Sandra M. Cardona

Control of microglial neurotoxicity by the fractalkine receptor

Microglia, the resident inflammatory cells of the CNS, are the only CNS cells that express the fractalkine receptor (CX3CR1). Using three different in vivo models, we show that CX3CR1 deficiency dysregulates microglial responses, resulting in neurotoxicity. Following peripheral lipopolysaccharide injections, Cx3cr1−/− mice showed cell-autonomous microglial neurotoxicity. In a toxic model of Parkinson disease and a transgenic model of amyotrophic lateral sclerosis, Cx3cr1−/− mice showed more extensive neuronal cell loss than Cx3cr1+ littermate controls. Augmenting CX3CR1 signaling may protect against microglial neurotoxicity, whereas CNS penetration by pharmaceutical CX3CR1 antagonists could increase neuronal vulnerability.

Scavenging roles of chemokine receptors: chemokine receptor deficiency is associated with increased levels of ligand in circulation and tissues

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.

The Role of Microglia in Diabetic Retinopathy

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 patients susceptibility to diabetic retinopathy.

Regulation of Adaptive Immunity by the Fractalkine Receptor during Autoimmune Inflammation

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.

Disruption of Fractalkine Signaling Leads to Microglial Activation and Neuronal Damage in the Diabetic Retina

Fractalkine (CX3CL1 or FKN) is a membrane-bound chemokine expressed on neuronal membranes and is proteolytically cleaved to shed a soluble chemoattractant domain. FKN signals via its unique receptor CX3CR1 expressed on microglia and other peripheral leukocytes. The aim of this study is to determine the role of CX3CR1 in inflammatory-mediated damage to retinal neurons using a model of diabetic retinopathy. For this, we compared neuronal, microglial, and astroglial densities and inflammatory response in nondiabetic and diabetic (Ins2Akita) CX3CR1-wild-type and CX3CR1-deficient mice at 10 and 20 weeks of age. Our results show that Ins2Akita CX3CR1-knockout mice exhibited (a) decreased neuronal cell counts in the retinal ganglion cell layer, (b) increased microglial cell numbers, and (c) decreased astrocyte responses comparable with Ins2Akita CX3CR1-Wild-type mice at 20 weeks of age. Analyses of the inflammatory response using PCR arrays showed several inflammatory genes differentially regulated in diabetic tissues. From those, the response in Ins2Akita CX3CR1-deficient mice at 10 weeks of age revealed a significant upregulation of IL-1β at the transcript level that was confirmed by enzyme-linked immunosorbent assay in soluble retinal extracts. Overall, IL-1β, VEGF, and nitrite levels as a read out of nitric oxide production were abundant in Ins2Akita CX3CR1-deficient retina. Notably, double immunofluorescence staining shows that astrocytes act as a source of IL-1β in the Ins2Akita retina, and CX3CR1-deficient microglia potentiate the inflammatory response via IL-1β release. Collectively, these data demonstrate that dysregulated microglial responses in absence of CX3CR1 contribute to inflammatory-mediated damage of neurons in the diabetic retina.

Isolation and Analysis of Mouse Microglial Cells

Microglia are mononuclear phagocytes that make up about 10% of the central nervous system (CNS). They are known for their surveillant behavior, which involves continuous monitoring of neural tissue by extending and retracting their processes. Microglial cells are derived from myeloid progenitor cells and play important roles in homeostasis as well as inflammatory and immune responses in the brain. This unit describes several microglial cell isolation protocols that can be easily adapted for projects requiring a rapid and efficient analysis of mouse microglial cells by flow cytometry. Methods for visualizing microglial cells using in situ immunohistochemistry and immunochemistry in free‐floating sections are also included. Curr. Protoc. Immunol. 104:14.35.1‐14.35.15.

Fractalkine Signaling Attenuates Perivascular Clustering of Microglia and Fibrinogen Leakage during Systemic Inflammation in Mouse Models of Diabetic Retinopathy

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.

Region specific knock-out reveals distinct roles of chromatin modifiers in adult neurogenic niches

ABSTRACT 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.

Elimination of intravascular thrombi prevents early mortality and reduces gliosis in hyper-inflammatory experimental cerebral malaria

BackgroundCerebral 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.MethodsUsing 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.ResultsMany 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.ConclusionsThese 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.

Role of the Fractalkine Receptor in CNS Autoimmune Inflammation: New Approach Utilizing a Mouse Model Expressing the Human CX3CR1I249/M280 Variant

Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS) is the leading cause of non-traumatic neurological disability in young adults. Immune mediated destruction of myelin and oligodendrocytes is considered the primary pathology of MS, but progressive axonal loss is the major cause of neurological disability. In an effort to understand microglia function during CNS inflammation, our laboratory focuses on the fractalkine/CX3CR1 signaling as a regulator of microglia neurotoxicity in various models of neurodegeneration. Fractalkine (FKN) is a transmembrane chemokine expressed in the CNS by neurons and signals through its unique receptor CX3CR1 present in microglia. During experimental autoimmune encephalomyelitis (EAE), CX3CR1 deficiency confers exacerbated disease defined by 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 influence 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 due to impaired 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 exhibited exacerbated EAE correlating with severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and ciliary neurotrophic factor during EAE in contrast to wild type 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.