Expression of Inducible Nitric Oxide Synthase (iNOS) in Microglia of the Developing Quail Retina

Inducible nitric oxide synthase (iNOS), which produce large amounts of nitric oxide (NO), is induced in macrophages and microglia in response to inflammatory mediators such as LPS and cytokines. Although iNOS is mainly expressed by microglia that become activated in different pathological and experimental situations, it was recently reported that undifferentiated amoeboid microglia can also express iNOS during normal development. The aim of this study was to investigate the pattern of iNOS expression in microglial cells during normal development and after their activation with LPS by using the quail retina as model. iNOS expression was analyzed by iNOS immunolabeling, western-blot, and RT-PCR. NO production was determined by using DAR-4M AM, a reliable fluorescent indicator of subcellular NO production by iNOS. Embryonic, postnatal, and adult in situ quail retinas were used to analyze the pattern of iNOS expression in microglial cells during normal development. iNOS expression and NO production in LPS-treated microglial cells were investigated by an in vitro approach based on organotypic cultures of E8 retinas, in which microglial cell behavior is similar to that of the in situ retina, as previously demonstrated in our laboratory. We show here that amoeboid microglia in the quail retina express iNOS during normal development. This expression is stronger in microglial cells migrating tangentially in the vitreal part of the retina and is downregulated, albeit maintained, when microglia differentiate and become ramified. LPS treatment of retina explants also induces changes in the morphology of amoeboid microglia compatible with their activation, increasing their lysosomal compartment and upregulating iNOS expression with a concomitant production of NO. Taken together, our findings demonstrate that immature microglial cells express iNOS during normal development, suggesting a certain degree of activation. Furthermore, LPS treatment induces overactivation of amoeboid microglia, resulting in a significant iNOS upregulation.     

The type 1 interleukin‐1 receptor is essential for multiple aspects of brain inflammation

Interleukin‐1 (IL‐1) is induced immediately after brain imjury and elevated levels of IL‐1 have been strongly implicated in the neurodegeneration that accompanies stroke, Alzheimer's disease and Multiple Sclerosis. Antagonizing IL‐1 reduces cell death; however, the basis for this protection has not been elucidated. Here we analyzed the response to penetrating brain injury in mice lacking the type 1 interleukin receptor (IL‐1R1) to determine which cellular and molecular mediators of tissue damage require IL‐1 signaling. At the cellular level fewer amoeboid microglia/macrophages appeared adjacent to the injured brain tissue in IL‐1R1 null mice, and those microglia present at early postinjury intervals retained their resting morphology. Astrogliosis also was mildly abrogated. At the molecular level, cyclooxygenase 2 and IL‐6 expression were depressed and delayed. Interestingly, basal levels of cyclooxygenase 2, IL‐1 and IL‐6 were significantly lower in the IL‐1R1 null mice. Additionally, stimulation of VCAM‐1 mRNA was depressed in the IL‐1R1 null mice, and correspondingly, there was reduced migration of peripheral macrophages into the IL‐1R1 null brain after injury. This observation correlated with a reduced number of cyclooxygenase 2+ amoeboid phagocytes adjacent to the injury. By contrast, the production of nerve growth factor was only mildly affected. Since antagonizing IL‐1 protects neural cells in experimental models of stroke and multiple sclerosis, our data suggest that cell preservation is achieved by abrogating microglial/macrophage activation and the subsequent self‐propagating cycle of inflammation. Acknowledgements: Supported by NMSS Award #RG 3837.     

Characteristics of the alternative phenotype of microglia/macrophages and its modulation in experimental gliomas

Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b(+) cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b(+) cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas.     

Myelin-Derived Lipids Modulate Macrophage Activity by Liver X Receptor Activation

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis.     

Enhanced microglial pro‐inflammatory response to lipopolysaccharide correlates with brain infiltration and blood–brain barrier dysregulation in a mouse model of telomere shortening

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self‐renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as ‘priming’. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first‐generation G1 mTerc^?/?)‐ and late‐generation (third‐generation G3 and G4 mTerc^?/?) telomerase‐deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late‐generation mTerc^?/? microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc^?/? microglia are comparable with microglia derived from G1 mTerc^?/? mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc^?/? microglia mice show an enhanced pro‐inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age‐associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood–brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening.     

A dual role of lipocalin 2 in the apoptosis and deramification of activated microglia

Activated microglia are thought to undergo apoptosis as a self-regulatory mechanism. To better understand molecular mechanisms of the microglial apoptosis, apoptosis-resistant variants of microglial cells were selected and characterized. The expression of lipocalin 2 (lcn2) was significantly down-regulated in the microglial cells that were resistant to NO-induced apoptosis. lcn2 expression was increased by inflammatory stimuli in microglia. The stable expression of lcn2 as well as the addition of rLCN2 protein augmented the sensitivity of microglia to the NO-induced apoptosis, while knockdown of lcn2 expression using short hairpin RNA attenuated the cell death. Microglial cells with increased lcn2 expression were more sensitive to other cytotoxic agents as well. Thus, inflammatory activation of microglia may lead to up-regulation of lcn2 expression, which sensitizes microglia to the self-regulatory apoptosis. Additionally, the stable expression of lcn2 in BV-2 microglia cells induced a morphological change of the cells into the round shape with a loss of processes. Treatment of primary microglia cultures with the rLCN2 protein also induced the deramification of microglia. The deramification of microglia was closely related with the apoptosis-prone phenotype, because other deramification-inducing agents such as cAMP-elevating agent forskolin, ATP, and calcium ionophore also rendered microglia more sensitive to cell death. Taken together, our results suggest that activated microglia may secrete LCN2 protein, which act in an autocrine manner to sensitize microglia to the self-regulatory apoptosis and to endow microglia with an amoeboid form, a canonical morphology of activated microglia in vivo.     

Development of ramified microglia from early macrophages in the zebrafish optic tectum

Microglia, the resident macrophage precursors of the brain, are necessary for the maintenance of tissue homeostasis and activated by a wide range of pathological stimuli. They have a key role in immune and inflammatory responses. Early microglia stem from primitive macrophages, however the transition from early motile forms to the ramified mature resident microglia has not been assayed in real time. In order to provide such an assay, we used zebrafish transgenic lines in which fluorescent reporter expression is driven by the promoter of 1 (mpeg1; Ellet et al. [2011]: Blood 117(4): e49–e56,). This enabled the investigation of the development of these cells in live, intact larvae. We show that microglia develop from highly motile amoeboid cells that are engaged in phagocytosis of apoptotic cell bodies into a microglial cell type that rapidly morphs back and forth between amoeboid and ramified morphologies. These morphing microglia eventually settle into a typical mature ramified morphology. Developing microglia frequently come into contact with blood capillaries in the brain, and also frequently contact each other. Up to 10 days postfertilization, microglia were observed to undergo symmetric division. In the adult optic tectum, the microglia are highly branched, resembling mammalian microglia. In addition, the mpeg1 transgene also labeled highly branched cells in the skin overlying the optic tectum from 8–9 days postfertilization, which likely represent Langerhans cells. Thus, the development of zebrafish microglia and their cellular interactions was studied in the intact developing brain in real time and at cellular resolution. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Microglial replacement in the aged brain restricts neuroinflammation following intracerebral hemorrhage

Aged microglia display augmented inflammatory activity after neural injury. Although aging is a risk factor for poor outcome after brain insults, the precise impact of aging-related alterations in microglia on neural injury remains poorly understood. Microglia can be eliminated via pharmacological inhibition of the colony–stimulating factor 1 receptor (CSF1R). Upon withdrawal of CSF1R inhibitors, microglia rapidly repopulate the entire brain, leading to replacement of the microglial compartment. In this study, we investigated the impact of microglial replacement in the aged brain on neural injury using a mouse model of intracerebral hemorrhage (ICH) induced by collagenase injection. We found that replacement of microglia in the aged brain reduced neurological deficits and brain edema after ICH. Microglial replacement-induced attenuation of ICH injury was accompanied with alleviated blood-brain barrier disruption and leukocyte infiltration. Notably, newly repopulated microglia had reduced expression of IL-1β, TNF-α and CD86, and upregulation of CD206 in response to ICH. Our findings suggest that replacement of microglia in the aged brain restricts neuroinflammation and brain injury following ICH.Subject terms: Neuroimmunology, Cognitive ageing     

Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation

Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.KEY WORDS: Brain, Intravital microscopy, Leukocytes, Microglia, Neurodegeneration, Zebrafish     

Tubulin cofactor B regulates microtubule densities during microglia transition to the reactive states

Microglia are highly dynamic cells of the CNS that continuously survey the welfare of the neural parenchyma and play key roles modulating neurogenesis and neuronal cell death. In response to injury or pathogen invasion parenchymal microglia transforms into a more active cell that proliferates, migrates and behaves as a macrophage. The acquisition of these extra skills implicates enormous modifications of the microtubule and actin cytoskeletons. Here we show that tubulin cofactor B (TBCB), which has been found to contribute to various aspects of microtubule dynamics in vivo, is also implicated in microglial cytoskeletal changes. We find that TBCB is upregulated in post-lesion reactive parenchymal microglia/macrophages, in interferon treated BV-2 microglial cells, and in neonate amoeboid microglia where the microtubule densities are remarkably low. Our data demonstrate that upon TBCB downregulation both, after microglia differentiation to the ramified phenotype in vivo and in vitro, or after TBCB gene silencing, microtubule densities are restored in these cells. Taken together these observations support the view that TBCB functions as a microtubule density regulator in microglia during activation, and provide an insight into the understanding of the complex mechanisms controlling microtubule reorganization during microglial transition between the amoeboid, ramified, and reactive phenotypes.     

Distinct Features of Brain-Resident Macrophages: Microglia and Non-Parenchymal Brain Macrophages

Tissue-resident macrophages play an important role in maintaining tissue homeostasis and innate immune defense against invading microbial pathogens. Brain-resident macrophages can be classified into microglia in the brain parenchyma and non-parenchymal brain macrophages, also known as central nervous system-associated or border-associated macrophages, in the brain-circulation interface. Microglia and non-parenchymal brain macrophages, including meningeal, perivascular, and choroid plexus macrophages, are mostly produced during embryonic development, and maintained their population by self-renewal. Microglia have gained much attention for their dual roles in the maintenance of brain homeostasis and the induction of neuroinflammation. In particular, diverse phenotypes of microglia have been increasingly identified under pathological conditions. Single-cell phenotypic analysis revealed that microglia are highly heterogenous and plastic, thus it is difficult to define the status of microglia as M1/M2 or resting/activated state due to complex nature of microglia. Meanwhile, physiological function of non-parenchymal brain macrophages remain to be fully demonstrated. In this review, we have summarized the origin and signatures of brain-resident macrophages and discussed the unique features of microglia, particularly, their phenotypic polarization, diversity of subtypes, and inflammasome responses related to neurodegenerative diseases.     

Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit

Microglia cells are the brain counterpart of macrophages and function as the first defense in the brain. Although they are neuroprotective in the young brain, microglia cells may be primed to react abnormally to stimuli in the aged brain and to become neurotoxic and destructive during neurodegeneration. Aging-induced immune senescence occurs in the brain as age-associated microglia senescence, which renders microglia to function abnormally and may eventually promote neurodegeneration. Microglia senescence is manifested by both morphological changes and alterations in immunophenotypic expression and inflammatory profile. These changes are likely caused by microinvironmental factors, but intrinsic factors cannot yet be completely excluded. Microglia senescence appears to underlie the switching of microglia from neuroprotective in the young brain to neurotoxic in the aged brain. The hypothesis of microglia senescence during aging offers a novel perspective on their roles in aging-related neurodegeneration. In Parkinson's disease and Alzheimer's disease, over-activation of microglia may play an active role in the pathogenesis because microglia senescence primes them to be neurotoxic during the development of the diseases.     

Sex differences in microglial colonization of the developing rat brain

Microglia are the resident immune cells within the brain and their production of immune molecules such as cytokines and chemokines is critical for the processes of normal brain development including neurogenesis, axonal migration, synapse formation, and programmed cell death. Notably, sex differences exist in many of these processes throughout brain development; however, it is unknown whether a sex difference concurrently exists in the colonization, number, or morphology of microglia within the developing brain. We demonstrate for the first time that the number and morphology of microglia throughout development is dependent upon the sex and age of the individual, as well as the brain region of interest. Males have overall more microglia early in postnatal development [postnatal day (P) 4], whereas females have more microglia with an activated/amoeboid morphology later in development, as juveniles and adults (P30-60). Finally, gene expression of a large number of cytokines, chemokines and their receptors shifts dramatically over development, and is highly dependent upon sex. Taken together, these data warrant further research into the role that sex-dependent mechanisms may play in microglial colonization, number, and function, and their potential contribution to neural development, function, or potential dysfunction.     

Involvement of heparanase in migration of microglial cells

Heparanase, a matrix-degrading enzyme that cleaves heparan sulfate side chains from heparan sulfate proteoglycans (HSPGs), has been shown to facilitate cell invasion, migration, and extravasation of metastatic tumor cells or immune cells. In this study, the expression and functions of heparanase were investigated using rat primary cultured microglia, the resident macrophages in the brain. The microglia were found to express heparanase mRNA and protein. Microglia treated with lipopolysaccharide (LPS) were activated, expressed induced nitric oxide synthase and elevated the expression of heparanase. Heparanase has two molecular weights: a 65 kDa latent form and an active 50 kDa. Both forms were expressed by LPS-treated activated microglia; however, untreated microglia primarily expressed the latent form. Cell lysates from microglia actually degraded Matrigel containing HSPG. Heparanase was colocalized with the actin cytoskeleton in microglial leading edges or ruffled membranes. Microglia transmigrated through a Matrigel-coated pored membrane. This process was inhibited by SF-4, a specific heparanase inhibitor, in a concentration-dependent manner. Degraded HSPG was generated when microglia transmigrated through the coated membrane, and this was also inhibited by SF-4. The results suggest the involvement of heparanase in the migration or invasion of microglia or brain macrophages across basement membrane around brain vasculature.     

Down regulation of CD4 expression in cultured microglia by immunosuppressants and lipopolysaccharide.

Microglia, brain macrophages, are thought to be the primary target of HIV-1 infection in the brain, because they exclusively express the CD4 antigen which is effectively used for viral entry. The expression of CD4 mRNA in cultured microglia could be detected by the reverse-PCR method. Using this and immunohistochemical staining, we found that the immunosuppressants cyclosporin A and FK506 decreased CD4 expression in cultured murine microglia without causing any significant decrease in cell viability. FK506 was more potent than cyclosporin A. Lipopolysaccharide also decreased CD4 mRNA expression in microglia. The effects of immunosuppressants and lipopolysaccharide seemed to be specific for microglia since these chemicals did not alter the CD4 expression in lymphocytes or peritoneal macrophages. These agents, if modified to pass through the blood-brain barrier, may prevent viral spread of HIV-1 infection in the central nervous system and the AIDS-dementia complex.     

Differential effects of albumin on microglia and macrophages; implications for neurodegeneration following blood–brain barrier damage

Microglial activation by blood-borne factors following blood–brain barrier damage may play a significant role in subsequent neuropathogenesis of several neurodegenerative diseases. Exposure of primary cultured rat brain microglia to pure, fatty acid- and lipid-deficient rat serum albumin or fraction V, (fatty acid and lipid-containing rat serum albumin), caused inducible nitric oxide synthase (iNOS) expression, glutamate release, tumour necrosis factor alpha (TNFα) and transforming growth factor-beta1 release. iNOS expression was attenuated by the MAPK/extracellular signal-regulated kinase pathway inhibitor U0126 and the phosphorylated forms of extracellular signal-regulated kinase 1 and 2 were detectable in microglia treated with albumin or fraction V. Glutamate release was prevented by l -α-aminoadipate and glutathione levels in microglia rose on exposure to albumin. Conditioned medium from microglia exposed to albumin or fraction V was neurotoxic. Peripheral macrophages were resistant to the effects of albumin but both microglia and macrophages responded to lipopolysaccharide, which induced interleukin-1 beta and tumour necrosis factor alpha release, cyclooxygenase-2 and iNOS expression in both cell types, indicating a discrete desensitised pathway in macrophages for albumin which was not desensitised in microglia. Thus, exposure of microglia in the brain to albumin may contribute to neuronal damage following blood–brain barrier breakdown and point to resident microglia rather than infiltrating macrophages as therapeutic targets.