EAE) mice, where these astrocyte subtypes showed distinct responses. While gene expression signatures associated with blood-brain barrier maintenance were lost, signatures involved in neuroinflammation and neurotoxicity were increased in spinal cord astrocytes, especially during acute disease stages. In chronic stages of EAE, this reactive astrocyte signature was slightly decreased, while obtaining a more proliferative profile, which might be relevant for glia scar formation and tissue regeneration. Morphological heterogeneity of astrocytes previously indicated the presence of astrocyte subtypes, and here we show diversity based on transcriptome variation associated with brain regions and differential responsiveness to a neuroinflammatory insult (EAE).

Exploring the VISTA of microglia: immune checkpoints in CNS inflammation.

Negative checkpoint regulators (NCR) are intensely pursued as targets to modulate the immune response in cancer and autoimmunity. A large variety of NCR is expressed by central nervous system (CNS)-resident cell types and is associated with CNS homeostasis, interactions with peripheral immunity and CNS inflammation and disease. Immunotherapy blocking NCR affects the CNS as patients can develop neurological issues including encephalitis and multiple sclerosis (MS). How these treatments affect the CNS is incompletely understood, since expression and function of NCR in the CNS are only beginning to be unravelled. V-type immunoglobulin-like suppressor of T cell activation (VISTA) is an NCR that is expressed primarily in the haematopoietic system by myeloid and T cells. VISTA regulates T cell quiescence and activation and has a variety of functions in myeloid cells including efferocytosis, cytokine response and chemotaxis. In the CNS, VISTA is predominantly expressed by microglia and macrophages of the CNS. In this review, we summarize the role of NCR in the CNS during health and disease. We highlight expression of VISTA across cell types and CNS diseases and discuss the function of VISTA in microglia and during CNS ageing, inflammation and neurodegeneration. Understanding the role of VISTA and other NCR in the CNS is important considering the adverse effects of immunotherapy on the CNS, and in view of their therapeutic potential in CNS disease.

VISTA expression by microglia decreases during inflammation and is differentially regulated in CNS diseases.

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator (NCR) involved in inhibition of T cell-mediated immunity. Expression changes of other NCRs (PD-1, PD-L1/L2, CTLA-4) during inflammation of the central nervous system (CNS) were previously demonstrated, but VISTA expression in the CNS has not yet been explored. Here, we report that in the human and mouse CNS, VISTA is most abundantly expressed by microglia, and to lower levels by endothelial cells. Upon TLR stimulation, VISTA expression was reduced in primary neonatal mouse and adult rhesus macaque microglia in vitro. In mice, microglial VISTA expression was reduced after lipopolysaccharide (LPS) injection, during experimental autoimmune encephalomyelitis (EAE), and in the accelerated aging Ercc1 Δ/- mouse model. After LPS injection, decreased VISTA expression in mouse microglia was accompanied by decreased acetylation of lysine residue 27 in histone 3 in both its promoter and enhancer region. ATAC-sequencing indicated a potential regulation of VISTA expression by Pu.1 and Mafb, two transcription factors crucial for microglia function. Finally, our data suggested that VISTA expression was decreased in microglia in multiple sclerosis lesion tissue, whereas it was increased in Alzheimer's disease patients. This study is the first to demonstrate that in the CNS, VISTA is expressed by microglia, and that VISTA is differentially expressed in CNS pathologies.

The Kaleidoscope of Microglial Phenotypes.

Gene expression analyses of microglia, the tissue-resident macrophages of the central nervous system (CNS), led to the identification of homeostatic as well as neurological disease-specific gene signatures of microglial phenotypes. Upon alterations in the neural microenvironment, either caused by local insults from within the CNS (during neurodegenerative diseases) or by macroenvironmental incidents, such as social stress, microglia can switch phenotypes-generally referred to as "microglial activation." The interplay between the microenvironment and its influence on microglial phenotypes, regulated by (epi)genetic mechanisms, can be imagined as the different colorful crystal formations (microglial phenotypes) that change upon rotation (microenvironmental changes) of a kaleidoscope. In this review, we will discuss microglial phenotypes in relation to neurodevelopment, homeostasis, in vitro conditions, aging, and neurodegenerative diseases based on transcriptome studies. By overlaying these disease-specific microglial signatures, recent publications have identified a specific set of genes that is differentially expressed in all investigated diseases, called a microglial core gene signature with multiple diseases. We will conclude this review with a discussion about the complexity of this microglial core gene signature associated with multiple diseases.

Identification of Variants in RET and IHH Pathway Members in a Large Family With History of Hirschsprung Disease.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. METHODS: We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. RESULTS: Within the linkage region, we identified 1 putative splice variant in the lipopolysaccharide responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on messenger RNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the Indian hedgehog gene (IHH) and its mediator, the transcription factor GLI family zinc finger 3 (GLI3). When expressed in cells, the RET-P399L variant disrupted protein glycosylation and had altered phosphorylation following activation by GDNF. The deletion in GDNF prevented secretion of its gene product, reducing RET activation, and the IHH-Q51K variant reduced expression of the transcription factor GLI1. Injection of morpholinos that target ihh reduced the number of enteric neurons to 13% ± 1.4% of control zebrafish. CONCLUSIONS: In a study of a large family with history of HSCR, we identified variants in LRBA, RET, the gene encoding the RET ligand (GDNF), IHH, and a gene encoding a mediator of IHH signaling (GLI3). These variants altered functions of the gene products when expressed in cells and knockout of ihh reduced the number of enteric neurons in the zebrafish gut.

Maternal inflammation induces immune activation of fetal microglia and leads to disrupted microglia immune responses, behavior, and learning performance in adulthood.

Maternal inflammation during pregnancy can have detrimental effects on embryonic development that persist during adulthood. However, the underlying mechanisms and insights in the responsible cell types are still largely unknown. Here we report the effect of maternal inflammation on fetal microglia, the innate immune cells of the central nervous system (CNS). In mice, a challenge with LPS during late gestation stages (days 15-16-17) induced a pro-inflammatory response in fetal microglia. Adult whole brain microglia of mice that were exposed to LPS during embryonic development displayed a persistent reduction in pro-inflammatory activation in response to a re-challenge with LPS. In contrast, hippocampal microglia of these mice displayed an increased inflammatory response to an LPS re-challenge. In addition, a reduced expression of brain-derived neurotrophic factor (BDNF) was observed in hippocampal microglia of LPS-offspring. Microglia-derived BDNF has been shown to be important for learning and memory processes. In line with these observations, behavioral- and learning tasks with mice that were exposed to maternal inflammation revealed reduced home cage activity, reduced anxiety and reduced learning performance in a T-maze. These data show that exposure to maternal inflammation during late gestation results in long term changes in microglia responsiveness during adulthood, which is different in nature in hippocampus compared to total brain microglia.

Immune hyperreactivity of Aß plaque-associated microglia in Alzheimer's disease.

Alzheimer's disease (AD) is strongly associated with microglia-induced neuroinflammation. Particularly, Aß plaque-associated microglia take on an "activated" morphology. However, the function and phenotype of these Aß plaque-associated microglia are not well understood. We show hyperreactivity of Aß plaque-associated microglia upon systemic inflammation in transgenic AD mouse models (i.e., 5XFAD and APP23). Gene expression profiling of Aß plaque-associated microglia (major histocompatibility complex II+ microglia) isolated from 5XFAD mice revealed a proinflammatory phenotype. The upregulated genes involved in the biological processes (gene ontology terms) included: "immune response to external stimulus" such as Axl, Cd63, Egr2, and Lgals3, "cell motility", such as Ccl3, Ccl4, Cxcr4, and Sdc3, "cell differentiation", and "system development", such as St14, Trpm1, and Spp1. In human AD tissue with similar Braak stages, expression of phagocytic markers and AD-associated genes, including HLA-DRA, APOE, AXL, TREM2, and TYROBP, was higher in laser-captured early-onset AD (EOAD) plaques than in late-onset AD plaques. Interestingly, the nonplaque parenchyma of both EOAD and late-onset AD brains, the expression of above-mentioned markers were similarly low. Here, we provide evidence that Aß plaque-associated microglia are hyperreactive in their immune response and phagocytosis in the transgenic AD mice as well as in EOAD brain tissue. We suggest that Aß plaque-associated microglia are the primary source of neuroinflammation related to AD pathology.

Isolation of Microglia and Immune Infiltrates from Mouse and Primate Central Nervous System.

Microglia are the innate immune cells of the central nervous system (CNS) and play an important role in the maintenance of tissue homeostasis, providing neural support and neuroprotection. Microglia constantly survey their environment and quickly respond to homeostatic perturbations. Microglia are increasingly implicated in neuropathological and neurodegenerative conditions, such as Alzheimer's disease, Parkinson's disease, and glioma progression. Here, we describe a detailed isolation protocol for microglia and immune infiltrates, optimized for large amounts of post mortem tissue from human and rhesus macaque, as well as smaller tissue amounts from mouse brain and spinal cord, that yield a highly purified microglia population (up to 98 % purity). This acute isolation protocol is based on mechanical dissociation and a two-step density gradient purification, followed by fluorescence-activated cell sorting (FACS) to obtain pure microglia and immune infiltrate populations.

Identification of highly connected hub genes in the protective response program of human macrophages and microglia activated by alpha B-crystallin.

The glial stress protein alpha B-crystallin (HSPB5) is an endogenous agonist for Toll-like receptor 2 in CD14+ cells. Following systemic administration, HSPB5 acts as a potent inhibitor of neuroinflammation in animal models and reduces lesion development in multiple sclerosis patients. Here, we show that systemically administered HSPB5 rapidly crosses the blood-brain barrier, implicating microglia as additional targets for HSPB5 along with peripheral monocytes and macrophages. To compare key players in the HSPB5-induced protective response of human macrophages and microglia, we applied weighted gene co-expression network analysis on transcript expression data obtained 1 and 4 h after activation. This approach identified networks of genes that are co-expressed in all datasets, thus reducing the complexity of the nonsynchronous waves of transcripts that appear after activation by HSPB5. In both cell types, HSPB5 activates a network of highly connected genes that appear to be functionally equivalent and consistent with the therapeutic effects of HSPB5 in vivo, since both networks include factors that suppress apoptosis, the production of proinflammatory factors, and the development of adaptive immunity. Yet, hub genes at the core of the network in either cell type were strikingly different. They prominently feature the well-known tolerance-promoting programmed-death ligand 1 as a key player in the macrophage response to HSPB5, and the immune-regulatory enzyme cyclooxygenase-2 (COX-2) in that of microglia. This latter finding indicates that despite its reputation as a potential target for nonsteroidal anti-inflammatory drugs, microglial COX-2 plays a central role in the therapeutic effects of HSPB5 during neuroinflammation. GLIA 2017;65:460-473.

Aging, microglia and cytoskeletal regulation are key factors in the pathological evolution of the APP23 mouse model for Alzheimer's disease.

Aging is the key risk factor for Alzheimer's disease (AD). In addition, the amyloid-beta (Aß) peptide is considered a critical neurotoxic agent in AD pathology. However, the connection between these factors is unclear. We aimed to provide an extensive characterization of the gene expression profiles of the amyloidosis APP23 model for AD and control mice and to evaluate the effect of aging on these profiles. We also correlated our findings to changes in soluble Aß-levels and other pathological and symptomatic features of the model. We observed a clear biphasic expression profile. The first phase displayed a maturation profile, which resembled features found in young carriers of familial AD mutations. The second phase reflected aging processes and showed similarities to the progression of human AD pathology. During this phase, the model displayed a clear upregulation of microglial activation and lysosomal pathways and downregulation of neuron differentiation and axon guidance pathways. Interestingly, the changes in expression were all correlated to aging in general, but appeared more extensive/accelerated in APP23 mice.

Identification of a conserved and acute neurodegeneration-specific microglial transcriptome in the zebrafish.

Microglia are brain resident macrophages important for brain development, connectivity, homeostasis and disease. However, it is still largely unclear how microglia functions and their identity are regulated at the molecular level. Although recent transcriptomic studies have identified genes specifically expressed in microglia, the function of most of these genes in microglia is still unknown. Here, we performed RNA sequencing on microglia acutely isolated from healthy and neurodegenerative zebrafish brains. We found that a large fraction of the mouse microglial signature is conserved in the zebrafish, corroborating the use of zebrafish to help understand microglial genetics in mammals in addition to studying basic microglia biology. Second, our transcriptome analysis of microglia following neuronal ablation suggested primarily a proliferative response of microglia, which we confirmed by immunohistochemistry and in vivo imaging. Together with the recent improvements in genome editing technology in zebrafish, these data offer opportunities to facilitate functional genetic research on microglia in vivo in the healthy as well as in the diseased brain. GLIA 2016;65:138-149.

Lymphocryptovirus Infection of Nonhuman Primate B Cells Converts Destructive into Productive Processing of the Pathogenic CD8 T Cell Epitope in Myelin Oligodendrocyte Glycoprotein.

EBV is the major infectious environmental risk factor for multiple sclerosis (MS), but the underlying mechanisms remain obscure. Patient studies do not allow manipulation in vivo. We used the experimental autoimmune encephalomyelitis (EAE) models in the common marmoset and rhesus monkey to model the association of EBV and MS. We report that B cells infected with EBV-related lymphocryptovirus (LCV) are requisite APCs for MHC-E-restricted autoaggressive effector memory CTLs specific for the immunodominant epitope 40-48 of myelin oligodendrocyte glycoprotein (MOG). These T cells drive the EAE pathogenesis to irreversible neurologic deficit. The aim of this study was to determine why LCV infection is important for this pathogenic role of B cells. Transcriptome comparison of LCV-infected B cells and CD20(+) spleen cells from rhesus monkeys shows increased expression of genes encoding elements of the Ag cross-presentation machinery (i.e., of proteasome maturation protein and immunoproteasome subunits) and enhanced expression of MHC-E and of costimulatory molecules (CD70 and CD80, but not CD86). It was also shown that altered expression of endolysosomal proteases (cathepsins) mitigates the fast endolysosomal degradation of the MOG40-48 core epitope. Finally, LCV infection also induced expression of LC3-II(+) cytosolic structures resembling autophagosomes, which seem to form an intracellular compartment where the MOG40-48 epitope is protected against proteolytic degradation by the endolysosomal serine protease cathepsin G. In conclusion, LCV infection induces a variety of changes in B cells that underlies the conversion of destructive processing of the immunodominant MOG40-48 epitope into productive processing and cross-presentation to strongly autoaggressive CTLs.