Several Alzheimer's disease (AD) risk genes are specifically expressed by microglia within the CNS. However, the mechanisms by which microglia regulate the pathological hallmarks of AD--extracellular deposition of ?-amyloid (A?) and intraneuronal hyperphosphorylation of microtubule-associated protein tau (MAPT)--remain to be established. Notably, deficiency for the microglial CX3CR1 receptor has opposing effects on A? and MAPT pathologies. CX3CL1, the neuronally derived cognate ligand for CX3CR1, signals both in membrane-anchored and soluble forms. In this study, we sought to determine the relative contribution on membrane-anchored versus soluble CX3CL1 in regulating the microglia-mediated amelioration of A? pathology, as well as provide insight into the potential downstream microglial-based mechanisms. As expected, CX3CL1 deficiency reduced A? deposition in APPPS1 animals in a similar manner to CX3CR1 deficiency. Surprisingly, however, CX3CL1-deficient APPPS1 animals exhibited enhanced neuronal MAPT phosphorylation despite reduced amyloid burden. Importantly, neither of these phenotypes was altered by transgenic expression of the soluble CX3CL1 isoform, suggesting that it is the membrane-anchored version of CX3CL1 that regulates microglial phagocytosis of A? and neuronal MAPT phosphorylation. Analysis of transcript levels in purified microglia isolated from APPPS1 mice with the various CX3CL1/CX3CR1 genotypes revealed increased expression of inflammatory cytokines and phagocytic markers, which was associated with activation of p38 mitogen-activated protein kinase and A? internalization within microglia. Together, these studies challenge the "frustrated phagocytosis" concept and suggest that neuronal-microglial communication link the two central AD pathologies.
Microglia, the only nonneuroepithelial cells found in the parenchyma of the CNS, originate during embryogenesis from the yolk sac and enter the CNS quite early (embryonic day 9.5-10 in mice). Thereafter, microglia are maintained independently of any input from the blood and, in particular, do not require hematopoietic stem cells as a source of replacement for senescent cells. Monocytes are hematopoietic cells, derived from bone marrow. The ontogeny of microglia and monocytes is important for understanding CNS pathologies. Microglial functions are distinct from those of blood-derived monocytes, which invade the CNS only under pathological conditions. Recent data reveal that microglia play an important role in managing neuronal cell death, neurogenesis, and synaptic interactions. In this article, we discuss the physiology of microglia and the functions of monocytes in CNS pathology. We address the roles of microglia and monocytes in neurodegenerative diseases as an example of CNS pathology.
Since the initial cloning of fractalkine/CX3CL1, it was proposed that the only known member of the CX3C or ? subfamily of chemotactic cytokines could play some significant role in the nervous system, due to its high expression on neurons. The pivotal description of the localization of the unique CX3CL1 receptor, CX3CR1, on microglial cells, firmed up by the generation of cx3cr1(GFP/GFP) mice, opened the road to the hypothesis of some specific key interactions between microglia and neurons mediated by this pair. This expectation has been indeed supported by recent exciting evidence indicating that CX3CL1-mediated microglia-neuron interaction modulates basic physiological activities during development, adulthood and aging, including: synaptic pruning; promoting survival of neurons and neural precursors; modulating synaptic transmission and plasticity; enhancing synapse and network maturation; and facilitating the establishment of neuropathic pain circuits. Beyond playing such fascinating roles in physiological conditions, CX3CL1 signaling has been implicated in different neuropathologies. Early papers demonstrated that the levels of CX3CL1 may be modulated by various toxic stimuli in vitro and that CX3CL1 signaling is positively or negatively regulated in EAE and MS, in HIV infection and LPS challenge, in epilepsy, in brain tumors, and in other neuropathologies. In this review we focus on the experimental evidence of CX3CL1 involvement in neuroprotection and survey the common molecular and cellular mechanisms described in different brain diseases.
Leukocyte transendothelial migration (TEM; diapedesis) is a critical event in immune surveillance and inflammation. Most TEM occurs at endothelial cell borders (paracellular). However, there is indirect evidence to suggest that, at the tight junctions of the blood-brain barrier (BBB), leukocytes migrate directly through the endothelial cell body (transcellular). Why leukocytes migrate through the endothelial cell body rather than the cell borders is unknown. To test the hypothesis that the tightness of endothelial cell junctions influences the pathway of diapedesis, we developed an in vitro model of the BBB that possessed 10-fold higher electrical resistance than standard culture conditions and strongly expressed the BBB tight junction proteins claudin-5 and claudin-3. We found that paracellular TEM was still the predominant pathway (?98%) and TEM was dependent on PECAM-1 and CD99. We show that endothelial tight junctions expressing claudin-5 are dynamic and undergo rapid remodeling during TEM. Membrane from the endothelial lateral border recycling compartment is mobilized to the exact site of tight junction remodeling. This preserves the endothelial barrier by sealing the intercellular gaps with membrane and engaging the migrating leukocyte with unligated adhesion molecules (PECAM-1 and CD99) as it crosses the cell border. These findings provide new insights into leukocyte-endothelial interactions at the BBB and suggest that tight junctions are more dynamic than previously appreciated.
In the human disorder multiple sclerosis (MS) and in the model experimental autoimmune encephalomyelitis (EAE), macrophages predominate in demyelinated areas and their numbers correlate to tissue damage. Macrophages may be derived from infiltrating monocytes or resident microglia, yet are indistinguishable by light microscopy and surface phenotype. It is axiomatic that T cell-mediated macrophage activation is critical for inflammatory demyelination in EAE, yet the precise details by which tissue injury takes place remain poorly understood. In the present study, we addressed the cellular basis of autoimmune demyelination by discriminating microglial versus monocyte origins of effector macrophages. Using serial block-face scanning electron microscopy (SBF-SEM), we show that monocyte-derived macrophages associate with nodes of Ranvier and initiate demyelination, whereas microglia appear to clear debris. Gene expression profiles confirm that monocyte-derived macrophages are highly phagocytic and inflammatory, whereas those arising from microglia demonstrate an unexpected signature of globally suppressed cellular metabolism at disease onset. Distinguishing tissue-resident macrophages from infiltrating monocytes will point toward new strategies to treat disease and promote repair in diverse inflammatory pathologies in varied organs.
Axonal degeneration is a primary cause of permanent neurological disability in individuals with the CNS demyelinating disease multiple sclerosis. Dysfunction of axonal mitochondria and imbalanced energy demand and supply are implicated in degeneration of chronically demyelinated axons. The purpose of this study was to define the roles of mitochondrial volume and distribution in axonal degeneration following acute CNS demyelination. We show that the axonal mitochondrial volume increase following acute demyelination of WT CNS axons does not occur in demyelinated axons deficient in syntaphilin, an axonal molecule that immobilizes stationary mitochondria to microtubules. These findings were supported by time-lapse imaging of WT and syntaphilin-deficient axons in vitro. When demyelinated, axons deficient in syntaphilin degenerate at a significantly greater rate than WT axons, and this degeneration can be rescued by reducing axonal electrical activity with the Na(+) channel blocker flecainide. These results support the concept that syntaphilin-mediated immobilization of mitochondria to microtubules is required for the volume increase of axonal mitochondria following acute demyelination and protects against axonal degeneration in the CNS.
In vitro blood-brain barrier (BBB) models can be useful for understanding leukocyte-endothelial interactions at this unique vascular-tissue interface. Desirable features of such a model include shear stress, non-transformed cells and co-cultures of brain microvascular endothelial cells with astrocytes. Recovery of transmigrated leukocytes for further analysis is also appealing.
Aminoglycoside antibiotics are highly effective agents against gram-negative bacterial infections, but they cause adverse effects on hearing and balance dysfunction as a result of toxicity to hair cells of the cochlea and vestibular organs. While ototoxicity has been comprehensively studied, the contributions of the immune system, which controls the host response to infection, have not been studied in antibiotic ototoxicity. Recently, it has been shown that an inflammatory response is induced by hair cell injury. In this study, we found that lipopolysaccharide (LPS), an important component of bacterial endotoxin, when given in combination with kanamycin and furosemide, augmented the inflammatory response to hair cell injury and exacerbated hearing loss and hair cell injury. LPS injected into the peritoneum of experimental mice induced a brisk cochlear inflammatory response with recruitment of mononuclear phagocytes into the spiral ligament, even in the absence of ototoxic agents. While LPS alone did not affect hearing, animals that received LPS prior to ototoxic agents had worse hearing loss compared to those that did not receive LPS pretreatment. The poorer hearing outcome in LPS-treated mice did not correlate to changes in endocochlear potential. However, LPS-treated mice demonstrated an increased number of CCR2(+) inflammatory monocytes in the inner ear when compared with mice treated with ototoxic agents alone. We conclude that LPS and its associated inflammatory response are harmful to the inner ear when coupled with ototoxic medications and that the immune system may contribute to the final hearing outcome in subjects treated with ototoxic agents.
Low-grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. Here, we investigate how Toll-like receptor-MyD88 signaling in myeloid and endothelial cells coordinately participates in the initiation and progression of high fat diet-induced systemic inflammation and metabolic inflammatory diseases. MyD88 deficiency in myeloid cells inhibits macrophage recruitment to adipose tissue and their switch to an M1-like phenotype. This is accompanied by substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. MyD88 deficiency in endothelial cells results in a moderate reduction in diet-induced adipose macrophage infiltration and M1 polarization, selective insulin sensitivity in adipose tissue, and amelioration of spontaneous atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to differentiate into M1-like inflammatory macrophages. Collectively, these results implicate a critical MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases.
After traumatic spinal cord injury, functional deficits increase as axons die back from the center of the lesion and the glial scar forms. Axonal dieback occurs in two phases: an initial axon intrinsic stage that occurs over the first several hours and a secondary phase which takes place over the first few weeks after injury. Here, we examine the secondary phase, which is marked by infiltration of macrophages. Using powerful time-lapse multi-photon imaging, we captured images of interactions between Cx3cr1(+/GFP) macrophages and microglia and Thy-1(YFP) axons in a mouse dorsal column crush spinal cord injury model. Over the first few weeks after injury, axonal retraction bulbs within the lesion are static except when axonal fragments are lost by a blebbing mechanism in response to physical contact followed by phagocytosis by mobile Cx3Cr1(+/GFP) cells. Utilizing a radiation chimera model to distinguish marrow-derived cells from radio-resistant CNS-resident microglia, we determined that the vast majority of accumulated cells in the lesion are derived from the blood and only these are associated with axonal damage. Interestingly, CNS-resident Cx3Cr1(+/GFP) microglia did not increasingly accumulate nor participate in neuronal destruction in the lesion during this time period. Additionally, we found that the blood-derived cells consisted mainly of singly labeled Ccr2(+/RFP) macrophages, singly labeled Cx3Cr1(+/GFP) macrophages and a small population of double-labeled cells. Since all axon destructive events were seen in contact with a Cx3Cr1(+/GFP) cell, we infer that the CCR2 single positive subset is likely not robustly involved in axonal dieback. Finally, in our model, deletion of CCR2, a chemokine receptor, did not alter the position of axons after dieback. Understanding the in vivo cellular interactions involved in secondary axonal injury may lead to clinical treatment candidates involving modulation of destructive infiltrating blood monocytes.
The interface between the blood circulation and the neural tissue features unique characteristics that are encompassed by the term blood-brain barrier (BBB). The main functions of this barrier, namely maintenance of brain homeostasis, regulation of influx and efflux transport, and protection from harm, are determined by its specialized multicellular structure. Every constituent cell type makes an indispensable contribution to the BBBs integrity. But if one member of the BBB fails, and as a result the barrier breaks down, there can be dramatic consequences and neuroinflammation and neurodegeneration can occur. In this Review, we highlight recently gained mechanistic insights into the development and maintenance of the BBB. We then discuss how BBB disruption can cause or contribute to neurological disease. Finally, we examine how this knowledge can be used to explore new possibilities for BBB repair.
Interleukin 17 (IL-17) is a signature cytokine of Th17 cells. We previously reported that deletion of NF-?B activator 1 (Act1), the key transducer of IL-17 receptor signaling, from the neuroectodermal lineage in mice (neurons, oligodendrocytes and astrocytes) results in attenuated severity of experimental autoimmune encephalomyelitis (EAE). Here we examined the cellular basis of this observation. EAE disease course was unaffected by deletion of Act1 in neurons or mature oligodendrocytes, and Act1 deletion in astrocytes only modestly affected disease course. Deletion of Act1 in NG2(+) glia resulted in markedly reduced EAE severity. Furthermore, IL-17 induced characteristic inflammatory mediator expression in NG2(+) glial cells. IL-17 also exhibited strong inhibitory effects on the maturation of oligodendrocyte lineage cells in vitro and reduced their survival. These data identify NG2(+) glia as the major CNS cellular target of IL-17 in EAE. The sensitivity of oligodendrocyte lineage cells to IL-17-mediated toxicity further suggests a direct link between inflammation and neurodegeneration in multiple sclerosis.
Fractalkine, a chemokine anchored to neurons or peripheral endothelial cells, serves as an adhesion molecule or as a soluble chemoattractant. Fractalkine binds CX3CR1 on microglia and circulating monocytes, dendritic cells, and NK cells. The aim of this study is to determine the role of CX3CR1 in the trafficking and function of myeloid cells to the CNS during experimental autoimmune encephalomyelitis (EAE). Our results show that, in models of active EAE, Cx3cr1(-/-) mice exhibited more severe neurologic deficiencies. Bone marrow chimeric mice confirmed that CX3CR1 deficiency in bone marrow enhanced EAE severity. Notably, CX3CR1 deficiency was associated with an increased accumulation of CD115(+)Ly6C(-)CD11c(+) dendritic cells into EAE-affected brains that correlated with enhanced demyelination and neuronal damage. Furthermore, higher IFN-? and IL-17 levels were detected in cerebellar and spinal cord tissues of CX3CR1-deficient mice. Analyses of peripheral responses during disease initiation revealed a higher frequency of IFN-?- and IL-17-producing T cells in lymphoid tissues of CX3CR1-deficient as well as enhanced T cell proliferation induced by CX3CR1-deficient dendritic cells. In addition, adoptive transfer of myelin oligodendrocyte glycoprotein35-55-reactive wild-type T cells induced substantially more severe EAE in CX3CR1-deficient recipients when compared with wild-type recipients. Collectively, the data demonstrate that besides its role in chemoattraction, CX3CR1 is a key regulator of myeloid cell activation contributing to the establishment of adaptive immune responses.
Massive neuronal loss is a key pathological hallmark of Alzheimers disease (AD). However, the mechanisms are still unclear. Here we demonstrate that neuroinflammation, cell autonomous to microglia, is capable of inducing neuronal cell cycle events (CCEs), which are toxic for terminally differentiated neurons. First, oligomeric amyloid-beta peptide (A?O)-mediated microglial activation induced neuronal CCEs via the tumor-necrosis factor-? (TNF?) and the c-Jun Kinase (JNK) signaling pathway. Second, adoptive transfer of CD11b+ microglia from AD transgenic mice (R1.40) induced neuronal cyclin D1 expression via TNF? signaling pathway. Third, genetic deficiency of TNF? in R1.40 mice (R1.40-Tnf?(-/-)) failed to induce neuronal CCEs. Finally, the mitotically active neurons spatially co-exist with F4/80+ activated microglia in the human AD brain and that a portion of these neurons are apoptotic. Together our data suggest a cell-autonomous role of microglia, and identify TNF? as the responsible cytokine, in promoting neuronal CCEs in the pathogenesis of AD.
Microglia are myeloid cells of the CNS that participate both in normal CNS function and in disease. We investigated the molecular signature of microglia and identified 239 genes and 8 microRNAs that were uniquely or highly expressed in microglia versus myeloid and other immune cells. Of the 239 genes, 106 were enriched in microglia as compared with astrocytes, oligodendrocytes and neurons. This microglia signature was not observed in microglial lines or in monocytes recruited to the CNS, and was also observed in human microglia. We found that TGF-? was required for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia and that microglia were absent in the CNS of TGF-?1-deficient mice. Our results identify a unique microglial signature that is dependent on TGF-? signaling and provide insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease.
Identifying and blocking chemokine inflammatory mediators in pediatric opsoclonus-myoclonus syndrome (OMS) is critical to the treatment of this autoimmune, paraneoplastic, neurological disorder. In a prospective, case-control, clinico-scientific study of children with OMS compared to non-inflammatory neurological controls and other inflammatory neurological disorders, CCL19 (n=369) and CCL21 (n=312) were quantified in CSF and serum, respectively, by ELISA. Both cross-sectional and longitudinal effects of OMS and various immunotherapies were evaluated. Significant upregulation of CCL21 concentration (mean ± SD) (+32%) was found in serum of untreated OMS (630 ± 133 pg/mL), compared to controls (478 ± 168 pg/mL), (p<0.0001). Both corticosteroids and ACTH (corticotropin) significantly lowered CCL21 to control levels, as they did in combination with IVIg, rituximab, cyclophosphamide or other treatments, without additional reduction attributable to the other agents. In a pilot longitudinal study of ACTH-based triple therapy, the mean serum CCL21 concentration fell 59% from elevated to less than 1 SD below controls 1 week after high-dose ACTH, gradually returning to the control mean with ACTH tapering by 3 weeks and out to 12 weeks (p<0.0001). In contrast, CCL19, detectable in CSF, was not significantly altered by OMS or various immunotherapies. In the "high" CCL21 subgroup, higher serum concentrations of CCL22 (+57%) and CXCL13 (+40%), as well as the CSF concentration of BAFF (+64%), also were found. Elevated serum CCL21, not CSF CCL19, correlates with OMS severity and duration in pediatric OMS. Corticosteroids and ACTH were the only immunotherapies evaluated that down-regulated CCL21 production. Validation studies are needed to assess treatment biomarker status.
The presentation of myelopathy in patients with the concomitant diagnosis of cervical stenosis (CS) and multiple sclerosis (MS) complicates both diagnosis and treatment because of the similarities of presentation and disease progression. There are only a few published case series that examine this unique patient population.
Type 2 CXC chemokine receptor CXCR2 plays roles in development, tumorigenesis, and inflammation. CXCR2 also promotes demyelination and decreases remyelination by actions toward hematopoietic cells and nonhematopoietic cells. Germline CXCR2 deficient (Cxcr2(-/-) ) mice reported in 1994 revealed the complexity of CXCR2 function and its differential expression in varied cell-types. Here, we describe Cxcr2(fl/fl) mice for which the targeting construct was generated by recombineering based on homologous recombination in E. coli. Without recombination Cxcr2(fl/fl) mice have CXCR2 expression on neutrophils in peripheral blood, bone marrow and spleen. Cxcr2(fl/fl) mice were crossed to Mx-Cre mice in which Cre recombinase is induced by Type I interferons, elicited by injection with polyinosinic-polycytidylic acid (poly(I:C)). CXCR2-deficient neutrophils were observed in poly(I:C) treated Cxcr2(fl/fl) ::Mx-Cre(+) (Cxcr2-CKO) mice, but not in poly(I:C) treated Cxcr2(f//+) ::Mx-Cre(+) mice. CXCR2 deletion was mainly observed peripherally but not in the CNS. Cxcr2-CKO mice showed impaired neutrophil migration in sterile peritonitis. Cxcr2-CKO mice reported here will provide a genetic reagent to dissect roles of CXCR2 in the neutrophil granulocyte lineage. Furthermore Cxcr2(fl/fl) mice will provide useful genetic models to evaluate CXCR2 function in varied cell populations.
The study was undertaken to determine the pathologic basis of subtle abnormalities in magnetization transfer ratio (MTR) and diffusion tensor imaging (DTI) parameters observed in normal-appearing white matter (NAWM) in multiple sclerosis brains.
Cortical disease has emerged as a critical aspect of the pathogenesis of multiple sclerosis, being associated with disease progression and cognitive impairment. Most studies of cortical lesions have focused on autopsy findings in patients with long-standing, chronic, progressive multiple sclerosis, and the noninflammatory nature of these lesions has been emphasized. Magnetic resonance imaging studies indicate that cortical damage occurs early in the disease.
The mechanism of IFN-? therapy in relapsing-remitting multiple sclerosis (RRMS) is not well understood, but induction of apoptosis in specific leukocyte subsets is likely to be important. Enhanced expression of TNFSF10 or TNF-related apoptosis-inducing ligand (TRAIL) mRNA in unseparated leukocytes has been put forward as a therapeutic response marker, but it is unclear which leukocyte subsets express TRAIL. We investigated the basis of TRAIL expression in response to IFN-? by studying activation of STATs 1, 3, and 5, p38 MAPK, and NF-?B in different leukocyte subsets of patients with RRMS. Monocytes, B cells, and T cells showed substantial differences in the activation of p38 and the STATs in response to i.m. injection of IFN-?1a or stimulation in vitro. Induction of cell-surface TRAIL, analyzed in nine leukocyte subsets, was observed only on monocytes and granulocytes and correlated with the activation of p38 and/or NF-?B in these subsets only, in agreement with previous work in fibroblasts showing that the induction of TRAIL in response to IFN-? depends on the activation of p38 and NF-?B as well as STATs 1 and 2. We propose that, in myeloid cells, the differential activation of p38 and NF-?B and induction of TRAIL, which sensitizes cells to apoptosis, can help to explain differences in responsiveness to IFN-? therapy among patients with RRMS and, furthermore, that such differential patterns of activation and expression may also be important in understanding the therapeutic responses to IFN-?/? in hepatitis and cancer.
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.
Lymphatic endothelial cells are important for efficient flow of antigen-bearing fluid and antigen-presenting cells (APCs) from peripheral sites to lymph nodes (LNs). APC movement to LNs is dependent on the constitutive chemokine receptor CCR7, although how conflicting inflammatory and constitutive chemokine cues are integrated at lymphatic surfaces during this process is not understood. Here we reveal a previously unrecognized aspect of the regulation of this process. The D6 chemokine-scavenging receptor, which is expressed on lymphatic endothelial cells (LECs), maintains lymphatic surfaces free of inflammatory CC-chemokines and minimizes interaction of inflammatory leukocytes with these surfaces. D6 does not alter the level of CCR7 ligands on LECs, thus ensuring selective presentation of homeostatic chemokines for interaction with CCR7(+) APCs. Accordingly, in D6-deficient mice, inflammatory CC-chemokine adherence to LECs results in inappropriate perilymphatic accumulation of inflammatory leukocytes at peripheral inflamed sites and draining LNs. This results in lymphatic congestion and impaired movement of APCs, and fluid, from inflamed sites to LNs. We propose that D6, by suppressing inflammatory chemokine binding to lymphatic surfaces, and thereby preventing inappropriate inflammatory leukocyte adherence, is a key regulator of lymphatic function and a novel, and indispensable, contributor to the integration of innate and adaptive immune responses.
The diseased brain hosts a heterogeneous population of myeloid cells, including parenchymal microglia, perivascular cells, meningeal macrophages and blood-borne monocytes. To date, the different types of brain myeloid cells have been discriminated solely on the basis of their localization, morphology and surface epitope expression. However, recent data suggest that resident microglia may be functionally distinct from bone marrow- or blood-derived phagocytes, which invade the CNS under pathological conditions. During the last few years, research on brain myeloid cells has been markedly changed by the advent of new tools in imaging, genetics and immunology. These methodologies have yielded unexpected results, which challenge the traditional view of brain macrophages. On the basis of these new studies, we differentiate brain myeloid subtypes with regard to their origin, function and fate in the brain and illustrate the divergent features of these cells during neurodegeneration.
Aberrant microglial activation has been proposed to contribute to the cognitive decline in Alzheimer disease (AD), but the underlying molecular mechanisms remain enigmatic. Fractalkine signaling, a pathway mediating the communication between microglia and neurons, is deficient in AD brains and down-regulated by amyloid-?. Although fractalkine receptor (CX3CR1) on microglia was found to regulate plaque load, no functional effects have been reported. Our study demonstrates that CX3CR1 deficiency worsens the AD-related neuronal and behavioral deficits. The effects were associated with cytokine production but not with plaque deposition. Ablation of CX3CR1 in mice overexpressing human amyloid precursor protein enhanced Tau pathology and exacerbated the depletion of calbindin in the dentate gyrus. The levels of calbindin in the dentate gyrus correlated negatively with those of tumor necrosis factor ? and interleukin 6, suggesting neurotoxic effects of inflammatory factors. Functionally, removing CX3CR1 in human amyloid precursor protein mice worsened the memory retention in passive avoidance and novel object recognition tests, and their memory loss in the novel object recognition test is associated with high levels of interleukin 6. Our findings identify CX3CR1 as a key microglial pathway in protecting against AD-related cognitive deficits that are associated with aberrant microglial activation and elevated inflammatory cytokines.
Macrophages exert divergent effects in the injured CNS, causing either neurotoxicity or regeneration. The mechanisms regulating these divergent functions are not understood but can be attributed to the recruitment of distinct macrophage subsets and the activation of specific intracellular signaling pathways. Here, we show that impaired signaling via the chemokine receptor CX3CR1 promotes recovery after traumatic spinal cord injury (SCI) in mice. Deficient CX3CR1 signaling in intraspinal microglia and monocyte-derived macrophages (MDMs) attenuates their ability to synthesize and release inflammatory cytokines and oxidative metabolites. Also, impaired CX3CR1 signaling abrogates the recruitment or maturation of MDMs with presumed neurotoxic effects after SCI. Indeed, in wild-type mice, Ly6C(lo)/iNOS(+)/MHCII(+)/CD11c(-) MDMs dominate the lesion site, whereas CCR2(+)/Ly6C(hi)/MHCII(-)/CD11c(+) monocytes predominate in the injured spinal cord of CX3CR1-deficient mice. Replacement of wild-type MDMs with those unable to signal via CX3CR1 resulted in anatomical and functional improvements after SCI. Thus, blockade of CX3CR1 signaling represents a selective anti-inflammatory therapy that is able to promote neuroprotection, in part by reducing inflammatory signaling in microglia and MDMs and recruitment of a novel monocyte subset.
CC chemokine ligand 2 (CCL2), a ligand of CC chemokine receptor 2 (CCR2), is essential to mount an adequate inflammatory response to repair acute skeletal muscle injury. We studied the mechanisms by which CCL2 regulates muscle inflammation and regeneration. Mobilization of monocytes/macrophages (MOs/MPs) but not lymphocytes or neutrophils was impaired from bone marrow to blood and from blood to injured muscles in Ccl2(-/-) mice. This was accompanied by poor phagocytosis, reduced up-regulation of insulin-like growth factor-1 (IGF-1), and impaired muscle regeneration. Bone marrow transfer from wild-type mice to irradiated Ccr2(-/-) but not Ccl2(-/-) mice restored muscle inflammation. Intravenously injected CCL2-deficient bone marrow monocytes could not enter wild-type injured muscles as well as wild-type bone marrow monocytes. Intravenously injected wild-type bone marrow monocytes could not enter CCL2-deficient injured muscles as well as wild-type injured muscles. CCL2 stimulated IGF-1 expression by wild-type but not CCR2-deficient intramuscular macrophages. A single intramuscular injection of IGF-1, but not PBS, markedly improved muscle regeneration in Ccl2(-/-) mice. We conclude that CCL2 is a major ligand of CCR2 to recruit MOs/MPs into injured muscles to conduct phagocytosis and produce IGF-1 for injury repair. CCL2 needs to be expressed by bone marrow cells, circulating monocytes, and injured muscle tissue cells to recruit MOs/MPs into injured muscles. CCL2/CCR2 signaling also up-regulates IGF-1 expression by intramuscular macrophages to promote acute skeletal muscle injury repair.
Infiltration of activated lymphocytes into the central nervous system (CNS) is potentially harmful by damaging resident cells through release of cytokines. Among these is IFN-? that is secreted by activated natural killer (NK) cells and T lymphocytes and can exert a cytotoxic effect on resident glial populations including oligodendrocytes. Here we show that treatment of mouse oligodendrocyte progenitor cell (OPC)-enriched cultures with IFN-? resulted in a dose-dependent increase in apoptosis. IFN-?-induced apoptosis is mediated, in part, through induction of the CXC chemokine ligand 10 (CXCL10; IP-10) from cultured OPCs. Treatment of OPCs with CXCL10 resulted in cell death in a concentration-dependent manner and IFN-?-treatment of CXCL10-/- OPCs resulted in >50% reduction in cell death. Further, treatment of CXCR3-/- OPC cultures with either IFN-? or CXCL10 resulted in reduced cell death supporting an important role for CXCL10 signaling in IFN-?-mediated OPC apoptosis. Data is also provided demonstrating that signaling through CXCR2 protects either IFN-? or CXCL10-treated OPC cultures from apoptosis and this effect is abolished in CXCR2-/- OPCs. CXCR2-mediated protection from apoptosis is associated with impaired cleavage of caspase 3 and elevated expression of the anti-apoptotic protein Bcl-2. These findings demonstrate a previously unappreciated role for CXCL10 in contributing to neuropathology by promoting oligodendrocyte apoptosis and emphasize the potential relevance in targeting CXCL10 in treating human demyelinating diseases including multiple sclerosis (MS).
Immunoglobulin in cerebral spinal fluid and antibody secreting cells (ASC) within the central nervous system (CNS) parenchyma are common hallmarks of microbial infections and autoimmune disorders. However, the signals directing ASC migration into the inflamed CNS are poorly characterized. This study demonstrates that CXCR3 mediates CNS accumulation of ASC during neurotropic coronavirus-induced encephalomyelitis. Expansion of CXCR3-expressing ASC in draining lymph nodes prior to accumulation within the CNS was consistent with their recruitment by sustained expression of CXCR3 ligands during viral persistence. Both total and virus-specific ASC were reduced greater than 80% in the CNS of infected CXCR3(-/-) mice. Similar T cell CNS recruitment and local T cell-dependent antiviral activity further indicated that the ASC migration defect was T cell independent. Furthermore, in contrast to the reduction of ASC in the CNS, neither virus-specific ASC trafficking to bone marrow nor antiviral serum antibody was reduced relative to levels in control mice. Impaired ASC recruitment into the CNS of infected CXCR3(-/-) mice coincided with elevated levels of persisting viral RNA, sustained infectious virus, increased clinical disease, and mortality. These results demonstrate that CXCR3 ligands are indispensable for recruitment of activated ASC into the inflamed CNS and highlight their local protective role during persistent infection.
Interferon-beta (IFN?) is used to inhibit disease activity in multiple sclerosis (MS), but its mechanisms of action are incompletely understood, individual treatment response varies, and biological markers predicting response to treatment have yet to be identified.
CXC chemokine receptor CXCR4 is expressed in vitro in both human and rodent adult neural progenitor cells (NPCs). It has been suggested that the CXCL12-CXCR4 axis potentially enhances the proliferation of NPCs. However, whether CXCR4 is expressed in the neural stem cells (NSCs), a subset of self-renewing and multipotent NPCs, and whether CXCR4 signaling is directly required for their proliferation are not clear. In this study, we report that CXCR4 is expressed in a subpopulation of NPCs in the early embryonic ventricular zone. In studies of a CXCR4(eGFP) bacterial artificial chromosomal (BAC) transgenic mouse line, we further isolated NPCs from E12.5 transgenic telencephalon and GFP(+) cells demonstrated self-renewal and multipotency in neurosphere assays in vitro. Consistent with these observations, we enriched GFP(+)/CXCR4(+) cells by fluorescence activated cell sorting (FACS) with either CXCR4 antibody 12G5 or GFP. Furthermore, we observed that CXCL12 alone did not activate the self-renewal of NPCs or increase the proliferation of NPCs that are induced by bFGF/EGF. However, we found that blocking CXCR4 receptor with antagonist AMD3100 impaired the bFGF/EGF-induced expansion of GFP(+) NPCs through modulating their cell cycling. In addition, AMD3100 treatment of pregnant mice reduced the generation of neurospheres from E12.5 embryos. Our data suggest that CXCR4 is a potential cell surface marker for early embryonic NSCs and modulates growth-factor signaling.
We previously demonstrated a correlation between the frequency of CX3CR1-expressing human natural killer (NK) cells and disease activity in multiple sclerosis and showed that CX3CR1(high) NK cells were more cytotoxic than their CX3CR1(neg/low) counterparts. Here we aimed to determine whether human NK cell fractions defined by CX3CR1 represent distinct subtypes. Phenotypic and functional NK cell analyses revealed that, distinct from CX3CR1(high), CX3CR1(neg/low) NK cells expressed high amounts of type 2 cytokines, proliferated robustly in response to interleukin-2 and promoted a strong up-regulation of the key co-stimulatory molecule CD40 on monocytes. Co-expression analyses of CX3CR1 and CD56 demonstrated the existence of different NK cell fractions based on the surface expression of these two surface markers, the CX3CR1(neg) CD56(bright), CX3CR1(neg) CD56(dim) and CX3CR1(high) CD56(dim) fractions. Additional investigations on the expression of NK cell receptors (KIR, NKG2A, NKp30 and NKp46) and the maturation markers CD27, CD62L and CD57 indicated that CX3CR1 expression of CD56(dim) discriminated between an intermediary CX3CR1(neg) ?CD56(dim) and fully mature CX3CR1(high) CD56(dim) NK cell fractions. Hence, CX3CR1 emerges as an additional differentiation marker that may link NK cell maturation with the ability to migrate to different organs including the central nervous system.
Expression of matrix metalloproteinases (MMPs), especially MMP9 correlates with blood-brain barrier (BBB) disruption during many neuroinflammatory diseases. During neurotropic coronavirus virus (JHMV) induced encephalomyelitis, MMP9 activity is restricted to neutrophils. Furthermore, myeloid cell depletion implicated MMP9 in facilitating leukocyte central nervous system (CNS) infiltration via loss of BBB integrity. The requirement of MMP9 in BBB disruption was thus assessed in JHMV infected MMP9 deficient (MMP9(-/-)) mice. Depletion of neutrophils reduced CNS accumulation of monocytes and T cells, albeit without affecting overall pathogenesis. By contrast, infected MMP9(-/-) mice revealed no differences in CNS leukocyte infiltration, composition or localization, consistent with BBB disruption similar to wild-type (WT) mice. Unimpaired T cell mediated virus control supported an unexpectedly redundant role of MMP9 in promoting leukocyte access to the brain parenchyma. Although MMP9 deficiency did not expand the overall limited pattern of MMP expression during JHMV infection, it coincided with MMP3 upregulation. MMP3 expression remained largely confined to astrocytes, similar to WT mice. These data demonstrate that neutrophil-derived MMP9 is not the sole mediator facilitating parenchymal leukocyte entry via BBB disruption during viral encephalomyelitis. Moreover, significantly enhanced MMP3 expression by astrocytes in infected MMP9(-/-) mice suggests an active role of resident cells in participating and potentially collaborating with infiltrating cells in regulating BBB permeability. Overall, these results highlight the complexity of targeting individual MMPs as a strategy to regulate inflammation.
Muscle fibrosis is a prominent pathological feature that directly causes muscle dysfunction in Duchenne muscular dystrophy (DMD). The DMD mouse models, mdx mice and mdx mice with haploinsufficiency of the utrophin gene (mdx/utrn(+/-) ), display progressive diaphragm fibrosis. We performed unrestrained whole-body plethysmography (WBP) in mdx and mdx/utrn(+/-) mice, and compared them with wild-type controls. Respiratory function gauged by respiratory frequency, tidal volume, minute volume, peak inspiratory flow, and peak expiratory flow was significantly impaired in the mdx mice. Consistent with more severe diaphragm fibrosis in the mdx/utrn(+/-) mice, respiratory impairment was worse than in mdx mice at 6 months. WBP is useful for monitoring in vivo respiratory function of mdx and mdx/utrn(+/-) mice, and it may serve as an outcome measurement for therapies that target diaphragm fibrosis. The mdx/utrn(+/-) mouse model may be better than the mdx model for testing antifibrotic therapies, especially at the severe stage.
Emergency mobilization of neutrophil granulocytes (neutrophils) from the bone marrow (BM) is a key event of early cellular immunity. The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) stimulates this process, but it is unknown how individual neutrophils respond in situ. We show by intravital 2-photon microscopy that a systemic dose of human clinical-grade G-CSF rapidly induces the motility and entry of neutrophils into blood vessels within the tibial BM of mice. Simultaneously, the neutrophil-attracting chemokine KC (Cxcl1) spikes in the blood. In mice lacking the KC receptor Cxcr2, G-CSF fails to mobilize neutrophils and antibody blockade of Cxcr2 inhibits the mobilization and induction of neutrophil motility in the BM. KC is expressed by megakaryocytes and endothelial cells in situ and is released in vitro by megakaryocytes isolated directly from BM. This production of KC is strongly increased by thrombopoietin (TPO). Systemic G-CSF rapidly induces the increased production of TPO in BM. Accordingly, a single injection of TPO mobilizes neutrophils with kinetics similar to G-CSF, and mice lacking the TPO receptor show impaired neutrophil mobilization after short-term G-CSF administration. Thus, a network of signaling molecules, chemokines, and cells controls neutrophil release from the BM, and their mobilization involves rapidly induced Cxcr2-mediated motility controlled by TPO as a pacemaker.
Autoimmune mechanisms, particularly through generation of autoantibodies, may contribute to the pathophysiology of idiopathic dilated cardiomyopathy (iDCM). The precise role of cellular autoimmune responses to cardiac-specific antigens has not been well described in humans. The purpose of this study was to characterize the cellular autoimmune response to cardiac troponin I (cTnI), specifically, the release of cytokines by peripheral blood mononuclear cells (PBMCs), in subjects with iDCM and healthy control subjects.
This article discusses the multiple roles of CXCL12 and its receptor, CXCR4, in bone marrow (BM) hematopoietic stem cell (HSC) development and regulation. CXCL12 interaction with CXCR4 results in effects as varied as cell migration, proliferation and survival or apoptosis. The selective signaling pathways that mediate these varied outcomes are summarized briefly. The CXCL12/CXCR4 pair is crucially involved in homing and repopulation of HSCs in the specific BM niches. Mechanisms of HSC mobilization to the peripheral circulation in response to physiological requests and therapeutic stimulations, as well as recent data on the novel receptor for CXCL12, CXCR7, are reviewed.
A microglial cell is both a glial cell of the central nervous system and a mononuclear phagocyte, which belongs to the haematopoietic system and is involved in inflammatory and immune responses. As such, microglia face a challenging task. The neurons of the central nervous system cannot divide and be replenished, and therefore need to be protected against pathogens, which is a key role of the immune system, but without collateral damage. In addition, after physical injury, neural cells need restorative support, which is provided by inflammatory responses. Excessive or chronic inflammatory responses can, however, be harmful. How microglia balance these demands, and how their behaviour can be modified to ameliorate disorders of the central nervous system, is becoming clear.
Treatment of cell lines with type I IFNs activates the formation of IFN-stimulated gene factor 3 (STAT1/STAT2/IFN regulatory factor-9), which induces the expression of many genes. To study this response in primary cells, we treated fresh human blood with IFN-? and used flow cytometry to analyze phosphorylated STAT1, STAT3, and STAT5 in CD4(+) and CD8(+) T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes and CD4(+) and CD8(+) T cells, few B cells activated STAT1 in response to IFN-?, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of suppressor of cytokine signaling 1 or relevant protein tyrosine phosphatases in B cells. Microarray and real-time PCR analyses revealed the induction of STAT1-dependent proapoptotic mRNAs in monocytes but not in B cells. These data show that IFN-stimulated gene factor 3 or STAT1 homodimers are not the main activators of gene expression in primary B cells of healthy humans. Notably, in B cells and, especially in CD4(+) T cells, IFN-? activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-? increases the survival of primary human B cells and CD4(+) T cells but enhances the apoptosis of monocytes, as well as to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections and by type I IFN treatment of patients with multiple sclerosis, hepatitis, or cancer.
CC chemokine receptor 2 (CCR2) is essential to acute skeletal muscle injury repair. We studied the subpopulation of inflammatory cells recruited via CCR2 signaling and their cellular functions with respect to muscle regeneration. Mobilization of monocytes/macrophages (MOs/MPs), but not lymphocytes or neutrophils, was impaired from bone marrow to blood and from blood to injured muscle in Ccr2(-/-) mice. While the Ly-6C(+) but not the Ly-6C(-) subset of MOs/MPs was significantly reduced in blood, both subsets were drastically reduced in injured muscle of Ccr2(-/-) mice. Expression of insulin-like growth factor-1 (IGF-I) was markedly up-regulated in injured muscle of wild-type but not Ccr2(-/-) mice. IGF-I was strongly expressed by macrophages within injured muscle, more prominently by the Ly-6C(-) subset. A single injection of IGF-I, but not PBS, into injured muscle to replace IGF-I remarkably improved muscle regeneration in Ccr2(-/-) mice. CCR2 was not detected in myogenic cells or capillary endothelial cells in injured muscle to suggest its direct involvement in muscle regeneration or angiogenesis. We conclude that CCR2 is essential to acute skeletal muscle injury repair primarily by recruiting Ly-6C(+) MOs/MPs. Within injured muscle, these cells conduct phagocytosis, contribute to accumulation of intramuscular Ly-6C(-) macrophages, and produce a high level of IGF-I to promote muscle regeneration.
Microglia, the primary immune effector cells in the brain, continually monitor the tissue parenchyma for pathological alterations and become activated in Alzheimers disease. Loss of signaling between neurons and microglia via deletion of the microglial receptor, CX3CR1, worsens phenotypes in various models of neurodegenerative diseases. In contrast, CX3CR1 deficiency ameliorates pathology in murine stroke models. To examine the role of CX3CR1 in Alzheimers disease-related ?-amyloid pathology, we generated APPPS1 and R1.40 transgenic mouse models of Alzheimers disease deficient for CX3CR1. Surprisingly, CX3CR1 deficiency resulted in a gene dose-dependent reduction in ?-amyloid deposition in both the APPPS1 and R1.40 mouse models of AD. Immunohistochemical analysis revealed reduced staining for CD68, a marker of microglial activation. Furthermore, quantitative immunohistochemical analysis revealed reduced numbers of microglia surrounding ?-amyloid deposits in the CX3CR1-deficient APPPS1 animals. The reduced ?-amyloid pathology correlated with reduced levels of TNF? and CCL2 mRNAs, but elevated IL1? mRNA levels, suggesting an altered neuroinflammatory milieu. Finally, to account for these seemingly disparate results, both in vitro and in vivo studies provided evidence that CX3CL1/CX3CR1 signaling alters the phagocytic capacity of microglia, including the uptake of A? fibrils. Taken together, these results demonstrate that loss of neuron-microglial fractalkine signaling leads to reduced ?-amyloid deposition in mouse models of AD that is potentially mediated by altered activation and phagocytic capability of CX3CR1-deficient microglia.
Aggregates of the hyperphosphorylated microtubule-associated protein tau (MAPT) are an invariant neuropathological feature of tauopathies. Here, we show that microglial neuroinflammation promotes MAPT phosphorylation and aggregation. First, lipopolysaccharide-induced microglial activation promotes hyperphosphorylation of endogenous mouse MAPT in nontransgenic mice that is further enhanced in mice lacking the microglial-specific fractalkine receptor (CX3CR1) and is dependent upon functional toll-like receptor 4 and interleukin-1 (IL-1) receptors. Second, humanized MAPT transgenic mice lacking CX3CR1 exhibited enhanced MAPT phosphorylation and aggregation as well as behavioral impairments that correlated with increased levels of active p38 MAPK. Third, in vitro experiments demonstrate that microglial activation elevates the level of active p38 MAPK and enhances MAPT hyperphosphorylation within neurons that can be blocked by administration of an interleukin-1 receptor antagonist and a specific p38 MAPK inhibitor. Taken together, our results suggest that CX3CR1 and IL-1/p38 MAPK may serve as novel therapeutic targets for human tauopathies.
The infiltration of leukocytes into the central nervous system (CNS) is an essential step in the neuropathogenesis of multiple sclerosis (MS). Leukocyte extravasation from the bloodstream is a multistep process that depends on several factors including fluid dynamics within the vasculature and molecular interactions between circulating leukocytes and the vascular endothelium. An important step in this cascade is the presence of chemokines on the vascular endothelial cell surface. Chemokines displayed along the endothelial lumen bind chemokine receptors on circulating leukocytes, initiating intracellular signaling that culminates in integrin activation, leukocyte arrest, and extravasation. The presence of chemokines at the endothelial lumen can help guide the movement of leukocytes through peripheral tissues during normal immune surveillance, host defense or inflammation. The expression and display of homeostatic or inflammatory chemokines therefore critically determine which leukocyte subsets extravasate and enter the peripheral tissues. Within the CNS, however, infiltrating leukocytes that cross the endothelium face additional boundaries to parenchymal entry, including the abluminal presence of localizing cues that prevent egress from perivascular spaces. This review focuses on the differential display of chemokines along endothelial surfaces and how they impact leukocyte extravasation into parenchymal tissues, especially within the CNS. In particular, the display of chemokines by endothelial cells of the blood brain barrier may be altered during CNS autoimmune disease, promoting leukocyte entry into this immunologically distinct site. Recent advances in microscopic techniques, including two-photon and intravital imaging have provided new insights into the mechanisms of chemokine-mediated capture of leukocytes within the CNS.
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS and remyelination in MS ultimately fails. Although strategies to promote myelin repair are eagerly sought, mechanisms underlying remyelination in vivo have been elusive. CXCR2 is expressed on neutrophils and oligodendrocyte lineage cells in the CNS. CXCR2-positive neutrophils facilitate inflammatory demyelination in demyelination models such as experimental autoimmune encephalomyelitis (EAE) and cuprizone intoxication. Systemic injection of a small molecule CXCR2 antagonist at the onset of EAE decreased demyelinated lesions. These results left the cellular target of the CXCR2 antagonist uncertain and did not clarify whether CXCR2 blockade prevented demyelination or promoted remyelination. Here, we show that the actions of CXCR2 on nonhematopoietic cells unexpectedly delay myelin repair. Bone marrow chimeric mice (Cxcr2(+/-)-->Cxcr2(-/-) and Cxcr2(+/-)-->Cxcr2(+/+)) were subjected to two distinct models of myelin injury. In all cases, myelin repair was more efficient in Cxcr2(+/-)-->Cxcr2(-/-) animals. Oligodendrocyte progenitor cells (OPCs) in demyelinated lesions of Cxcr2(+/-)-->Cxcr2(-/-) mice proliferated earlier and more vigorously than in tissues from Cxcr2(+/-)--> Cxcr2(+/+) animals. In vitro demyelinated CNS slice cultures also showed better myelin repair when CXCR2 was blocked with neutralizing antibodies or was genetically deleted. Our results suggest that CXCR2 inactivation permits optimal spatiotemporal positioning of OPCs in demyelinating lesions to receive local proliferative and differentiating signals. Given that CXCR2 exerts dual functions that promote demyelination and decrease remyelination by actions toward hematopoietic cells and nonhematopoietic cells, respectively, our findings identify CXCR2 as a promising drug target for clinical demyelinating disorders.
The functional role of ELR-positive CXC chemokines during viral-induced demyelination was assessed. Inoculation of the neuroattenuated JHM strain of mouse hepatitis virus (JHMV) into the CNS of susceptible mice results in an acute encephalomyelitis that evolves into a chronic demyelinating disease, modeling white matter pathology observed in the human demyelinating disease Multiple Sclerosis.
The etiology of delayed cerebral vasospasm (DCV) after aneurysmal subarachnoid hemorrhage (SAH) has remained elusive. Growing evidence supports a role for inflammation in the pathogenesis of DCV. We showed that CSF neutrophils predict which patients will develop DCV.
Leukocyte access into the central nervous system (CNS) parenchyma is tightly regulated by the blood-brain barrier (BBB). Leukocyte migration through the endothelial cell wall into the perivascular space is well characterized; however, mechanisms regulating their penetration through the glia limitans into the parenchyma are less well studied, and the role of monocytes relative to neutrophils is poorly defined. Acute viral encephalitis was thus induced in CCL2-deficient (CCL2(-/-)) mice to specifically abrogate monocyte recruitment. Impaired monocyte recruitment prolonged T cell retention in the perivascular space, although no difference in overall CNS accumulation of CD4 or CD8 T cells was detected by flow cytometry. Delayed penetration to the CNS parenchyma was not associated with reduced or altered expression of either matrix metalloproteinases (MMP) or the T cell chemoattractants CXCL10 and CCL5. Nevertheless, decreased parenchymal leukocyte infiltration delayed T cell-mediated control of virus replication as well as clinical disease. These data are the first to demonstrate that the rapid monocyte recruitment into the CNS during viral encephalitis is dispensable for T cell migration across the blood vessel endothelium. However, monocytes facilitate penetration through the glia limitans. Thus, the rapid monocyte response to viral encephalitis constitutes an indirect antiviral pathway by aiding access of effector T cells to the site of viral infection.
Multiple sclerosis is an inflammatory demyelinating disorder of the CNS. Recent studies have suggested diverse mechanisms as underlying demyelination, including a subset of lesions induced by an interaction between metabolic insult to oligodendrocytes and inflammatory mediators. For mice of susceptible strains, cuprizone feeding results in oligodendrocyte cell loss and demyelination of the corpus callosum. Remyelination ensues and has been extensively studied. Cuprizone-induced demyelination remains incompletely characterized. We found that mice lacking the type 2 CXC chemokine receptor (CXCR2) were relatively resistant to cuprizone-induced demyelination and that circulating CXCR2-positive neutrophils were important for cuprizone-induced demyelination. Our findings support a two-hit process of cuprizone-induced demyelination, supporting the idea that multiple sclerosis pathogenesis features extensive oligodendrocyte cell loss. These data suggest that cuprizone-induced demyelination is useful for modeling certain aspects of multiple sclerosis pathogenesis.
Interleukin-17 (IL-17) secreted by T helper 17 (Th17) cells is essential in the development of experimental autoimmune encephalomyelitis (EAE). However, it remains unclear how IL-17-mediated signaling in different cellular compartments participates in the central nervous system (CNS) inflammatory process. We examined CNS inflammation in mice with specific deletion of Act1, a critical component required for IL-17 signaling, in endothelial cells, macrophages and microglia, and neuroectoderm (neurons, astrocytes, and oligodendrocytes). In Act1-deficient mice, Th17 cells showed normal infiltration into the CNS but failed to recruit lymphocytes, neutrophils, and macrophages. Act1 deficiency in endothelial cells or in macrophages and microglia did not substantially impact the development of EAE. However, targeted Act1 deficiency in neuroectoderm-derived CNS-resident cells resulted in markedly reduced severity in EAE. Specifically, Act1-deficient astrocytes showed impaired IL-17-mediated inflammatory gene induction. Thus, astroctyes are critical in IL-17-Act1-mediated leukocyte recruitment during autoimmune-induced inflammation of the CNS.
Activation of chemokine genes in response to interferon (IFN)-gamma or NF-kappaB is an important aspect of inflammation. Using the chemokine gene ip-10 in mouse embryonic fibroblast cells as an example, we show that the response to IFN-gamma is long lasting but secondary: initial STAT1 activation drives IRF1 synthesis, and IRF1 then binds to IFN-stimulated regulatory elements (ISREs) in the ip-10 promoter. The promoters of most IKK-beta-dependent IFN-stimulated genes (ISGs) also contain ISREs. In response to IFN-gamma, inhibitor of NF-kappaB (IkappaB) kinase beta (IKK-beta) is required to activate both newly synthesized IRF1 and the p65 subunit of NF-kappaB, which contributes to ip-10 expression by binding to kappaB sites in the ip-10 promoter, with little or no activation of classical NF-kappaB. In contrast to IFN-gamma, IL-1beta induces ip-10 expression rapidly but transiently, by activating classical NF-kappaB and increasing the synthesis of IRF1. Together, IL-1beta and IFN-gamma induce ip-10 synergistically. IFN-gamma does not affect the transient activation of classical NF-kappaB by IL-1beta and synergistic induction of ip-10 expression by IFN-gamma and IL-1beta occurs even after the activation of classical NF-kappaB has returned to basal levels. Therefore, IKK-beta has a novel role in IFN-gamma-dependent activation of chemokine gene expression through its activation of IRF1 and p65.
The role of innate immunity and macrophage recruitment to the inner ear after hair cell injury is a subject where little is known. In this paper, we demonstrate recruitment of monocytes and macrophages to the inner ear after kanamycin. We also examined the effect of fractalkine receptor (CX3CR1) deletion in kanamycin ototoxicity. We observed more functional and structural damage in CX3CR1 null mice compared to wild-type and heterozygous littermates. In order to determine if increased susceptibility to kanamycin resulted from CX3CR1 deletion from cochlear leukocytes, we created bone marrow chimeras by transplanting CX3CR1-null bone marrow into wild-type mice whose native bone marrow was ablated by lethal irradiation. These mice were then treated with kanamycin sulfate. Auditory brainstem responses (ABR), hair cell counts, and numbers of macrophages recruited to the cochlea were recorded in irradiated mice that received either wild-type, CX3CR1 heterozygous, or CX3CR1 knockout bone marrow. A strong correlation was present between numbers of macrophages and hair cell death in recipients transplanted with CX3CR1 null marrow. No correlation between macrophage number and hair cell loss was present in mice transplanted with wild-type or CX3CR1 heterozygous marrow. We suggest that CX3CR1 plays a role in modulating the detrimental effects of cochlear macrophages after kanamycin ototoxicity. Our data point to the possibility that CX3CR1-deficient cochlear macrophages exacerbate kanamycin ototoxicity while CX3CR1-expressing monocytes do not.
The mouse spinal cord is an important site for autoimmune and injury models. Skull thinning surgery provides a minimally invasive window for microscopy of the mouse cerebral cortex, but there are no parallel methods for the spinal cord. We introduce a novel, facile and inexpensive method for two-photon laser scanning microscopy of the intact spinal cord in the mouse by taking advantage of the naturally accessible intervertebral space. These are powerful methods when combined with gene-targeted mice in which endogenous immune cells are labeled with green fluorescent protein (GFP). We first demonstrate that generation of the intervertebral window does not elicit a reaction of GFP(+) microglial cells in CX3CR1(gfp/+) mice. We next demonstrate a distinct rostrocaudal migration of GFP(+) immune cells in the spinal cord of CXCR6(gfp/+) mice during active experimental autoimmune encephalomyelitis (EAE). Interestingly, infiltration of the cerebral cortex by GFP(+) cells in these mice required three conditions: EAE induction, cortical injury and expression of CXCR6 on immune cells.
There are several molecular entities common to the immune and nervous systems. Salient among them are the chemokines and their receptors, which play remarkably varied and potent roles in immunobiology and neurobiology. This review limns several illustrative examples and presents general principles of chemokine action that are manifest in both systems. These include the following: (1) chemokines tend equally to arrest cells and to make them move, in the process of positioning target cells with spatiotemporal precision; (2) signaling and nonsignaling receptors collaborate to adjust the chemokine environment for maximal efficacy; and (3) expression of a single chemokine receptor on circulating blood cells and parenchymal cells is often used to coordinate complex tissue responses. The challenge is to integrate knowledge of the roles of key receptors (as well as their ligands) into a coherent account of events during pathologic processes, in order to guide therapeutic development.
The formation of blood vessels in embryos and tumors are different processes but under the control of common molecular mechanisms. Chemokine CXCL12 involved in both embryonic and tumor angiogenesis. In this review, we summarize recent advances in understanding the roles of CXCL12 in brain tumor angiogenesis/vasculogenesis. CXCL12 and its cognate receptors are abnormally induced in brain tumors, in particular in tumor cells and endothelium. Pathologically enhanced CXCL12 signaling may promote the formation of new vessels through recruiting circulating endothelial progenitor cells or directly enhancing the migration/growth of endothelial cells. Therefore, CXCL12 signaling represents an important mechanism that regulates brain tumor angiogenesis/vasculogenesis and may provide potential targets for anti-angiogenic therapy in malignant gliomas.
The onset of neurological signs in experimental autoimmune encephalomyelitis is tightly associated with infiltration and reactivation of T cells in the central nervous system. The anatomic localization of the initial T cell-antigen-presenting cell (APC) interactions leading to reactivation of T cells in the central nervous system is, however, still unclear. We hypothesized that activated CD4(+) T cells gain direct access to the subarachnoid space and become reactivated on encounter with cognate antigen in this compartment.
Chemokines are a family of mainly-secreted proteins, traditionally associated with regulation of leukocyte trafficking during host defense and pathological immune/inflammatory reactions. All chemokines signal to G protein-coupled receptors. Recent studies show that chemokines and their receptors are also expressed by neuroepithelial cells, and govern developmental, physiological and pathological processes through actions towards these cells, as well as infiltrating and resident hematopoietic cells. Understanding chemokine action at the tissue level therefore requires defining which cells express chemokine receptors. At a first level of approximation (and lacking appropriate immunohistochemical reagents) this determination can be made by in situ hybridization (ISH), which localizes mRNA expression for chemokines and their receptors at the cellular level. Here we provide a protocol for ISH and demonstrate its application for localizing mRNA encoding two chemokine receptors, CXCR4 and CXCR7 in murine CNS tissues.
The functional role of ELR-positive CXC chemokines in host defense during acute viral-induced encephalomyelitis was determined. Inoculation of the neurotropic JHM strain of mouse hepatitis virus (JHMV) into the central nervous system (CNS) of mice resulted in the rapid mobilization of PMNs expressing the chemokine receptor CXCR2 into the blood. Migration of PMNs to the CNS coincided with increased expression of transcripts specific for the CXCR2 ELR-positive chemokine ligands CXCL1, CXCL2, and CXCL5 within the brain. Treatment of JHMV-infected mice with anti-CXCR2 blocking antibody reduced PMN trafficking into the CNS by >95%, dampened MMP-9 activity, and abrogated blood-brain-barrier (BBB) breakdown. Correspondingly, CXCR2 neutralization resulted in diminished infiltration of virus-specific T cells, an inability to control viral replication within the brain, and 100% mortality. Blocking CXCR2 signaling did not impair the generation of virus-specific T cells, indicating that CXCR2 is not required to tailor anti-JHMV T cell responses. Evaluation of mice in which CXCR2 is genetically silenced (CXCR2-/- mice) confirmed that PMNs neither expressed CXCR2 nor migrated in response to ligands CXCL1, CXCL2, or CXCL5 in an in vitro chemotaxis assay. Moreover, JHMV infection of CXCR2-/- mice resulted in an approximate 60% reduction of PMN migration into the CNS, yet these mice survived infection and controlled viral replication within the brain. Treatment of JHMV-infected CXCR2-/- mice with anti-CXCR2 antibody did not modulate PMN migration nor alter viral clearance or mortality, indicating the existence of compensatory mechanisms that facilitate sufficient migration of PMNs into the CNS in the absence of CXCR2. Collectively, these findings highlight a previously unappreciated role for ELR-positive chemokines in enhancing host defense during acute viral infections of the CNS.
Interferons (IFNs) are widely used in therapy for viral, neoplastic, and inflammatory disorders, but clinical response varies among patients. The biological basis for variable clinical response is not known. We determined the primary molecular response to IFN-beta (IFN-beta) injections in 35 treatment-naïve multiple sclerosis (MS) patients using a customized cDNA macroarray with 186 interferon-stimulated genes (ISGs). Our results revealed striking interindividual heterogeneity, both in the magnitude as well as the nature of the primary molecular response to IFN-beta injections. Despite marked between-subject variability in the molecular response, responses within individual subjects were stable over a 6-month interval. Our data suggest that clinical response to IFN-beta therapy for MS differs among patients because of qualitative rather than quantitative variability in the primary molecular response to the drug.
Microglia, the macrophages of the central nervous system parenchyma, have in the normal healthy brain a distinct phenotype induced by molecules expressed on or secreted by adjacent neurons and astrocytes, and this phenotype is maintained in part by virtue of the blood-brain barriers exclusion of serum components. Microglia are continually active, their processes palpating and surveying their local microenvironment. The microglia rapidly change their phenotype in response to any disturbance of nervous system homeostasis and are commonly referred to as activated on the basis of the changes in their morphology or expression of cell surface antigens. A wealth of data now demonstrate that the microglia have very diverse effector functions, in line with macrophage populations in other organs. The term activated microglia needs to be qualified to reflect the distinct and very different states of activation-associated effector functions in different disease states. Manipulating the effector functions of microglia has the potential to modify the outcome of diverse neurological diseases.
Myelopathy is an uncommon complication of VZV infection and may develop in the absence of rash. We report the rare recurrence of myelopathy in an immunocompetent adult who initially developed myelopathy after ophthalmic-distribution zoster. Recurrent myelopathy two years later caused by VZV was verified by the presence of new-onset clinical symptoms and signs consistent with myelopathy, new lesions in the spinal cord detected by MRI, and evidence of intrathecal synthesis of anti-VZV IgG antibody in CSF. After virological verification and antiviral therapy, myelopathy did not recur and anti-VZV IgG antibody could not be detected in CSF. In contrast to earlier cases of recurrent VZV myelopathy in immunocompetent adults that developed weeks to months after the first episode of myelopathy, this is the first instance of recurrence years later.
Mouse models take advantage of genetic manipulations that can be achieved in this species. There are currently two accepted mouse models of subarachnoid hemorrhage (SAH) and cerebral vasospasm (CVs). Both are technically demanding and labor intensive. In this study, we report a reproducible and technically feasible method to induce SAH, and subsequently CVs, in mice. We tested this model in multiple strains of mice that are commonly used for genetic manipulation.
Duchenne-Meryon muscular dystrophy (DMD) is the most common and lethal genetic muscle disease. Ameliorating muscle necrosis, inflammation, and fibrosis represents an important therapeutic approach for DMD. Imatinib, an antineoplastic agent, demonstrated antiinflammatory and antifibrotic effects in liver, kidney, lung, and skin of various animal models. This study tested antiinflammatory and antifibrotic effects of imatinib in mdx mice, a DMD mouse model. We treated mdx mice with intraperitoneal injections of imatinib at the peak of limb muscle inflammation and the onset of diaphragm fibrosis. Controls received PBS vehicle or were left untreated. Muscle necrosis, inflammation, fibrosis, and function were evaluated by measuring serum CK activity, endomysial CD45 immunoreactive inflammation area, endomysial collagen III deposition, and hind limb grip strength. Phosphorylation of the tyrosine kinase targets of imatinib was assessed by Western blot in diaphragm tissue and in primary cultures of peritoneal macrophages and skeletal muscle fibroblasts. Imatinib markedly reduced muscle necrosis, inflammation, and fibrosis, and significantly improved hind limb grip strength in mdx mice. Reduced clinical disease was accompanied by inhibition of c-abl and PDGFR phosphorylation and suppression of TNF-alpha and IL-1beta expression. Imatinib therapy for DMD may hold promise for ameliorating muscle necrosis, inflammation, and fibrosis by inhibiting c-abl and PDGFR signaling pathways and downstream inflammatory cytokine and fibrotic gene expression.
Accumulation of aggregated amyloid-beta (Abeta) peptide was studied as an initial step for Alzheimers disease (AD) pathogenesis. Following amyloid plaque formation, reactive microglia and astrocytes accumulate around plaques and cause neuroinflammation. Here brain chemokines play a major role for the glial accumulation. We have previously shown that transgenic overexpression of chemokine CCL2 in the brain results in increased microglial accumulation and diffuse amyloid plaque deposition in a transgenic mouse model of AD expressing Swedish amyloid precursor protein (APP) mutant. Here, we report that adeno-associated virus (AAV) serotype 1 and 2 hybrid efficiently deliver 7ND gene, a dominant-negative CCL2 mutant, in a dose-response manner and express >1,000-fold higher recombinant CCL2 than basal levels after a single administration. AAV1/2 hybrid virus principally infected neurons without neuroinflammation with sustained expression for 6-months. 7ND expressed in APP/presenilin-1 (APP/PS1) bigenic mice reduced astro/microgliosis, beta-amyloidosis, including suppression of both fibrillar and oligomer Abeta accumulation, and improved spatial learning. Our data support the idea that the AAV1/2 system is a useful tool for CNS gene delivery, and suppression of CCL2 may be a therapeutic target for the amelioration of AD-related neuroinflammation.
Insights into sequential leukocyte-endothelial interactions during leukocyte trafficking have been obtained through experiments using human umbilical vein endothelial cells (HUVEC) under flow conditions. To investigate leukocyte-brain endothelial cell interactions, we developed a dynamic in vitro system, using Transfected Human Brain Microvascular Endothelial Cells (THBMEC) and a parallel plate flow chamber. Human peripheral blood mononuclear cells (PBMC) were perfused across confluent THBMEC cultures at a velocity that approximates the rate found in human brain capillaries. Leukocyte-THBMEC interactions were visualized by phase-contrast microscopy, and images were captured on a CCD camera. To simulate inflammatory conditions, we activated THBMEC with the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma), which up-regulated chemokine and adhesion molecule expression in THBMEC without affecting the distribution of immunoreactivity for tight junction-associated proteins. PBMC adhesion was enhanced by cytokine-mediated activation of THBMEC. G protein-coupled receptor (GPCR) activation was essential for leukocyte-THBMEC interaction, as pertussis toxin (PTX) treatment of PBMC abrogated PBMC adhesion to activated THBMEC. The anti-alpha4 integrin antibody, natalizumab, infused into MS patients, significantly reduced the adhesion of their ex vivo PBMC to activated THBMEC under flow conditions. Further study showed that alternatively spliced fibronectin containing the CS1 region (FN-CS1), but not Vascular Cell Adhesion Molecule type 1 (VCAM-1), was the ligand of alpha4 integrin on activated THBMEC. Blocking FN-CS1 abrogated PBMC adhesion on activated THBMEC, while anti-VCAM-1 antibodies had no effect. These results established a novel in vitro dynamic BBB model. We also demonstrated the dependence of leukocyte-endothelial interactions in this model on alpha4 integrins and FN-CS1.
The linkages between neuroinflammation and Alzheimers disease (AD) pathogenesis are well established. What is not, however, is how specific immune pathways and proteins affect the disease. To this end, we previously demonstrated that transgenic over-expression of CCL2 enhanced microgliosis and induced diffuse amyloid plaque deposition in Tg2576 mice. This rodent model of AD expresses a Swedish beta-amyloid (Abeta) precursor protein mutant.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.