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Microglia have been implicated in multiple sclerosis (MS) pathogenesis. The fractalkine receptor CX3CR1 limits the activation of pathogenic microglia and the human polymorphic CX3CR1

Microglia have been implicated in multiple sclerosis (MS) pathogenesis. The fractalkine receptor CX3CR1 regulates the activation of pathogenic microglia in models of MS and the human polymorphic CX3CR1I249/M280 (hCX3CR1I249/M280) variant increases MS disease progression. However, the role of hCX3CR1I249/M280 on microglial activation and central nervous system repair and regenerative mechanisms remain unknown. Therefore, using transgenic mice expressing the hCX3CR1I249/M280 variant, we aimed to determine the contribution of defective CX3CR1 signaling to remyelination and neurogenesis in the cuprizone model of focal demyelination. Here, we report that mice expressing hCX3CR1I249/M280 exhibit marked demyelination and microgliosis follow acute cuprizone treatment. Cuprizone-treated CX3CR1-deficient and fractalkine-deficient mice displayed a comparable phenotype. Nanostring gene expression analysis in demyelinated lesions showed that hCX3CR1I249/M280 upregulates genes associated with inflammation, oxidative stress and disease-associated microglia. In addition, gene expression analysis in the subgranular zone (SGZ) of the hippocampus in hCX3CR1I249/M280 mice was associated with a significant downregulation of gene networks linked to neurogenesis following acute demyelination. Confocal microscopy showed that hCX3CR1I249/M280 or loss of CX3CR1 signaling inhibits the generation of progeny from the neurogenic niche, including cells involved in myelin repair. These results provide evidence for the pathogenic capacity of hCX3CR1I249/M280 on microglia dysfunction and therapeutic targeting of CX3CR1 to promote CNS repair in MS.

Microglia-mediated inflammation plays a significant role in neuronal and vascular damage in diabetic retinopathy, but the mechanism linking inflammation, neurodegeneration, and impaired vascular integrity is still unclear. Our previous studies from diabetic mouse models showed accumulation of fibrinogen, at vessel lesions surrounded by perivascular microglial clusters. In this study, we evaluated whether the pathological hallmarks of gliosis and vascular aberrations characterized in diabetic animal models are consistent with those in diabetic human retinas. Postmortem human retinas were analyzed by immunohistochemistry for markers of gliosis, vascular integrity, and fibrinogen deposition. Immunohistochemical and gene expression analyses of human postmortem retinas revealed evidence of an inflammatory microenvironment, with microgliosis and impaired vasculature. To define the therapeutic potential of reducing fibrinogen in DR, the defibrinogenating agent ancrod was administered in a two-hit inflammatory diabetic mouse model, after which retinal pathology and visual acuity were assessed. Histopathological analyses revealed microglial activation, vascular aberrations, and fibrinogen deposition in the diabetic murine retina. Notably, after treatment with ancrod, diabetic mice appeared to improve visual acuity, which was associated with reduced microglia activation and less fibrinogen deposition in the retina. This study shows that fibrinogen-mediated microglial activation, blood-retinal barrier damage, and vision loss, can be ameliorated by reducing fibrinogen levels. Overall, these findings suggest that that fibrinogen contributes to microglia-mediated inflammation in the diabetic retina. Supported by R01 EY029913

Histone methyltransferases (HMTs) are present in heterogeneous cell populations within the adult brain including neurogenic niches. Yet the question remains whether loss of HMTs and the resulting changes in histone methylation alter cell fate in a region-specific manner. We utilized stereotaxic injection of Cre recombinant protein into the adult neurogenic niches, the subventricular zone (SVZ) adjacent to the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus. We confirmed that Cre protein was enzymatically active in vivo and recombination events were restricted to the vicinity of injection areas. In this study, we focus on using Cre mediated recombination in mice harboring floxed HMT: enhancer of zeste homolog 2 (EZH2) or suppressor of variegation homolog (Suv4-20h). Injectable Cre protein successfully knocked out either EZH2 or Suv4-20h, allowing assessment of long-term effects in a region-specific fashion. We performed meso-scale imaging and flow cytometry for phenotype analysis and unbiased quantification. We demonstrated that regional loss of EZH2 affects the differentiation paradigm of neural stem progenitor cells as well as the maintenance of stem cell population. We further demonstrated that regional loss of Suv4-20h influences the cell cycle but does not affect stem cell differentiation patterns. Therefore, Cre protein mediated knock-out a given HMT unravel their distinguishable and important roles in adult neurogenic niches. This Cre protein-based approach offers tightly-controlled knockouts in multiple cell types simultaneously for studying diverse regulatory mechanisms and is optimal for region-specific manipulation within complex, heterogeneous brain architectures.

It is becoming increasingly clear that neuroinflammation has a causal role in the pathogenesis of central nervous system (CNS)-related diseases, and therefore therapeutic strategies targeting the regulation or availability of inflammatory mediators can be used to prevent or mitigate pathology. Interestingly, the proinflammatory cytokine, interleukin-1 beta (IL-1β), has been implicated in perpetuating immune responses and contributing to disease severity in a variety of CNS diseases ranging from multiple sclerosis, neurodegenerative diseases, traumatic brain injury, and diabetic retinopathy. Moreover, pharmacological blockade of IL-1 signaling has shown to be beneficial in some autoimmune and autoinflammatory diseases, making IL-1β a promising therapeutic target in neuroinflammatory conditions. This review highlights recent advances of our understanding on the multifaceted roles of IL-1β in neuroinflammatory diseases.

Neural stem cells (NSCs) exist throughout life in the ventricular–subventricular zone (V-SVZ) of the mammalian forebrain. During aging NSC function is diminished through an unclear mechanism. In this study, we establish microglia, the immune cells of the brain, as integral niche cells within the V-SVZ that undergo age-associated repositioning in the V-SVZ. Microglia become activated early before NSC deficits during aging resulting in an antineurogenic microenvironment due to increased inflammatory cytokine secretion. These age-associated changes were not observed in non-neurogenic brain regions, suggesting V-SVZ microglia are specialized. Using a sustained inflammatory model in young adult mice, we induced microglia activation and inflammation that was accompanied by reduced NSC proliferation in the V-SVZ. Furthermore, in vitro studies revealed secreted factors from activated microglia reduced proliferation and neuron production compared to secreted factors from resting microglia. Our results suggest that age-associated chronic inflammation contributes to declines in NSC function within the aging neurogenic niche.

Background Fractalkine (CX3CL1) and its receptor (CX3CR1) play an important role in regulating microglial function. We have previously shown that Cx 3 cr1 deficiency exacerbated tau pathology and led to cognitive impairment. However, it is still unclear if the chemokine domain of the ligand CX3CL1 is essential in regulating neuronal tau pathology. Methods We used transgenic mice lacking endogenous Cx 3 cl1 (Cx 3 cl1 −/−) and expressing only obligatory soluble form (with only chemokine domain) and lacking the mucin stalk of CX3CL1 (referred to as Cx 3 cl1 105Δ mice) to assess tau pathology and behavioral function in both lipopolysaccharide (LPS) and genetic (hTau) mouse models of tauopathy. Results First, increased basal tau levels accompanied microglial activation in Cx 3 cl1 105Δ mice compared to control groups. Second, increased CD45+ and F4/80+ neuroinflammation and tau phosphorylation were observed in LPS, hTau/Cx 3 cl1 −/−, and hTau/Cx 3 cl1 105Δ mouse models of tau pathology, which correlated with impaired spatial learning. Finally, microglial cell surface expression of CX3CR1 was reduced in Cx 3 cl1 105Δ mice, suggesting enhanced fractalkine receptor internalization (mimicking Cx 3 cr1 deletion), which likely contributes to the elevated tau pathology. Conclusions Collectively, our data suggest that overexpression of only chemokine domain of CX3CL1 does not protect against tau pathology.

Background Cerebral malaria (CM) is the most lethal outcome of Plasmodium infection. There are clear correlations between expression of inflammatory cytokines, severe coagulopathies, and mortality in human CM. However, the mechanisms intertwining the coagulation and inflammation pathways, and their roles in CM, are only beginning to be understood. In mice with T cells deficient in the regulatory cytokine IL-10 (IL-10 KO), infection with Plasmodium chabaudi leads to a hyper-inflammatory response and lethal outcome that can be prevented by anti-TNF treatment. However, inflammatory T cells are adherent within the vasculature and not present in the brain parenchyma, suggesting a novel form of cerebral inflammation. We have previously documented behavioral dysfunction and microglial activation in infected IL-10 KO animals suggestive of neurological involvement driven by inflammation. In order to understand the relationship of intravascular inflammation to parenchymal dysfunction, we studied the congestion of vessels with leukocytes and fibrin(ogen) and the relationship of glial cell activation to congested vessels in the brains of P. chabaudi-infected IL-10 KO mice. Methods Using immunofluorescence microscopy, we describe severe thrombotic congestion in these animals. We stained for immune cell surface markers (CD45, CD11b, CD4), fibrin(ogen), microglia (Iba-1), and astrocytes (GFAP) in the brain at the peak of behavioral symptoms. Finally, we investigated the roles of inflammatory cytokine tumor necrosis factor (TNF) and coagulation on the pathology observed using neutralizing antibodies and low-molecular weight heparin to inhibit both inflammation and coagulation, respectively. Results Many blood vessels in the brain were congested with thrombi containing adherent leukocytes, including CD4 T cells and monocytes. Despite containment of the pathogen and leukocytes within the vasculature, activated microglia and astrocytes were prevalent in the parenchyma, particularly clustered near vessels with thrombi. Neutralization of TNF, or the coagulation cascade, significantly reduced both thrombus formation and gliosis in P. chabaudi-infected IL-10 KO mice. Conclusions These findings support the contribution of cytokines, coagulation, and leukocytes within the brain vasculature to neuropathology in malaria infection. Strikingly, localization of inflammatory leukocytes within intravascular clots suggests a mechanism for interaction between the two cascades by which cytokines drive local inflammation without considerable cellular infiltration into the brain parenchyma.

Pathological aggregation of tau is a hallmark of Alzheimer's disease and related tauopathies. We have previously shown that the deficiency of the microglial fractalkine receptor (CX3CR1) led to the acceleration of tau pathology and memory impairment in an hTau mouse model of tauopathy. Here, we show that microglia drive tau pathology in a cell-autonomous manner. First, tau hyperphosphorylation and aggregation occur as early as 2 months of age in hTauCx3cr1(-/-) mice. Second, CD45(+) microglial activation correlates with the spatial memory deficit and spread of tau pathology in the anatomically connected regions of the hippocampus. Third, adoptive transfer of purified microglia derived from hTauCx3cr1(-/-) mice induces tau hyperphosphorylation within the brains of non-transgenic recipient mice. Finally, inclusion of interleukin 1 receptor antagonist (Kineret®) in the adoptive transfer inoculum significantly reduces microglia-induced tau pathology. Together, our results suggest that reactive microglia are sufficient to drive tau pathology and correlate with the spread of pathological tau in the brain.

Monocytes are derived from hematopoietic stem cells through a series of intermediate progenitor stages, but the factors that regulate this process are incompletely defined. Using a Ccr2/Cx(3)cr1 dual-reporter system to model murine monocyte ontogeny, we conducted a small molecule screen that identified an essential role of mechanistic target of rapamycin complex 1 (mTORC1) in the development of monocytes and other myeloid cells. Confirmatory studies using mice with inducible deletion of the mTORC1 component Raptor demonstrated absence of mature circulating monocytes, as well as disruption in neutrophil and dendritic cell development, reflecting arrest of terminal differentiation at the granulocyte-monocyte progenitor (GMP) stage. Conversely, excess activation of mTORC1 through deletion of the mTORC1 inhibitor tuberous sclerosis complex 2 (Tsc2) promoted spontaneous myeloid cell development and maturation. Inhibitor studies and stage-specific expression profiling identified failure to downregulate the transcription factor Myc by the mTORC1 target ribosomal S6 kinase 1 (S6K1) as the mechanistic basis for disrupted myelopoiesis. Together, these findings define the mTORC1-S6K1-Myc pathway as a key checkpoint in terminal myeloid development.

Fractalkine (FKN) is a chemokine expressed constitutively by healthy neurons and signals to microglia upon interaction with the FKN receptor, CX3CR1. Signaling between FKN and CX3CR1 transduces inhibitory signals that ameliorate microglial activation and proinflammatory cytokine release in neuroinflammatory conditions. The aim of this study is to determine the mechanisms associated with microglial activation and vascular leakage during diabetic retinopathy (DR) and under conditions of low-level endotoxemia, common in diabetic patients. Utilizing the Ins2Akita strain (Akita), a mouse model of type 1 diabetes, our results show that leakage of the blood-protein fibrin(ogen) into the retina occurs as a result of chronic (4 months) but not acute (1.5 months) hyperglycemia. Conversely, inducing endotoxin-mediated systemic inflammation during acute diabetes resulted in fibrinogen deposition in the retina, a phenotype that was exacerbated in mice lacking CX3CR1 signaling. Systemic inflammation in Cx3cr1−/− mice led to robust perivascular clustering of proliferating microglia in areas of fibrinogen extravasation, and induced IL-1β expression in microglia and astrocytes. Lastly, we determined a protective effect of modulating FKN/CX3CR1 signaling in the diabetic retina. We show that intravitreal (iv) administration of recombinant FKN into diabetic FKN-KO mice, reduced fibrinogen deposition and perivascular clustering of microglia in the retina during systemic inflammation. These data suggest that dysregulated microglial activation via loss of FKN/CX3CR1 signaling disrupts the vascular integrity in retina during systemic inflammation.

[This corrects the article DOI: 10.1371/journal.pone.0013693.].

Induction of experimental autoimmune encephalomyelitis (EAE) in susceptible animals requires reactivation of encephalitogenic CD4+ T cells by APCs in the CNS. However, it has remained unresolved from where APCs in the CNS acquire myelin Ag for T cell activation and under which conditions, that is, whether only during EAE or also in the naive CNS. In this study, we investigated the kinetics of myelin Ag uptake by CNS APCs during EAE and in the naive CNS. Our results show that during EAE CX3CR1+CD11b+ microglia were the first APCs in the CNS to contain myelin Ag upon induction of disease, albeit in very small numbers. Dendritic cells (DCs) arrived in the CNS in sizable numbers significantly later (day 5 postimmunization), without detectable myelin Ag, but acquired it by day 7 postimmunization. Furthermore, a sharp increase in neuroantigen-containing DCs coincided with the onset of EAE symptoms. Importantly, in naive mice a low but consistent number of microglia contained myelin Ag, suggesting release by oligodendrocytes under steady state conditions. Although microglia isolated from naive brain and spinal cord did not elicit a strong CD4+ T cell response in vitro, myelin Ag-containing microglia may still play a local role in modulating encephalitogenic CD4+ T cell responses in early EAE prior to the arrival of other professional APCs, such as DCs. Finally, newly arriving DCs in the CNS not yet loaded with myelin Ag before the onset of EAE may be a potential therapeutic target.

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-γ 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-γ– 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.

Neurocysticercosis (NCC) is a central nervous system (CNS) infection caused by the metacestode stage of the parasite Taenia solium . During NCC, the parasites release immunodominant glycan antigens in the CNS environment, invoking immune responses. The majority of the associated pathogenesis is attributed to the immune response against the parasites. Glycans from a number of pathogens, including helminths, act as pathogen-associated molecular pattern molecules (PAMPs), which are recognized by pattern recognition receptors (PRRs) known as C-type lectin receptors (CLRs). Using a mouse model of NCC by infection with the related parasite Mesocestoides corti , we have investigated the role of mannose receptor C type 1 (MRC1), a CLR which recognizes high-mannose-containing glycan antigens. Here we show that MRC1 −/− mice exhibit increased survival times after infection compared with their wild-type (WT) counterparts. The decreased disease severity correlates with reduced levels of expression of markers implicated in NCC pathology, such as interleukin-1β (IL-1β), IL-6, CCL5, and matrix metalloproteinase 9 (MMP9), in addition to induction of an important repair marker, fibroblast growth factor 2 (FGF2). Furthermore, the immune cell subsets that infiltrate the brain of MRC1 −/− mice are dramatically altered and characterized by reduced numbers of T cells and the accumulation of granulocytic cells with an immune phenotype resembling granulocytic myeloid-dependent suppressor cells (gMDSCs). The results suggest that MRC1 plays a critical role in myeloid plasticity, which in turn affects the adaptive immune response and immunopathogenesis during murine NCC.

Fractalkine receptor (CX3CR1)-deficient mice develop very severe experimental autoimmune encephalomyelitis (EAE), associated with impaired NK cell recruitment into the CNS. Yet, the precise implications of NK cells in autoimmune neuroinflammation remain elusive. Here, we investigated the pattern of NK cell mobilization and the contribution of CX3CR1 to NK cell dynamics in the EAE. We show that in both wild-type and CX3CR1-deficient EAE mice, NK cells are mobilized from the periphery and accumulate in the inflamed CNS. However, in CX3CR1-deficient mice, the infiltrated NK cells displayed an immature phenotype contrasting with the mature infiltrates in WT mice. This shift in the immature/mature CNS ratio contributes to EAE exacerbation in CX3CR1-deficient mice, since transfer of mature WT NK cells prior to immunization exerted a protective effect and normalized the CNS NK cell ratio. Moreover, mature CD11b(+) NK cells show higher degranulation in the presence of autoreactive 2D2 transgenic CD4(+) T cells and kill these autoreactive cells more efficiently than the immature CD11b(-) fraction. Together, these data suggest a protective role of mature NK cells in EAE, possibly through direct modulation of T cells inside the CNS, and demonstrate that mature and immature NK cells are recruited into the CNS by distinct chemotactic signals.

Summary 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 CX3CR1high NK cells were more cytotoxic than their CX3CR1neg/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 CX3CR1high, CX3CR1neg/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 CX3CR1neg CD56bright, CX3CR1neg CD56dim and CX3CR1high CD56dim 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 CD56dim discriminated between an intermediary CX3CR1neg CD56dim and fully mature CX3CR1high CD56dim 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.

Microglia, the primary immune effector cells in the brain, continually monitor the tissue parenchyma for pathological alterations and become activated in Alzheimer’s 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 Alzheimer’s disease–related β-amyloid pathology, we generated APPPS1 and R1.40 transgenic mouse models of Alzheimer’s disease deficient for CX3CR1. Surprisingly, CX3CR1 deficiency resulted in a gene dose-dependent reduction in β-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 β-amyloid deposits in the CX3CR1-deficient APPPS1 animals. The reduced β-amyloid pathology correlated with reduced levels of TNFα and CCL2 mRNAs, but elevated IL1β 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 Aβ fibrils. Taken together, these results demonstrate that loss of neuron-microglial fractalkine signaling leads to reduced β-amyloid deposition in mouse models of AD that is potentially mediated by altered activation and phagocytic capability of CX3CR1-deficient microglia.

Neurocysticercosis (NCC) is the leading cause of acquired epilepsy worldwide caused by Taenia solium larvae. Disease progression involves an initial asymptomatic phase followed by a symptomatic period, in which dying organisms induce inflammation and symptoms such as seizures, intracranial hypertension, or cognitive decline. A mixed Th1/Th2 response develops in the brain, contrasting the canonical Th2 pathway associated with peripheral helminthic infections. We found that disease progression was more severe in BALB/c mice, correlating with increased parasite burdens and enhanced eosinophil and macrophage infiltration, although comparable numbers of T cells were detected. Moreover, elevated IL-1β levels were significantly elevated in BALB/c mice. Notably, Mrc1-deficient mice which exhibited a more resistant phenotype with decreased mortality and morbidity displayed low IL-1β expression. Therefore, we hypothesize that early up regulation of IL-1β in response to antigen recognition by macrophages plays a key role in damage to the nervous tissue. Comparison of glial and neuronal distribution in naïve and infected wild type C57BL6 and Balb/c mice, and also in strains with defective trafficking of eosinophils to the CNS, revealed that expression of IL-1β was detected early during infection and dramatically increased by 2 wks, colocalizing to macrophages and astrocytes. Notably, calbindin+ neurons were also significantly reduced in cerebellar regions where severe pockets of inflammatory cells are detected. Thus, our results suggest that IL-1β is a central proinflammatory mediator with neurotoxic potential during NCC infection.

Multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS is the leading cause of nontraumatic neurological disability in young adults. Immune mediated destruction of the myelin and oligodendrocytes are considered its primary pathology, but progressive axonal loss is the major cause of neurological disability. In an effort to understand microglia function in CNS inflammation, our laboratory showed that Fractalkine/CX3CR1 signaling regulates microglia neurotoxicity during neurodegeneration. Fractalkine (FKN), a transmembrane chemokine expressed in the CNS by neurons signals through its unique receptor, CX3CR1 present in microglia. During EAE, CX3CR1 deficiency confers exacerbated disease, severe inflammation and neuronal loss. The CX3CR1 human polymorphism I249/M280 present in ~20% of the population exhibits reduced adhesion for FKN conferring defective signaling whose role in microglia function and effect on neurons during MS remains unsolved. The aim of this study is to assess the effect of weaker signaling through hCX3CR1I249/M280 during EAE. We hypothesize that dysregulated microglial responses in absence of CX3CR1 signaling enhance neuronal/axonal damage. We generated an animal model replacing the mouse CX3CR1 locus for the hCX3CR1I249/M280 variant. Upon EAE induction, these mice exhibit exacerbated EAE defined by severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and CNTF during EAE as WT mice. Our results provide validation of defective function of the hCX3CR1I249/M280 variant and the foundation to broaden the understanding of microglia dysfunction during neuroinflammation.

Inflammatory demyelination is a hallmark of the pathology of multiple sclerosis (MS) and microglia are considered responsible for exacerbating myelin loss. The link between inflammation and neural stem cell (NSC) development, key events during the remyelination process, is being increasingly recognized. However, the mechanisms that connect microglia, myelination and inflammatory-mediated oxidative damage are unclear. Fractalkine (FKN), a neuronal derived chemokine, and its unique receptor, CX3CR1, expressed by microglia, are highly abundant in the healthy brain, and our laboratory has reported severe CNS demyelination and neuronal damage in EAE-induced CX3CR1-KO mice. Our hypothesis is that CX3CR1-defective signaling promotes inflammatory-mediated damage to neuron and myelin-forming cells. We found that expression of the adhesive defective human variant hCX3CR1-M280 caused enhanced disease severity, increased demyelination and exacerbated neuronal loss, mirroring the findings we observed in CX3CR1KO mice. Also, enhanced demyelination was observed after 4 wk of cuprizone treatment. Densities of NG2+ glia were comparable between WT and CX3CR1KO mice, but a significant reduction in DCX+ cells in the subgranular zone of the hippocampus was observed. In addition, lipocalin-2 an acute phase protein, linked to resolution of inflammation by reducing iron-induced toxicity, was found upregulated in cuprizone-induced demyelinated lesions of WT but not CX3CR1KO or hCX3CR1M280 mice. Thus, the FKN/CX3CR1 signaling pathway in microglia plays an under-appreciated role in MS, perhaps via anti-inflammatory processes that enhance neurogenesis through regulation of oxidative damage.

In vitro studies have implicated chemokine receptors in consumption and clearance of specific ligands. We studied the role that various signaling chemokine receptors play during ligand homeostasis in vivo. We examined the levels of ligands in serum and CNS tissue in mice lacking chemokine receptors. Compared with receptor-sufficient controls, Cx3cr1−/− mice exhibited augmented levels of CX3CL1 both in serum and brain, and circulating levels of CXCL1 and CXCL2 were increased in Cxcr2−/− mice. CCR2-deficient mice showed significantly increased amounts of circulating CCL2 compared with wild-type mice. Cxcr3−/− mice revealed increased levels of circulating and brain CXCL10 after experimental autoimmune encephalomyelitis (EAE) induction. CCR2-deficient peripheral blood and resident peritoneal cells exhibited reduced binding capacity and biologic responses to the CCR1 ligand CCL3, suggesting that elevated levels of CCR2 ligands had down-regulated CCR1. The results indicate that signaling chemokine receptors clear chemokines from circulation and tissues. These homeostatic functions of signaling chemokine receptors need to be integrated into safety and efficacy calculations when considering therapeutic receptor blockade.

Actions of chemokines and the interaction with specific receptors go beyond their original, defined role of recruiting leukocytes to inflamed tissues. Chemokine receptor expression in peripheral elements and resident cells of the central nervous system (CNS) represents a relevant communication system during neuroinflammatory conditions. The following examples are described in this review: Chemokine receptors play important homeostatic properties by regulating levels of specific ligands in blood and tissues during healthy and pathological conditions; chemokines and their receptors are clearly involved in leukocyte extravasation and recruitment to the CNS, and current studies are directed toward understanding the interaction between chemokine receptors and matrix metalloproteinases in the process of blood brain barrier breakdown. We also propose novel functions of chemokine receptors during demyelination/remyelination, and developmental processes.

Neurocysticercosis (NCC) is one of the most common helminth parasitic diseases of the central nervous system (CNS) and the leading cause of acquired epilepsy worldwide. NCC is caused by the presence of the metacestode larvae of the tapeworm Taenia solium within brain tissues. NCC patients exhibit a long asymptomatic phase followed by a phase of symptoms including increased intra-cranial pressure and seizures. While the asymptomatic phase is attributed to the immunosuppressive capabilities of viable T. solium parasites, release of antigens by dying organisms induce strong immune responses and associated symptoms. Previous studies in T. solium-infected pigs have shown that the inflammatory response consists of various leukocyte populations including eosinophils, macrophages, and T cells among others. Because the role of eosinophils within the brain has not been investigated during NCC, we examined parasite burden, disease susceptibility and the composition of the inflammatory reaction in the brains of infected wild type (WT) and eosinophil-deficient mice (ΔdblGATA) using a murine model of NCC in which mice were infected intracranially with Mesocestoides corti, a cestode parasite related to T. solium. In WT mice, we observed a time-dependent induction of eosinophil recruitment in infected mice, contrasting with an overall reduced leukocyte infiltration in ΔdblGATA brains. Although, ΔdblGATA mice exhibited an increased parasite burden, reduced tissue damage and less disease susceptibility was observed when compared to infected WT mice. Cellular infiltrates in infected ΔdblGATA mice were comprised of more mast cells, and αβ T cells, which correlated with an abundant CD8+ T cell response and reduced CD4+ Th1 and Th2 responses. Thus, our data suggest that enhanced inflammatory response in WT mice appears detrimental and associates with increased disease susceptibility, despite the reduced parasite burden in the CNS. Overall reduced leukocyte infiltration due to absence of eosinophils correlates with attenuated tissue damage and longer survival of ΔdblGATA mice. Therefore, our study suggests that approaches to clear NCC will require strategies to tightly control the host immune response while eradicating the parasite with minimal damage to brain tissue., Author Summary Eosinophils are known to mediate a protective response against several parasitic infections. This is largely accomplished by eosinophil degranulation (direct killing) and modulating effective adaptive immune responses. Consequently, eosinophils can also contribute to host pathology via a bystander effect. However, the outcome of infection varies depending upon the parasite species. In the case of neurocysticercosis (NCC), the role of eosinophils in disease progression has not been investigated despite the known eosinophilic response in patients. NCC is one of the most common parasitic diseases of the brain which is caused by the metacestode (larva) of the tapeworm Taenia solium. To determine the role of eosinophils in NCC disease outcome, we used a murine model of NCC in which wildtype (WT) or eosinophil deficient mice (ΔdblGATA) were infected intracranially with Mesocestoides corti, a cestode parasite related to T. solium. Our data show that murine NCC is characterized by a robust eosinophil response that correlates with lower parasite burden in the brain. Comparison of T cell response reveals a mixed Th1/Th2 in the WT brain, and ΔdblGATA mice showed a significant decrease in both population but in particular in the Th2 response. In addition, the strong eosinophil reaction observed in WT brains correlates with exacerbated pathology and increased morbidity. Thus, our study suggest that eosinophils act as a double-edged sword playing a role in controlling the infection but worsening the disease outcome by contributing to host pathology.

Neuroinflammation is one of the neuropathological hallmarks of Alzheimer's disease (AD) and related tauopathies. Activated microglia spatially coexist with microtubule-associated protein tau (Mapt or tau)-burdened neurons in the brains of human AD and non-AD tauopathies. Numerous studies have suggested that neuroinflammation precedes tau pathology and that induction or blockage of neuroinflammation via lipopolysaccharide (LPS) or anti-inflammatory compounds (such as FK506) accelerate or block tau pathology, respectively in several animal models of tauopathy. We have previously demonstrated that microglia-mediated neuroinflammation via deficiency of the microglia-specific chemokine (fractalkine) receptor, CX3CR1, promotes tau pathology and neurodegeneration in a mouse model of LPS-induced systemic inflammation. Here, we demonstrate that tau mediates the neurotoxic effects of LPS in Cx3cr1 (-/-) mice. First, Mapt (+/+) neurons displayed elevated levels of Annexin V (A5) and TUNEL (markers of neurodegeneration) when co-cultured with LPS-treated Cx3cr1 (-/-)microglia, which is rescued in Mapt (-/-) neurons. Second, a neuronal population positive for phospho-S199 (AT8) tau in the dentate gyrus is also positive for activated or cleaved caspase (CC3) in the LPS-treated Cx3cr1 (-/-) mice. Third, genetic deficiency for tau in Cx3cr1 (-/-) mice resulted in reduced microglial activation, altered expression of inflammatory genes and a significant reduction in the number of neurons positive for CC3 compared to Cx3cr1 (-/-)mice. Finally, Cx3cr1 (-/-)mice exposed to LPS displayed a lack of inhibition in an open field exploratory behavioral test, which is rescued by tau deficiency. Taken together, our results suggest that pathological alterations in tau mediate inflammation-induced neurotoxicity and that deficiency of Mapt is neuroprotective. Thus, therapeutic approaches toward either reducing tau levels or blocking neuroinflammatory pathways may serve as a potential strategy in treating tauopathies.

There is increasing interest in the isolation of adult microglia to study their functions at a morphological and molecular level during normal and neuroinflammatory conditions. Microglia have important roles in brain homeostasis, and in disease states they exert neuroprotective or neurodegenerative functions. To assay expression profiles or functions of microglia, we have developed a method to isolate microglial cells and infiltrating leukocytes from adult mouse brain. This protocol uses a digestion cocktail containing collagenase and dispase, and it involves separation over discontinuous percoll gradients. Isolated cells can be used for RNA analysis, including RNase protection analysis (RPA), quantitative RT-PCR, high-density microarray, proteomic or flow cytometric characterization of cell surface markers or adoptive transfer. Cell isolation can be completed in less than 4 h.

Paracoccidiodes brasiliensis es el agente etiológico de la Paracoccidioidomicosis (PCM), una de las micosis sistémicas mas importantes en Latinoamérica. Dos genes hsp70 (Clones 2.2 y 1DB5) fueron clonados, caracterizados y secuenciados. El análisis de secuencia mostró que los clones 2.2 y 1DB5 poseen una alta homología con la familia de genes de choque térmico hsp70. Mediante la utilización de fragmentos de ADN provenientes de ambos clones, se determinó la expresión por Northern blot durante la fase de transición a levadura. Nosotros encontramos los niveles mas altos de transcriptos ocurre entre los 30 min y 6 h después de cambiar la temperatura a 37°C. Entre las 36 y 48 h, se reducen los niveles de ARNm. Sin embargo, los niveles de ARNm aumentan a las 72 h hasta alcanzar la fase de levadura. Como resultado del aumento de la temperatura durante la fase de transición micelio a levadura, se incrementa la expresión de genes hsp70 sugiriendo que las proteínas de choque térmico tienen un papel durante el proceso de diferenciación.

Histone 3 lysine 4 trimethylation (H3K4me3) is known to be associated with transcriptionally active or poised genes and required for postnatal neurogenesis within the subventricular zone (SVZ) in the rodent model. Previous comparisons have shown significant correlation between baboon (Papio anubis) and human brain. In this study, we demonstrate that chromatin activation mark H3K4me3 is present in undifferentiated progenitor cells within the SVZ of adult baboon brain. To identify the targets and regulatory role of H3K4me3 within the baboon SVZ, we developed a technique to purify undifferentiated SVZ cells while preserving the endogenous nature without introducing culture artifact to maintain the in vivo chromatin state for genome-wide studies (ChIP-Seq and RNA-Seq). Overall, H3K4me3 is significantly enriched for genes involved in cell cycle, metabolism, protein synthesis, signaling pathways, and cancer mechanisms. Additionally, we found elevated levels of H3K4me3 in the MRI-classified SVZ-associated Glioblastoma Multiforme (GBM), which has a transcriptional profile that reflects the H3K4me3 modifications in the undifferentiated progenitor cells of the baboon SVZ. Our findings highlight the importance of H3K4me3 in coordinating distinct networks and pathways for life-long neurogenesis, and suggest that subtypes of GBM could occur, at least in part, due to aberrant H3K4me3 epigenetic regulation.

Microglia are mononuclear phagocytes that make up about 10% of the central nervous system (CNS). They are known for their surveillant behavior which comprises continuously monitoring neural tissue by extending and retracting their processes. Microglial cells are derived from myeloid progenitor cells and play important roles in homeostasis, inflammatory and immune responses in the brain. This Unit describes several microglial cell isolation protocols (Basic Protocol 1, Alternate Protocol, and Basic Protocol 2) that can be easily adapted for projects requiring a rapid and efficient analysis of mouse microglial cells by flow cytometry (Support Protocol 1). Methods for visualizing microglial cells using in situ immunohistochemistry (Basic Protocol 3) and immunochemistry in free-floating sections (Basic Protocol 4) are also included.

There is growing evidence that chronic inflammation plays a role in both the development and progression of diabetic retinopathy. There is also evidence that molecules produced as a result of hyperglycemia can activate microglia. However the exact contribution of microglia, the resident immune cells of the central nervous system, to retinal tissue damage during diabetes remains unclear. Current data suggest that dysregulated microglial responses are linked to their deleterious effects in several neurological diseases associated with chronic inflammation. As inflammatory cytokines and hyperglycemia disseminate through the diabetic retina, microglia can change to an activated state, increase in number, translocate through the retina, and themselves become the producers of inflammatory and apoptotic molecules or alternatively exert anti-inflammatory effects. In addition, microglial genetic variations may account for some of the individual differences commonly seen in patient’s susceptibility to diabetic retinopathy.

Microglia are best known as the mononuclear phagocytes of the central nervous system (CNS) parenchyma. As a resident glial cell population, microglia play key roles during the initiation, propagation, and/or resolution of inflammation. Recently, the discovery that microglial cells continuously survey their local CNS environment in vivo improved our understanding of their immune-surveillance properties in health and disease. Microglial interactions with other elements of the immune system and resident cells of the CNS define a fine balance between neuroprotection and irreparable tissue damage. In this chapter we highlight the innate immune properties of microglia, with a focus on events that initiate an inflammatory response within the brain proper including, Toll-like receptors, inflammasomes, cytokines, and chemokines, and their relationship to immune-mediated disease exacerbation or resolution.

Neurocysticercosis is the most common parasitic disease of the central nervous system worldwide. It is caused by the metacestode form of the helminth Taenia solium. Study of the immune response in the human brain has been limited by the chronic progression of the disease, the influence of corticosteroid treatment, and the scarcity of patients who undergo surgical intervention. To better understand the immune response and associated pathology in neurocysticercosis, a mouse model was developed by intracranial infection of BALB/c mice with Mesocestoides corti, a cestode organism related to T. solium. The immune response reveals the presence of abundant inflammatory infiltrates appearing as early as 2 days postinfection in extraparenchymal regions. In contrast, infiltration of immune cells into parenchymal tissue is significantly delayed. There is a natural progression of innate (neutrophils and macrophages), early induced (NK cells and γδ T cells), and adaptive immune responses (αβ T cells and B cells) in infected mice. γδ T cells are the predominant T cell population. A cell-mediated Th1 pathway of cytokine expression is evident in contrast to the previously described Th2 phenotype induced in the periphery.

Massive neuronal loss is a key pathological hallmark of Alzheimer's 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 (AβO)-mediated microglial activation induced neuronal CCEs via the tumor-necrosis factor-α (TNFα) 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 TNFα signaling pathway. Third, genetic deficiency of TNFα in R1.40 mice (R1.40-Tnfα(-/-)) 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 TNFα as the responsible cytokine, in promoting neuronal CCEs in the pathogenesis of AD.