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International audience; Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as "resting versus activated" and "M1 versus M2." This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper.

Central nervous system (CNS) injury and infection can result in profound tissue remodeling in the brain, the mechanism and purpose of which is poorly understood. Infection with the protozoan parasite Toxoplasma gondii causes chronic infection and inflammation in the brain parenchyma. Control of parasite replication requires the continuous presence of IFNγ-producing T cells to keep T. gondii in its slowly replicating cyst form. During infection, a network of extracellular matrix fibers, revealed using multiphoton microscopy, forms in the brain. The origin and composition of these structures are unknown but the fibers have been observed to act as a substrate for migrating T cells. In this study, we show a critical regulator of extracellular matrix (ECM) remodeling, Secreted Protein, Acidic, Rich in Cysteine (SPARC), is upregulated in the brain during the early phases of infection in the frontal cortex. In the absence of SPARC, a reduced and disordered fibrous network, increased parasite burden, and reduced antigen-specific T cell entry into the brain points to a role for SPARC in T cell recruitment to and migration within the brain. We also report SPARC can directly bind to CCR7 ligands CCL19 and CCL21 but not CXCL10, and enhance migration toward a chemokine gradient. Measurement of T cell behavior points to tissue remodeling being important for access of immune cells to the brain and facilitating cellular locomotion. Together, these data identify SPARC as an important regulatory component of immune cell trafficking and access to the inflamed CNS.

Author(s): Burr, A.C.; Kamath, R; Velazquez, J; Peng, X; Maltz, M; Botthoff, J; Cocker, D; Aronson, E; Carson, M; Lo, D; Nordgren, T.M.

The Research Centers in Minority Institutions (RCMI) Program was congressionally mandated in 1985 to build research capacity at institutions that currently and historically recruit, train, and award doctorate degrees in the health professions and health-related sciences, primarily to individuals from underrepresented and minority populations. RCMI grantees share similar infrastructure needs and institutional goals. Of particular importance is the professional development of multidisciplinary teams of academic and community scholars (the “workforce”) and the harnessing of the heterogeneity of thought (the “thinkforce”) to reduce health disparities. The purpose of this report is to summarize the presentations and discussion at the RCMI Investigator Development Core (IDC) Workshop, held in conjunction with the RCMI Program National Conference in Bethesda, Maryland, in December 2019. The RCMI IDC Directors provided information about their professional development activities and Pilot Projects Programs and discussed barriers identified by new and early-stage investigators that limit effective career development, as well as potential solutions to overcome such obstacles. This report also proposes potential alignments of professional development activities, targeted goals and common metrics to track productivity and success.

Seventy‐five percent of all traumatic brain injuries are mild and do not cause readily visible abnormalities on routine medical imaging making it difficult to predict which individuals will develop unwanted clinical sequelae. Microglia are brain‐resident macrophages and early responders to brain insults. Their activation is associated with changes in morphology or expression of phenotypic markers including P2Y12 and major histocompatibility complex class II. Using a murine model of unrestrained mild closed head injury (mCHI), we used microglia as reporters of acute brain injury at sites of impact versus sites experiencing rotational stress 24 h post‐mCHI. Consistent with mild injury, a modest 20% reduction in P2Y12 expression was detected by quantitative real‐time PCR (qPCR) analysis but only in the impacted region of the cortex. Furthermore, neither an influx of blood‐derived immune cells nor changes in microglial expression of CD45, TREM1, TREM2, major histocompatibility complex class II or CD40 were detected. Using magnetic resonance imaging (MRI), small reductions in T2 weighted values were observed but only near the area of impact and without overt tissue damage (blood deposition, edema). Microglial morphology was quantified without cryosectioning artifacts using ScaleA 2 clarified brains from CX3CR1‐green fluorescence protein (GFP) mice. The cortex rostral to the mCHI impact site receives greater rotational stress but neither MRI nor molecular markers of microglial activation showed significant changes from shams in this region. However, microglia in this rostral region did display signs of morphologic activation equivalent to that observed in severe CHI. Thus, mCHI‐triggered rotational stress is sufficient to cause injuries undetectable by routine MRI that could result in altered microglial surveillance of brain homeostasis. Acute changes in microglial morphology reveal brain responses to unrestrained mild traumatic brain injury In areas subjected to rotational stress distant from impact siteIn the absence of detectable changes in standard molecular indicators of brain damage, inflammation or microglial activation.That might result in decreased surveillance of brain function and increased susceptibility to subsequent brain insults.

Increasing prevalence in obesity has become a significant public concern. C57BL/6J mice are prone to diet-induced obesity (DIO) when fed high-fat diet (HFD), and develop chronic inflammation and metabolic syndrome, making them a good model to analyze mechanisms whereby obesity elicits pathologies. DIO mice demonstrated profound sex differences in response to HFD with respect to inflammation and hypothalamic function. First, we determined that males are prone to DIO, while females are resistant. Ovariectomized females, on the other hand, are susceptible to DIO, implying protection by ovarian hormones. Males, but not females, exhibit changes in hypothalamic neuropeptide expression. Surprisingly, ovariectomized females remain resistant to neuroendocrine changes, showing that ovarian hormones are not necessary for protection. Second, obese mice exhibit sex differences in DIO-induced inflammation. Microglial activation and peripheral macrophage infiltration is seen in the hypothalami of males, while females are protected from the increase in inflammatory cytokines and do not exhibit microglia morphology changes nor monocyte-derived macrophage infiltration, regardless of the presence of ovarian hormones. Strikingly, the anti-inflammatory cytokine IL-10 is increased in the hypothalami of females but not males. Third, this study posits a potential mechanism of obesity-induced impairment of hypothalamic function whereby obese males exhibit reduced levels of synaptic proteins in the hypothalamus and fewer spines in GnRH neurons, located in the areas exhibiting macrophage infiltration. Our studies suggest that inflammation-induced synaptic remodeling is potentially responsible for hypothalamic impairment that may contribute to diminished levels of gonadotropin hormones, testosterone, and sperm numbers, which we observe and corresponds to the observations in obese humans. Taken together, our data implicate neuro-immune mechanisms underlying sex-specific differences in obesity-induced impairment of the hypothalamic function with potential consequences for reproduction and fertility.

Mild traumatic brain injury (mTBI) has become an increasing public health concern as subsequent injuries can exacerbate existing neuropathology and result in neurological deficits. This study investigated the temporal development of cortical lesions using magnetic resonance imaging (MRI) to assess two mTBIs delivered to opposite cortical hemispheres. The controlled cortical impact model was used to produce an initial mTBI on the right cortex followed by a second injury induced on the left cortex at 3 (rmTBI 3d) or 7 (rmTBI 7d) days later. Histogram analysis was combined with a novel semi-automated computational approach to perform a voxel-wise examination of extravascular blood and edema volumes within the lesion. Examination of lesion volume 1d post last injury revealed increased tissue abnormalities within rmTBI 7d animals compared to other groups, particularly at the site of the second impact. Histogram analysis of lesion T2 values suggested increased edematous tissue within the rmTBI 3d group and elevated blood deposition in the rm TBI 7d animals. Further quantification of lesion composition for blood and edema containing voxels supported our histogram findings, with increased edema at the site of second impact in rmTBI 3d animals and elevated blood deposition in the rmTBI 7d group at the site of the first injury. Histological measurements revealed spatial overlap of regions containing blood deposition and microglial activation within the cortices of all animals. In conclusion, our findings suggest that there is a window of tissue vulnerability where a second distant mTBI, induced 7d after an initial injury, exacerbates tissue abnormalities consistent with hemorrhagic progression., Highlights ► rmTBI 7d apart resulted in an increased total lesion volume 1d post last injury. ► Lesion volume was increased at the distant second injury site in rmTBI 7d animals. ► Increased edema was seen at the distant second injury site in rmTBI 3d animals. ► Voxel-wise analysis detected hemorrhagic progression in rmTBI 7d animals. ► Increased microglia were observed in the rmTBI 7d animals.

Injury, infection and autoimmune triggers increase CNS expression of the chemokine CCL21. Outside the CNS, CCL21 contributes to chronic inflammatory disease and autoimmunity by three mechanisms: recruitment of lymphocytes into injured or infected tissues, organization of inflammatory infiltrates into lymphoid-like structures and promotion of homeostatic CD4+ T-cell proliferation. To test if CCL21 plays the same role in CNS inflammation, we generated transgenic mice with astrocyte-driven expression of CCL21 (GFAP-CCL21 mice). Astrocyte-produced CCL21 was bioavailable and sufficient to support homeostatic CD4+ T-cell proliferation in cervical lymph nodes even in the absence of endogenous CCL19/CCL21. However, lymphocytes and glial-activation were not detected in the brains of uninfected GFAP-CCL21 mice, although CCL21 levels in GFAP-CCL21 brains were higher than levels expressed in inflamed Toxoplasma-infected non-transgenic brains. Following Toxoplasma infection, T-cell extravasation into submeningeal, perivascular and ventricular sites of infected CNS was not CCL21-dependent, occurring even in CCL19/CCL21-deficient mice. However, migration of extravasated CD4+, but not CD8+ T cells from extra-parenchymal CNS sites into the CNS parenchyma was CCL21-dependent. CD4+ T cells preferentially accumulated at perivascular, submeningeal and ventricular spaces in infected CCL21/CCL19-deficient mice. By contrast, greater numbers of CD4+ T cells infiltrated the parenchyma of infected GFAP-CCL21 mice than in wild-type or CCL19/CCL21-deficient mice. Together these data indicate that CCL21 expression within the CNS has the potential to contribute to T cell-mediated CNS pathology via: (a) homeostatic priming of CD4+ T-lymphocytes outside the CNS and (b) by facilitating CD4+ T-cell migration into parenchymal sites following pathogenic insults to the CNS.

Increased polyamine production is observed in a variety of chronic neuroinflammatory disorders, but in vitro and in vivo studies yield conflicting data on the immunomodulatory consequences of their production. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in endogenous polyamine production. To identify the role of polyamine production in CNS-intrinsic inflammatory responses, we defined CNS sites of ODC expression and the consequences of inhibiting ODC in response to intracerebral injection of LPS±IFNγ. In situ hybridization analysis revealed that both neurons and non-neuronal cells rapidly respond to LPS±IFNγ by increasing ODC expression. Inhibiting ODC by co-injecting DFMO decreased LPS-induced CCL2 expression and macrophage influx into the CNS, without altering LPS-induced microglial or macrophage activation. Conversely, intracerebral injection of polyamines was sufficient to trigger macrophage influx into the CNS of wild-type but not CCL2KO mice, demonstrating the dependence of macrophage influx on CNS expression of CCL2. Consistent with these data, addition of putrescine and spermine to mixed glial cultures dramatically increased CCL2 expression and to a much lesser extent, TNF expression. Addition of all three polyamines to mixed glial cultures also decreased the numbers and percentages of oligodendrocytes present. However, in vivo, inhibiting the basal levels of polyamine production was sufficient to induce expression of apolipoprotein D, a marker of oxidative stress, within white matter tracts. Considered together, our data indicate that: (1) CNS-resident cells including neurons play active roles in recruiting pro-inflammatory TREM1-positive macrophages into the CNS via polyamine-dependent induction of CCL2 expression and (2) modulating polyamine production in vivo may be a difficult strategy to limit inflammation and promote repair due to the dual homeostatic and pro-inflammatory roles played by polyamines.

We investigated the properties and regulation of P2X receptors in immortalized C8-B4 cells of cerebellar microglial origin. Resting C8-B4 cells expressed virtually no functional P2X receptors, but largely increased functional expression of P2X4 receptors within 2–6 h of entering the activated state. Using real-time polymerase chain reaction, we found that P2X4 transcripts were increased during the activated state by 2.4-fold, but this increase was not reflected by a parallel increase in total P2X4 proteins. In resting C8-B4 cells, P2X4 subunits were mainly localized within intracellular compartments, including lysosomes. We found that cell surface P2X4 receptor levels increased by ∼3.5-fold during the activated state. This change was accompanied by a decrease in the lysosomal pool of P2X4 proteins. We next exploited our findings with C8-B4 cells to investigate the mechanism by which antidepressants reduce P2X4 responses. We found little evidence to suggest that several antidepressants were antagonists of P2X4 receptors in C8-B4 cells. However, we found that moderate concentrations of the same antidepressants reduced P2X4 responses in activated microglia by affecting lysosomal function, which indirectly reduced cell surface P2X4 levels. In summary, our data suggest that activated C8-B4 cells express P2X4 receptors when the membrane insertion of these proteins by lysosomal secretion exceeds their removal, and that antidepressants indirectly reduce P2X4 responses by interfering with lysosomal trafficking.

Neurons have been the natural focus of discussion for most of the history of research on seizures and epilepsy. Simply stated, epilepsy is a disease of sporadic, progressive disruption of neuronal activity. Thus causes and therapies for epilepsy have been naturally aimed at the obvious manifestation of disease: neuronal dysfunction. However, over the last two decades a new view is beginning to emerge that is defining the dependence of neuronal function and seizure susceptibility on glia. This view changes the definition of epilepsy as a disease of neurons to a disease of a heterogeneous neuronal-glial network. This new glial focus is suggesting new opportunities to treat the nearly 1/3 of individuals who do not respond to traditional antiepileptic drug (AEDs) therapies as well as suggesting ways to reduce the many unwanted side effects of AEDs.

The chemokines CCL21 and CCL19, and cell bound TNF family ligand lymphotoxin beta (LTbeta), have been associated with numerous chronic inflammatory diseases. A general role in chronic inflammatory diseases cannot be assumed however; in the case of allergic inflammatory disease, CCL21/CCL19 and LTbeta have not been associated with the induction, recruitment, or effector function of Th2 cells nor dendritic cells to the lung. We have examined the induction of allergic inflammatory lung disease in mice deficient in CCL21/CCL19 or LTbeta and found that both kinds of mice can develop allergic lung inflammation. To control for effects of priming differences in knockout mice, adoptive transfers of Th2 cells were also performed, and they showed that such effector cells had equivalent effects on airway hyper-responsiveness in both knockout background recipients. Moreover, class II positive antigen presenting cells (B cells and CD11c+ dendritic cells) showed normal recruitment to the peribronchial spaces along with CD4 T cells. Thus, the induction of allergic responses and recruitment of both effector Th2 cells and antigen presenting cells to lung peribronchial spaces can develop independently of CCL21/CCL19 and LTbeta.

The chemokines CCL21 and CCL19, and cell bound TNF family ligand lymphotoxin beta (LTβ), have been associated with numerous chronic inflammatory diseases. A general role in chronic inflammatory diseases cannot be assumed however; in the case of allergic inflammatory disease, CCL21/CCL19 and LTβ have not been associated with the induction, recruitment, or effector function of Th2 cells nor dendritic cells to the lung. We have examined the induction of allergic inflammatory lung disease in mice deficient in CCL21/CCL19 or LTβ and found that both kinds of mice can develop allergic lung inflammation. To control for effects of priming differences in knockout mice, adoptive transfers of Th2 cells were also performed, and they showed that such effector cells had equivalent effects on airway hyper-responsiveness in both knockout background recipients. Moreover, class II positive antigen presenting cells (B cells and CD11c+ dendritic cells) showed normal recruitment to the peribronchial spaces along with CD4 T cells. Thus, the induction of allergic responses and recruitment of both effector Th2 cells and antigen presenting cells to lung peribronchial spaces can develop independently of CCL21/CCL19 and LTβ.

Two different macrophage populations contribute to CNS neuroinflammation: CNS-resident microglia and CNS-infiltrating peripheral macrophages. Markers distinguishing these two populations in tissue sections have not been identified. Therefore, we compared gene expression between LPS (lipopolysaccharide)/interferon (IFN)γ-treated microglia from neonatal mixed glial cultures and similarly treated peritoneal macrophages. Fifteen molecules were identified by quantative PCR (qPCR) as being enriched from 2-fold to 250-fold in cultured neonatal microglia when compared with peritoneal macrophages. Only three of these molecules (C1qA, Trem2, and CXCL14) were found by qPCR to be also enriched in adult microglia isolated from LPS/IFNγ-injected CNS when compared with infiltrating peripheral macrophages from the same CNS. The discrepancy between the in vitro and in vivo qPCR data sets was primarily because of induced expression of the 'microglial' molecules (such as the tolerance associated transcript, Tmem176b) in CNS-infiltrating macrophages. Bioinformatic analysis of the ∼19000 mRNAs detected by TOGA gene profiling confirmed that LPS/IFNγ-activated microglia isolated from adult CNS displayed greater similarity in total gene expression to CNS-infiltrating macrophages than to microglia isolated from unmanipulated healthy adult CNS. In situ hybridization analysis revealed that nearly all microglia expressed high levels of C1qA, while subsets of microglia expressed Trem2 and CXCL14. Expression of C1qA and Trem2 was limited to microglia, while large numbers of GABA+ neurons expressed CXCL14. These data suggest that (i) CNS-resident microglia are heterogeneous and thus a universal microglia-specific marker may not exist; (ii) the CNS micro-environment plays significant roles in determining the phenotypes of both CNS-resident microglia and CNS-infiltrating macrophages; (iii) the CNS microenvironment may contribute to immune privilege by inducing macrophage expression of anti-inflammatory molecules. © 2009 International Society for Neurochemistry.

Microglial activation and macrophage infiltration into the CNS are common features of CNS autoimmune disease and of chronic neurodegenerative diseases. Because these cells largely express an overlapping set of common macrophage markers, it has been difficult to separate their respective contributions to disease onset and progression. This problem is further confounded by the many types of macrophages that have been termed microglia. Several approaches, ranging from molecular profiling of isolated cells to the generation of irradiation chimeric rodent models, are now beginning to generate rudimentary definitions distinguishing the various types of microglia and macrophages found within the CNS and the potential roles that these cells may play in health and disease.

Inflammation mediated by macrophages is increasingly found to play a central role in diseases and disorders that affect a myriad of organs, prominent among these are diseases of the CNS. The neurotoxicant-induced, cuprizone model of demyelination is ideally suited for the analysis of inflammatory events. Demyelination on exposure to cuprizone is accompanied by predictable microglial activation and astrogliosis, and, after cuprizone withdrawal, this activation reproducibly diminishes during remyelination. This study demonstrates enhanced expression of lymphotoxin β receptor (LtβR) during the demyelination phase of this model, and LtβR is found in areas enriched with microglial and astroglial cells. Deletion of the LtβR gene (LtβR−/−) resulted in a significant delay in demyelination but also a slight delay in remyelination. Inhibition of LtβR signaling by an LtβR–Ig fusion decoy protein successfully delayed demyelination in wild-type mice. Unexpectedly, this LtβR–Ig decoy protein dramatically accelerated the rate of remyelination, even after the maximal pathological disease state had been reached. This strongly indicates the beneficial role of LtβR–Ig in the delay of demyelination and the acceleration of remyelination. The discrepancy between remyelination rates in these systems could be attributed to developmental abnormalities in the immune systems ofLtβR−/−mice. These findings bode well for the use of an inhibitory LtβR–Ig as a candidate biological therapy in demyelinating disorders, because it is beneficial during both demyelination and remyelination.

The immune system is a host defense system comprised of both innate mechanisms able to rapidly recognize and respond to conserved pathogen associated molecular patterns (PAMPs) as well as adaptive mechanisms able to respond to a wide variety of non-conserved and conserved pathogen associated molecules. In vitro and in vivo studies have demonstrated that the kinetics and type of immune response triggered by pathogenic insults is a function of both the nature of the insult and the subsequent cross-regulatory interactions between the responding immune cells. In this context, the potential immunomodulatory influences of the nervous system have been often viewed as exerting minimal modulatory effects and thus of being largely irrelevant in the development of immune responses. Here, using a Saturday Night Live (SNL)-styled point:counterpoint format, we discuss whether and to what extent the nervous system can shape the responses of the immune system. Finally, we examine whether primary degenerative disorders of the CNS are likely to lead to alterations in immune function.

Increasing evidence suggests that Alzheimer's disease pathogenesis is not restricted to the neuronal compartment but strongly interacts with immunological mechanisms in the brain. Misfolded and aggregated proteins bind to pattern recognition receptors on micro- and astroglia and trigger an innate immune response, characterized by the release of inflammatory mediators, which contribute to disease progression and severity. Genome wide analysis suggests that several genes, which increase the risk for sporadic Alzheimer's disease en-code for factors that regulate glial clearance of misfolded proteins and the inflammatory reaction. External factors, including systemic inflammation and obesity are likely to interfere with the immunological processes of the brain and further promote disease progression. This re-view provides an overview on the current knowledge and focuses on the most recent and exciting findings. Modulation of risk factors and intervention with the described immune mechanisms are likely to lead to future preventive or therapeutic strategies for Alzheimer's disease.

Neurological complications of human immunodeficiency virus (HIV) infection are a public health problem despite the availability of active antiretroviral therapies. The neuropathogenesis of HIV infection revolves around a complex cascade of events that include viral infection and glial immune activation, monocyte–macrophage brain infiltration, and secretion of a host of viral and cellular inflammatory and neurotoxic molecules. Although there is evidence that HIV-infected drug abusers experience more severe neurological disease, the biological basis for this finding is unknown. A scientific workshop organized by the National Institute on Drug Abuse (NIDA) was held on March 23–24, 2006 to address this question. The goal of the meeting was to bring together basic science and clinical researchers who are experts in NeuroAIDS, glial immunity, drugs of abuse, and/or pharmacology in order to find new approaches to understanding interactions between drug abuse and neuroAIDS. The format of the meeting was designed to stimulate open discussion and forge new multidisciplinary research collaborations. This report includes transcripts of active discussions and short presentations from invited participants. The presentations were separated into sections that included: Glial Biology, Inflammation, and HIV; Pharmacology, Neurotoxicology, and Neuroprotection; NeuroAIDS and Virology; and Virus–Drug and Immune–Drug Interactions. Research priorities were identified. Additional information about this meeting is available through links from the NIDA AIDS Research Program website ( http://www.nida.nih.gov/about/organization/arp/arp-websites.htm ).

Cuprizone-induced demyelination is a mouse model of multiple sclerosis (MS) as cuprizone-fed mice exhibit neuroin-flammation and demyelination in the brain. Upon removal of cuprizone from the diet, inflammation is resolved and reparative remyelination occurs. In an Affymetrix Gene-Chip analysis, the stress-associated gene p8 was strongly upregulated (>10×) during cuprizone-induced demyelination but not remyelination. We verified this upregulation (>15×) of p8 in the CNS during demyelination by real-time polymerase chain reaction (PCR). This upregulation is brain-specific, as p8 is not elevated in the liver, lung, kidney, spleen, and heart of cuprizone-treated mice. We also localized the cellular source of p8 during cuprizone treatment, and further found elevated expression during embryogenesis but not in normal adult brain. Compared with wild-type controls, the death of oligodendrocytes in p8−/− mice is delayed, as is microglial recruitment to areas of demyelination. The corpus callosum of p8−/− mice demyelinates at a slower rate than wild-type mice, suggesting that p8 exacerbates CNS inflammation and demyelination. Enhanced expression of p8 is also observed in the spinal cords of mice with acute experimental autoimmune encephalomyelitis (EAE) induced by PLP139–151 peptide (10×). Increased expression is detected during disease onset and expression wanes during the remission phase. Finally, p8 is found upregulated (8×) in post-mortem tissue from MS patients and is higher in the plaque tissue compared with adjacent normal-appearing white and gray matter. Thus, p8 is an excellent candidate as a novel biomarker of demyelination.

Catecholamines and adipokines function as hormones; catecholamines as neurotransmitters in the sympathetic nervous system, and adipokines as mediators of metabolic processes. It has become increasingly clear, however, that both also function as immunomodulators of innate and adaptive immune cells, including macrophages. Macrophages can respond to, as well as produce their own catecholamines. Dopamine, noradrenaline, and adrenaline are the most abundant catecholamines in the body, and can induce both pro-inflammatory and anti-inflammatory immune responses in macrophages, as well as non-immune processes such as thermogenesis. Though they are responsive to adipokines, particularly lipoproteins, leptin, and adiponectin, macrophages generally do not synthesize their own adipokines, with the exception being resistin-like molecules. Adipokines contribute to adverse metabolic and immune responses by stimulating lipid accumulation, foam cell formation and pro-inflammatory cytokine production in macrophages. Adipokines can also promote balance or resolution during metabolic and immune processes by promoting reverse lipid transport and expression of Th2 cytokines. This review will explore the mechanisms by which catecholamines and adipokines influence macrophage function in neural pathways, immunity and metabolism.

Microglia are the tissue macrophage of the central nervous system (CNS) and their activation is among the earliest signs of CNS dysfunction and disease. Because microglia express many macrophage markers, they are presumed to act primarily as effectors of CNS inflammation and destruction. While such responses are beneficial to the extent that they destroy CNS pathogens, these responses do have the potential to have neurotoxic outcomes. Consequently, therapies for many CNS neurodegenerative and inflammatory diseases have been directed at suppressing microglial function. There is evidence to suggest that microglia play an important role during CNS development and maintenance of CNS function that may go beyond simple defense against pathogens. Molecular analysis of microglial phenotypes and function has revealed three striking findings: (i) that microglia are a unique CNS-specific type of tissue macrophage; (ii) that they are highly heterogeneous within the healthy CNS; and (iii) that microglial responses are exquisitely tailored to specific regions of the CNS and specific pathological insults. We suggest that ubiquitous suppression (rather than targeted manipulation) of microglial function may fail to fully ameliorate CNS pathology and may even ultimately promote maladaptive outcomes.

Microglial activation is an early and common feature of almost all neuropathologies, including multiple sclerosis, Alzheimer's disease and mechanical injury. To better understand the relative contributions microglia make toward neurodegeneration and neuroprotection, we used TOGA(R) to identify molecules expressed by microglia and regulated by inflammatory signals. Triggering receptor expressed on myeloid cells-2 (TREM-2) was among the mRNAs identified as being expressed by unactivated microglia, but down-regulated by lipopolysaccharide/interferon gamma. In the healthy CNS, not all microglia expressed TREM-2. Microglial expression of TREM-2 varied not only between brain regions but also within each brain region. Brain regions with an incomplete blood-brain barrier had the lowest percentages of TREM-2- expressing microglia, whereas the lateral entorhinal and cingulate cortex had the highest percentages. A novel form of TREM-2b that lacked a transmembrane domain was detected, perhaps indicating a soluble form of the protein. Taken together, these data suggest that (1) subsets of microglia are specialized to respond to defined extracellular signals; and (2) regional variations in TREM-2 expression may contribute to the varying sensitivities of different brain regions to similar pathological signals.

Microglia play critical roles in brain development, homeostasis, and neurological disorders. Here, we report that human microglial-like cells (iMGLs) can be differentiated from iPSCs to study their function in neurological diseases, like Alzheimer's disease (AD). We find that iMGLs develop in vitro similarly to microglia in vivo, and whole-transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia. Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates. iMGLs were used to examine the effects of Aβ fibrils and brain-derived tau oligomers on AD-related gene expression and to interrogate mechanisms involved in synaptic pruning. Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo. Together, these findings demonstrate that iMGLs can be used to study microglial function, providing important new insight into human neurological disease.

The role of IL-12 in the evolution of immunoinflammatory responses at a localized tissue level was investigated. Transgenic mice were developed with expression of either both the IL-12 subunits (p35 and p40) or only the IL-12 p40 subunit genes targeted to astrocytes in the mouse CNS. Glial fibrillary acidic protein (GF)-IL-12 mice, bigenic for the p35 and p40 genes, developed neurologic disease which correlated with the levels and sites of transgene-encoded IL-12 expression. In these mice, the brain contained numerous perivascular and parenchymal inflammatory lesions consisting of predominantly CD4+ and CD8+ T cells as well as NK cells. The majority of the infiltrating T cells had an activated phenotype (CD44high, CD45Rblow, CD62Llow, CD69high, VLA-4 high, and CD25+). Functional activation of the cellular immune response was also evident with marked cerebral expression of the IFN-γ, TNF, and IL-1αβ genes. Concomitant with leukocyte infiltration, the CNS expression of immune accessory molecules was induced or up-regulated, including ICAM-1, VCAM-1, and MHC class II and B7-2. Glial fibrillary acidic protein-p40 mice with expression of IL-12 p40 alone remained asymptomatic, with no inflammation evident at any age studied. The effect of local CNS production of IL-12 in the development of experimental autoimmune encephalomyelitis was studied. After immunization with myelin oligodendrocyte glycoprotein-peptides, GF-IL-12 mice had an earlier onset and higher incidence but not more severe disease. We conclude that localized expression of IL-12 by astrocytes can 1) promote the spontaneous development of activated type 1 T cell and NK cellular immunity and cytokine responses in the CNS, and 2) promote more effective Ag-specific T cell dynamics but not activity in experimental autoimmune encephalomyelitis.

We have investigated the functional coupling of the rat 5HT(5a) receptor subtype to adenylate cyclase in a rat C6 glioma cell line. In 5HT(5a) receptor-transfected cells, 5HT caused a concentration-dependent inhibition of forskolin-stimulated cAMP accumulation, with an EC(50) value of 41 nM and a maximal effect of 57% inhibition. This effect was dependent on the concentration of forskolin used to elevate cAMP levels. Methiothepin (1 mcM), which has high affinity for the 5HT(5a) receptor, antagonized the 5HT(5a) receptor-mediated inhibition, and unmasked a stimulation of cAMP formation similar to that observed in untransfected cells, whereas ketanserin (0.1 mcM) enhanced the inhibitory effect of 5HT. Pertussis toxin treatment (0.5 mcg/ml) completely blocked the inhibitory effect of 5HT on cAMP formation, also revealing increase in cAMP accumulation. Pretreatment of the transfected membranes with pertussis toxin abolished subsequent ADP-ribosylation of a 41 kDa protein, correlating the cAMP effect with a functional uncoupling of an inhibitory G protein from its receptor. These results demonstrate an efficient functional coupling of the rat 5HT(5a) receptor to the inhibition of adenylate cyclase via a pertussis toxin-sensitive G[alpha(i)], inhibitory G-protein.

Oleamide belongs to a family of amidated lipids with diverse biological activities, including sleep induction and signaling modulation of several 5-hydroxytryptamine (5-HT) receptor subtypes, including 5-HT1A, 5-HT2A/2C, and 5-HT7. The 5-HT7 receptor, predominantly localized in the hypothalamus, hippocampus, and frontal cortex, stimulates cyclic AMP formation and is thought to be involved in the regulation of sleep-wake cycles. Recently, it was proposed that oleamide acts at an allosteric site on the 5-HT7 receptor to regulate cyclic AMP formation. We have further investigated the interaction between oleamide and 5-HT7 receptors by performing radioligand binding assays with HeLa cells transfected with the 5-HT7 receptor. Methiothepin, clozapine, and 5-HT all displaced specific [3H]5-HT (100 nM) binding, with pK(D) values of 7.55, 7.85, and 8.39, respectively. Oleamide also displaced [3H]5-HT binding, but the maximum inhibition was only 40% of the binding. Taking allosteric (see below) cooperativity into account, a K(D) of 2.69 nM was calculated for oleamide. In saturation binding experiments, oleamide caused a 3-fold decrease in the affinity of [3H]5-HT for the 5-HT7 receptor, without affecting the number of binding sites. A Schild analysis showed that the induced shift in affinity of [3H]5-HT reached a plateau, unlike that of a competitive inhibitor, illustrating the allosteric nature of the interaction between oleamide and the 5-HT7 receptor. Oleic acid, the product of oleamide hydrolysis, had a similar effect on [3H]5-HT binding, whereas structural analogs of oleamide, trans-9,10-octadecenamide, cis-8,9-octadecenamide, and erucamide, did not alter [3H]5-HT binding significantly. The findings support the hypothesis that oleamide acts via an allosteric site on the 5-HT7 receptor regulating receptor affinity.

Immunological privilege of the central nervous system (CNS) has often been viewed as the summation of mechanisms that are protective of, but extrinsic to, the CNS. Their primary role has then been seen as isolating the CNS from the organism as a whole. Experiments in recent years indicate that the CNS itself may have an innate immune system comprised of astrocytes and microglia capable of regulating the initiation and progression of immune responses. Thus, immunological privilege should be considered as an intrinsic property of the CNS that could involve direct CNS:immune cell interactions. Malfunctions of these intrinsic mechanisms could play significant roles augmenting or even initiating CNS-directed autoimmunity and inflammation. J. Neurosci. Res. 55:1–8, 1999. © 1999 Wiley-Liss, Inc.

Owing to the difficulties of isolating adequate numbers of microglia from adult tissue, much of our understanding of their function is based on characterizations of microglia that develop in mixed glial cultures. To learn more about the nature of these cells in vivo, we have compared the phenotypes of murine microglia isolated from adults, neonates, and from mixed glial cultures with spleen cells from fetuses, neonates, and adults. In the adult CNS, the only resident population of cells that express CD45, a protein tyrosine phosphatase, are the F4/80+ and FcR+ cells: the microglia. In contrast to all other differentiated cells of hemopoietic origin, microglial CD45 levels fail to increase from the neonatal period through adulthood. Rather, their levels are indistinguishable from the low levels found on a small population of embryonic day 16 liver cells. Conversely, we find that the F4/80 values of microglia are elevated as compared to splenic macrophages. Strikingly, microglia that develop in mixed glial cultures display a more activated phenotype, with low F4/80 values, weak MHC class II expression, and the appearance of a subset of cells positive for the dendritic cell marker, NLDC145. Additionally, CD45 values are elevated to a level intermediate between that of adult microglia and adult spleen, a level similar to that found on microglia activated in vivo. Consistent with this activated phenotype, indomethacin revealed the ability of mixed glial culture microglia to present a peptide antigen to naive T-cells expressing a defined T-cell receptor. Although adult microglia did express costimulatory molecules, B7.2, ICAM-1, and CD40, and could be induced to express MHC class II, they failed to present antigen in the same assay. Interestingly, these same cells could stimulate T-cell proliferation in a mixed lymphocyte reaction but not in an allogeneic specific manner. Taken together these data suggest that adult microglia remain in a relatively immature and unactivated state of differentiation as compared to other tissue macrophages.

In response to central nervous system (CNS) injury and infection, astrocytes, neurons, and CNS vasculature express several chemokines, including CCL21. Quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemical methods can quantify mRNA and protein expression. However, these methods do not quantify chemokine bioavailability and bioactivity, variables modified by many environmental factors including composition of the extracellular matrix (ECM). Here we illustrate how two-photon microscopy and carboxyfluorescein succinimidyl ester (CFSE or CFDA SE) labeling of T cells coupled with flow cytometry can be used as tools to assess chemokine-mediated regulation of T cell proliferation, activation, and migration.

The mRNA for the 5-hydroxytryptamine receptor 5-HT5A was detected at embryonic day 18 in the rat central nervous system and peaked by postnatal day 20. At all time points examined, 5-HT5A immunoreactivity observed on astrocyte cell bodies and in the stellate processes not only colocalized with the astrocyte-specific marker glial fibrillary acidic protein (GFAP) but was coordinately regulated with GFAP, increasing during development and during gliosis. Transfection of 5-HT5A into glioma cells prevented the 5-HT-induced increase in cAMP observed in untransfected cells and decreased the relative forskolin response by approximately 20%, suggesting that the 5-HT5A receptor couples negatively to adenylyl cyclase in astrocytes. Together, these results indicate a neuron-to-astrocyte serotonergic signaling pathway mediating cAMP concentrations, which could provide a neuronally driven mechanism for regulating astrocyte physiology with relevance to gliosis.

Microglia are the resident immune cells of the brain. As such, their activation is associated with a range of conditions in the healthy and inflamed CNS. In the healthy brain, microglia maintain homeostasis by continuously sampling their environment [1]. When CNS infection and/or inflammation occurs, microglia respond appropriately by changing their activation state. Recent studies illustrate that microglial activation is not an “all or nothing” event. Instead, like peripheral macrophages, microglia exhibit a range of activation states associated with pathogen defense, wound healing, and tissue repair functions (Fig. 1.1) [2, 3]. In addition, although microglia can change their morphology in response to specific stimuli, morphology is now well demonstrated to be a poor discriminator between different types of activation (Fig. 1.1) [2–4].

Huntington's disease (HD) is caused by an expanded polyglutamine tract in the protein huntingtin (htt). Although HD has historically been viewed as a brain-specific disease, htt is expressed ubiquitously, and recent studies indicate that mutant htt might cause changes to the immune system that could contribute to pathogenesis. Monocytes from HD patients and mouse models are hyperactive in response to stimulation, and increased levels of inflammatory cytokines and chemokines are found in pre-manifest patients that correlate with pathogenesis. In this study, wild-type (WT) bone marrow cells were transplanted into two lethally irradiated transgenic mouse models of HD that ubiquitously express full-length htt (YAC128 and BACHD mice). Bone marrow transplantation partially attenuated hypokinetic and motor deficits in HD mice. Increased levels of synapses in the cortex were found in HD mice that received bone marrow transplants. Importantly, serum levels of interleukin-6, interleukin-10, CXC chemokine ligand 1, and interferon-γ were significantly higher in HD than WT mice but were normalized in mice that received a bone marrow transplant. These results suggest that immune cell dysfunction might be an important modifier of pathogenesis in HD.

Publisher Summary This chapter provides an overview on the multiple mechanisms by which the nervous system regulates and directs immune function towards what is needed and tolerated by the nervous system. The immune system plays two essential roles necessary for the survival of complex organisms, including tissue homeostasis and tissue defense against pathogens. These immune functions are essential to maintain the func­tions of all organs in the body and are studied as a part of the general field of immunology. The immune system is divided into two arms: a rapid “innate” immune response system and a slow “adaptive” immune response system. The receptors for the types of “alarm” signals detected by the innate immune system are preformed and stably encoded in the genome. By contrast, the receptors for the types of signals that trigger the adaptive immune system are in part stochastically generated and in part shaped by the types and frequency of the pathogens encountered.

The chemokine receptor CCR7 is a well-established homing receptor for dendritic cells and T cells. Interactions with its ligands, CCL19 and CCL21, facilitate priming of immune responses in lymphoid tissue, yet CCR7-independent immune responses can be generated in the presence of sufficient antigen. In these studies, we investigated the role of CCR7 signaling in the generation of protective immune responses to the intracellular protozoan parasite Toxoplasma gondii . The results demonstrated a significant increase in the expression of CCL19, CCL21, and CCR7 in peripheral and central nervous system (CNS) tissues over the course of infection. Unexpectedly, despite the presence of abundant antigen, CCR7 was an absolute requirement for protective immunity to T. gondii , as CCR7 −/− mice succumbed to the parasite early in the acute phase of infection. Although serum levels of interleukin 12 (IL-12), IL-6, tumor necrosis factor alpha (TNF-α), and IL-10 remained unchanged, there was a significant decrease in CCL2/monocyte chemoattractant protein 1 (MCP-1) and inflammatory monocyte recruitment to the site of infection. In addition, CCR7 −/− mice failed to produce sufficient gamma interferon (IFN-γ), a critical Th1-associated effector cytokine required to control parasite replication. As a result, there was increased parasite dissemination and a significant increase in parasite burden in the lungs, livers, and brains of infected mice. Adoptive-transfer experiments revealed that expression of CCR7 on the T-cell compartment alone is sufficient to enable T-cell priming, increase IFN-γ production, and allow the survival of CCR7 −/− mice. These data demonstrate an absolute requirement for T-cell expression of CCR7 for the generation of protective immune responses to Toxoplasma infection.

The CNS (central nervous system) is unquestionably the central organ that regulates directly or indirectly all physiological systems in the mammalian body. Yet, when considering the defence of the CNS from pathogens, the CNS has often been considered passive and subservient to the pro-inflammatory responses of the immune system. In this view, neuroinflammatory disorders are examples of when the tail (the immune system) wags the dog (the CNS) to the detriment of an individual's function and survival.

Trem2 is an orphan, DAP12 associated receptor constitutively expressed in vivo by subsets of microglia in the healthy adult murine CNS and in vitro by subsets of oligodendrocytes in neonatal mixed glial cultures. Loss of a functional Trem2 signaling pathway is the genetic cause of Nasu-Hakola disease. Whether the early onset cognitive dementia and myelin-pallor associated with this disorder are due to deficits in functional Trem2 signaling in microglia and/or oligodendrocytes is still being debated. Here, we find that Trem2/DAP12 expression is detected in embryonic day 14 CNS mRNA. Using dual immunohistochemistry/in situ hybridization, we find that both Trem2 and DAP12 expression always co-localized with markers of microglia/macrophages. However, Trem2/DAP12 positive microglia are found in very close apposition with CNP+ oligodendrocytes prior to myelination (post-natal day 1). In addition, CNS expression of TREM2 and DAP12 are not detected in PU.1KO which lack microglia and macrophages. Our data provide continuing support for Nasu-Hakola disease being identified as a cognitive disorder caused by a primary dysfunction of CNS microglia.