Macrophages in Spinal Cord Injury: Phenotypic and Functional Change From Exposure to Myelin Debris

ArticleinGlia 63(4) · April 2015with 523 Reads 
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DOI: 10.1002/glia.22774 · Source: PubMed
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Abstract
Macrophage activation and persistent inflammation contribute to the pathological process of spinal cord injury (SCI). It was reported that M2 macrophages were induced at 3-7 days after SCI but M2 markers were reduced or eliminated after 1 week. By contrast, M1 macrophage response is rapidly induced and then maintained at injured spinal cord. However, factors that modulate macrophage phenotype and function are poorly understood. We developed a model to distinguish bone-marrow derived macrophages (BMDMs) from residential microglia and explored how BMDMs change their phenotype and functions in response to the lesion-related factors in injured spinal cord. Infiltrating BMDMs expressing higher Mac-2 and lower CX3CR1 migrate to the epicenter of injury, while microglia expressing lower Mac-2 but higher CX3CR1 distribute to the edges of lesion. Myelin debris at the lesion site switches BMDMs from M2 phenotype towards M1-like phenotype. Myelin debris activates ATP-binding cassette transporter A1 (ABCA1) for cholesterol efflux in response to myelin debris loading in vitro. However, this homeostatic mechanism in injured site is overwhelmed, leading to the development of foamy macrophages and lipid plaque in the lesion site. The persistence of these cells indicates a pro-inflammatory environment, associated with enhanced neurotoxicity and impaired wound healing. These foamy macrophages have poor capacity to phagocytose apoptotic neutrophils resulting in uningested neutrophils releasing their toxic contents and further tissue damage. In conclusion, these data demonstrate for the first time that myelin debris generated in injured spinal cord modulates macrophage activation. Lipid accumulation following macrophage phenotype switch contributes to SCI pathology. GLIA 2014. © 2014 Wiley Periodicals, Inc.

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  • Article
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    We report, for the first time, the detection and specific localization of long-chain acylcarnitines (LC ACs) along the lesion margins in an experimental model of spinal cord injury (SCI) using 3D mass spectrometry imaging (MSI). Acylcarnitines palmitoylcarnitine (AC(16:0)), palmitoleoylcarnitine (AC(16:1)), elaidic carnitine (AC(18:1)) and tetradecanoylcarnitine (AC(14:1)) were detected as early as 3 days post injury, and were present along the lesion margins 7 and 10 days after SCI induced by balloon compression technique in the rat. 3D MSI revealed the heterogeneous distribution of these lipids across the injured spinal cord, appearing well-defined at the lesion margins rostral to the lesion center, and becoming widespread and less confined to the margins at the region located caudally. The assigned acylcarnitines co-localize with resident microglia/macrophages detected along the lesion margins by immunofluorescence. Given the reported pro-inflammatory role of these acylcarnitines, their specific spatial localization along the lesion margin could hint at their potential pathophysiological roles in the progression of SCI.
  • Article
    Spinal cord injury (SCI) affects over 17,000 individuals in the United States per year, resulting in sudden motor, sensory and autonomic impairments below the level of injury. These deficits may be due at least in part to the loss of oligodendrocytes and demyelination of spared axons as it leads to slowed or blocked conduction through the lesion site. It has long been accepted that progenitor cells form new oligodendrocytes after SCI, resulting in the acute formation of new myelin on demyelinated axons. However, the chronicity of demyelination and the functional significance of remyelination remain contentious. Here we review work examining demyelination and remyelination after SCI as well as the current understanding of oligodendrocyte lineage cell responses to spinal trauma, including the surprisingly long‐lasting response of NG2+ oligodendrocyte progenitor cells (OPCs) to proliferate and differentiate into new myelinating oligodendrocytes for months after SCI. OPCs are highly sensitive to microenvironmental changes, and therefore respond to the ever‐changing post‐SCI milieu, including influx of blood, monocytes and neutrophils; activation of microglia and macrophages; changes in cytokines, chemokines and growth factors such as ciliary neurotrophic factor and fibroblast growth factor‐2; glutamate excitotoxicity; and axon degeneration and sprouting. We discuss how these changes relate to spontaneous oligodendrogenesis and remyelination, the evidence for and against demyelination being an important clinical problem and if remyelination contributes to motor recovery. Oligodendrocyte formation persists for months after spinal cord injury, resulting in the formation of new myelin on demyelinated axons, in spite of fluctuating intraspinal conditions over time. The chronicity and significance are discussed herein.
  • Article
    Microglia are activated after spinal cord injury (SCI), but their phagocytic mechanisms and link to neuroprotection remain incompletely characterized. Docosahexaenoic acid (DHA) has been shown to have significant neuroprotective effects after hemisection and compression SCI and can directly affect microglia in these injury models. In rodent contusion SCI, we demonstrate that DHA (500 nmol/kg) administered acutely post-injury confers neuroprotection and enhances locomotor recovery, and also exerts a complex modulation of the microglial response to injury. In rodents, at 7 days after SCI, the level of phagocytosed myelin within Iba1-positive or P2Y12-positive cells was significantly lower after DHA treatment, and this occurred in parallel with an increase in intracellular miR-124 expression. Furthermore, intraspinal administration of a miR-124 inhibitor significantly reduced the DHA-induced decrease in myelin phagocytosis in mice at 7 days post-SCI. In rat spinal primary microglia cultures, DHA reduced the phagocytic response to myelin, which was associated with an increase in miR-124, but not miR-155. A similar response was observed in a microglia cell line (BV2) treated with DHA, and the effect was blocked by a miR-124 inhibitor. Furthermore, the phagocytic response of BV2 cells to stressed neurones was also reduced in the presence of DHA. In peripheral monocyte-derived macrophages, the expression of the M1, but not the M0 or M2 phenotype, was reduced by DHA, but the phagocytic activation was not altered. These findings show that DHA induces neuroprotection in contusion injury. Furthermore, the improved outcome is via a miR-124-dependent reduction in the phagocytic response of microglia.
  • Article
    Unlabelled: Acute oligodendrocyte (OL) death after traumatic spinal cord injury (SCI) is followed by robust neuron-glial antigen 2 (NG2)-positive OL progenitor proliferation and differentiation into new OLs. Inflammatory mediators are prevalent during both phases and can influence the fate of NG2 cells and OLs. Specifically, toll-like receptor (TLR) 4 signaling induces OL genesis in the naive spinal cord, and lack of TLR4 signaling impairs white matter sparing and functional recovery after SCI. Therefore, we hypothesized that TLR4 signaling may regulate oligodendrogenesis after SCI. C3H/HeJ (TLR4-deficient) and control (C3H/HeOuJ) mice received a moderate midthoracic spinal contusion. TLR4-deficient mice showed worse functional recovery and reduced OL numbers compared with controls at 24 h after injury through chronic time points. Acute OL loss was accompanied by reduced ferritin expression, which is regulated by TLR4 and needed for effective iron storage. TLR4-deficient injured spinal cords also displayed features consistent with reduced OL genesis, including reduced NG2 expression, fewer BrdU-positive OLs, altered BMP4 signaling and inhibitor of differentiation 4 (ID4) expression, and delayed myelin phagocytosis. Expression of several factors, including IGF-1, FGF2, IL-1β, and PDGF-A, was altered in TLR4-deficient injured spinal cords compared with wild types. Together, these data show that TLR4 signaling after SCI is important for OL lineage cell sparing and replacement, as well as in regulating cytokine and growth factor expression. These results highlight new roles for TLR4 in endogenous SCI repair and emphasize that altering the function of a single immune-related receptor can dramatically change the reparative responses of multiple cellular constituents in the injured CNS milieu. Significance statement: Myelinating cells of the CNS [oligodendrocytes (OLs)] are killed for several weeks after traumatic spinal cord injury (SCI), but they are replaced by resident progenitor cells. How the concurrent inflammatory signaling affects this endogenous reparative response is unclear. Here, we provide evidence that immune receptor toll-like receptor 4 (TLR4) supports OL lineage cell sparing, long-term OL and OL progenitor replacement, and chronic functional recovery. We show that TLR4 signaling is essential for acute iron storage, regulating cytokine and growth factor expression, and efficient myelin debris clearance, all of which influence OL replacement. Importantly, the current study reveals that a single immune receptor is essential for repair responses after SCI, and the potential mechanisms of this beneficial effect likely change over time after injury.
  • Preprint
    Full-text available
    Traumatic spinal cord injury (SCI) elicits a robust intraspinal inflammatory reaction that is dominated by at least two major subpopulations of macrophages, i.e., those derived from resident microglia and another from monocytes that infiltrate the injury site from the circulation. Previously, we implicated monocyte-derived macrophages (MDMs) as effectors of acute post-injury pathology after SCI; however, it is still unclear whether microglia also contribute to lesion pathology. Assigning distinct functional roles to microglia and MDMs in vivo has been difficult because these CNS macrophage subsets are morphologically and phenotypically similar. Here, to characterize the role that microglia play in experimental models of thoracic spinal contusion or lumbar crush injury, mice were fed vehicle chow or chow laced with a CSF1R receptor antagonist, PLX5622. Feeding PLX5622 depletes microglia. In both groups, spontaneous recovery of hindlimb motor function was evaluated for up to 8 weeks post-SCI using open-field and horizontal ladder tests. Histopathological assessment of intraspinal pathology was assessed in 8 week post-injury tissues. In both SCI models, microglia depletion exacerbated lesion pathology and impaired spontaneous recovery of hind limb function. Notably, the loss of microglia prevented astroglial encapsulation of the lesion core, which was associated with larger lesions, enhanced demyelination and neuron loss and a larger inflammatory response that was dominated by monocyte-derived macrophages. The neuroprotective and healing properties of microglia become obvious in the subacute phases of recovery; microglia depletion up to 7 days post-injury (dpi) had no apparent effect on recovery while delayed depletion from 8-28dpi exacerbated lesion pathology and significantly impaired functional recovery. These data suggest that microglia have essential tissue repair functions after SCI. Selective enhancement of microglial activities may be a novel strategy to preserve tissue and promote recovery of function after neurotrauma. All rights reserved. No reuse allowed without permission. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
  • Article
    Full-text available
    Peripheral nerve injury causes neuropathic pain accompanied by remarkable microgliosis in the spinal cord dorsal horn. However, it is still debated whether infiltrated monocytes contribute to injury-induced expansion of the microglial population. Here, we found that spinal microgliosis predominantly results from local proliferation of resident microglia but not from infiltrating monocytes after spinal nerve transection (SNT) by using two genetic mouse models (CCR2RFP/+:CX3CR1GFP/+ and CX3CR1creER/+:R26tdTomato/+ mice) as well as specific staining of microglia and macrophages. Pharmacological inhibition of SNT-induced microglial proliferation correlated with attenuated neuropathic pain hypersensitivities. Microglial proliferation is partially controlled by purinergic and fractalkine signaling, as CX3CR1−/− and P2Y12−/− mice show reduced spinal microglial proliferation and neuropathic pain. These results suggest that local microglial proliferation is the sole source of spinal microgliosis, which represents a potential therapeutic target for neuropathic pain management.
  • Article
    Full-text available
    Spinal cord injury (SCI) is a devastating event that results in significant physical disabilities for affected individuals. Apart from local injury within the spinal cord, SCI patients develop a variety of complications characterized by multiple organ dysfunction or failure. These disorders, such as neurogenic pain, depression, lung injury, cardiovascular disease, liver damage, kidney dysfunction, urinary tract infection, and increased susceptibility to pathogen infection, are common in injured patients, hinder functional recovery, and can even be life threatening. Multiple lines of evidence point to pathological connections emanating from the injured spinal cord, post-injury systemic inflammation, and immune suppression as important multifactorial mechanisms underlying post-SCI complications. SCI triggers systemic inflammatory responses marked by increased circulation of immune cells and pro-inflammatory mediators, which result in the infiltration of inflammatory cells into secondary organs and persistence of an inflammatory microenvironment that contributes to organ dysfunction. SCI also induces immune deficiency through immune organ dysfunction, resulting in impaired responsiveness to pathogen infection. In this review, we summarize current evidence demonstrating the relevance of inflammatory conditions and immune suppression in several complications frequently seen following SCI. In addition, we highlight the potential pathways by which inflammatory and immune cues contribute to multiple organ failure and dysfunction and discuss current anti-inflammatory approaches used to alleviate post-SCI complications. A comprehensive review of this literature may provide new insights into therapeutic strategies against complications after SCI by targeting systemic inflammation.
  • Article
    Full-text available
    Spinal cord injury (SCI) causes functional impairment as a result of the initial injury followed by secondary injury mechanism. SCI provokes an inflammatory response that causes secondary tissue damage and neurodegeneration. While, the use of neural stem cell (NSC) engraftment to mitigate secondary injury has been of interest to many researchers, it still faces several limitations. As such, we investigated if NSC-conditioned media (NSC-M) possesses therapeutic potential for the treatment of SCI. It has been proposed that many of the beneficial effects attributed to stem cell therapies are due to secreted factors. Utilizing primary cell culture and murine models of SCI, we determined that systemic treatment with NSC-M was able to significantly improve motor function and lesion healing. In addition, NSC-M demonstrated significant anti-inflammatory potential in vitro and in vivo, reducing inflammatory cytokine expression in both activated macrophages and injured spinal cord tissues. NSC-M was also able to reduce the expression of inducible nitric oxide synthase (iNOS) within the spleen of injured animals, indicating an ability to reduce systemic inflammation. Thus, we believe that NSC-M offers a possible alternative to direct stem cell engraftment for the treatment of SCI.
  • Article
    Full-text available
    Background: Although myelin is composed of mostly lipids, the pathological role of myelin lipids in demyelinating diseases remains elusive. The principal lipid of the myelin sheath is β-galactosylceramide (β-Galcer). Its α-anomer (α-Galcer) has been demonstrated to be antigenically presented by macrophages via CD1d, a MHC class I-like molecule. Myelin, which is mostly composed of β-Galcer, has been long considered as an immunologically-inert neuron insulator, because the antigen-binding cleft of CD1d is highly α-form-restricted. Results: Here, we report that CD1d-mediated antigenic presentation of myelin-derived galactosylceramide (Mye-GalCer) by macrophages contributed significantly to the progression of experimental autoimmune encephalomyelitis (EAE). Surprisingly, this presentation was recognizable by α-Galcer:CD1d-specific antibody (clone L363), but incapable of triggering expansion of iNKT cells and production of iNKT signature cytokines (IFNγ and IL-4). Likewise, a synthesized analog of Mye-Galcer, fluorinated α-C-GalCer (AA2), while being efficiently presented via CD1d on macrophages, failed to stimulate production of IFNγ and IL-4. However, AA2 significantly exacerbated EAE progression. Further analyses revealed that the antigenic presentations of both Mye-GalCer and its analog (AA2) in α-form via CD1d promoted IL-17 production from T cells, leading to elevated levels of IL-17 in EAE spinal cords and sera. The IL-17 neutralizing antibody significantly reduced the severity of EAE symptoms in AA2-treated mice. Furthermore, D-sphingosine, a lipid possessing the same hydrophobic base as ceramide but without a carbohydrate residue, efficiently blocked this glycolipid antigen presentation both in vitro and in spinal cords of EAE mice, and significantly decreased IL-17 and ameliorated the pathological symptoms. Conclusion: Our findings reveal a novel pathway from the presentation of Mye-GalCer to IL-17 production, and highlight the promising therapeutic potential of D-sphingosine for the human disorder of multiple sclerosis.
  • Article
    In this study, we presented a facile functionalization of carbon nanotubes (CNTs) via dopamine self-polymerization on the outer surface of multi-walled CNTs (MWCNTs). A CNT nanocomposite was constructed through layer-by-layer assembly of polydopamine (PDA)-functionalized MWCNTs (CNT@PDA) and poly-l-lysine (PLL). Measurements by transmission electron microscopy and X-ray photoelectron spectroscopy confirmed PDA deposition on MWCNTs, and assessments by UV–vis spectroscopy indicated the sequential deposition of PLL and CNT@PDA in a linear growth of (PLL-CNT@PDA) films. We intensively investigated the activation profile and corresponding cellular events of macrophages upon their interfacing with CNT@PDA and the (PLL-CNT@PDA) film, including cell viability, intracellular reactive oxygen species production, phagocytic capacity, inflammatory responses and gene expressions. The results suggested that PDA functionalization significantly decreased inflammatory responses of MWCNTs in vitro when compared with the widely-applied carboxylic acid functionalization (COOH). Interestingly, RNAseq transcriptome analysis revealed that, unlike the control (PLL-CNT-COOH) film, the (PLL-CNT@PDA) film exerted a minor impact on gene expressions in macrophages, affecting mainly those associated with cell cycle. Moreover, no foreign-body response was found in mice bearing (PLL-CNT@PDA) film transplantation, confirming its in vivo biocompatibility. Collectively, our proposed method of PDA functionalization and particularly fabrication of CNT@PDA multilayered nanofilms may have numerous translational applications in biomedicine.
  • Article
    Full-text available
    Zhijian Cheng, Xijing He Department of Orthopedics, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China Abstract: Spinal cord injury (SCI) is a traumatic event that involves not just an acute physical injury but also inflammation-driven secondary injury. Macrophages play a very important role in secondary injury. The effects of macrophages on tissue damage and repair after SCI are related to macrophage polarization. Stem cell transplantation has been studied as a promising treatment for SCI. Recently, increasing evidence shows that stem cells, including mesenchymal stem, neural stem/progenitor, and embryonic stem cells, have an anti-inflammatory capacity and promote functional recovery after SCI by inducing macrophages M1/M2 phenotype transformation. In this review, we will discuss the role of stem cells on macrophage polarization and its role in stem cell-based therapies for SCI. Keywords: stem cells, macrophages, spinal cord injury, polarization
  • Article
    Full-text available
    Significance statement: The intrinsic molecular mechanisms that regulate macrophage function after spinal cord injury are poorly understood. We obtained macrophage-specific mRNA directly from the injured spinal cord, and performed RNA sequencing to investigate their transcriptional profile. Our data show that at 7 days after SCI, macrophages are best described as foam cells, with lipid catabolism representing the main biological process, and canonical nuclear receptor pathways as their potential mediators. Genetic deletion of a lipoprotein receptor, CD36, reduces macrophage lipid content and improves lesion size and locomotor recovery. Therefore, we report the first macrophage-specific transcriptional profile after SCI and highlight the lipid catabolic pathway as an important macrophage function that can be therapeutically targeted after SCI.
  • Article
    Oligodendrocyte progenitor cells (OPCs) are present throughout the adult brain and spinal cord and can replace oligodendrocytes lost to injury, aging, or disease. Their differentiation, however, is inhibited by myelin debris, making clearance of this debris an important step for cellular repair following demyelination. In models of peripheral nerve injury, TLR4 activation by lipopolysaccharide (LPS) promotes macrophage phagocytosis of debris. Here we tested whether the novel synthetic TLR4 agonist E6020, a Lipid A mimetic, promotes myelin debris clearance and remyelination in spinal cord white matter following lysolecithin-induced demyelination. In vitro, E6020 induced TLR4-dependent cytokine expression (TNFα, IL1β, IL-6) and NF-κB signaling, albeit at ∼10-fold reduced potency compared to LPS. Microinjection of E6020 into the intact rat spinal cord gray/white matter border induced macrophage activation, OPC proliferation, and robust oligodendrogenesis, similar to what we described previously using an intraspinal LPS microinjection model. Finally, a single co-injection of E6020 with lysolecithin into spinal cord white matter increased axon sparing, accelerated myelin debris clearance, enhanced Schwann cell infiltration into demyelinated lesions, and increased the number of remyelinated axons. In vitro assays confirmed that direct stimulation of macrophages by E6020 stimulates myelin phagocytosis. These data implicate TLR4 signaling in promoting repair after CNS demyelination, likely by stimulating phagocytic activity of macrophages, sparing axons, recruiting myelinating cells, and promoting remyelination. This work furthers our understanding of immune-myelin interactions and identifies a novel synthetic TLR4 agonist as a potential therapeutic avenue for white matter demyelinating conditions such as spinal cord injury and multiple sclerosis.
  • Article
    Full-text available
    Impaired signaling via CX3CR1, the fractalkine receptor, promotes recovery after traumatic spinal contusion injury in mice, a benefit achieved in part by reducing macrophage-mediated injury at the lesion epicenter. Here, we tested the hypothesis that CX3CR1-dependent changes in microglia and macrophage functions also will enhance neuroplasticity, at and several segments below the injury epicenter. New data show that in the presence of inflammatory stimuli, CX3CR1-deficient (CX3CR1-/-) microglia and macrophages adopt a reparative phenotype and increase expression of genes that encode neurotrophic and gliogenic proteins. At the lesion epicenter (midthoracic spinal cord), the microenvironment created by CX3CR1-/- microglia/macrophages enhances NG2 cell responses, axon sparing, and sprouting of serotonergic axons. In lumbar spinal cord, inflammatory signaling is reduced in CX3CR1-/- microglia. This is associated with reduced dendritic pathology and improved axonal and synaptic plasticity on ventral horn motor neurons. Together, these data indicate that CX3CR1, a microglia-specific chemokine receptor, is a novel therapeutic target for enhancing neuroplasticity and recovery after SCI. Interventions that specifically target CX3CR1 could reduce the adverse effects of inflammation and augment activity-dependent plasticity and restoration of function. Indeed, limiting CX3CR1-dependent signaling could improve rehabilitation and spinal learning.
  • Chapter
    Traumatic spinal cord injury (SCI) is a drama in two acts. The first part represents the trauma itself, causing the destruction of neural tissue, i.e., the elimination of neuronal and glial cells at the primary lesion site, as well as the transection of axons transiting through the lesioned area. Additionally, damage to the vascular system will provoke hemorrhage and the disruption of the blood–spinal cord barrier. Together, these damages will induce secondary cascades responsible for cell death, enlargement of lesioned area, and further loss of neurological functions. Edema will develop in the early ischemic period triggering a phase of glutamate excitotoxicity and ionic imbalance. The ensuing mitochondrial failure is thereafter responsible for an energy depletion and oxidative stress. The rapid inflammatory response to spinal cord injury is provided by the resident microglia, but foremost by the infiltrating neutrophils and macrophages. At the end of the acute phase, the lesioned area will get enclosed and stabilized by a fibroglial scar. This chapter reviews the sequence of pathophysiological processes occurring after traumatic spinal cord injury, which constitute targets for potential protective or regenerative interventions.
  • Article
    Spinal cord injury (SCI) triggers chronic intraspinal inflammation consisting of activated resident and infiltrating immune cells (especially microglia/macrophages). The environmental factors contributing to this protracted inflammation are not well understood; however, myelin lipid debris is a hallmark of SCI. Myelin is also a potent macrophage stimulus and target of complement-mediated clearance and inflammation. The downstream effects of these neuroimmune interactions have the potential to contribute to ongoing pathology or facilitate repair. This depends in large part on whether myelin drives pathological or reparative macrophage activation states, commonly referred to as M1 (proinflammatory) or M2 (alternatively) macrophages, respectively. Here we review the processes by which myelin debris may be cleared through macrophage surface receptors and the complement system, how this differentially influences macrophage and microglial activation states, and how the cellular functions of these myelin macrophages and complement proteins contribute to chronic inflammation and secondary injury after SCI.
  • Article
    Individuals that suffer injury to the spinal cord can result in long-term, debilitating sequelae. Spinal cord injured patients have increased risk for the development of metabolic disease which can further hinder the effectiveness of treatments to rehabilitate the cord and improve quality of life. In the present study, we sought to understand the impact of high-fat diet induced obesity on spinal cord injury (SCI) by examining transcriptome changes in the area of the injury and rostral and caudal to site of damage 12 weeks after injury. Adult, male Long Evans rats received either thoracic level contusion of the spinal cord or sham laminectomy and then were allowed to recover on normal rat chow for 4 weeks and further on HFD for an additional 8 weeks. Spinal cord tissues harvested from the rats were processed for Affymetrix microarray and further transcriptomic analysis. Diverse changes in gene expression were identified in the injured cord in genes such as MMP12, APOC4, GPNMB and IGF1 and 2. The greatest signaling changes occurred in pathways involved in cholesterol biosynthesis and immune cell trafficking. Taken together, the cord changes in the chronically obese rat following thoracic spinal cord injury (SCI) injury reveal further potential targets for therapy. These could be further explored as they overlap with genes involved in metabolic disease.
  • Thesis
    Macrophages play an important role during the neuroinflammatory response of spinal cord injury (SCI). The dominant M1 macrophages induce axonal retraction while the transient M2 macrophages induce regeneration. Histone deacetylase 3 (HDAC3) influences the macrophage polarization; inhibition of HDAC3 would shift the polarization towards the M2 phenotype. Therefore, the hypothesis of this study is that inhibition of HDAC3, as a central regulator of macrophage polarization, will improve functional recovery in a mouse model of SCI. In vitro analysis of several M1 and M2 markers reveals that HDAC3 specific inhibitor RGFP966 boosts the Arg1-correlated M2 phenotype. RGFP966 does not affect the LPS-induced gene expression of M1 markers. LPS- and IL-4-primed macrophages phagocytose spinal cord debris in vitro and become foamy as a result. Administration of RGFP966 reduces the formation of foamy macrophages in vitro regardless of their activation state. These in vitro results strengthen our hypothesis. However, RGFP966 does not improve functional recovery in the mouse model of SCI nor does it modulate the macrophage polarization on acute or on chronic term in vivo. This is possibly due to the complex pathophysiology which might overcome the potential effect of RGFP966.
  • Article
    The nature of traumatic spinal cord injury (SCI) often involves limited recovery and long-term quality of life complications. The initial injury sets off a variety of secondary cascades, which result in an expanded lesion area. Ultimately, the native tissue fails to regenerate. As treatments are developed in the laboratory, the management of this secondary cascade is an important first step in achieving recovery of normal function. Current literature identifies four broad targets for intervention: inflammation, oxidative stress, disruption of the blood-spinal cord barrier, and formation of an inhibitory glial scar. Because of the complexity and interconnected nature of these events, strategies that combine multiple therapies together show much promise. Specifically, approaches that rely on biomaterials to perform a variety of functions are generating intense research interest. In this review, we examine each target and discuss how biomaterials are currently used to address them. Overall, we show that there are an impressive amount of biomaterials and combinatorial treatments which show good promise for slowing secondary events and improving outcomes. If more emphasis is placed on growing our understanding of how materials can manage secondary events, treatments for SCI can be designed in an increasingly rational manner, ultimately improving their potential for translation to the clinic.
  • Article
    In humans, vitamin B12 deficiency causes peripheral and CNS manifestations. Loss of myelin in the peripheral nerves and the spinal cord (SC) contributes to peripheral neuropathy and motor deficits. The metabolic basis for the demyelination and brain disorder is unknown. The transcobalamin receptor-knockout mouse ( Cd320-/-) develops cobalamin (Cbl) deficiency in the nervous system, with mild anemia. A decreased S-adenosylmethionine: S-adenosylhomocysteine ratio and increased methionine were seen in the brain with no significant changes in neurotransmitter metabolites. The structural pathology in the SC presented as loss of myelin in the axonal tracts with inflammation. The sciatic nerve (SN) showed increased nonuniform, internodal segments suggesting demyelination, and remyelination in progress. Consistent with these changes, the Cd320-/- mouse showed an increased latency to thermal nociception. Further, lower amplitude of compound action potential in the SN suggested that the functional capacity of the heavily myelinated axons were preferentially compromised, leading to loss of peripheral sensation. Although the metabolic basis for the demyelination and the structural and functional alterations of the nervous system in Cbl deficiency remain unresolved, the Cd320-/- mouse provides a unique model to investigate the pathologic consequences of vitamin B12 deficiency. -Arora, K., Sequeira, J. M., Alarcon, J. M., Wasek, B., Arning, E., Bottiglieri, T., Quadros, E. V. Neuropathology of vitamin B12 deficiency in the Cd320-/- mouse.
  • Article
    Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.
  • Preprint
    Full-text available
    Traumatic spinal cord injury (SCI) elicits a robust intraspinal inflammatory reaction that is dominated by at least two major subpopulations of macrophages, i.e., those derived from resident microglia and another from monocytes that infiltrate the injury site from the circulation. Previously, we implicated monocyte-derived macrophages (MDMs) as effectors of acute post-injury pathology after SCI; however, it is still unclear whether microglia also contribute to lesion pathology. Assigning distinct functional roles to microglia and MDMs in vivo has been difficult because these CNS macrophage subsets are morphologically and phenotypically similar. Here, to characterize the role that microglia play in experimental models of thoracic spinal contusion or lumbar crush injury, mice were fed vehicle chow or chow laced with a CSF1R receptor antagonist, PLX5622. Feeding PLX5622 depletes microglia. In both groups, spontaneous recovery of hindlimb motor function was evaluated for up to 8 weeks post-SCI using open-field and horizontal ladder tests. Histopathological assessment of intraspinal pathology was assessed in 8 week post-injury tissues. In both SCI models, microglia depletion exacerbated lesion pathology and impaired spontaneous recovery of hind limb function. Notably, the loss of microglia prevented astroglial encapsulation of the lesion core, which was associated with larger lesions, enhanced demyelination and neuron loss and a larger inflammatory response that was dominated by monocyte-derived macrophages. The neuroprotective and healing properties of microglia become obvious in the subacute phases of recovery; microglia depletion up to 7 days post-injury (dpi) had no apparent effect on recovery while delayed depletion from 8-28dpi exacerbated lesion pathology and significantly impaired functional recovery. These data suggest that microglia have essential tissue repair functions after SCI. Selective enhancement of microglial activities may be a novel strategy to preserve tissue and promote recovery of function after neurotrauma.
  • Article
    Full-text available
    Spinal cord injury (SCI) is a devastating type of central nervous system (CNS) trauma with limited therapeutic treatments. The polarization of microglia into the M1 or M2 state has been documented to play important roles in the pathogenesis of SCI, although the complete repertoire of underlying factors has not been identified. Interestingly, the time point at which hematomyelia (intramedullary spinal cord hemorrhage) is alleviated coincides with a decrease in the number of M2 microglia. Here the function of Hemopexin (Hpx), a hematogenous glycoprotein, was examined in the crush model of SCI. Hpx levels were elevated at the lesion site during hematomyelia and were synchronously correlated with the level of the M2 marker Arginase-1 (Arg-1). Ablation of Hpx in vivo affected the polarization state of lipopolysaccharide (LPS)-stimulated microglia, as mirrored by a lower percentage of M2 microglia and a higher percentage of M1 microglia in the lesion site, which delayed the recovery and exacerbated the behavioral dysfunction after SCI. However, Hpx induced a rapid switch from the M1 to M2 phenotype in LPS-stimulated primary cultured microglia in a heme scavenging-independent manner. The supernant of Hpx-treated microglia ameliorated neuronal degeneration, alleviated demyelination, and promoted oligodendrocyte precursor cell (OPC) maturation. This modulatory effect of Hpx on microglia polarization was at least partially mediated by the LRP-1 receptor. Based on these results, Hpx is considered a novel modulator of the polarization of microglia during the pathogenesis of SCI and may play a crucial role in the recovery from SCI.
  • Article
    Background and purpose: Interleukin-19 (IL-19) skews the immune response towards a Th2 type and appears to stimulate angiogenesis. In the current study, we tested if IL-19 treatment could reduce secondary injury and improve functional recovery after contusion spinal cord injury (SCI). Experimental approach: Firstly, mice were given a moderate-severe thoracic SCI at the T9-10level and expression of IL-19 and its receptor was measured in the injured spinal cord. Then, SCI mice were treated with mouse recombinant IL-19 and its blocking antibody to investigate the therapeutic effect of IL-19. Key results: Protein expression of IL-19 and its receptor IL-20R1 and IL-20R2 was upregulated in the injured spinal cord of mice. IL-19 treatment significantly promoted the recovery of locomotor function dose-dependently, and reduced loss of motor neurons and microglial and glial activation following SCI. Treatment of SCI mice with IL-19 attenuated macrophage accumulation, reduced protein levels of TNF-α and MCP-1, and promoted Th2 response and M2 macrophage activation in the injured region. Treatment of SCI mice with IL-19 promoted angiogenesis through upregulating VEGF in the injured region. Treatment of SCI mice with IL-19 upregulated HO-1 expression and abated oxidative stress in the injured region. The beneficial effect of IL-19 was abolished by coadministration of the blocking antibody. Additionally, IL-19 deficiency in mice delayed the recovery of locomotor function following SCI. Conclusions and implications: IL-19 treatment reduced secondary injuries and improved locomotor functional recovery after contusion SCI, with diverse mechanisms including immune cell polarization, angiogenesis and anti-oxidative response.
  • Article
    Widespread traumatic axonal injury (TAI) results in brain network dysfunction which commonly leads to persisting cognitive and behavioral impairments following traumatic brain injury (TBI). TBI induces a complex neuroinflammatory response, frequently located at sites of axonal pathology. The role of the pro-inflammatory cytokine interleukin-1β (IL-1β) has not been established in TAI. An IL-1β-neutralizing or a control antibody was administered intraperitoneally at 30 min following central fluid percussion injury (cFPI), a mouse model of widespread TAI. Animals subjected to moderate cFPI (n=41) were compared to sham-injured controls (n=20) and untreated, naive animals (n=9). The anti-IL-1β antibody reached the target brain regions in adequate therapeutic concentrations (up to ~30μg /g brain tissue) at 24h post-injury in both cFPI-injured (n=5) and sham-injured animals (n=3) with lower concentrations at 72 h post-injury, (up to ~18μg /g brain tissue in three cFPI-injured mice). Functional outcome was analyzed using the multivariate concentric square field (MCSF) test at 2 and 9 days post-injury and the Morris water maze (MWM) at 14-21 days post-injury. Following TAI, the IL-1β-neutralizing antibody resulted in an improved behavioral outcome, including normalized behavioral profiles in the MCSF test. The performance in the MWM probe (memory) trial was improved, although not in the learning trials. The IL1β neutralizing treatment did not influence cerebral ventricle size or the number of microglia/macrophages. These findings support the hypothesis that IL-1β is an important contributor to the processes causing complex cognitive and behavioral disturbances following TAI. This article is protected by copyright. All rights reserved.
  • Article
    Macrophages display remarkable plasticity and can change their physiology in response to environmental cues. These changes can give rise to different populations of cells with distinct functions. In this Review we suggest a new grouping of macrophage populations based on three different homeostatic activities - host defence, wound healing and immune regulation. We propose that similarly to primary colours, these three basic macrophage populations can blend into various other 'shades' of activation. We characterize each population and provide examples of macrophages from specific disease states that have the characteristics of one or more of these populations.
  • Article
    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.
  • Article
    Oligodendrocyte precursor cells (OPCs) originate from restricted regions of the brain and migrate into the developing white matter, where they differentiate into oligodendrocytes and myelinate axons in the central nervous system (CNS). The molecular mechanisms that orchestrate these long distance trips of OPCs to populate throughout the CNS are poorly understood. Emerging evidence has argued the expression of N-methyl-d-aspartic acid (NMDA) receptors (NMDARs) in oligodendrocyte lineage cells in vivo, but their physiological function remains elusive. We have previously demonstrated the expression and function of NMDARs in OPC differentiation and myelination/remyelination. Here, we show that NMDARs stimulation promotes OPC migration both by chemotaxis and chemokinesis as demonstrated by various cell migration systems including Boyden transwell, single cell, matrix-gel cell mass, and SVZ tissue explants assays. The pro-migration effect of NMDAR can be abolished by either pharmacological inhibition or shRNA knock down of the T lymphoma invasion and metastasis 1 (Tiam1), a Rac1 guanine nucleotide exchange factor (Rac1-GEF) which is coexpressed and interacts with NMDAR in OPCs. Moreover, NMDAR stimulation evokes cascade activation of the Tiam1/Rac1/ERK signaling pathway which mediates its effect on OPC migration. We also show that glutamate released from cultured cortical neuron promotes OPCs migration via NMDAR, and that antagonism of NMDAR or inhibition of Tiam1 blocks the endogenous glutamate-induced OPCs migration from SVZ to cortical plate in the embryonic brain slice culture. Thus, our result suggests a critical role of NMDAR in regulation of OPCs migration during CNS development by coupling to and activating the Tiam1/Rac1 pathway. GLIA 2013.
  • Article
    Full-text available
    The seven-transmembrane receptor CX3CR1 is a specific receptor for the novel CX3C chemokine fractalkine (FKN) (neurotactin). In vitro data suggest that membrane anchoring of FKN, and the existence of a shed, soluble FKN isoform allow for both adhesive and chemoattractive properties. Expression on activated endothelium and neurons defines FKN as a potential target for therapeutic intervention in inflammatory conditions, particularly central nervous system diseases. To investigate the physiological function of CX3CR1-FKN interactions, we generated a mouse strain in which the CX3CR1 gene was replaced by a green fluorescent protein (GFP) reporter gene. In addition to the creation of a mutant CX3CR1 locus, this approach enabled us to assign murine CX3CR1 expression to monocytes, subsets of NK and dendritic cells, and the brain microglia. Analysis of CX3CR1-deficient mice indicates that CX3CR1 is the only murine FKN receptor. Yet, defying anticipated FKN functions, absence of CX3CR1 interferes neither with monocyte extravasation in a peritonitis model nor with DC migration and differentiation in response to microbial antigens or contact sensitizers. Furthermore, a prominent response of CX3CR1-deficient microglia to peripheral nerve injury indicates unimpaired neuronal-glial cross talk in the absence of CX3CR1.
  • Article
    Full-text available
    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.
  • Article
    Full-text available
    Although stem cell therapy holds promise as a potential treatment in a number of diseases, the tumorigenicity of embryonic stem cells (ESC) and induced pluripotent stem cells remains a major obstacle. In vitro predifferentiation of ESCs can help prevent the risk of teratoma formation, yet proliferating neural progenitors can generate tumors, especially in the presence of immunosuppressive therapy. In this study, we investigated the effects of the microenvironment on stem cell growth and teratoma development using undifferentiated ESCs. Syngeneic ESC transplantation triggered an inflammatory response that involved the recruitment of bone marrow (BM)-derived macrophages. These macrophages differentiated into an M2 or angiogenic phenotype that expressed multiple angiogenic growth factors and proteinases, such as macrophage migration inhibitory factor (MIF), VEGF, and matrix metalloproteinase 9, creating a microenvironment that supported the initiation of teratoma development. Genetic deletion of MIF from the host but not from ESCs specifically reduced angiogenesis and teratoma growth, and MIF inhibition effectively reduced teratoma development after ESC transplantation. Together, our findings show that syngeneic ESC transplantation provokes an inflammatory response that involves the rapid recruitment and activation of BM-derived macrophages, which may be a crucial driving force in the initiation and progression of teratomas.
  • Article
    Full-text available
    Microglial cells are difficult to track during development because of the lack of specific reagents for myeloid subpopulations. To further understand how myeloid lineages differentiate during development to create microglial cells, we investigated CX3CR1 and CCR2 transcription unit activation in Cx3cr1(+/GFP)CCR2(+/RFP) knockin fluorescent protein reporter mice. The principal findings include: 1) CX3CR1(+) cells localized to the aorta-gonad-mesonephros region, and visualized at embryonic day (E)9.0 in the yolk sac and neuroectoderm; 2) at E10.5, CX3CR1 single-positive microglial cells were visualized penetrating the neuroepithelium; and 3) CX3CR1 and CCR2 distinguished infiltrating macrophages from resident surveillant or activated microglia within tissue sections and by flow cytometric analyses. Our results support the contribution of the yolk sac as a source of microglial precursors. We provide a novel model to monitor chemokine receptor expression changes in microglia and myeloid cells early (E8.0-E10.5) in development and during inflammatory conditions, which have been challenging to visualize in mammalian tissues.
  • Article
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    Plexins are cell surface receptors widely studied in the nervous system, where they mediate migration and morphogenesis though the Rho family of small GTPases. More recently, plexins have been implicated in immune processes including cell-cell interaction, immune activation, migration, and cytokine production. Plexin-B2 facilitates ligand induced cell guidance and migration in the nervous system, and induces cytoskeletal changes in overexpression assays through RhoGTPase. The function of Plexin-B2 in the immune system is unknown. This report shows that Plexin-B2 is highly expressed on cells of the innate immune system in the mouse, including macrophages, conventional dendritic cells, and plasmacytoid dendritic cells. However, Plexin-B2 does not appear to regulate the production of proinflammatory cytokines, phagocytosis of a variety of targets, or directional migration towards chemoattractants or extracellular matrix in mouse macrophages. Instead, Plxnb2(-/-) macrophages have greater cellular motility than wild type in the unstimulated state that is accompanied by more active, GTP-bound Rac and Cdc42. Additionally, Plxnb2(-/-) macrophages demonstrate faster in vitro wound closure activity. Studies have shown that a closely related family member, Plexin-B1, binds to active Rac and sequesters it from downstream signaling. The interaction of Plexin-B2 with Rac has only been previously confirmed in yeast and bacterial overexpression assays. The data presented here show that Plexin-B2 functions in mouse macrophages as a negative regulator of the GTPases Rac and Cdc42 and as a negative regulator of basal cell motility and wound healing.
  • Article
    The phenotype of macrophages in atherosclerotic lesions can vary dramatically, from a large lipid laden foam cell to a small inflammatory cell. Classically, the concept of macrophage heterogeneity discriminates between two extremes called either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. Polarisation of plaque macrophages is predominantly determined by the local micro-environment present in the atherosclerotic lesion and is rather more complex than typically described by the M1/M2 paradigm. In this review we will discuss the role of various polarising factors in regulating the phenotypical state of plaque macrophages. We will focus on two main levels of phenotype regulation, one determined by differentiation factors produced in the lesion and the other determined by T-cell-derived polarising cytokines. With foam cell formation being a key characteristic of macrophages during atherosclerosis initiation and progression, these polarisation factors will also be linked to lipid handling of macrophages.
  • Article
    The nuclear receptors known as PPARs and LXRs are lipid-activated transcription factors that have emerged as key regulators of lipid metabolism and inflammation. PPARs and LXRs are activated by non-esterified fatty acids and cholesterol metabolites, respectively, and both exert positive and negative control over the expression of a range of metabolic and inflammatory genes. The ability of these nuclear receptors to integrate metabolic and inflammatory signalling makes them attractive targets for intervention in human metabolic diseases, such as atherosclerosis and type 2 diabetes, as well as for the modulation of inflammation and immune responses.
  • Article
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    Macrophages from the peripheral circulation and those derived from resident microglia are among the main effector cells of the inflammatory response that follows spinal cord trauma. There has been considerable debate in the field as to whether the inflammatory response is good or bad for tissue protection and repair. Recent studies on macrophage polarization in non-neural tissues have shed much light on their changing functional states. In the context of this literature, we discuss the activation of macrophages and microglia following spinal cord injury, and their effects on repair. Harnessing their anti-inflammatory properties could pave the way for new therapeutic strategies for spinal cord trauma.
  • Article
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    Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck & Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.
  • Article
    Oligodendrocytes (OLs) are particularly susceptible to the toxicity of the acute lesion environment after spinal cord injury (SCI). They undergo both necrosis and apoptosis acutely, with apoptosis continuing at chronic time points. Loss of OLs causes demyelination and impairs axon function and survival. In parallel, a rapid and protracted OL progenitor cell proliferative response occurs, especially at the lesion borders. Proliferating and migrating OL progenitor cells differentiate into myelinating OLs, which remyelinate demyelinated axons starting at 2 weeks post-injury. The progression of OL lineage cells into mature OLs in the adult after injury recapitulates development to some degree, owing to the plethora of factors within the injury milieu. Although robust, this endogenous oligogenic response is insufficient against OL loss and demyelination. First, in this review we analyze the major spatial-temporal mechanisms of OL loss, replacement, and myelination, with the purpose of highlighting potential areas of intervention after SCI. We then discuss studies on OL protection and replacement. Growth factors have been used both to boost the endogenous progenitor response, and in conjunction with progenitor transplantation to facilitate survival and OL fate. Considerable progress has been made with embryonic stem cell-derived cells and adult neural progenitor cells. For therapies targeting oligogenesis to be successful, endogenous responses and the effects of the acute and chronic lesion environment on OL lineage cells must be understood in detail, and in relation, the optimal therapeutic window for such strategies must also be determined.
  • Article
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    Monocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track in vivo, partly due to lack of CCR2 reagents. We created CCR2-red fluorescent protein (RFP) knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6C(hi)/CCR2(hi) monocytes. Surprisingly, neutrophils, not Ly6C(lo) monocytes, largely replaced Ly6C(hi) cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia. These results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations.
  • Article
    Macrophages play an important role in demyelination in multiple sclerosis (MS). Activated macrophages ingest myelin particles, thereby acquiring a foamy appearance. Foamy macrophages in MS lesions were described as being anti-inflammatory. Therefore, these cells might play a role in modulating the inflammatory state of an active lesion. Here, we investigated the mechanism by which myelin uptake leads to skewing of macrophages toward an anti-inflammatory phenotype. Macrophages were incubated with myelin, leading to the development of foamy macrophages. Afterwards, the cells were stimulated with the TLR-4 ligand lipopolysaccharide (LPS), and cytokine production was determined. Interestingly, foamy macrophages appeared to have a reduced cytokine secretion and were LPS insensitive only when generated with one of the myelin preparations. The factor responsible for the different outcomes between different myelin batches turned out to be LPS. We demonstrated that LPS contamination induced insensitivity to LPS in foamy macrophages. On the contrary, foamy macrophages generated in the presence of LPS-free myelin were able to secrete cytokines upon activation. To conclude, myelin-laden macrophages were not LPS insensitive, indicating that they had not acquired an anti-inflammatory phenotype.
  • Article
    The shift between pro-inflammatory (M1) and anti-inflammatory (M2) states of macrophage polarization allows the resolution of inflammatory processes as well as the maintenance of a basal anti-inflammatory environment in tissues continuously exposed to harmless antigens (e.g., lung and gut). To identify markers for the anti-inflammatory state of macrophages, expression profiling was performed on human macrophages polarized by either GM-CSF or M-CSF, which lead to the generation of TNF-alpha and IL-12p40-producing pro-inflammatory macrophages [M1 (GM-CSF)] or IL-10-producing anti-inflammatory macrophages [M2 (M-CSF)] upon exposure to LPS, respectively. A different iron metabolism gene signature was detected in both macrophage types, with the heme regulatory molecules CD163 and Heme Oxygenase-1 (HO-1) being preferentially expressed by M2 (M-CSF) macrophages. M1-polarizing cytokines (GM-CSF, IFNgamma) inhibited, while IL-4 enhanced, the M-CSF-driven HO-1 expression. In agreement with this in vitro data, HO-1 expression in metastatic melanoma was primarily detected in CD163(+) tumor-associated macrophages, which are known to exhibit an M2-skewed polarization phenotype. In contrast to the HO-1 inhibitor tin protoporphyrin (SnPP), the administration of cobalt protoporphyrin (CoPP), a potent inducer of HO-1 resulted in increased LPS-triggered IL-10 release from M2 (M-CSF) macrophages. The data suggests that HO-1 is important for the anti-inflammatory activities of M-CSF-polarized M2 macrophages. Moreover, since M2 (M-CSF) macrophages also express higher levels of the CD163 scavenger receptor, the CD163/HO-1/IL-10 axis appears to contribute to the generation of an immunosuppressive environment within the tumor stroma.
  • Article
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    Inflammatory response following central nervous system (CNS) injury contributes to progressive neuropathology and reduction in functional recovery. Axons are sensitive to mechanical injury and toxic inflammatory mediators, which may lead to demyelination. Although it is well documented that degenerated myelin triggers undesirable inflammatory responses in autoimmune diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), there has been very little study of the direct inflammatory consequences of damaged myelin in spinal cord injury (SCI), i.e., there is no direct evidence to show that myelin debris from injured spinal cord can trigger undesirable inflammation in vitro and in vivo. Our data showed that myelin can initiate inflammatory responses in vivo, which is complement receptor 3 (CR3)-dependent via stimulating macrophages to express pro-inflammatory molecules and down-regulates expression of anti-inflammatory cytokines. Mechanism study revealed that myelin-increased cytokine expression is through activation of FAK/PI3K/Akt/NF-kappaB signaling pathways and CR3 contributes to myelin-induced PI3K/Akt/NF-kappaB activation and cytokine production. The myelin induced inflammatory response is myelin specific as sphingomyelin (the major lipid of myelin) and myelin basic protein (MBP, one of the major proteins of myelin) are not able to activate NF-kappaB signaling pathway. In conclusion, our results demonstrate a crucial role of myelin as an endogenous inflammatory stimulus that induces pro-inflammatory responses and suggest that blocking myelin-CR3 interaction and enhancing myelin debris clearance may be effective interventions for treating SCI.
  • Article
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    An increasing number of therapies for spinal cord injury (SCI) are emerging from the laboratory and seeking translation into human clinical trials. Many of these are administered as soon as possible after injury with the hope of attenuating secondary damage and maximizing the extent of spared neurologic tissue. In this article, we systematically review the available pre-clinical research on such neuroprotective therapies that are administered in a non-invasive manner for acute SCI. Specifically, we review treatments that have a relatively high potential for translation due to the fact that they are already used in human clinical applications, or are available in a form that could be administered to humans. These include: erythropoietin, NSAIDs, anti-CD11d antibodies, minocycline, progesterone, estrogen, magnesium, riluzole, polyethylene glycol, atorvastatin, inosine, and pioglitazone. The literature was systematically reviewed to examine studies in which an in-vivo animal model was utilized to assess the efficacy of the therapy in a traumatic SCI paradigm. Using these criteria, 122 studies were identified and reviewed in detail. Wide variations exist in the animal species, injury models, and experimental designs reported in the pre-clinical literature on the therapies reviewed. The review highlights the extent of investigation that has occurred in these specific therapies, and points out gaps in our knowledge that would be potentially valuable prior to human translation.
  • Article
    Full-text available
    Traumatic injury to the central nervous system results in the disruption of the blood brain/spinal barrier, followed by the invasion of cells and other components of the immune system that can aggravate injury and affect subsequent repair and regeneration. Although studies of chronic neuroinflammation in the injured spinal cord of animals are clinically relevant to most patients living with traumatic injury to the brain or spinal cord, very little is known about chronic neuroinflammation, though several studies have tested the role of neuroinflammation in the acute period after injury. The present study characterizes a novel cell preparation method that assesses, quickly and effectively, the changes in the principal immune cell types by flow cytometry in the injured spinal cord, daily for the first 10 days and periodically up to 180 days after spinal cord injury. These data quantitatively demonstrate a novel time-dependent multiphasic response of cellular inflammation in the spinal cord after spinal cord injury and are verified by quantitative stereology of immunolabelled spinal cord sections at selected time points. The early phase of cellular inflammation is comprised principally of neutrophils (peaking 1 day post-injury), macrophages/microglia (peaking 7 days post-injury) and T cells (peaking 9 days post-injury). The late phase of cellular inflammation was detected after 14 days post-injury, peaked after 60 days post-injury and remained detectable throughout 180 days post-injury for all three cell types. Furthermore, the late phase of cellular inflammation (14-180 days post-injury) did not coincide with either further improvements, or new decrements, in open-field locomotor function after spinal cord injury. However, blockade of chemoattractant C5a-mediated inflammation after 14 days post-injury reduced locomotor recovery and myelination in the injured spinal cord, suggesting that the late inflammatory response serves a reparative function. Together, these data provide new insight into cellular inflammation of spinal cord injury and identify a surprising and extended multiphasic response of cellular inflammation. Understanding the role of this multiphasic response in the pathophysiology of spinal cord injury could be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions.
  • Article
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    Macrophage activation comprises a continuum of functional states critically determined by cytokine microenvironment. Activated macrophages have been functionally grouped according to their response to pro-Th1/proinflammatory stimuli [lipopolysaccharide, IFNgamma, granulocyte macrophage colony-stimulating factor (GM-CSF); M1] or pro-Th2/anti-inflammatory stimuli [interleukin (IL)-4, IL-10, M-CSF; M2]. We report that folate receptor beta (FRbeta), encoded by the FOLR2 gene, is a marker for macrophages generated in the presence of M-CSF (M2), but not GM-CSF (M1), and whose expression correlates with increased folate uptake ability. The acquisition of folate uptake ability by macrophages is promoted by M-CSF, maintained by IL-4, prevented by GM-CSF, and reduced by IFNgamma, indicating a link between FRbeta expression and M2 polarization. In agreement with in vitro data, FRbeta expression is detected in tumor-associated macrophages (TAM), which exhibit an M2-like functional profile and exert potent immunosuppressive functions within the tumor environment. FRbeta is expressed, and mediates folate uptake, by CD163(+) CD68(+) CD14(+) IL-10-producing TAM, and its expression is induced by tumor-derived ascitic fluid and conditioned medium from fibroblasts and tumor cell lines in an M-CSF-dependent manner. These results establish FRbeta as a marker for M2 regulatory macrophage polarization and indicate that folate conjugates of therapeutic drugs are a potential immunotherapy tool to target TAM.
  • During injury to the nervous system, innate immune cells mediate phagocytosis of debris, cytokine production, and axon regeneration. In the neuro-degenerative disease amyotrophic lateral sclerosis (ALS), innate immune cells in the CNS are activated. However, the role of innate immunity in the peripheral nervous system (PNS) has not been well defined. In this study, we characterized robust activation of CD169/CD68/Iba1+ macrophages throughout the PNS in mutant SOD1(G93A) and SOD1(G37R) transgenic mouse models of ALS. Macrophage activation occurred pre-symptomatically, and expanded from focal arrays within nerve bundles to a tissue-wide distribution following symptom onset. We found a striking dichotomy for immune cells within the spinal cord and PNS. Flow cytometry and GFP bone marrow chimeras showed that spinal cord microglia were mainly tissue resident derived, dendritic-like cells, whereas in peripheral nerves, the majority of activated macrophages infiltrated from the circulation. Humoral antibodies and complement localized to PNS tissue in tandem with macrophage recruitment, and deficiency in complement C4 led to decreased macrophage activation. Therefore, cross-talk between nervous and immune systems occurs throughout the PNS during ALS disease progression. These data reveal a progressive innate and humoral immune response in peripheral nerves that is separate and distinct from spinal cord immune activation in ALS transgenic mice.
  • Article
    Full-text available
    Macrophages dominate sites of CNS injury in which they promote both injury and repair. These divergent effects may be caused by distinct macrophage subsets, i.e., "classically activated" proinflammatory (M1) or "alternatively activated" anti-inflammatory (M2) cells. Here, we show that an M1 macrophage response is rapidly induced and then maintained at sites of traumatic spinal cord injury and that this response overwhelms a comparatively smaller and transient M2 macrophage response. The high M1/M2 macrophage ratio has significant implications for CNS repair. Indeed, we present novel data showing that only M1 macrophages are neurotoxic and M2 macrophages promote a regenerative growth response in adult sensory axons, even in the context of inhibitory substrates that dominate sites of CNS injury (e.g., proteoglycans and myelin). Together, these data suggest that polarizing the differentiation of resident microglia and infiltrating blood monocytes toward an M2 or "alternatively" activated macrophage phenotype could promote CNS repair while limiting secondary inflammatory-mediated injury.
  • Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in arteries, but the mechanisms linking cholesterol accumulation in macrophage foam cells to inflammation are poorly understood. Macrophage cholesterol efflux occurs at all stages of atherosclerosis and protects cells from free cholesterol and oxysterol-induced toxicity. The ATP-binding cassette transporters ABCA1 and ABCG1 are responsible for the major part of macrophage cholesterol efflux to serum or HDL in macrophage foam cells, but other less efficient pathways such as passive efflux are also involved. Recent studies have shown that the sterol efflux activities of ABCA1 and ABCG1 modulate macrophage expression of inflammatory cytokines and chemokines as well as lymphocyte proliferative responses. In macrophages, transporter deficiency causes increased signaling via various Toll-like receptors including TLR4. These studies have shown that the traditional roles of HDL and ABC transporters in cholesterol efflux and reverse cholesterol transport are mechanistically linked to antiinflammatory and immunosuppressive functions of HDL. The underlying mechanisms may involve modulation of sterol levels and lipid organization in cell membranes.
  • Article
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    Although macrophages (MΦ) are known as essential players in wound healing, their contribution to recovery from spinal cord injury (SCI) is a subject of debate. The difficulties in distinguishing between different MΦ subpopulations at the lesion site have further contributed to the controversy and led to the common view of MΦ as functionally homogenous. Given the massive accumulation in the injured spinal cord of activated resident microglia, which are the native immune occupants of the central nervous system (CNS), the recruitment of additional infiltrating monocytes from the peripheral blood seems puzzling. A key question that remains is whether the infiltrating monocyte-derived MΦ contribute to repair, or represent an unavoidable detrimental response. The hypothesis of the current study is that a specific population of infiltrating monocyte-derived MΦ is functionally distinct from the inflammatory resident microglia and is essential for recovery from SCI.
  • Article
    Microglia cells exhibit two forms of motility, constant movement of filopodia probing surrounding brain tissue, and outgrowth of larger processes in response to nearby damage. The mechanisms and functions of filopodia sensing and process outgrowth are not well characterized but are likely critical for normal immune function in the brain. Using two photon laser scanning microscopy we investigated microglia process outgrowth in response to damage, and explored the relationship between process outgrowth and filopodia movement. Further, we examined the roles of Cl(-) or K(+) channel activation, as well as actin polymerization in these two distinct processes, because mechanistic understanding could provide a strategy to modulate microglia function. We found that volume sensitive Cl(-) channel blockers (NPPB, tamoxifen, DIDS) prevented the rapid process outgrowth of microglia observed in response to damage. In contrast, filopodia extension during sensing was resistant to Cl(-) channel inhibitors, indicating that these motile processes have different cellular mechanisms. However, both filopodia sensing and rapid process outgrowth were blocked by inhibition of actin polymerization. Following lesion formation under control conditions, rapidly outgrowing processes contacted the damaged area and this was associated with a 37% decrease in lesion volume. Inhibition of process outgrowth by Cl(-) channel block, prevention of actin polymerization, or by selectively ablating microglia all allowed lesion volume to increase and spread into the surrounding tissue. Therefore, process outgrowth in response to focal brain damage is beneficial by preventing lesion expansion and suggests microglia represent a front line defence against damage in the brain.
  • Article
    Full-text available
    Spinal cord injury (SCI) triggers inflammation with activation of innate immune responses that contribute to secondary injury including oligodendrocyte apoptosis, demyelination, axonal degeneration, and neuronal death. Macrophage activation, accumulation, and persistent inflammation occur in SCI. Macrophages are heterogeneous cells with extensive functional plasticity and have the capacity to switch phenotypes by factors present in the inflammatory microenvironment of the injured spinal cord. This review will discuss the role of different polarized macrophages and the potential effect of macrophage-based therapies for SCI.
  • Article
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    Sulfatide, a major lipid component of myelin sheath, participates in diverse cellular events of the CNS, and its cellular level has recently been implicated in many inflammation-associated neuronal diseases. Herein, we report that sulfatide alone can trigger pathological inflammatory responses in glia, brain-resident immune cells. We show that sulfatide changed the morphology of primary microglia to their activated form, and it significantly induced the production of various inflammatory mediators in primary microglia and astrocytes. Moreover, sulfatide rapidly triggered the phosphorylation of p38, ERK, and JNK within 30 min, and it markedly enhanced the NF binding activity to NF-kappaB and AP-1 binding elements. However, nonsulfated galactocerebroside, another major lipid component of myelin, had no effect on activation of glia. We further reveal that CD1d did not contribute to sulfatide-stimulated activation of MAPKs, although its expression was enhanced by sulfatide and sulfatide-treated microglial cells actually stimulated type II NKT cells. Sulfatide significantly stimulated the phosphorylation of MAPKs in glia from CD1d-deficient mice, and the phosphorylation levels were similar to those in wild-type littermates. Sulfatide-triggered inflammatory events appear to occur at least in part through an L-selectin-dependent mechanism. L-selectin was dramatically down-regulated upon exposure to sulfatide, and inhibition of L-selectin resulted in suppression of sulfatide-triggered responses. Collectively, these results show that abnormally released sulfatide at demyelinated regions may act as an endogenous stimulator in the brain immune system, thus causing and further exacerbating pathological conditions in the brain.
  • Article
    Biological systems are integrated networks constantly responding to internal and external stimulators. Understanding the intrinsic response to an imbalanced system provides the opportunity to develop therapeutic approaches to reinstate the natural balanced state. Increasing evidence suggests that members of the nuclear receptor superfamily integrate both inflammatory and metabolic signals to maintain homeostasis in immune cells such as macrophages and lymphocytes. PPAR and LXR are nuclear receptors activated by fatty acid and cholesterol derivatives respectively that control the expression of an array of genes involved in lipid metabolism and inflammation. Recent studies have uncovered distinct mechanisms for transcriptional regulation of metabolic and inflammatory target genes by PPAR and LXR and have expanded the biology of these receptors to include roles in alternative macrophage activation and adaptive immunity.
  • Article
    Contact-dependent axon growth inhibitory activity is present in CNS myelin, but the inhibitory proteins have not been fully characterized. We report here that at least two peaks of inhibitory activity can be separated by fractionating solubilized CNS myelin proteins by DEAE chromatography. A major peak of inhibitory activity corresponded to the elution profile of myelin-associated glycoprotein (MAG). Immunodepletion of MAG from these inhibitory fractions removed neurite growth inhibition, whereas recombinant MAG (ectodomain) was a potent inhibitor of neurite outgrowth. Immunodepletion of MAG from total extracts of CNS myelin restored neurite growth up to 63% of control levels. These results establish that MAG is a significant, and possibly the major, inhibitor in CNS myelin; this has broad implications for axonal regeneration in the injured mammalian CNS.
  • Article
    We examined whether cholesterol needed for myelin formation is locally synthesized or whether it comes from the circulation. The experimental design was to inject [3H]water and to use incorporation of label into brain cholesterol as a measure of the rate of accumulation of newly synthesized cholesterol in brain. The contribution of the circulation to this labeled cholesterol pool was minimized by repressing liver synthesis of cholesterol with a high cholesterol diet. The rate of accumulation of total cholesterol was calculated from the increasing amounts of sterol in brain regions at successive time intervals during development. Thus, accumulating cholesterol not explained as being newly synthesized (radioactive) could be assumed to have come from the circulation. Long-Evans rats, ranging in age from birth to 35 days, were injected intraperitoneally with [3H]water (0.3-1.0 mCi/g of body weight) and killed 2 h later. The brain was dissected into brainstem, cerebellum, and cerebral hemispheres, and total lipids were extracted. Cholesterol and its precursors were quantified by HPLC. The radioactivity associated with the sterol fractions and the specific activity of body water determined from serum were used to calculate the absolute amount of newly synthesized sterol. The rates of cholesterol synthesis were compared with the rates of accumulation of total cholesterol in each brain region. The rate of accumulation of total sterol (cholesterol and desmosterol) closely followed that of newly synthesized total sterol in all brain regions from the second through the fifth postnatal weeks.(ABSTRACT TRUNCATED AT 250 WORDS)
  • Article
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    Demyelination of axons in the central nervous system (CNS) during multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE) is a result of phagocytosis and digestion by macrophages (M phi) and the local release of inflammatory mediators like tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO). We have investigated the process of myelin phagocytosis by M phi in vitro using flow cytometric analysis. The binding and uptake of CNS-derived myelin was dose dependent, was abolished in the presence of EDTA and was enhanced after opsonization with complement. The phagocytosis of opsonized myelin could be inhibited by antibodies directed against complement receptor type 3 (CR3). Furthermore, CR3 also contributes to phagocytosis of non-opsonized myelin, e.g. under serum-free conditions. The phagocytosis of CNS-derived myelin induced the production of substantial amounts of TNF-alpha and NO by the M phi. Our results indicate an important role for CR3 in myelin phagocytosis. The induction of TNF-alpha and NO which accompanies this phagocytosis may further contribute to the overall process of demyelination during MS or EAE.
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