Social defeat-induced increase in the conditioned rewarding effects of cocaine: Role of CX3CL1.

Social stress is associated with higher vulnerability to drug use, as it enhances the reinforcing effects of psychostimulants in rodents. Furthermore, continued or severe stress induces a proinflammatory state of microglial activation and augmented cytokine production. The aim of the present work was to evaluate the role of fractalkine [C-X3-C motif ligand 1 (CX3CL1)], an inflammatory chemokine, in the increased conditioned rewarding effects of cocaine in animals exposed to social defeat stress. In addition, we measured the signaling cascade pathway of CX3CL1 in the hippocampus (HPC) (including p-ERK/ERK, p-p38/p38 MAPK, p-p65/p65 NFκB and p-CREB/CREB ratios). The glutamate receptor subunits NR1, NR2B and GluA1 were also assessed. A total of 102 adult male C57BL/6 J wild-type (WT) and Cx3cr1 knockout (KO) mice were divided into different experimental groups according to stress condition (exploration or social defeat). Three weeks after the last social defeat, conditioned place preference (CPP) was induced by a subthreshold dose of cocaine (1 mg/kg). Brain tissue samples were taken 24 h after the CPP procedure to determine the levels of the proteins and transcription factors. Our results showed that, in WT animals, repeated social defeat (RSD) decreased CX3CL1 striatal levels without producing changes in the HPC. In addition, RSD induced an increase in the conditioned rewarding effects of cocaine, regardless of the genotype. After CPP induced by cocaine, defeated Cx3cr1-deficient mice showed a decrease in the p-p65/p65 NFκB and pCREB/CREB ratio in the HPC, and an increase in the hippocampal levels of CX3CL1 and p-p38/p38 MAPK relation. In all defeated mice, there was a decrease in the ionotropic glutamate receptor subunit NR1. In conclusion, these results suggest that the lack of CX3CL1/Cx3cr1 signaling under stress conditions induces changes in protein and transcription factors, indicating that CX3CL1 is needed to shield the response to social defeat.

Systemic exposure to CpG-ODN elicits low-grade inflammation in the retina.

Previous studies have reported that topical exposure to the toll-like receptor (TLR) 9 ligand CpG-ODN causes widespread ocular inflammation, including retinal microglial activation and posterior segment inflammation. Here we sought to determine the effects of systemic exposure to CpG-ODN in the retina and whether this inflammatory response was altered with Cx3cr1 deficiency or hyperglycemia. Male non-diabetic Cx3cr1+/gfp and Cx3cr1gfp/gfp littermates (normoglycemic controls) and Cx3cr1+/gfpIns2Akitaand Cx3cr1gfp/gfpIns2Akita diabetic mice were injected intraperitoneally with 40 µg CpG-ODN. Immunofluorescence staining was performed 1 week later to assess the expression of MHC Class II and glial fibrillary acidic protein (GFAP), as well as to identify morphological changes to microglia and changes in retinal macrophage cell density. Systemic exposure to CpG-ODN induced the upregulated expression of both GFAP on retinal Müller cells and MHC Class II on the retinal vasculature. Additionally, there was an increased accumulation of macrophages in the subretinal space 1 week after exposure to systemic CpG-ODN as well as characteristic morphological changes to microglia indicative of an activated phenotype. These preliminary studies demonstrate that low-grade inflammatory changes were not enhanced in Cx3cr1-deficient or diabetic mice, indicating that the inflammatory response to systemic CpG-ODN in the retina is unaltered in the context of Cx3cr1 deficiency or prolonged hyperglycemia.

Prevention of lipopolysaccharide-induced preterm labor by the lack of CX3CL1-CX3CR1 interaction in mice.

Preterm labor (PTL) is the most common cause of neonatal death and long-term adverse outcome. The pharmacological agents for PTL prevention are palliative and frequently fail to prevent PTL and improve neonatal outcome. It is essential to fully understand the molecular mechanisms of PTL in order to develop novel therapeutic methods against PTL. Several lines of evidence indicate some chemokines are expressed in gestational tissues during labor or PTL. To reveal the pathophysiological roles of the CX3CL1-CX3CR1 axis in PTL, we performed present study using LPS-induced PTL mice model in CX3CR1-deficient (Cx3cr1-/-) mice. We indicated that PTL was suppressed in Cx3cr1-/- mice and immunoneutralization of CX3CL1 in WT mice. From immunohistochemical and the gene expression analyses, the CX3CL1-CX3CR1 axis has detrimental roles in PTL through intrauterine recruitment of macrophages and the enhancement of macrophage-derived inflammatory mediators. Thus, the CX3CL1-CX3CR1 axis may be a good molecular target for preventing PTL.

Cx3cr1-deficiency exacerbates alpha-synuclein-A53T induced neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the degeneration of dopaminergic neurons of the substantia nigra and the accumulation of protein aggregates, called Lewy bodies, where the most abundant is alpha-synuclein (α-SYN). Mutations of the gene that codes for α-SYN (SNCA), such as the A53T mutation, and duplications of the gene generate cases of PD with autosomal dominant inheritance. As a result of the association of inflammation with the neurodegeneration of PD, we analyzed whether overexpression of wild-type α-SYN (α-SYNWT ) or mutated α-SYN (α-SYNA53T ) are involved in the neuronal dopaminergic loss and inflammation process, along with the role of the chemokine fractalkine (CX3CL1) and its receptor (CX3CR1). We generated in vivo murine models overexpressing human α-SYNWT or α-SYNA53T in wild type (Cx3cr1+/+ ) or deficient (Cx3cr1-/- ) mice for CX3CR1 using unilateral intracerebral injection of adeno-associated viral vectors. No changes in CX3CL1 levels were observed by immunofluorescence or analysis by qRT-PCR in this model. Interestingly, the expression α-SYNWT induced dopaminergic neuronal death to a similar degree in both genotypes. However, the expression of α-SYNA53T produced an exacerbated neurodegeneration, enhanced in the Cx3cr1-/- mice. This neurodegeneration was accompanied by an increase in neuroinflammation and microgliosis as well as the production of pro-inflammatory markers, which were exacerbated in Cx3cr1-/- mice overexpressing α-SYNA53T . Furthermore, we observed that in primary microglia CX3CR1 was a critical factor in the modulation of microglial dynamics in response to α-SYNWT or α-SYNA53T . Altogether, our study reveals that CX3CR1 plays an essential role in neuroinflammation induced by α-SYNA53T .

Essential involvement of the CX3CL1-CX3CR1 axis in bleomycin-induced pulmonary fibrosis via regulation of fibrocyte and M2 macrophage migration.

The potential role of macrophages in pulmonary fibrosis (PF) prompted us to evaluate the roles of CX3CR1, a chemokine receptor abundantly expressed in macrophages during bleomycin (BLM)-induced PF. Intratracheal BLM injection induced infiltration of leukocytes such as macrophages into the lungs, which eventually resulted in fibrosis. CX3CR1 expression was mainly detected in the majority of macrophages and in a small portion of α-smooth muscle actin-positive cells in the lungs, while CX3CL1 was expressed in macrophages. BLM-induced fibrotic changes in the lungs were reduced without any changes in the number of leukocytes in Cx3cr1 -/- mice, as compared with those in the wild-type (WT) mice. However, intrapulmonary CX3CR1+ macrophages displayed pro-fibrotic M2 phenotypes; lack of CX3CR1 skewed their phenotypes toward M1 in BLM-challenged lungs. Moreover, fibrocytes expressed CX3CR1, and were increased in BLM-challenged WT lungs. The number of intrapulmonary fibrocytes was decreased in Cx3cr1 -/- mice. Thus, locally-produced CX3CL1 can promote PF development primarily by attracting CX3CR1-expressing M2 macrophages and fibrocytes into the lungs.

Brown adipose tissue in obesity: Fractalkine-receptor dependent immune cell recruitment affects metabolic-related gene expression.

Brown adipose tissue (BAT) plays essential role in metabolic- and thermoregulation and displays morphological and functional plasticity in response to environmental and metabolic challenges. BAT is a heterogeneous tissue containing adipocytes and various immune-related cells, however, their interaction in regulation of BAT function is not fully elucidated. Fractalkine is a chemokine synthesized by adipocytes, which recruits fractalkine receptor (CX3CR1)-expressing leukocytes into the adipose tissue. Using transgenic mice, in which the fractalkine receptor, Cx3cr1 gene was replaced by Gfp, we evaluated whether deficiency in fractalkine signaling affects BAT remodeling and function in high-fat-diet - induced obesity. Homo- and heterozygote male CX3CR1-GFP mice were fed with normal or fat enriched (FatED) diet for 10weeks. Interscapular BAT was collected for molecular biological analysis. Heterozygous animals in which fractalkine signaling remains intact, gain more weight during FatED than CX3CR1 deficient gfp/gfp homozygotes. FatED in controls resulted in macrophage recruitment to the BAT with increased expression of proinflammatory mediators (Il1a, b, Tnfa and Ccl2). Local BAT inflammation was accompanied by increased expression of lipogenic enzymes and resulted in BAT "whitening". By contrast, fractalkine receptor deficiency prevented accumulation of tissue macrophages, selectively attenuated the expression of Tnfa, Il1a and Ccl2, increased BAT expression of lipolytic enzymes (Atgl, Hsl and Mgtl) and upregulated genes involved thermo-metabolism (Ucp1, Pparg Pgc1a) in response to FatED. These results highlight the importance of fractalkine-CX3CR1 interaction in recruitment of macrophages into the BAT of obese mice which might contribute to local tissue inflammation, adipose tissue remodeling and regulation of metabolic-related genes.

Retinal Phenotype following Combined Deletion of the Chemokine Receptor CCR2 and the Chemokine CX3CL1 in Mice.

PURPOSE: Conflicting data were reported with respect to the retinal phenotype of mice with dual perturbation of the CCL2 and CX3CR1 genes. We report the generation and retinal phenotype of mice with a reverse CCR2/CX3CL1 gene deficiency as a suggested model for age-related macular degeneration (AMD). METHODS: Crossing of single-deficient mice generated CCR2/CX3CL1 DKO mice. DKO mice were compared with age-matched C57BL6J mice. Evaluation included color fundus photographs, electroretinography (ERG), histology and morphometric analysis. Immunohistochemistry for CD11b in retinal cross-sections and retinal pigment epithelium (RPE)-choroid flat mounts was performed to assess microglia and macrophage recruitment. RESULTS: A minority of DKO mice showed yellowish subretinal deposits at 10 months. ERG recordings showed reduced cone sensitivity in young, but not older DKO mice. Compared to wild-type mice, DKO mice exhibited 11% reduction in the number of outer nuclear layer nuclei. Old DKO mice had an increased number of CD11b-positive cells across the retina, and on RPE-choroid flat mounts. CONCLUSIONS: In the absence of the rd8 allele, deficiency of CCR2 and CX3CL1 in mice leads to a mild form of retinal degeneration which is associated with the recruitment of macrophages, particularly to the subretinal space. This model enables to assess consequences of perturbed chemokine signaling, but it does not recapitulate cardinal AMD features.

Altered microglia morphology and higher resilience to stress-induced depression-like behavior in CX3CR1-deficient mice.

Microglia are suggested to be involved in several neuropsychiatric diseases. Indeed changes in microglia morphology have been reported in different mouse models of depression. A crucial regulatory system for microglia function is the well-defined CX3C axis. Thus, we aimed to clarify the role of microglia and CX3CR1 in depressive behavior by subjecting CX3CR1-deficient mice to a particular chronic despair model (CDM) paradigm known to exhibit face validity to major depressive disorder. In wild-type mice we observed the development of chronic depressive-like behavior after 5days of repetitive swim stress. 3D-reconstructions of Iba-1-labeled microglia in the dentate molecular layer revealed that behavioral effects were associated with changes in microglia morphology towards a state of hyper-ramification. Chronic treatment with the anti-depressant venlafaxine ameliorated depression-like behavior and restored microglia morphology. In contrast, CX3CR1 deficient mice showed a clear resistance to either (i) stress-induced depressive-like behavior, (ii) changes in microglia morphology and (iii) antidepressant treatment. Our data point towards a role of hyper-ramified microglia in the etiology of chronic depression. The lack of effects in CX3CR1 deficient mice suggests that microglia hyper-ramification is controlled by neuron-microglia signaling via the CX3C axis. However, it remains to be elucidated how hyper-ramified microglia contribute to depressive-like behavior.

The soluble isoform of CX3CL1 is necessary for neuroprotection in a mouse model of Parkinson's disease.

The chemokine CX3CL1/fractalkine is expressed by neurons as a transmembrane-anchored protein that can be cleaved to yield a soluble isoform. However, the roles for these two types of endogenous CX3CL1 in neurodegenerative pathophysiology remain elusive. As such, it has been difficult to delineate the function of the two isoforms of CX3CL1, as both are natively present in the brain. In this study we examined each isoform's ability to regulate neuroinflammation in a mouse model of Parkinson's disease initiated by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We were able to delineate the function of both CX3CL1 isoforms by using adeno-associated virus-mediated gene therapy to selectively express synthetic variants of CX3CL1 that remain either permanently soluble or membrane bound. In the present study we injected each CX3CL1 variant or a GFP-expressing vector directly into the substantia nigra of CX3CL1(-/-) mice. Our results show that only the soluble isoform of CX3CL1 is sufficient for neuroprotection after exposure to MPTP. Specifically, we show that the soluble CX3CL1 isoform reduces impairment of motor coordination, decreases dopaminergic neuron loss, and ameliorates microglial activation and proinflammatory cytokine release resulting from MPTP exposure. Furthermore, we show that the membrane-bound isoform provides no neuroprotective capability to MPTP-induced pathologies, exhibiting similar motor coordination impairment, dopaminergic neuron loss, and inflammatory phenotypes as MPTP-treated CX3CL1(-/-) mice, which received the GFP-expressing control vector. Our results reveal that the neuroprotective capacity of CX3CL1 resides solely upon the soluble isoform in an MPTP-induced model of Parkinson's disease.

CX3CL1 is neuroprotective in permanent focal cerebral ischemia in rodents.

The chemokine CX3CL1 and its receptor CX3CR1 are constitutively expressed in the nervous system. In this study, we used in vivo murine models of permanent middle cerebral artery occlusion (pMCAO) to investigate the protective potential of CX3CL1. We report that exogenous CX3CL1 reduced ischemia-induced cerebral infarct size, neurological deficits, and caspase-3 activation. CX3CL1-induced neuroprotective effects were long lasting, being observed up to 50 d after pMCAO in rats. The neuroprotective action of CX3CL1 in different models of brain injuries is mediated by its inhibitory activity on microglia and, in vitro, requires the activation of adenosine receptor 1 (A1R). We show that, in the presence of the A1R antagonist 1,3-dipropyl-8-cyclopentylxanthine and in A1R⁻/⁻ mice, the neuroprotective effect of CX3CL1 on pMCAO was abolished, indicating the critical importance of the adenosine system in CX3CL1 protection also in vivo. In apparent contrast with the above reported data but in agreement with previous findings, cx3cl1⁻/⁻ and cx3cr1(GFP/GFP) mice, respectively, deficient in CX3CL1 or CX3CR1, had less severe brain injury on pMCAO, and the administration of exogenous CX3CL1 increased brain damage in cx3cl1⁻/⁻ ischemic mice. We also report that CX3CL1 induced a different phagocytic activity in wild type and cx3cl1⁻/⁻ microglia in vitro during cotreatment with the medium conditioned by neurons damaged by oxygen-glucose deprivation. Together, these data suggest that acute administration of CX3CL1 reduces ischemic damage via an adenosine-dependent mechanism and that the absence of constitutive CX3CL1-CX3CR1 signaling changes the outcome of microglia-mediated effects during CX3CL1 administration to ischemic brain.

Fractalkine/CX3CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death.

BACKGROUND: Fractalkine/CX3CL1 and its cognate receptor CX3CR1 are abundantly expressed in the CNS. Fractalkine is an unusual C-X3-C motif chemokine that is important in neuron-microglial communication, a co-receptor for HIV infection, and can be neuroprotective. To assess the effects of fractalkine on opiate-HIV interactive neurotoxicity, wild-type murine striatal neurons were co-cultured with mixed glia from the striata of wild-type or Cx3cr1 knockout mice ± HIV-1 Tat and/or morphine. Time-lapse digital images were continuously recorded at 20 min intervals for up to 72 h using computer-aided microscopy to track the same cells repeatedly. RESULTS: Co-exposure to Tat and morphine caused synergistic increases in neuron death, dendritic pruning, and microglial motility as previously reported. Exogenous fractalkine prevented synergistic Tat and morphine-induced dendritic losses and neuron death even though the inflammatory mediator TNF-α remained significantly elevated. Antibody blockade of CX3CR1 mimicked the toxic effects of morphine plus Tat, but did not add to their toxicity; while fractalkine failed to protect wild-type neurons co-cultured with Cx3cr1-/--null glia against morphine and Tat toxicity. Exogenous fractalkine also normalized microglial motility, which is elevated by Tat and morphine co-exposure, presumably limiting microglial surveillance that may lead to toxic effects on neurons. Fractalkine immunofluorescence was expressed in neurons and to a lesser extent by other cell types, whereas CX3CR1 immunoreactivity or GFP fluorescence in cells cultured from the striatum of Cx3cr1-/- (Cx3cr1GFP/GFP) mice were associated with microglia. Immunoblotting shows that fractalkine levels were unchanged following Tat and/or morphine exposure and there was no increase in released fractalkine as determined by ELISA. By contrast, CX3CR1 protein levels were markedly downregulated. CONCLUSIONS: The results suggest that deficits in fractalkine-CX3CR1 signaling contribute to the synergistic neurotoxic effects of opioids and Tat. Importantly, exogenous fractalkine can selectively protect neurons from the injurious effects of chronic opioid-HIV-1 Tat co-exposure, and this suggests a potential therapeutic course for neuroAIDS. Although the cellular mechanisms underlying neuroprotection are not certain, findings that exogenous fractalkine reduces microglial motility and fails to protect neurons co-cultured with Cx3cr1-/- mixed glia suggest that fractalkine may act by interfering with toxic microglial-neuron interactions.

CX3CR1 is expressed by human B lymphocytes and mediates [corrected] CX3CL1 driven chemotaxis of tonsil centrocytes.

BACKGROUND: Fractalkine/CX(3)CL1, a surface chemokine, binds to CX(3)CR1 expressed by different lymphocyte subsets. Since CX(3)CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX(3)CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX(3)CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX(3)CR1 but only germinal centre B cells were attracted by soluble CX(3)CL1 in a transwell assay. CX(3)CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX(3)CR1(+) germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX(3)CL1. ELISA assay showed that soluble CX(3)CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX(3)CL1 did not attract spleen B cells from wild type mice. OVA immunized CX(3)CR1(-/-) or CX(3)CL1(-/-) mice showed significantly decreased specific IgG production compared to wild type mice. CONCLUSION/SIGNIFICANCE: We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX(3)CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.

Fractalkine deficiency markedly reduces macrophage accumulation and atherosclerotic lesion formation in CCR2-/- mice: evidence for independent chemokine functions in atherogenesis.

BACKGROUND: Monocyte-derived foam cells are the hallmark of early atherosclerosis, and recent evidence indicates that chemokines play important roles in directing monocyte migration from the blood to the vessel wall. Genetic deletions of monocyte chemoattractant protein-1 (MCP-1, CCL2), fractalkine (CX3CL1), or their cognate receptors, CCR2 and CX3CR1, markedly reduce atherosclerotic lesion size in murine models of atherosclerosis. The aim of this study was to determine whether these 2 chemokines act independently or redundantly in promoting atherogenesis. METHODS AND RESULTS: We crossed CX3CL1(-/-)ApoE(-/-) and CCR2(-/-)ApoE(-/-) mice to create CX3CL1(-/-)CCR2(-/-)ApoE(-/-) triple knockouts and performed a 4-arm atherosclerosis study. Here, we report that deletion of CX3CL1 in CCR2(-/-) mice dramatically reduced macrophage accumulation in the artery wall and the subsequent development of atherosclerosis. Deletion of CX3CL1 did not reduce the number of circulating monocytes in either "wild-type" ApoE(-/-) mice or CCR2(-/-)ApoE(-/-) mice, which suggests a role for CX3CL1 in the direct recruitment and/or capture of CCR2-deficient monocytes. CONCLUSIONS: These data provide the first in vivo evidence for independent roles for CCR2 and CX3CL1 in macrophage accumulation and atherosclerotic lesion formation and suggest that successful therapeutic strategies may need to target multiple chemokines or chemokine receptors.

Increased macrophage infiltration and fractalkine expression in cisplatin-induced acute renal failure in mice.

Inflammatory mechanisms contribute to cisplatin-induced acute renal failure (CisARF). Our first aim was to determine renal macrophage infiltration in CisARF. A more than 2-fold increase in CD11b-positive macrophages in the kidney on day 2 preceded the increase in blood urea nitrogen (BUN) and serum creatinine (SCr). Our next aim was to determine the chemoattractant for macrophage infiltration in CisARF. Fractalkine (CX(3)CL1) is expressed on activated endothelial cells and is a potent chemoattractant for macrophages that express its receptor (CX(3)CR1). Immunoblotting showed that whole-kidney CX(3)CL1 expression on days 1, 2, and 3 after cisplatin administration was increased. On immunofluorescence, the intensity of renal endothelial staining of CX(3)CL1 in blood vessels was significantly increased on day 2. Circulating von Willebrand factor (vWF), a measure of systemic endothelial injury, was increased on day 2. Next we determined whether macrophages played an injurious role in CisARF. Macrophages were depleted with injections of liposome-encapsulated clodronate (LEC). LEC resulted in a decrease in renal CD11b-positive macrophages on day 3. However, LEC-treated mice were not protected from CisARF on day 3. To determine the role of CX(3)CR1, both a specific anti-CX(3) CR1 antibody and CX(3) CR1(-/-) mice were used. Administration of the CX(3)CR1 antibody and CX(3) CR1(-/-) mice was not protected against CisARF. In summary, in CisARF, macrophage infiltration in the kidney, CX(3)CL1 expression in whole kidney and blood vessels, and the increase in circulating vWF precede BUN and SCr increase. However, inhibition of macrophage infiltration in the kidney or CX(3)CR1 blockade is not sufficient to prevent CisARF.