IL-4 shapes microglia-dependent pruning of the cerebellum during postnatal development.

Interleukin-4 (IL-4) is a type 2 cytokine with pleiotropic functions in adaptive immunity, allergies, and cognitive processes. Here, we show that low levels of IL-4 in the early postnatal stage delineate a critical period in which microglia extensively prune cerebellar neurons. Elevating the levels of this cytokine via peripheral injection, or using a mouse model of allergic asthma, leads to defective pruning, permanent increase in cerebellar granule cells, and circuit alterations. These animals also show a hyperkinetic and impulsive-like phenotype, reminiscent of attention-deficit hyperactivity disorder (ADHD). These alterations are blocked in Il4rαfl/fl::Cx3cr1-CreER mice, which are deficient in IL-4 receptor signaling in microglia. These findings demonstrate a previously unknown role for IL-4 during a neuroimmune critical period of cerebellar maturation and provide a first putative mechanism for the comorbidity between allergic disease and ADHD observed in humans.

Microglia states and nomenclature: A field at its crossroads.

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.

N-3 PUFA Deficiency Affects the Ultrastructural Organization and Density of White Matter Microglia in the Developing Brain of Male Mice.

Over the last century, westernization of dietary habits has led to a dramatic reduction in dietary intake of n-3 polyunsaturated fatty acids (n-3 PUFAs). In particular, low maternal intake of n-3 PUFAs throughout gestation and lactation causes defects in brain myelination. Microglia are recognized for their critical contribution to neurodevelopmental processes, such as myelination. These cells invade the white matter in the first weeks of the post-natal period, where they participate in oligodendrocyte maturation and myelin production. Therefore, we investigated whether an alteration of white matter microglia accompanies the myelination deficits observed in the brain of n-3 PUFA-deficient animals. Macroscopic imaging analysis shows that maternal n-3 PUFA deficiency decreases the density of white matter microglia around post-natal day 10. Microscopic electron microscopy analyses also revealed alterations of microglial ultrastructure, a decrease in the number of contacts between microglia and myelin sheet, and a decreased amount of myelin debris in their cell body. White matter microglia further displayed increased mitochondrial abundance and network area under perinatal n-3 PUFA deficiency. Overall, our data suggest that maternal n-3 PUFA deficiency alters the structure and function of microglial cells located in the white matter of pups early in life, and this could be the key to understand myelination deficits during neurodevelopment.

The Immune System's Role in the Consequences of Mild Traumatic Brain Injury (Concussion).

Mild traumatic brain injury (mild TBI), often referred to as concussion, is the most common form of TBI and affects millions of people each year. A history of mild TBI increases the risk of developing emotional and neurocognitive disorders later in life that can impact on day to day living. These include anxiety and depression, as well as neurodegenerative conditions such as chronic traumatic encephalopathy (CTE) and Alzheimer's disease (AD). Actions of brain resident or peripherally recruited immune cells are proposed to be key regulators across these diseases and mood disorders. Here, we will assess the impact of mild TBI on brain and patient health, and evaluate the recent evidence for immune cell involvement in its pathogenesis.

B cells on the brain: meningeal IgA and a novel gut-brain firewall.

Fitzpatrick et al. describe how IgA secretion by B cells and plasma cells in the mengines is crucial for protection against microbial invasion into the brain and the CNS.

Benefits of physical exercise on cognition and glial white matter pathology in a mouse model of vascular cognitive impairment and dementia.

White matter (WM) pathology is a clinically predictive feature of vascular cognitive impairment and dementia (VCID). Mice overexpressing transforming growth factor-ß1 (TGF) with an underlying cerebrovascular pathology when fed a high cholesterol diet (HCD) develop cognitive deficits (VCID mice) that we recently found could be prevented by physical exercise (EX). Here, we further investigated cognitive and WM pathology in VCID mice and examined the cellular substrates of the protective effects of moderate aerobic EX focusing on WM alterations. Six groups were studied: Wild-type (WT) and TGF mice (n = 20-24/group) fed standard lab chow or a 2% HCD, with two HCD-fed groups given concurrent access to running wheels. HCD had a significant negative effect in TGF mice that was prevented by EX on working and object recognition memory, the latter also altered in WT HCD mice. Whisker-evoked increases in cerebral blood flow (CBF) were reduced in HCD-fed mice, deficits that were countered by EX, and baseline WM CBF was similarly affected. VCID mice displayed WM functional deficits characterized by lower compound action potential amplitude not found in EX groups. Moreover, there was an increased number of collapsing capillaries, galectin-3-expressing microglial cells, as well as a reduced number of oligodendrocytes in the WM of VCID mice; all of which were prevented by EX. Our findings indicate that a compromised cerebral circulation precedes reduced WM vascularization, enhanced WM inflammation and impaired oligodendrogenesis that all likely account for the increased susceptibility to memory impairments in VCID mice, which can be prevented by EX. MAIN POINTS: A compromised cerebral circulation increases susceptibility to anatomical and functional white matter changes that develop alongside cognitive deficits when challenged with a high cholesterol diet; preventable by a translational regimen of exercise.

Immune cell regulation of glia during CNS injury and disease.

Glial cells are abundant in the CNS and are essential for brain development and homeostasis. These cells also regulate tissue recovery after injury and their dysfunction is a possible contributing factor to neurodegenerative and psychiatric disease. Recent evidence suggests that microglia, which are also the brain's major resident immune cells, provide disease-modifying regulation of the other major glial populations, namely astrocytes and oligodendrocytes. In addition, peripheral immune cells entering the CNS after injury and in disease may directly affect microglial, astrocyte and oligodendrocyte function, suggesting an integrated network of immune cell-glial cell communication.

Peripherally derived macrophages modulate microglial function to reduce inflammation after CNS injury.

Infiltrating monocyte-derived macrophages (MDMs) and resident microglia dominate central nervous system (CNS) injury sites. Differential roles for these cell populations after injury are beginning to be uncovered. Here, we show evidence that MDMs and microglia directly communicate with one another and differentially modulate each other's functions. Importantly, microglia-mediated phagocytosis and inflammation are suppressed by infiltrating macrophages. In the context of spinal cord injury (SCI), preventing such communication increases microglial activation and worsens functional recovery. We suggest that macrophages entering the CNS provide a regulatory mechanism that controls acute and long-term microglia-mediated inflammation, which may drive damage in a variety of CNS conditions.

Myeloid cell responses after spinal cord injury.

The past decade has revealed much about the complexity of the local inflammatory response after spinal cord injury (SCI). A major challenge is to distinguish between microglia and monocyte-derived macrophages (MDMs) to determine their phenotype and function. Transcriptome studies have revealed microglia-selective genes but are still limited in scope because many markers are downregulated after injury. Additionally, new genetic reporter mice are available to study microglia and MDMs. There is more evidence now for the plasticity and heterogeneity of microglia and MDMs. We also discuss the role of neutrophils that are the first peripheral cells to enter the injured CNS.

Neutrophil infiltration to the brain is platelet-dependent, and is reversed by blockade of platelet GPIbα.

Neutrophils are key components of the innate immune response, providing host defence against infection and being recruited to non-microbial injury sites. Platelets act as a trigger for neutrophil extravasation to inflammatory sites but mechanisms and tissue-specific aspects of these interactions are currently unclear. Here, we use bacterial endotoxin in mice to trigger an innate inflammatory response in different tissues and measure neutrophil invasion with or without platelet reduction. We show that platelets are essential for neutrophil infiltration to the brain, peritoneum and skin. Neutrophil numbers do not rise above basal levels in the peritoneum and skin and are decreased (~60%) in the brain when platelet numbers are reduced. In contrast neutrophil infiltration in the lung is unaffected by platelet reduction, up-regulation of CXCL-1 (2·4-fold) and CCL5 (1·4-fold) acting as a compensatory mechanism in platelet-reduced mice during lung inflammation. In brain inflammation targeting platelet receptor GPIbα results in a significant decrease (44%) in platelet-mediated neutrophil invasion, while maintaining platelet numbers in the circulation. These results suggest that therapeutic blockade of platelet GPIbα could limit the harmful effects of excessive inflammation while minimizing haemorrhagic complications of platelet reduction in the brain. The data also demonstrate the ability to target damaging brain inflammation in stroke and related disorders without compromising lung immunity and hence risk of pneumonia, a major complication post stroke. In summary, our data reveal an important role for platelets in neutrophil infiltration to various tissues, including the brain, and so implicate platelets as a key, targetable component of cerebrovascular inflammatory disease or injury.

Microglia and macrophages differ in their inflammatory profile after permanent brain ischemia.

We studied the expression of pro- and anti-inflammatory molecules in microglia and infiltrating monocyte-derived macrophages after permanent Middle Cerebral Artery Occlusion (pMCAO). LysM-EGFP knock-in mice were used to distinguish between these two cell types, as peripheral myeloid cells are LysM-EGFP+, while microglia are not. This was confirmed with P2ry12 (a microglial specific marker), Iba-1 and EGFP immunostaining. The peak of LysM-EGFP+ myeloid cell infiltration was 72h post-ischemia, and were distributed evenly in the lesion core, surrounded by a dense region of microglia. Flow cytometry showed that a higher percentage of microglia expressed TNF-α at 3 (24.3% vs 1.4%) and 7 (18.8% vs 3.4%) days post-pMCAO as compared to infiltrating macrophages. Microglia and macrophages were purified by fluorescence activated cell sorting 72h post-ischemia to assess the mRNA expression of inflammatory markers. Macrophages upregulated expression of mRNA for arginase-1 (Arg-1) by 1000-fold, and IL-1ß by 90-fold as compared to microglia. At the protein level, a significantly number of macrophages expressed Arg-1, while few if any microglia expressed Arg-1. However, IL-1ß protein was not detected in macrophages by flow cytometry or immunofluorescence labeling of tissue sections. It was, however, detected in astrocytes along the lesion border. A PCR-array screen of 84 inflammatory genes revealed that pro-inflammatory chemokines and cytokines were predominantly upregulated in macrophages but down-regulated in microglia in the ischemic brain. Our results show clear differences in the inflammatory expression profiles between microglia and macrophages 72h post-ischemia which may shape repair and pro-regenerative mechanisms after stroke.

Novel Influences of IL-10 on CNS Inflammation Revealed by Integrated Analyses of Cytokine Networks and Microglial Morphology.

Coordinated interactions between cytokine signaling and morphological dynamics of microglial cells regulate neuroinflammation in CNS injury and disease. We found that pro-inflammatory cytokine gene expression in vivo showed a pronounced recovery following systemic LPS. We performed a novel multivariate analysis of microglial morphology and identified changes in specific morphological properties of microglia that matched the expression dynamics of pro-inflammatory cytokine TNFα. The adaptive recovery kinetics of TNFα expression and microglial soma size showed comparable profiles and dependence on anti-inflammatory cytokine IL-10 expression. The recovery of cytokine variations and microglial morphology responses to inflammation were negatively regulated by IL-10. Our novel morphological analysis of microglia is able to detect subtle changes and can be used widely. We implemented in silico simulations of cytokine network dynamics which showed-counter-intuitively, but in line with our experimental observations-that negative feedback from IL-10 was sufficient to impede the adaptive recovery of TNFα-mediated inflammation. Our integrative approach is a powerful tool to study changes in specific components of microglial morphology for insights into their functional states, in relation to cytokine network dynamics, during CNS injury and disease.

Docosahexaenoic acid-containing choline phospholipid modulates LPS-induced neuroinflammation in vivo and in microglia in vitro.

BACKGROUND: Neuroinflammatory processes are considered a double-edged sword, having both protective and detrimental effects in the brain. Microglia, the brain's resident innate immune cells, are a key component of neuroinflammatory response. There is a growing interest in developing drugs to target microglia and control neuroinflammatory processes. In this regard, docosahexaenoic acid (DHA), the brain's n-3 polyunsaturated fatty acid, is a promising molecule to regulate pro-inflammatory microglia and cytokine production. Several works reported that the bioavailability of DHA to the brain is higher when DHA is acylated to phospholipid. In this work, we analyzed the anti-inflammatory activity of DHA-phospholipid, either acetylated at the sn-1 position (AceDoPC, a stable form thought to have superior access to the brain) or acylated with palmitic acid at the sn-1 position (PC-DHA) using a lipopolysaccharide (LPS)-induced neuroinflammation model both in vitro and in vivo. METHODS: In vivo, adult C57Bl6/J mice were injected intravenously (i.v.) with either AceDoPC or PC-DHA 24 h prior to LPS (i.p.). For in vitro studies, immortalized murine microglia cells BV-2 were co-incubated with DHA forms and LPS. AceDoPC and PC-DHA effect on brain or BV-2 PUFA content was assessed by gas chromatography. LPS-induced pro-inflammatory cytokines interleukin IL-1ß, IL-6, and tumor necrosis factor (TNF) α production were measured by quantitative PCR (qPCR) or multiplex. IL-6 receptors and associated signaling pathway STAT3 were assessed by FACS analysis and western-blot in vitro. RESULTS: In vivo, a single injection of AceDoPC or PC-DHA decreased LPS-induced IL-6 production in the hippocampus of mice. This effect could be linked to their direct effect on microglia, as revealed in vitro. In addition, AceDoPC or PC-DHA reduced IL-6 receptor while only AceDoPC decreased IL-6-induced STAT3 phosphorylation. CONCLUSIONS: These results highlight the potency of administered DHA-acetylated to phospholipids-to rapidly regulate LPS-induced neuroinflammatory processes through their effect on microglia. In particular, both IL-6 production and signaling are targeted by AceDoPC in microglia.

Microglial TNF-α Suppresses Cocaine-Induced Plasticity and Behavioral Sensitization.

Repeated administration of cocaine results in the development of behavioral sensitization, accompanied by a decrease in excitatory synaptic strength in the nucleus accumbens (NAc) through an unknown mechanism. Furthermore, glial cells in the NAc are activated by drugs of abuse, but the contribution of glia to the development of addictive behaviors is unknown. Tumor necrosis factor alpha (TNF-α), an inflammatory cytokine released by activated glia, can drive the internalization of synaptic AMPA receptors on striatal medium spiny neurons. Here we show that repeated administration of cocaine activates striatal microglia and induces TNF-α production, which in turn depresses glutamatergic synaptic strength in the NAc core and limits the development of behavioral sensitization. Critically, following a period of abstinence, a weak TLR4 agonist can reactivate microglia, increase TNF-α production, depress striatal synaptic strength, and suppress cocaine-induced sensitization. Thus, cytokine signaling from microglia can regulate both the induction and expression of drug-induced behaviors.

Arginase-1 is expressed exclusively by infiltrating myeloid cells in CNS injury and disease.

Resident microglia and infiltrating myeloid cells play important roles in the onset, propagation, and resolution of inflammation in central nervous system (CNS) injury and disease. Identifying cell type-specific mechanisms will help to appropriately target interventions for tissue repair. Arginase-1 (Arg-1) is a well characterised modulator of tissue repair and its expression correlates with recovery after CNS injury. Here we assessed the cellular localisation of Arg-1 in two models of CNS damage. Using microglia specific antibodies, P2ry12 and Fc receptor-like S (FCRLS), we show the LysM-EGFP reporter mouse is an excellent model to distinguish infiltrating myeloid cells from resident microglia. We show that Arg-1 is expressed exclusively in infiltrating myeloid cells but not microglia in models of spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). Our in vitro studies suggest that factors in the CNS environment prevent expression of Arg-1 in microglia in vivo. This work suggests different functional roles for these cells in CNS injury and repair and shows that such repair pathways can be switched on in infiltrating myeloid cells in pro-inflammatory environments.

Dosimetric characteristics of brass mesh as bolus under megavoltage photon irradiation.

OBJECTIVE: This article presents a set of dosimetric measurements describing the properties of brass mesh (Whiting and Davis, Attleboro Falls, MA) under megavoltage photon irradiation conditions, with particular relevance to its use in breast radiotherapy. METHODS: The effectiveness of brass mesh as a bolus material was investigated using 6-, 15- and 6-MV flattening filter-free photon beams. The effect on dose build-up at the entrance surface, build-down at the beam-exit surface, dose with surface entrance obliquity, beam profiles, penumbra and percentage depth doses were investigated. RESULTS: One layer of the brass mesh produces a build-up effect equivalent to 1.1 mm of water at 6 MV and 1.9 mm at 15 MV. The brass generates a backscattered component of dose, if the photon beam exits through it. Percentage depth-dose curves are largely unaffected by the mesh and are shown to be equivalent to plain-field data. Beam penumbra and profiles are unchanged by the brass except within the first millimetre (mm) of phantom, where a periodic pattern of dose enhancement is seen. CONCLUSION: The data presented demonstrate that one layer of brass mesh provides a similar dose build-up effect equivalent to only a few millimetres of water. However, backscatter from the high atomic number (Z) mesh, at the beam exit, contributes appreciably to the overall dose surface enhancement. This dosimetric consequence cannot be neglected and indeed should be considered and accounted for, when determining the bolus effect of the brass mesh in the case of tangential breast irradiation. Advance in knowledge: This article provides dosimetric data necessary for the introduction of brass mesh bolus into the clinical setting for external-beam breast radiotherapy.

Computational modeling of cytokine signaling in microglia.

Neuroinflammation due to glial activation has been linked to many CNS diseases. We developed a computational model of a microglial cytokine interaction network to study the regulatory mechanisms of microglia-mediated neuroinflammation. We established a literature-based cytokine network, including TNFα, TGFß, and IL-10, and fitted a mathematical model to published data from LPS-treated microglia. The addition of a previously unreported TGFß autoregulation loop to our model was required to account for experimental data. Global sensitivity analysis revealed that TGFß- and IL-10-mediated inhibition of TNFα was critical for regulating network behavior. We assessed the sensitivity of the LPS-induced TNFα response profile to the initial TGFß and IL-10 levels. The analysis showed two relatively shifted TNFα response profiles within separate domains of initial condition space. Further analysis revealed that TNFα exhibited adaptation to sustained LPS stimulation. We simulated the effects of functionally inhibiting TGFß and IL-10 on TNFα adaptation. Our analysis showed that TGFß and IL-10 knockouts (TGFß KO and IL-10 KO) exert divergent effects on adaptation. TFGß KO attenuated TNFα adaptation whereas IL-10 KO enhanced TNFα adaptation. We experimentally tested the hypothesis that IL-10 KO enhances TNFα adaptation in murine macrophages and found supporting evidence. These opposing effects could be explained by differential kinetics of negative feedback. Inhibition of IL-10 reduced early negative feedback that results in enhanced TNFα-mediated TGFß expression. We propose that differential kinetics in parallel negative feedback loops constitute a novel mechanism underlying the complex and non-intuitive pro- versus anti-inflammatory effects of individual cytokine perturbations.

Requirement for interleukin-1 to drive brain inflammation reveals tissue-specific mechanisms of innate immunity.

The immune system is implicated in a wide range of disorders affecting the brain and is, therefore, an attractive target for therapy. Interleukin-1 (IL-1) is a potent regulator of the innate immune system important for host defense but is also associated with injury and disease in the brain. Here, we show that IL-1 is a key mediator driving an innate immune response to inflammatory challenge in the mouse brain but is dispensable in extracerebral tissues including the lung and peritoneum. We also demonstrate that IL-1α is an important ligand contributing to the CNS dependence on IL-1 and that IL-1 derived from the CNS compartment (most likely microglia) is the major source driving this effect. These data reveal previously unknown tissue-specific requirements for IL-1 in driving innate immunity and suggest that IL-1-mediated inflammation in the brain could be selectively targeted without compromising systemic innate immune responses that are important for resistance to infection. This property could be exploited to mitigate injury- and disease-associated inflammation in the brain without increasing susceptibility to systemic infection, an important complication in several neurological disorders.

TNF and increased intracellular iron alter macrophage polarization to a detrimental M1 phenotype in the injured spinal cord.

Macrophages and microglia can be polarized along a continuum toward a detrimental (M1) or a beneficial (M2) state in the injured CNS. Although phagocytosis of myelin in vitro promotes M2 polarization, macrophage/microglia in the injured spinal cord retain a predominantly M1 state that is detrimental to recovery. We have identified two factors that underlie this skewing toward M1 polarization in the injured CNS. We show that TNF prevents phagocytosis-mediated conversion from M1 to M2 cells in vitro and in vivo in spinal cord injury (SCI). Additionally, iron that accumulates in macrophages in SCI increases TNF expression and the appearance of a macrophage population with a proinflammatory mixed M1/M2 phenotype. In addition, transplantation experiments show that increased loading of M2 macrophages with iron induces a rapid switch from M2 to M1 phenotype. The combined effect of this favors predominant and prolonged M1 macrophage polarization that is detrimental to recovery after SCI.

Differences in the phagocytic response of microglia and peripheral macrophages after spinal cord injury and its effects on cell death.

Macrophages in the injured spinal cord arise from resident microglia and infiltrating, peripherally derived monocytes. It is still not clear if macrophages derived from these two populations differ in their roles after CNS injury. The aims of this study are to investigate the phagocytic response and clearance of damaged axons and tissue debris by these distinct subsets of macrophages and assess their viability after spinal cord injury (SCI). The lysozyme M EGFP-knockin mouse tags hematogenous macrophages, but not microglia. Using a combination of immunofluorescence, flow cytometry, and neuronal tracing techniques, we show that microglia contact damaged axons early (24 h) after SCI and are the main type of macrophage to contain phagocytic material at 3 d. Thereafter, infiltrating macrophages become the predominant cell in contact with degenerating axons and contain more phagocytic material, which in contrast to microglia, persists for up to 42 d. Furthermore, after phagocytosis of myelin in vitro, bone marrow-derived macrophages are much more susceptible to apoptotic and necrotic cell death than CNS microglia, which is mirrored in vivo with apoptotic TUNEL-positive cells of infiltrating macrophage origin. This work suggests that microglia play a major role in the early response to SCI, by phagocytosing damaged and degenerating tissue, processing phagocytic material efficiently, and remaining viable. Later, macrophages of peripheral origin contribute predominantly to phagocytosis but are less efficient at processing CNS debris, and their death, in situ, may contribute to the secondary damage after CNS injury.