Microglial Cell Function Research Articles

Activin A increases phagocytosis of Escherichia coli K1 by primary murine microglial cells activated by toll-like receptor agonists

BackgroundBacterial meningitis is associated with high mortality and long-term neurological sequelae. Increasing the phagocytic activity of microglia could improve the resistance of the CNS against infections. We studied the influence of activin A, a member of the TGF-β family with known immunoregulatory and neuroprotective effects, on the functions of microglial cells in vitro.MethodsPrimary murine microglial cells were treated with activin A (0.13 ng/ml–13 μg/ml) alone or in combination with agonists of TLR2, 4, and 9. Phagocytosis of Escherichia coli K1 as well as release of TNF-α, IL-6, CXCL1, and NO was assessed.ResultsActivin A dose-dependently enhanced the phagocytosis of Escherichia coli K1 by microglial cells activated by agonists of TLR2, 4, and 9 without further increasing NO and proinflammatory cytokine release. Cell viability of microglial cells was not affected by activin A.ConclusionsPriming of microglial cells with activin A could increase the elimination of bacteria in bacterial CNS infections. This preventive strategy could improve the resistance of the brain to infections, particularly in elderly and immunocompromised patients.

Curcumin Prevents Acute Neuroinflammation and Long-Term Memory Impairment Induced by Systemic Lipopolysaccharide in Mice.

Systemic lipopolysaccharide (LPS) induces an acute inflammatory response in the central nervous system (CNS) (“neuroinflammation”) characterized by altered functions of microglial cells, the major resident immune cells of the CNS, and an increased inflammatory profile that can result in long-term neuronal cell damage and severe behavioral and cognitive consequences. Curcumin, a natural compound, exerts CNS anti-inflammatory and neuroprotective functions mainly after chronic treatment. However, its effect after acute treatment has not been well investigated. In the present study, we provide evidence that 50 mg/kg of curcumin, orally administered for 2 consecutive days before a single intraperitoneal injection of a high dose of LPS (5 mg/kg) in young adult mice prevents the CNS immune response. Curcumin, able to enter brain tissue in biologically relevant concentrations, reduced acute and transient microglia activation, pro-inflammatory mediator production, and the behavioral symptoms of sickness. In addition, short-term treatment with curcumin, administered at the time of LPS challenge, anticipated the recovery from memory impairments observed 1 month after the inflammatory stimulus, when mice had completely recovered from the acute neuroinflammation. Together, these results suggest that the preventive effect of curcumin in inhibiting the acute effects of neuroinflammation could be of value in reducing the long-term consequences of brain inflammation, including cognitive deficits such as memory dysfunction.

Microglia, the missing link in maternal immune activation and fetal neurodevelopment; and a possible link in preeclampsia and disturbed neurodevelopment?

Disturbances in fetal neurodevelopment have extensively been related to neurodevelopmental disorders in early and later life. Fetal neurodevelopment is dependent on adequate functioning of the fetal immune system. During pregnancy, the maternal immune system is challenged to both tolerate the semi-allogenic fetus and to protect the mother and fetus from microbes. The fetal immune system is influenced by maternal immune disturbances; therefore, perturbations in maternal immunity likely do not only alter pregnancy outcome but also alter fetal neurodevelopment. A possible common pathway could be modulating the functioning of tissue macrophages in the placenta and brain.Maternal immune tolerance towards the fetus involves several complex adaptations. In this active maternal immune state, the fetus develops its own immunity. As cytokines and other players of the immune system -which can pass the placenta- are involved in neurodevelopment, disruptions in immune balance influence fetal neurodevelopment. Several studies reported an association between maternal immune activation, complications of pregnancy as preeclampsia, and altered neonatal neurodevelopment. A possible pathway involves dysfunctioning of microglia cells, the immune cells of the brain. Functionality of microglia cells during normal pregnancy is, however, poorly understood. The recent outbreak of ZIKA virus (ZKV), but also the literature on virus infections in general and its consequences on microglial cell function and fetal neurodevelopment show the devastating effects a virus infection during pregnancy can have.

A Tale of Two Genes: Microglial Apoe and Trem2

Microglial cell function is implicated in the etiology of Alzheimer's disease by human genetics. In this issue of Immunity, Krasemann etal. (2017) describe a gene expression signature associated with an APOE- and TREM2-dependent response of microglia to brain tissue damage that accumulates in aging and disease, defining an axis that might be amenable to therapeutic targeting.

Microglia in steady state.

Microglial cells are the resident tissue macrophages of the CNS and are widely recognized for their immune surveillance of the healthy CNS. In addition to this well-accepted function, recent findings point to major roles for microglia in instructing and regulating the proper function of the neuronal networks in the adult CNS, but these cells are also involved in creating neuronal networks by orchestrating construction of the whole network during development. In this Review, we highlight recent findings about the steady-state functions of microglial cells, the factors that are important for physiological microglial function, and how microglia help to maintain tissue homeostasis in the CNS.

Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol

BackgroundOpioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus.MethodsNeonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2–6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons.ResultsIn the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol’s ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons.ConclusionsThese results suggest that ethanol’s neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.

Cellular and Molecular Characterization of Microglia: A Unique Immune Cell Population.

Microglia are essential for the development and function of the adult brain. Microglia arise from erythro-myeloid precursors in the yolk sac and populate the brain rudiment early during development. Unlike monocytes that are constantly renewed from bone marrow hematopoietic stem cells throughout life, resident microglia in the healthy brain persist during adulthood via constant self-renewal. Their ontogeny, together with the absence of turnover from the periphery and the singular environment of the central nervous system, make microglia a unique cell population. Supporting this notion, recent genome-wide transcriptional studies revealed specific gene expression profiles clearly distinct from other brain and peripheral immune cells. Here, we highlight the breakthrough studies that, over the last decades, helped elucidate microglial cell identity, ontogeny, and function. We describe the main techniques that have been used for this task and outline the crucial milestones that have been achieved to reach our actual knowledge of microglia. Furthermore, we give an overview of the “microgliome” that is currently emerging thanks to the constant progress in the modern profiling techniques.

Protective Effect of Cactus Cladode Extracts on Peroxisomal Functions in Microglial BV-2 Cells Activated by Different Lipopolysaccharides.

In this study, we aimed to evaluate the antioxidant and anti-inflammatory properties of Opuntia ficus-indica cactus cladode extracts in microglia BV-2 cells. Inflammation associated with microglia activation in neuronal injury can be achieved by LPS exposure. Using four different structurally and biologically well-characterized LPS serotypes, we revealed a structure-related differential effect of LPS on fatty acid β-oxidation and antioxidant enzymes in peroxisomes: Escherichia coli-LPS decreased ACOX1 activity while Salmonella minnesota-LPS reduced only catalase activity. Different cactus cladode extracts showed an antioxidant effect through microglial catalase activity activation and an anti-inflammatory effect by reducing nitric oxide (NO) LPS-dependent production. These results suggest that cactus extracts may possess a neuroprotective activity through the induction of peroxisomal antioxidant activity and the inhibition of NO production by activated microglial cells.

GluNs Detection and Functions in Microglial Cells.

Proving endogenous GluN presence and functions in microglia require complementary steps to demonstrate (1) that GluN genes are transcripted and translated, (2) their cellular localization, (3) that the GluN are functional, and (4) the role of the functional GluN. The complete demonstration is performed by using mRNA detection technics, western blots, immunofluorescence, electrophysiology, calcium imaging, morphology studies, multiplex immunoassay together with conditional microglial Knock-Out mice and brain lesion models.

TRPC Channels and Brain Inflammation.

Nonresolving low-grade inflammation is supposed to underly the basis of chronic disorders including cardiovascular diseases, cancer, diabetes, obesity, and psychiatric disorders such as depression and Alzheimer's diseases. There is increasing evidence suggesting that pathophysiology of psychiatric disorders is related to the inflammatory responses mediated by microglial cells. Elevation of intracellular Ca2+ is important for the activation of microglial cell functions, including proliferation, release of NO, cytokines, and BDNF. It has been shown that alteration of intracellular Ca2+ signaling underlies the pathophysiology of psychiatric disorders, including depression. BDNF induces a sustained intracellular Ca2+ elevation through the upregulation of the surface expression of TRPC3 channels in rodent microglial cells. Microglial cells are able to respond to BDNF, which may be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of psychiatric disorders. We also need to study the effect of proBDNF on microglial cells especially by focusing on the TRPC channels.

Immortalization of primary microglia: a new platform to study HIV regulation in the central nervous system

The major reservoirs for HIV in the CNS are in the microglia, perivascular macrophages, and to a lesser extent, astrocytes. To study the molecular events controlling HIV expression in the microglia, we developed a reliable and robust method to immortalize microglial cells from primary glia from fresh CNS tissues and commercially available frozen glial cells. Primary human cells, including cells obtained from adult brain tissue, were transformed with lentiviral vectors expressing SV40 T antigen or a combination of SVR40 T antigen and hTERT. The immortalized cells have microglia-like morphology and express key microglial surface markers including CD11b, TGFβR, and P2RY12. Importantly, these cells were confirmed to be of human origin by sequencing. The RNA expression profiles identified by RNA-seq are also characteristic of microglial cells. Furthermore, the cells demonstrate the expected migratory and phagocytic activity, and the capacity to mount an inflammatory response characteristic of primary microglia. The immortalization method has also been successfully applied to a wide range of microglia from other species (macaque, rat, and mouse). To investigate different aspects of HIV molecular regulation in CNS, the cells have been superinfected with HIV reporter viruses and latently infected clones have been selected that reactive HIV in response to inflammatory signals. The cell lines we have developed and rigorously characterized will provide an invaluable resource for the study of HIV infection in microglial cells as well as studies of microglial cell function.

Genetic deletion of galectin-3 enhances neuroinflammation, affects microglial activation and contributes to sub-chronic injury in experimental neonatal focal stroke

The pathophysiology of neonatal stroke and adult stroke are distinct in many aspects, including the inflammatory response. We previously showed endogenously protective functions of microglial cells in acute neonatal stroke. We asked if galectin-3 (Gal3), a pleotropic molecule that mediates interactions between microglia/macrophages and the extracellular matrix (ECM), plays a role in early injury after transient middle cerebral occlusion (tMCAO) in postnatal day 9–10 mice. Compared to wild type (WT) pups, in Gal3 knockout pups injury was worse and cytokine/chemokine production altered, including further increase of MIP1α and MIP1β levels and reduced IL6 levels 72h after tMCAO. Lack of Gal3 did not affect morphological transformation or proliferation of microglia but markedly attenuated accumulation of CD11b+/CD45med-high cells after injury, as determined by multi-color flow cytometry. tMCAO increased expression of αV and β3 integrin subunits in CD11b+/CD45low microglial cells and cells of non-monocyte lineage (CD11b−/CD45−), but not in CD11b+/CD45med-high cells within injured regions of WT mice or Gal3−/− mice. αV upregulated in areas occupied and not occupied by CD68+ cells, most prominently in the ECM, lining blood vessels, with expanded αV coverage in Gal3−/− mice. Cumulatively, these data show that lack of Gal3 worsens subchronic injury after neonatal focal stroke, likely by altering the neuroinflammatory milieu, including an imbalance between pro- and anti-inflammatory molecules, effects on microglial activation, and deregulation of the composition of the ECM.

Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity.

Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing (NGS) approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden-death medication-free individuals postmortem. Using small RNA-seq, we also examined miRNA expression (9 samples per group). DeSeq2 identified thirty-five genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted p<0.1). In depression, altered genes include humanin like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted p<0.1). Hypothesis-driven GO analysis suggests lower expression of genes involved in oligodendrocyte differentiation, regulation of glutamatergic neurotransmission, and oxytocin receptor expression in both suicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted p<0.1) in depression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders.

Myelin and microglia in the aging brain

Myelin turnover that occurs normally with age or abnormally in disease overburdens the housekeeping function of microglial cells.

Novel Methods for Microglia Segmentation, Feature Extraction, and Classification.

Segmentation and analysis of histological images provides a valuable tool to gain insight into the biology and function of microglial cells in health and disease. Common image segmentation methods are not suitable for inhomogeneous histology image analysis and accurate classification of microglial activation states has remained a challenge. In this paper, we introduce an automated image analysis framework capable of efficiently segmenting microglial cells from histology images and analyzing their morphology. The framework makes use of variational methods and the fast-split Bregman algorithm for image denoising and segmentation, and of multifractal analysis for feature extraction to classify microglia by their activation states. Experiments show that the proposed framework is accurate and scalable to large datasets and provides a useful tool for the study of microglial biology.

Immune activation in the central nervous system throughout the course of HIV infection.

Robust and dynamic innate and adaptive responses characterize the acute central nervous system (CNS) response to HIV and other viral infections. In a state of chronic infection or viral latency, persistent immune activation associates with abnormality in the CNS. Understanding this process is critical, as immune-mediated abnormality in nonrenewable CNS cells may result in long-term neurologic sequelae for HIV-infected individuals. In humans, immune activation is reduced by suppressive combination antiretroviral therapy, but persists at abnormally elevated levels on treatment. CNS immune activation is initiated in acute infection and progressively increases until combination antiretroviral therapy is started. Newly identified characteristics of the CNS immune surveillance network include features of homeostasis and function of brain microglial cells, lymphatic drainage from CNS to cervical lymph nodes, and cells in cerebrospinal fluid associated with neurocognitive impairment. More research is required to determine whether early intervention to reduce infection limits the immunopathology established by sustained immune responses that ultimately fail to resolve infection, and to unravel mechanisms of persistent immune activation during treated HIV so that strategies can be developed to therapeutically protect the brain.

Severe chronic cerebral hypoperfusion induces microglial dysfunction leading to memory loss in APPswe/PS1 mice.

Cerebral vasculature plays a key role in controlling brain homeostasis. Cerebral vasculature dysfunction, associated to irregularities in cerebral blood perfusion, has been proposed to directly contribute to Alzheimer's disease (AD) pathogenesis. More precisely, chronic cerebral hypoperfusion, which impairs brain homeostasis, was demonstrated to take place even before cognitive decline. However, the mechanisms underlying the implication of chronic cerebral hypoperfusion in AD pathogenesis remain elusive. Therefore, this study aims at investigating the role of severe chronic cerebral hypoperfusion (SCCH) in AD pathogenesis. For this purpose, SCCH was induced in young APPswe/PS1 in order to evaluate the progression of AD-like pathology in these mice. We observed that SCCH accelerated the cognitive decline of young APPswe/PS1 mice, which was associated with an increased amyloid plaque number in brain parenchyma. In addition, SCCH reduced the activity of extracellular signal-regulated kinases 1/2 (ERK1/2), which has been shown to play an important role in the adaptive responses of neurons. Importantly, SCCH impaired the function of microglial cells, which are implicated in amyloid-β (Aβ) elimination. In vitro approaches underlined the ability of a low-glucose microenvironment to decrease the general activity and phagocytic capacity of microglia. By using a new model of SCCH, our study unravels new insights into the implication of severe chronic cerebral hypoperfusion in AD pathogenesis, mainly by altering microglial cell activity and consequently Aβ clearance.

Peripheral viral infection induced microglial sensome genes and enhanced microglial cell activity in the hippocampus of neonatal piglets

Although poorly understood, early-life infection is predicted to affect brain microglial cells, making them hypersensitive to subsequent stimuli. To investigate this, we assessed gene expression in hippocampal tissue obtained from a previously published study reporting increased microglial cell activity and reduced hippocampal-dependent learning in neonatal piglets infected with porcine reproductive and respiratory syndrome virus (PRRSV), a virus that induces interstitial pneumonia. Infection altered expression of 455 genes, of which 334 were up-regulated and 121 were down-regulated. Functional annotation revealed that immune function genes were enriched among the up-regulated differentially expressed genes (DEGs), whereas calcium binding and synaptic vesicle genes were enriched among the down-regulated DEGs. Twenty-six genes encoding part of the microglia sensory apparatus (i.e., the sensome) were up-regulated (e.g., IL1R1, TLR2, and TLR4), whereas 15 genes associated with the synaptosome and synaptic receptors (e.g., NPTX2, GABRA2, and SLC5A7) were down-regulated. As the sensome may foretell microglia reactivity, we next inoculated piglets with culture medium or PRRSV at PD 7 and assessed hippocampal microglia morphology and function at PD 28 when signs of infection were waning. Consistent with amplification of the sensome, microglia from PRRSV piglets had enhanced responsiveness to chemoattractants, increased phagocytic activity, and secreted more TNFα in response to lipopolysaccharide and Poly I:C. Immunohistochemical staining indicated PRRSV infection increased microglia soma length and length-to-width ratio. Bipolar rod-like microglia not evident in hippocampus of control piglets, were present in infected piglets. Collectively, this study suggests early-life infection alters the microglia sensome as well as microglial cell morphology and function.

Intrinsic determination of microglial cell function in health and disease - role of IRF8

Intrinsic determination of microglial cell function in health and disease - role of IRF8

Stress Granules Modulate SYK to Cause Microglial Cell Dysfunction in Alzheimer's Disease

Microglial cells in the brains of Alzheimer's patients are known to be recruited to amyloid-beta (Aβ) plaques where they exhibit an activated phenotype, but are defective for plaque removal by phagocytosis. In this study, we show that microglia stressed by exposure to sodium arsenite or Aβ(1–42) peptides or fibrils form extensive stress granules (SGs) to which the tyrosine kinase, SYK, is recruited. SYK enhances the formation of SGs, is active within the resulting SGs and stimulates the production of reactive oxygen and nitrogen species that are toxic to neuronal cells. This sequestration of SYK inhibits the ability of microglial cells to phagocytose Escherichia coli or Aβ fibrils. We find that aged microglial cells are more susceptible to the formation of SGs; and SGs containing SYK and phosphotyrosine are prevalent in the brains of patients with severe Alzheimer's disease. Phagocytic activity can be restored to stressed microglial cells by treatment with IgG, suggesting a mechanism to explain the therapeutic efficacy of intravenous IgG. These studies describe a mechanism by which stress, including exposure to Aβ, compromises the function of microglial cells in Alzheimer's disease and suggest approaches to restore activity to dysfunctional microglial cells.