Polysialylation and lipopolysaccharide‐induced shedding of E‐selectin ligand‐1 and neuropilin‐2 by microglia and THP‐1 macrophages

Microglia are tissue macrophages and mediators of innate immune responses in the brain. The protein‐modifying glycan polysialic acid (polySia) is implicated in modulating microglia activity. Cultured murine microglia maintain a pool of Golgi‐confined polySia, which is depleted in response to lipopolysaccharide (LPS)‐induced activation. Polysialylated neuropilin‐2 (polySia‐NRP2) contributes to this pool but further polySia protein carriers have remained elusive. Here, we use organotypic brain slice cultures to demonstrate that injury‐induced activation of microglia initiates Golgi‐confined polySia expression in situ. An unbiased glycoproteomic approach with stem cell‐derived microglia identifies E‐selectin ligand‐1 (ESL‐1) as a novel polySia acceptor. Together with polySia‐NRP2, polySia‐ESL‐1 is also detected in primary cultured microglia, in brain slice cultures and in phorbol ester‐induced THP‐1 macrophages. Induction of stem cell‐derived microglia, activated microglia in brain slice cultures and THP‐1 macrophages by LPS, but not interleukin‐4, causes polySia depletion and, as shown for stem cell‐derived microglia, a metalloproteinase‐dependent release of polySia‐ESL‐1 and polySia‐NRP2. Moreover, soluble polySia attenuates LPS‐induced production of nitric oxide and proinflammatory cytokines. Thus, shedding of polySia‐ESL‐1 and polySia‐NRP2 after LPS‐induced activation of microglia and THP‐1 macrophages may constitute a mechanism for negative feedback regulation. GLIA 2016 GLIA 2016;64:1314–1330     

Microglia stimulate naive T‐cell differentiation without stimulating T‐cell proliferation

A major question relevant to the initiation and progression of inflammation and autoimmune processes within the central nervous system (CNS) is whether resident microglia or only infiltrating macrophage can productively interact with T‐cells that enter the CNS either actively through extravasation or passively through defects in the blood brain barrier (BBB). We isolated microglia and macrophage from the brains of healthy adult mice and transgenic mice that displayed many features of multiple sclerosis and HIV leukoencephalopathy due to the astrocytic expression of interleukin (IL)‐3 and compared their antigen‐presenting cell (APC) functions. We found that unactivated microglia isolated from healthy nontransgenic mice and activated microglia isolated from transgenic siblings are relatively weak stimulators of naive T‐cell proliferation compared to macrophage populations. The APC function of activated, but not unactivated, microglia could be increased by treatment acutely with lipopolysaccharide (LPS)/interferon γ (IFNγ). However, this treatment also induced the apparent production of prostaglandins, which reduced T‐cell proliferation when indomethacin was absent from the assay cultures. Strikingly, even in the absence of stimulated T‐cell proliferation, both unactivated and activated microglia stimulated the differentiation of naive T‐cells into Th1 effector cells, although neither microglial population was a more effective inducer than macrophages or splenic APCs. Thus, while microglia are clearly capable of productively interacting with naive T‐cells, macrophages have a more robust APC function. J. Neurosci. Res. 55:127–134, 1999. © 1999 Wiley‐Liss, Inc.     

Microglia‐derived purines modulate mossy fibre synaptic transmission and plasticity through P2X4 and A1 receptors

Recent data have provided evidence that microglia, the brain‐resident macrophage‐like cells, modulate neuronal activity in both physiological and pathophysiological conditions, and microglia are therefore now recognized as synaptic partners. Among different neuromodulators, purines, which are produced and released by microglia, have emerged as promising candidates to mediate interactions between microglia and synapses. The cellular effects of purines are mediated through a large family of receptors for adenosine and for ATP (P2 receptors). These receptors are present at brain synapses, but it is unknown whether they can respond to microglia‐derived purines to modulate synaptic transmission and plasticity. Here, we used a simple model of adding immune‐challenged microglia to mouse hippocampal slices to investigate their impact on synaptic transmission and plasticity at hippocampal mossy fibre (MF) synapses onto CA3 pyramidal neurons. MF–CA3 synapses show prominent forms of presynaptic plasticity that are involved in the encoding and retrieval of memory. We demonstrate that microglia‐derived ATP differentially modulates synaptic transmission and short‐term plasticity at MF–CA3 synapses by acting, respectively, on presynaptic P2X₄ receptors and on adenosine A₁ receptors after conversion of extracellular ATP to adenosine. We also report that P2X₄ receptors are densely located in the mossy fibre tract in the dentate gyrus–CA3 circuitry. In conclusion, this study reveals an interplay between microglia‐derived purines and MF–CA3 synapses, and highlights microglia as potent modulators of presynaptic plasticity.     

Microglia and macrophages differentially modulate cell death after brain injury caused by oxygen‐glucose deprivation in organotypic brain slices

Macrophage can adopt several phenotypes, process call polarization, which is crucial for shaping inflammatory responses to injury. It is not known if microglia, a resident brain macrophage population, polarizes in a similar way, and whether specific microglial phenotypes modulate cell death in response to brain injury. In this study, we show that both BV2‐microglia and mouse bone marrow derived macrophages (BMDMs) were able to adopt different phenotypes after LPS (M1) or IL‐4 (M2) treatment in vitro, but regulated cell death differently when added to mouse organotypic hippocampal brain slices. BMDMs induced cell death when added to control slices and exacerbated damage when combined with oxygen–glucose deprivation (OGD), independently of their phenotype. In contrast, vehicle‐ and M2‐BV2‐microglia were protective against OGD‐induced death. Direct treatment of brain slices with IL‐4 (without cell addition) was protective against OGD and induced an M2 phenotype in the slice. In vivo, intracerebral injection of LPS or IL‐4 in mice induced microglial phenotypes similar to the phenotypes observed in brain slices and in cultured cells. After injury induced by middle cerebral artery occlusion, microglial cells did not adopt classical M1/M2 phenotypes, suggesting that another subtype of regulatory phenotype was induced. This study highlights functional differences between macrophages and microglia, in response to brain injury with fundamentally different outcomes, even if both populations were able to adopt M1 or M2 phenotypes. These data suggest that macrophages infiltrating the brain from the periphery after an injury may be cytotoxic, independently of their phenotype, while microglia may be protective.     

Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain

Microglia, the resident CNS macrophages, represent about 10% of the adult brain cell population. Although described a long time ago, their origin and developmental lineage is still debated. While del Rio-Hortega suggested that microglia originate from meningeal macrophages penetrating the brain during embryonic development, many authors claim that brain parenchymal microglia derive from circulating blood monocytes originating from bone marrow. We have previously reported that the late embryonic and adult mouse brain parenchyma contains potential microglial progenitors [F. Alliot, E. Lecain, B. Grima, B. Pessac, Microglial progenitors with a high proliferative capacity in the embryonic and the adult mouse brain, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 1541-1545]. We now report that they can be detected in the brain rudiment from embryonic day 8, after their appearance in the yolk sac and that their number increases until late gestation. We also show that microglia appear during embryonic development and that their number increases steadily during the first two postnatal weeks, when about 95% of microglia are born. Finally, the main finding of this study is that microglia is the result of in situ proliferation, as shown by the high proportion of parenchymal microglial cells that express PCNA, a marker of cell multiplication, in embryonic and postnatal brain. Taken together, our data support the hypothesis that terminally differentiated brain parenchymal microglia are derived from cells originating from the yolk sac whose progeny actively proliferates in situ during development.     

Microglia promote colonization of brain tissue by breast cancer cells in a Wnt‐dependent way

Although there is increasing evidence that blood‐derived macrophages support tumor progression, it is still unclear whether specialized resident macrophages, such as brain microglia, also play a prominent role in metastasis formation. Here, we show that microglia enhance invasion and colonization of brain tissue by breast cancer cells, serving both as active transporters and guiding rails. This is antagonized by inactivation of microglia as well as by the Wnt inhibitor Dickkopf‐2. Proinvasive microglia demonstrate altered morphology, but neither upregulation of M2‐like cytokines nor differential gene expression. Bacterial lipopolysacharide shifts tumor‐educated microglia into a classical M1 phenotype, reduces their proinvasive function, and unmasks inflammatory and Wnt signaling as the most strongly regulated pathways. Histological findings in human brain metastases underline the significance of these results. In conclusion, microglia are critical for the successful colonization of the brain by epithelial cancer cells, suggesting inhibition of proinvasive microglia as a promising antimetastatic strategy. © 2010 Wiley‐Liss, Inc.     

Endothelial nitric oxide synthase inhibition triggers inflammatory responses in the brain of male rats exposed to ischemia‐reperfusion injury

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) plays a role in preserving and maintaining the brain's microcirculation, inhibiting platelet aggregation, leukocyte adhesion, and migration. Inhibition of eNOS activity results in exacerbation of neuronal injury after ischemia by triggering diverse cellular mechanisms, including inflammatory responses. To examine the relative contribution of eNOS in stroke‐induced neuroinflammation, we analyzed the effects of systemic treatment with l‐N‐(1‐iminoethyl)ornithine (L‐NIO), a relatively selective eNOS inhibitor, on the expression of MiR‐155‐5p, a key mediator of innate immunity regulation and endothelial dysfunction, in the cortex of male rats subjected to transient middle cerebral artery occlusion (tMCAo) followed by 24 hr of reperfusion. Inducible NO synthase (iNOS) and interleukin‐10 (IL‐10) mRNA expression were evaluated by real‐time polymerase chain reaction in cortical homogenates and in resident and infiltrating immune cells isolated from ischemic cortex. These latter cells were also analyzed for their expression of CD40, a marker of M1 polarization of microglia/macrophages.tMCAo produced a significant elevation of miR155‐5p and iNOS expression in the ischemic cortex as compared with sham surgery. eNOS inhibition by L‐NIO treatment further elevated the cortical expression of these inflammatory mediators, while not affecting IL‐10 mRNA levels. Interestingly, modulation of iNOS occurred in resident and infiltrating immune cells of the ischemic hemisphere. Accordingly, L‐NIO induced a significant increase in the percentage of CD40⁺ events in CD68⁺ microglia/macrophages of the ischemic cortex as compared with vehicle‐injected animals. These findings demonstrate that inflammatory responses may underlie the detrimental effects due to pharmacological inhibition of eNOS in cerebral ischemia.     

Less neurogenesis and inflammation in the immature than in the juvenile brain after cerebral hypoxia-ischemia.

The effects of hypoxia-ischemia (HI) on proliferation and differentiation in the immature (postnatal day 9) and juvenile (postnatal day 21) mouse hippocampus were investigated by injecting bromodeoxyuridine (50 mg/kg) daily for 7 days after the insult and evaluating the labeling 5 weeks after HI. Phenotypic differentiation was evaluated using NeuN, Iba1, APC, and S100beta as markers of neurons, microglia, oligodendrocytes, and astrocytes, respectively. The basal proliferation, in particular neurogenesis, was higher in the immature than in the juvenile hippocampus. Hypoxia-ischemia did not increase neurogenesis significantly in the immature dentate gyrus (DG), but it increased several-fold in the juvenile brain, reaching the same level as in the normal, noninjured immature brain. This suggests that the immature hippocampus is already working at the top of its proliferative capacity and that even though basal neurogenesis decreased with age, the injury-induced generation of new neurons in the juvenile hippocampus could not increase beyond the basal level of the immature brain. Generation of glial cells of all three types after HI was significantly more pronounced in the cornu ammonis of the hippocampus region of the juvenile hippocampus. In the DG, only microglia production was greater in the juvenile brain. Increased microglia proliferation correlated with increased levels of the proinflammatory cytokines MCP-1 and IL-18 3 days after HI, indicating that the inflammatory response is stronger in the juvenile hippocampus. In summary, contrary to what has been generally assumed, our results indicate that the juvenile brain has a greater capacity for neurogenesis after injury than the immature brain.     

Intracerebral granulocyte‐macrophage colony‐stimulating factor induces functionally competent dendritic cells in the mouse brain

Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is a hematopoietic growth factor and a proinflammatory cytokine. While GM‐CSF is lacking in normal brain tissue, it is expressed under pathological conditions and correlates with the presence of dendritic cells (DC). However, the role of GM‐CSF for the onset of immune responses in the brain is still unclear. To analyze the role of GM‐CSF for the induction and functional activity of immune cells in the brain, we performed chronic intracerebroventricular administration of GM‐CSF to the brains of adult mice. After GM‐CSF administration, intracerebral leukocytes (ICL) were characterized by means of flow cytometry, immunohistochemistry, and an ex vivo functional assay. GM‐CSF treatment significantly increased the number of leukocytes expressing high levels of CD45, indicative of peripheral, blood‐derived cells. The infiltrating cells were preferentially DC of the myeloid lineage (CD45^high CD11c⁺ CD11b⁺) with an activated phenotype characterized by upregulated expression of MHCII and the costimulatory ligand CD80. Furthermore, DC from GM‐CSF treated mice were fully competent to activate naive allogeneic T cells in a mixed leukocyte reaction. In contrast, intracerebroventricular IFN‐γ administration stimulated MHCII expression on cells resembling resident microglia, but did not induce comparable presence of DC. Taken together, intracerebroventricular GM‐CSF treatment results in high numbers of DC in the brain. Moreover, these GM‐CSF‐induced DC display an activated phenotype and exhibit the capacity to act as fully competent DC even without a further inflammatory stimulus. © 2009 Wiley‐Liss, Inc.     

Hypoxia-ischemia upregulates TRAIL and TRAIL receptors in the immature rat brain

The immature brain is susceptible to inflammatory injury induced by hypoxia-ischemia (HI) or infection, which causes serious neurodevelopmental disabilities in the survivors of preterm births. Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (death receptor DR4/5 and decoy receptor DcR1/2) were reported to mediate various neuroinflammatory responses. However, little information is available regarding the role of TRAIL and its receptors in the immature brain after HI. The purpose of this study was to evaluate the expression of TRAIL and its receptors in the immature brain after HI and relate this expression to neurological function. We performed right common carotid artery ligation followed by hypoxia (6% O(2), 37°C) for 2.5 h to induce HI in postnatal day 3 rats. The distribution of TRAIL and its receptors, caspase-3 and CD68-labeled microglia/macrophages was evaluated 24 h after HI by immunostaining. The protein and mRNA expression of TRAIL and DR5 was measured by Western blot and real-time PCR, respectively. Delayed neuronal loss was evaluated by NeuN and Nissl staining 7 days after HI. Furthermore, neurological deficits were evaluated by a righting reflex test, time of eye opening and T-maze test. The expression of TRAIL, DR5 and DcR1/2 receptors and caspase-3 was more pronounced in the ipsilateral hemisphere compared with the contralateral part and the control group 24 h after HI. DR5/active caspase-3 double-positive cells were observed at 24 h after HI in the ipsilateral hemisphere but not in the contralateral hemisphere. The TRAIL and CD68 double-labeled cells were more pronounced in the ipsilateral cortical regions compared with the corresponding regions of the contralateral part. HI also resulted in a significant increase in TRAIL and DR5 protein and mRNA expression at 24 h, which corresponded to neuronal cell loss 7 days after HI. Furthermore, the HI group displayed impaired neurobehavioral development compared with the control group (p < 0.05). Altogether our results show that the TNF-α superfamily ligand TRAIL is induced on CD68+ microglia/macrophages after perinatal HI and that one of its receptors, DR5, is induced on neocortical neurons and glial cells. That many DR5+ cells were also caspase-3+ strongly supports the conclusion that these signaling molecules are involved in the delayed loss of neurons in the neocortex and in the neurobehavioral deficits that are often seen after perinatal HI.     

Chronic foot‐shock stress potentiates the influx of bone marrow‐derived microglia into hippocampus

For several years, a new population of microglia derived from bone marrow has been described in multiple settings such as infection, trauma, and neurodegenerative disease. The aim of this study was to investigate the migration of bone marrow‐derived cells to the brain parenchyma after stress exposure. Stress exposure was performed in mice that had received bone marrow transplantation from GFP mice, allowing identification of blood‐derived elements within the brain. Electric foot‐shock exposure was chosen because of its ability to serve as fundamental and physical stress in mice. Bone marrow‐derived GFP⁺ cells migrated to the ventral part of the hippocampus and acquired a ramified microglia‐like morphology. Microglia marker Iba1 was expressed by 100% of the ramified cells, whereas ramified cells were negative for the astrocyte marker GFAP. Compared with the case in the control group, ramified cells significantly increased after chronic exposure to stress (5 days). One month after 5 days of stress exposure, ramified cells significantly decreased in ventral hippocampus compared with the group examined immediately after the last stress exposure. We report for the first time the migration of bone marrow‐derived cells to the ventral hippocampus after stress exposure. These cells have the characteristics of microglia. Mechanisms responsible for this migration and their roles in the brain remain to be determined. © 2010 Wiley‐Liss, Inc.     

Microglia are less pro‐inflammatory than myeloid infiltrates in the hippocampus of mice exposed to status epilepticus

Activated microglia, astrogliosis, expression of pro‐inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro‐inflammatory cytokines with the activated morphology of microglia, attributing them a pro‐epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS‐based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine‐treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen‐presenting cell phenotype correlated with the presence of CD4^pos T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL‐1β and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL‐1β. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)?9 and 12 while microglia only expressed MMP‐12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile. GLIA 2016 GLIA 2016;64:1350–1362     

Microglia contributes to plaque growth by cell death due to uptake of amyloid β in the brain of Alzheimer's disease mouse model

Pathological hallmarks of Alzheimer's disease (AD) include extracellularly accumulated amyloid β (Aβ) plaques and intracellular neurofibrillary tangles in the brain. Activated microglia, brain‐resident macrophages, are also found surrounding Aβ plaques. The study of the brain of AD mouse models revealed that Aβ plaque formation is completed by the consolidation of newly generated plaque clusters in vicinity of existed plaques. However, the dynamics of Aβ plaque formation, growth and the mechanisms by which microglia contribute to Aβ plaque formation are unknown. In the present study, we confirmed how microglia are involved in Aβ plaque formation and their growth in the brain of 5XFAD mice, the Aβ‐overexpressing AD transgenic mouse model, and performed serial intravital two‐photon microscopy (TPM) imaging of the brains of 5XFAD mice crossed with macrophage/microglia‐specific GFP‐expressing CX3CR1^GFP/GFP mice. We found that activated microglia surrounding Aβ plaques take up Aβ, which are clusters developed inside activated microglia in vivo and this was followed by microglial cell death. These dying microglia release the accumulated Aβ into the extracellular space, which contributes to Aβ plaque growth. This process was confirmed by live TPM in vivo imaging and flow cytometry. These results suggest that activated microglia can contribute to formation and growth of Aβ plaques by causing microglial cell death in the brain. GLIA 2016;64:2274–2290