Repetitive Traumatic Brain Injury Causes Neuroinflammation before Tau Pathology in Adolescent P301S Mice.

Repetitive closed head injury (rCHI) is commonly encountered in young athletes engaged in contact and collision sports. Traumatic brain injury (TBI) including rCHI has been reported to be an important risk factor for several tauopathies in studies of adult humans and animals. However, the link between rCHI and the progression of tau pathology in adolescents remains to be elucidated. We evaluated whether rCHI can trigger the initial acceleration of pathological tau in adolescent mice and impact the long-term outcomes post-injury. To this end, we subjected adolescent transgenic mice expressing the P301S tau mutation to mild rCHI and assessed tau hyperphosphorylation, tangle formation, markers of neuroinflammation, and behavioral deficits at 40 days post rCHI. We report that rCHI did not accelerate tau pathology and did not worsen behavioral outcomes compared to control mice. However, rCHI induced cortical and hippocampal microgliosis and corpus callosum astrocytosis in P301S mice by 40 days post-injury. In contrast, we did not find significant microgliosis or astrocytosis after rCHI in age-matched WT mice or sham-injured P301S mice. Our data suggest that neuroinflammation precedes the development of Tau pathology in this rCHI model of adolescent repetitive mild TBI.

Glioblastoma hijacks microglial gene expression to support tumor growth.

BACKGROUND: Glioblastomas are the most common and lethal primary brain tumors. Microglia, the resident immune cells of the brain, survey their environment and respond to pathogens, toxins, and tumors. Glioblastoma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Despite the presence of large numbers of microglia in glioblastoma, the tumors continue to grow, and these neuroimmune cells appear incapable of keeping the tumor in check. To understand this process, we analyzed gene expression in microglia interacting with glioblastoma cells. METHODS: We used RNASeq of isolated microglia to analyze the expression patterns of genes involved in key microglial functions in mice with glioblastoma. We focused on microglia that had taken up tumor-derived EVs and therefore were within and immediately adjacent to the tumor. RESULTS: We show that these microglia have downregulated expression of genes involved in sensing tumor cells and tumor-derived danger signals, as well as genes used for tumor killing. In contrast, expression of genes involved in facilitating tumor spread was upregulated. These changes appear to be in part EV-mediated, since intracranial injection of EVs in normal mice led to similar transcriptional changes in microglia. We observed a similar microglial transcriptomic signature when we analyzed datasets from human patients with glioblastoma. CONCLUSION: Our data define a microgliaGlioblastoma specific phenotype, whereby glioblastomas have hijacked gene expression in the neuroimmune system to favor avoiding tumor sensing, suppressing the immune response, clearing a path for invasion, and enhancing tumor propagation. For further exploration, we developed an interactive online tool at http://www.glioma-microglia.com with all expression data and additional functional and pathway information for each gene.

Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease.

Microglia are the principal immune cells of the brain. In Alzheimer disease, these brain mononuclear phagocytes are recruited from the blood and accumulate in senile plaques. However, the role of microglia in Alzheimer disease has not been resolved. Microglia may be neuroprotective by phagocytosing amyloid-beta (Abeta), but their activation and the secretion of neurotoxins may also cause neurodegeneration. Ccr2 is a chemokine receptor expressed on microglia, which mediates the accumulation of mononuclear phagocytes at sites of inflammation. Here we show that Ccr2 deficiency accelerates early disease progression and markedly impairs microglial accumulation in a transgenic mouse model of Alzheimer disease (Tg2576). Alzheimer disease mice deficient in Ccr2 accumulated Abeta earlier and died prematurely, in a manner that correlated with Ccr2 gene dosage, indicating that absence of early microglial accumulation leads to decreased Abeta clearance and increased mortality. Thus, Ccr2-dependent microglial accumulation plays a protective role in the early stages of Alzheimer disease by promoting Abeta clearance.

Linking indirect effects of cytomegalovirus in transplantation to modulation of monocyte innate immune function.

Cytomegalovirus (CMV) is an important cause of morbidity and mortality in the immunocompromised host. In transplant recipients, a variety of clinically important "indirect effects" are attributed to immune modulation by CMV, including increased mortality from fungal disease, allograft dysfunction and rejection in solid organ transplantation, and graft-versus-host-disease in stem cell transplantation. Monocytes, key cellular targets of CMV, are permissive to primary, latent and reactivated CMV infection. Here, pairing unbiased bulk and single cell transcriptomics with functional analyses we demonstrate that human monocytes infected with CMV do not effectively phagocytose fungal pathogens, a functional deficit which occurs with decreased expression of fungal recognition receptors. Simultaneously, CMV-infected monocytes upregulate antiviral, pro-inflammatory chemokine, and inflammasome responses associated with allograft rejection and graft-versus-host disease. Our study demonstrates that CMV modulates both immunosuppressive and immunostimulatory monocyte phenotypes, explaining in part, its paradoxical "indirect effects" in transplantation. These data could provide innate immune targets for the stratification and treatment of CMV disease.

Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer's disease mice.

Early microglial accumulation in Alzheimer's disease (AD) delays disease progression by promoting clearance of beta-amyloid (Abeta) before formation of senile plaques. However, persistent Abeta accumulation despite increasing microglial numbers suggests that the ability of microglia to clear Abeta may decrease with age and progression of AD pathology. To determine the effects of aging and Abeta deposition on microglial ability to clear Abeta, we used quantitative PCR to analyze gene expression in freshly isolated adult microglia from 1.5-, 3-, 8-, and 14-month-old transgenic PS1-APP mice, an established mouse model of AD, and from their nontransgenic littermates. We found that microglia from old PS1-APP mice, but not from younger mice, have a twofold to fivefold decrease in expression of the Abeta-binding scavenger receptors scavenger receptor A (SRA), CD36, and RAGE (receptor for advanced-glycosylation endproducts), and the Abeta-degrading enzymes insulysin, neprilysin, and MMP9, compared with their littermate controls. In contrast, PS1-APP microglia had a 2.5-fold increase in the proinflammatory cytokines IL-1beta (interleukin-1beta) and tumor necrosis factor alpha (TNFalpha), suggesting that there is an inverse correlation between cytokine production and Abeta clearance. In support of this possibility, we found that incubation of cultured N9 mouse microglia with TNFalpha decreased the expression of SRA and CD36 and reduced Abeta uptake. Our data indicate that, although early microglial recruitment promotes Abeta clearance and is neuroprotective in AD, as disease progresses, proinflammatory cytokines produced in response to Abeta deposition downregulate genes involved in Abeta clearance and promote Abeta accumulation, therefore contributing to neurodegeneration. Antiinflammatory therapy for AD should take this dichotomous microglial role into consideration.

Analysis of the Microglial Sensome.

Sensing changes in the brain milieu is a major microglial function that regulates the ability of these cells to perform other tasks including host defense and homeostasis. Microglia express a cluster of genes that allow them to perform their sensing functions termed the sensome. It is important to be able to assess expression of these genes and their corresponding proteins in isolated microglia. Here we describe a step by step procedure to isolate microglia from adult mouse brains and provide examples of analyzing sensome genes and proteins by quantitative PCR and flow cytometry ex vivo. We also describe an in situ hybridization method to detect sensome RNA in the mouse brain. These approaches can be applied to all known sensome genes and can be used in analyzing sensome expression in physiological and pathological conditions.

Heterozygous CX3CR1 Deficiency in Microglia Restores Neuronal ß-Amyloid Clearance Pathways and Slows Progression of Alzheimer's Like-Disease in PS1-APP Mice.

CX3CR1 is a chemokine receptor expressed on microglia that binds Fractalkine (CX3CL1) and regulates microglial recruitment to sites of neuroinflammation. Full deletion of CX3CR1 in mouse models of Alzheimer's disease have opposing effects on amyloid-ß and tau pathologies raising concerns about the benefits of targeting CX3CR1 for treatment of this disease. Since most therapies achieve only partial blockade of their targets, we investigated the effects of partial CX3CR1 deficiency on the development and progression of amyloid-ß deposition in the PS1-APP Alzheimer's mouse model. We generated PS1-APP mice heterozygous for CX3CR1 (PS1-APP-CX3CR1+/-) and analyzed these mice for Alzheimer's-like pathology. We found that partial CX3CR1 deficiency was associated with a significant reduction in Aß levels and in senile-like plaque load in the brain as compared with age-matched PS1-APP mice. Reduced Aß level in the brain was associated with improved cognitive function. Levels of the neuronal-expressed Aß-degrading enzymes insulysin and matrix metalloproteinase 9, which are reduced in the brains of regular PS1-APP mice, were significantly higher in PS1-APP-CX3CR1+/- mice. Our data indicate that lowering CX3CR1 levels or partially inhibiting its activity in the brain may be a therapeutic strategy to increase neuronal Aß clearance, reduce Aß levels and delay progression of Alzheimer's-Like disease. Our findings also suggest a novel pathway where microglial CX3CR1 can regulates gene expression in neurons.

Time-Dependent Changes in Microglia Transcriptional Networks Following Traumatic Brain Injury.

The neuroinflammatory response to traumatic brain injury (TBI) is critical to both neurotoxicity and neuroprotection, and has been proposed as a potentially modifiable driver of secondary injury in animal and human studies. Attempts to broadly target immune activation have been unsuccessful in improving outcomes, in part because the precise cellular and molecular mechanisms driving injury and outcome at acute, subacute, and chronic time points after TBI remain poorly defined. Microglia play a critical role in neuroinflammation and their persistent activation may contribute to long-term functional deficits. Activated microglia are characterized by morphological transformation and transcriptomic changes associated with specific inflammatory states. We analyzed the temporal course of changes in inflammatory genes of microglia isolated from injured brains at 2, 14, and 60 days after controlled cortical impact (CCI) in mice, a well-established model of focal cerebral contusion. We identified a time dependent, injury-associated change in the microglial gene expression profile toward a reduced ability to sense tissue damage, perform housekeeping, and maintain homeostasis in the early stages following CCI, with recovery and transition to a specialized inflammatory state over time. This later state starts at 14 days post-injury and is characterized by a biphasic pattern of IFNγ, IL-4, and IL-10 gene expression changes, with concurrent proinflammatory and anti-inflammatory gene changes. Our transcriptomic data sets are an important step to understand microglial role in TBI pathogenesis at the molecular level and identify common pathways that affect outcome. More studies to evaluate gene expression at the single cell level and focusing on subacute and chronic timepoint are warranted.

TREM2 Acts Downstream of CD33 in Modulating Microglial Pathology in Alzheimer's Disease.

The microglial receptors CD33 and TREM2 have been associated with risk for Alzheimer's disease (AD). Here, we investigated crosstalk between CD33 and TREM2. We showed that knockout of CD33 attenuated amyloid beta (Aß) pathology and improved cognition in 5xFAD mice, both of which were abrogated by additional TREM2 knockout. Knocking out TREM2 in 5xFAD mice exacerbated Aß pathology and neurodegeneration but reduced Iba1+ cell numbers, all of which could not be rescued by additional CD33 knockout. RNA-seq profiling of microglia revealed that genes related to phagocytosis and signaling (IL-6, IL-8, acute phase response) are upregulated in 5xFAD;CD33-/- and downregulated in 5xFAD;TREM2-/- mice. Differential gene expression in 5xFAD;CD33-/- microglia depended on the presence of TREM2, suggesting TREM2 acts downstream of CD33. Crosstalk between CD33 and TREM2 includes regulation of the IL-1ß/IL-1RN axis and a gene set in the "receptor activity chemokine" cluster. Our results should facilitate AD therapeutics targeting these receptors.

Directly visualized glioblastoma-derived extracellular vesicles transfer RNA to microglia/macrophages in the brain.

BACKGROUND: To understand the ability of gliomas to manipulate their microenvironment, we visualized the transfer of vesicles and the effects of tumor-released extracellular RNA on the phenotype of microglia in culture and in vivo. METHODS: Extracellular vesicles (EVs) released from primary human glioblastoma (GBM) cells were isolated and microRNAs (miRNAs) were analyzed. Primary mouse microglia were exposed to GBM-EVs, and their uptake and effect on proliferation and levels of specific miRNAs, mRNAs, and proteins were analyzed. For in vivo analysis, mouse glioma cells were implanted in the brains of mice, and EV release and uptake by microglia and monocytes/macrophages were monitored by intravital 2-photon microscopy, immunohistochemistry, and fluorescence activated cell sorting analysis, as well as RNA and protein levels. RESULTS: Microglia avidly took up GBM-EVs, leading to increased proliferation and shifting of their cytokine profile toward immune suppression. High levels of miR-451/miR-21 in GBM-EVs were transferred to microglia with a decrease in the miR-451/miR-21 target c-Myc mRNA. In in vivo analysis, we directly visualized release of EVs from glioma cells and their uptake by microglia and monocytes/macrophages in brain. Dissociated microglia and monocytes/macrophages from tumor-bearing brains revealed increased levels of miR-21 and reduced levels of c-Myc mRNA. CONCLUSIONS: Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs.

TREM2 and the neuroimmunology of Alzheimer's disease.

Late-onset Alzheimer's disease (AD) is a sporadic disorder with increasing prevalence in aging. The ɛ4 allele of Apolipoprotein E(ApoEɛ4) was the only known major risk factor for late onset AD. Recently, two groups of investigators independently identified variants of the TREM2 gene, encoding triggering receptor expressed on myeloid cells 2 as causing increased susceptibility to late onset AD with an odds ratio similar to that of ApoEɛ4. TREM2 is a receptor expressed on innate immune cells. Using a novel technology called Direct RNA Sequencing wedetermined the quantitative transcriptome of microglia, the principal innate neuroimmune cells and confirmed that TREM2 is a major microglia-specific gene in the central nervous system. Over the past several years we have shown that microglia play a dichotomous role in AD. Microglia can be protective and promote phagocytosis, degradation and ultimately clearance of Aß, the pathogenic protein deposited in the brains of Alzheimer's patients. However, with disease progression, microglia become dysfunctional, release neurotoxins, lose their ability to clear Aß and produce pro-inflammatory cytokines that promote Aß production and accumulation. TREM2 has been shown to regulate the phagocytic ability of myeloid cells and their inflammatory response. Here we propose that the mechanism(s) by which TREM2 variants cause Alzheimer's disease are via down regulation of the Aß phagocytic ability of microglia and by dysregulation of the pro-inflammatory response of these cells. Based on our discussion we propose that TREM2 is a potential therapeutic target for stopping ordelaying progression of AD.

The neuroimmune system in Alzheimer's disease: the glass is half full.

It is well established that microglia, the neuroimmune cells of the brain, are associated with amyloid-ß (Aß) deposits in Alzheimer's disease (AD). However, the roles of these cells and other mononuclear phagocytes such as monocytes and macrophages in AD pathogenesis and progression have been elusive. Clues to mononuclear phagocyte involvement came with the demonstration that Aß directly activates microglia and monocytes to produce neurotoxins, signifying that a receptor mediated interaction of Aß with these cells may be critical for neurodegeneration seen in AD. Also, in AD brain, mononuclear phagocyte distribution changes from a uniform pattern that covers the brain parenchyma to distinct clusters intimately associated with areas of Aß deposition, but the driving force behind this choreography was unclear. Here, we review our recent work identifying mononuclear phagocyte receptors for Aß and unraveling mechanisms of recruitment of these cells to areas of Aß deposition. While our findings and those of others have added significantly to our understanding of the role of the neuroimmune system in AD, the glass remains half full (or half empty) and a lot remains to be uncovered.

Four-dimensional microglia response to anti-Aß treatment in APP/PS1xCX3CR1/GFP mice.

Senile plaques, mainly composed of amyloid-ß (Aß), are a major hallmark of Alzheimer disease (AD), and immunotherapy is a leading therapeutic approach for Aß clearance. Although the ultimate mechanisms for Aß clearance are not well known, characteristic microglia clusters are observed in the surround of senile plaques, and are implicated both in the elimination of Aß as well as the deleterious inflammatory effects observed in AD patients after active immunization. Therefore, analyzing the direct effect of immunotherapy on microglia, using longitudinal in vivo multiphoton microscopy can provide important information regarding the role of microglia in immunotherapy. While microglia were observed to surround senile plaques, topical anti-Aß antibody administration, which led to a reduction in plaque size, directed microglia toward senile plaques, and the overall size of microglia and number of processes were increased. In some cases, we observed clusters of microglia in areas of the brain that did not have detectable amyloid aggregates, but this did not predict the deposition of new plaques in the area within a week of imaging, indicating that microglia react to but do not precipitate amyloid aggregation. The long-term presence of large microglial clusters in the surrounding area of senile plaques suggests that microglia cannot effectively remove Aß unless anti-Aß antibody is administered. All together, these data suggest that although there is a role for microglia in Aß clearance, it requires an intervention like immunotherapy to be effective.

The microglial sensome revealed by direct RNA sequencing.

Microglia, the principal neuroimmune sentinels of the brain, continuously sense changes in their environment and respond to invading pathogens, toxins and cellular debris. Microglia exhibit plasticity and can assume neurotoxic or neuroprotective priming states that determine their responses to danger. We used direct RNA sequencing, without amplification or cDNA synthesis, to determine the quantitative transcriptomes of microglia of healthy adult and aged mice. We validated our findings using fluorescence dual in situ hybridization, unbiased proteomic analysis and quantitative PCR. We found that microglia have a distinct transcriptomic signature and express a unique cluster of transcripts encoding proteins for sensing endogenous ligands and microbes that we refer to as the sensome. With aging, sensome transcripts for endogenous ligand recognition were downregulated, whereas those involved in microbe recognition and host defense were upregulated. In addition, aging was associated with an overall increase in the expression of microglial genes involved in neuroprotection.

Scara1 deficiency impairs clearance of soluble amyloid-ß by mononuclear phagocytes and accelerates Alzheimer's-like disease progression.

In Alzheimer's disease, soluble amyloid-ß causes synaptic dysfunction and neuronal loss. Receptors involved in clearance of soluble amyloid-ß are not known. Here we use short hairpin RNA screening and identify the scavenger receptor Scara1 as a receptor for soluble amyloid-ß expressed on myeloid cells. To determine the role of Scara1 in clearance of soluble amyloid-ß in vivo, we cross Scara1 null mice with PS1-APP mice, a mouse model of Alzheimer's disease, and generate PS1-APP-Scara1-deficient mice. Scara1 deficiency markedly accelerates Aß accumulation, leading to increased mortality. In contrast, pharmacological upregulation of Scara1 expression on mononuclear phagocytes increases Aß clearance. This approach is a potential treatment strategy for Alzheimer's disease.

Mechanisms of mononuclear phagocyte recruitment in Alzheimer's disease.

Alzheimer's disease (AD) is associated with a significant neuroinflammatory component. Mononuclear phagocytes including monocytes and microglia are the principal cells involved, and they accumulate at perivascular sites of beta-amyloid (Abeta) deposition and in senile plaques. Recent evidence suggests that mononuclear phagocyte accumulation in the AD brain is dependent on chemokines. CCL2, a major monocyte chemokine, is upregulated in the AD brain. Interaction of CCL2 with its receptor CCR2 regulates mononuclear phagocyte accumulation in a mouse model of AD. CCR2 deficiency leads to lower mononuclear phagocyte accumulation and is associated with higher brain Abeta levels, specifically around blood vessels, suggesting that monocytes accumulate at sites of Abeta deposition in an initial attempt to clear these deposits and stop or delay their neurotoxic effects. Indeed, enhancing mononuclear phagocyte accumulation delays progression of AD. Here we review the mechanisms of mononuclear phagocyte accumulation in AD and discuss the potential roles of additional chemokines and their receptors in this process. We also propose a multi-step model for recruitment of mononuclear phagocytes into the brain. The first step involves egress of monocyte/microglial precursors from the bone marrow into the blood. The second step is crossing the blood-brain barrier to the perivascular areas and into the brain parenchyma. The final step includes movement of monocytes/microglia from areas of the brain that lack any amyloid deposition to senile plaques. Understanding the mechanism of recruitment of mononuclear phagocytes to the AD brain is necessary to further understand the role of these cells in the pathogenesis of AD and to identify any potential therapeutic use of these cells for the treatment of this disease.

Evolutionarily conserved recognition and innate immunity to fungal pathogens by the scavenger receptors SCARF1 and CD36.

Receptors involved in innate immunity to fungal pathogens have not been fully elucidated. We show that the Caenorhabditis elegans receptors CED-1 and C03F11.3, and their mammalian orthologues, the scavenger receptors SCARF1 and CD36, mediate host defense against two prototypic fungal pathogens, Cryptococcus neoformans and Candida albicans. CED-1 and C03F11.1 mediated antimicrobial peptide production and were necessary for nematode survival after C. neoformans infection. SCARF1 and CD36 mediated cytokine production and were required for macrophage binding to C. neoformans, and control of the infection in mice. Binding of these pathogens to SCARF1 and CD36 was beta-glucan dependent. Thus, CED-1/SCARF1 and C03F11.3/CD36 are beta-glucan binding receptors and define an evolutionarily conserved pathway for the innate sensing of fungal pathogens.