CX3 CL1 Research Articles

Rationally designed synthetic vectors for therapeutic nucleic acid delivery against human cytomegalovirus infection.

RNA therapy represents a great way to precisely regulate cellular processes by modulating the gene expression. Despite this potential, a profound gap exists in our knowledge of how to subsequently deliver these RNAs into the specific target cells and turn therapeutically active RNAs into practical medicines. An advanced series of interlocked, thermodynamically self-regulated processes that enable the precise assembly of functional synthetic carriers of siRNA to the target cells in vivo was developed. To demonstrate the efficacy of this delivery system, we used it to treat human cytomegalovirus (HCMV) infection in a humanized mouse model. In this study, we use small interfering RNA (siRNA) and small complementary RNA (scRNA) to inhibit the expressions of two HCMV genes, IE1 and IE2. The auto-regulated nanocarrier polywraplex with core-shell structure was designed to condense and package these RNAs for delivering. To allow these particles recognize the HCMV-infected cells, a ligand was coupled on the surface of nanoparticle, which would specifically target the HCMV-encoded CX3 CL1 chemokine receptor presented in the HCMV-infected cells. The results demonstrated that the polywraplex conjugated with the target molecule CX3 CL1 effectively and specifically delivered the siRNA/scRNA to HCMV infected cells and inhibited virus growth in vitro and in vivo.

Inhibition of the Progression of Skin Inflammation, Fibrosis, and Vascular Injury by Blockade of the CX3 CL1/CX3 CR1 Pathway in Experimental Mouse Models of Systemic Sclerosis.

To assess the preclinical efficacy and mechanism of action of an anti-CX3 CL1 monoclonal antibody (mAb) in systemic sclerosis (SSc). Cultured human dermal fibroblasts were used to evaluate the direct effect of anti-CX3 CL1 mAb on fibroblasts. In addition, bleomycin-induced and growth factor-induced models of SSc were used to investigate the effect of anti-CX3 CL1 mAb on leukocyte infiltration, collagen deposition, and vascular damage in the skin. Anti-CX3 CL1 mAb treatment significantly inhibited Smad3 phosphorylation (P < 0.05) and expression of type I collagen and fibronectin 1 (P < 0.01) in dermal fibroblasts stimulated with transforming growth factor β1 (TGFβ1). In the bleomycin model, daily subcutaneous bleomycin injection increased serum CX3 CL1 levels (P < 0.05) and augmented lesional CX3 CL1 expression. Simultaneous administration of anti-CX3 CL1 mAb or CX3 CR1 deficiency significantly suppressed the dermal thickness, collagen content, and capillary loss caused by bleomycin (P < 0.05). Injection of bleomycin induced expression of pSmad3 and TGFβ1 in the skin, which was inhibited by anti-CX3 CL1 mAb. Further, the dermal infiltration of CX3 CR1+ cells, macrophages (inflammatory and alternatively activated [M2-like] subsets), and CD3+ cells significantly decreased following anti-CX3 CL1 mAb therapy (P < 0.05), as did the enhanced skin expression of fibrogenic molecules, such as thymic stromal lymphopoietin and secreted phosphoprotein 1 (P < 0.05). However, the treatment did not significantly reduce established skin fibrosis. In the second model, simultaneous anti-mCX3 CL1 mAb therapy significantly diminished the skin fibrosis induced by serial subcutaneous injection of TGFβ and connective tissue growth factor (P < 0.01). Anti-CX3 CL1 mAb therapy may be a novel approach for treating early skin fibrosis in inflammation-driven fibrotic skin disorders such as SSc.

The influence of sex and neonatal stress on medullary microglia in rat pups.

What is the central question of the study? Does neonatal stress, in the form of neonatal maternal separation, influence the maturation of microglial density, morphology and neuronal signalling in medullary regions regulating cardiorespiratory function in rat pups? What is the main finding and its importance? Using Iba-1 immunohistochemistry, we show that neonatal maternal separation augments microglial density and the proportion of cells with an amoeboid morphology in the medulla. Although the current understanding of the effect of early life stress on medullary development is relatively limited, these data show that within this area, microglia are affected by neonatal stress. Microglia could therefore be important effectors in cardiorespiratory disorders resulting from maternal separation. Neonatal stress has wide-ranging consequences for the developing brain, including the medullary cardiorespiratory network. In rat pups, the reflexive cardiorespiratory inhibition triggered by the presence of liquids near the larynx is augmented by neonatal maternal separation (NMS), especially in males. Sex-specific enhancement of synaptic connectivity by NMS might explain this cardiorespiratory dysfunction. Microglia influence the formation, maturation, activity and elimination of developing synapses, but their role in the wiring of medullary networks is unknown. Owing to their sensitivity to sex hormones and stress hormones, microglial dysfunction could contribute to the abnormal cardiorespiratory phenotype observed in NMS pups. Here, we first used ionized calcium-binding adapter molecule-1(Iba-1) immunolabelling to compare the density and morphology of microglia in the medulla of male versus female rat pups (14-15days old) that were either undisturbed or subjected to NMS (3hday-1 ; postnatal days3-12). Neonatal maternal separation augmented the density of Iba-1+ cells (caudal region of the NTS), increased the size of the soma and reduced the arborization area (especially in the dorsal motor nucleus of the vagus). Sex-based differences were not observed. Given that the actions of microglia are regulated by neuronal fractalkine (CX3 CL1 ), we then used western blot analysis to compare the expression of CX3 CL1 and its microglial receptor (CX3 CR1 ) in medullary homogenates from control and NMS pups. Although CX3 CR1 expression was 59% greater in males versus females, NMS had no effect on CX3 CL1 /CX3 CR1 signalling. Given that an amoeboid morphology reflects an immature phenotype in developing microglia, NMS could interfere with synaptic pruning via a different mechanism.

Genetic disruption of fractalkine signaling leads to enhanced loss of cochlear afferents following ototoxic or acoustic injury.

Cochlear hair cells are vulnerable to a variety of insults like acoustic trauma and ototoxic drugs. Such injury can also lead to degeneration of spiral ganglion neurons (SGNs), but this occurs over a period of months to years. Neuronal survival is necessary for the proper function of cochlear prosthetics, therefore, it is of great interest to understand the mechanisms that regulate neuronal survival in deaf ears. We have recently demonstrated that selective hair cell ablation is sufficient to attract leukocytes into the spiral ganglion, and that fractalkine signaling plays a role in macrophage recruitment and in the survival of auditory neurons. Fractalkine (CX3 CL1), a chemokine that regulates adhesion and migration of leukocytes is expressed by SGNs and signals to leukocytes via its receptor CX3 CR1. The present study has extended the previous findings to more clinically relevant conditions of sensorineural hearing loss by examining the role of fractalkine signaling after aminoglycoside ototoxicity or acoustic trauma. Both aminoglycoside treatment and acoustic overstimulation led to the loss of hair cells as well as prolonged increase in the numbers of cochlear leukocytes. Lack of CX3 CR1 did not affect macrophage recruitment after injury, but resulted in increased loss of SGNs and enhanced expression of the inflammatory cytokine interleukin-1β, when compared to mice with intact CX3 CR1. These data indicate that the dysregulation of macrophage response caused by the absence of CX3 CR1 may contribute to inflammation-mediated neuronal loss in the deafened ear, suggesting a key role for inflammation in the long-term survival of target-deprived afferent neurons.

Regulation and consequences of monocytosis.

Monocytes are part of the vertebrate innate immune system. Blood monocytes are produced by bone marrow and splenic progenitors that derive from hematopoietic stem cells (HSCs). In cardiovascular disease, such as atherosclerosis and myocardial infarction, HSCs proliferate at higher levels that in turn increase production of hematopoietic cells, including monocytes. Once produced in hematopoietic niches, monocytes intravasate blood vessels, circulate, and migrate to sites of inflammation. Monocyte recruitment to atherosclerotic plaque and the ischemic heart depends on various chemokines, such as CCL2, CX3 CL1, and CCL5. Once in tissue, monocytes can differentiate into macrophages and dendritic cells. Macrophages are end effector cells that regulate the steady state and tissue healing, but they can also promote disease. At sites of inflammation, monocytes and macrophages produce inflammatory cytokines, which can exacerbate disease progression. Macrophages can also phagocytose tissue debris and produce pro-healing cytokines. Additionally, macrophages are antigen-presenting cells and can prime T cells. The tissue environment, including cytokines and types of inflammation, instructs macrophage specialization. Understanding monocytosis and its consequences in disease will reveal new therapeutic opportunities without compromising steady state functions.

Increased Accumulation of CD16+ Monocytes at Local Sites of Inflammation in Patients with Chronic Kidney Disease

Patients with chronic kidney disease (CKD) display a high prevalence of cardiovascular events and acute infections. Potential effector cells are the CD16(+) monocytes, known to be increased in the peripheral circulation in CKD. The aim of this study was to assess the expression of CD16 and CX3 CR1 on peripheral and in vivo extravasated monocytes in patients with CKD (GFR < 20 ml/min × 1.73 m²) using flow cytometry. In vivo extravasated monocytes were collected from a local inflammatory site, induced by a skin blistering technique. Soluble markers were assessed by Luminex. The number of CD16(+) monocytes was significantly higher in patients with CKD compared with healthy subjects, both in the peripheral circulation (P < 0.05) and at the site of induced inflammation (P < 0.001). Patients with CKD displayed significantly higher concentration of soluble CX3 CL1 both in the peripheral circulation (P < 0.01) and in the interstitial fluid (P < 0.001). In addition, patients with CKD had a significantly higher concentration of TNF-α in the peripheral circulation (P < 0.001). On the contrary, at the inflammatory site, concentrations of both TNF-α and IL-10 were significantly lower in patients with CKD compared with healthy controls (P < 0.05 for both). In conclusion, patients with CKD have an increased percentage of CD16(+) monocytes in both circulation and at the inflammatory site, and this finding is in concurrence with simultaneous changes in CX3 CR1. Together with distorted TNF-α and IL-10 levels, this may have potential impact on the altered inflammatory response in CKD.

Progesterone Activates Multiple Innate Immune Pathways in Chlamydia trachomatis‐Infected Endocervical Cells

Susceptibility to Chlamydia trachomatis infection is increased by oral contraceptives and modulated by sex hormones. We therefore sought to determine the effects of female sex hormones on the innate immune response to C. trachomatis infection. ECC-1 endometrial cells, pre-treated with oestradiol or progesterone, were infected with C. trachomatis and the host transcriptome analysed by Illumina Sentrix HumanRef-8 microarray. Primary endocervical epithelial cells, prepared at either the proliferative or secretory phase of the menstrual cycle, were infected with C. trachomatis and cytokine gene expression determined by quantitative RT-PCR analysis. Chlamydia trachomatis yield from progesterone-primed ECC-1 cells was significantly reduced compared with oestradiol-treated cells. Genes upregulated in progesterone-treated and Chlamydia-infected cells only included multiple CC and CXC chemokines, IL-17C, IL-29, IL-32, TNF-α, DEFB4B, LCN2, S100A7-9, ITGAM, NOD2, JAK1, IL-6ST, type I and II interferon receptors, numerous interferon-stimulated genes and STAT6. CXCL10, CXCL11, CX3 CL1 and IL-17C, which were also upregulated in infected secretory-stage primary cells, and there was a trend towards higher levels of immune mediators in infected secretory-phase compared with proliferative-phase cells. Progesterone treatment primes multiple innate immune pathways in hormone-responsive epithelial cells that could potentially increase resistance to chlamydial infection.

Role of ADAM‐17, p38 MAPK, Cathepsins, and the Proteasome Pathway in the Synthesis and Shedding of Fractalkine/CX3CL1 in Rheumatoid Arthritis

To evaluate the mechanism of fractalkine (FKN)/CX3 CL1 synthesis and shedding in rheumatoid arthritis synovial fibroblasts (RASFs) and in rat adjuvant-induced arthritis (AIA). The effect of tumor necrosis factor α (TNFα) and/or interferon-γ (IFNγ) on FKN synthesis and shedding in human RASFs was determined over time by immunostaining, quantitative reverse transcription-polymerase chain reaction, and Western blotting. The role of protease enzymes and signaling pathways was evaluated using chemical inhibitors and small interfering RNA (siRNA). The activity of 20S proteasome in the lysates and the DNA binding of NF-κB/p65 in the nuclear fractions were evaluated. The in vivo relevance of these findings was examined in rat AIA. In RASFs, stimulation with the combination of TNFα and IFNγ induced cellular expression of FKN within 24 hours. Activation of ADAM-17, but not ADAM-10, partly mediated the proteolytic shedding and release of soluble FKN (sFKN) following TNFα/IFNγ stimulation for 24-72 hours. Compared with control siRNA, ADAM-17 siRNA markedly inhibited TNFα/IFNγ-induced sFKN production (by ∼33%). TNFα/IFNγ-induced sFKN release was markedly suppressed by inhibitors of ADAM-17, p38 MAPK, proteasome, or cathepsin inhibitor but not by inhibitors of caspase 3 or calpain. TNFα/IFNγ-induced proteasome activity also correlated with rapid degradation of IκBα and p38 MAPK phosphorylation. In vivo findings showed increased FKN expression in the joints of rats with AIA, which correlated with increased expression of ADAM-17 and phospho-p38 MAPK. Our results provide new understanding of the role of ADAM-17, p38 MAPK, cathepsins, and the proteasome pathway in FKN expression and shedding. Regulating these pathways may suppress FKN-mediated inflammation and tissue destruction.

CX 3CL1-induced modulation at CA1 synapses reveals multiple mechanisms of EPSC modulation involving adenosine receptor subtypes

We characterized the role of adenosine receptor (AR) subtypes in the modulation of glutamatergic neurotransmission by the chemokine fractalkine (CX 3CL1) in mouse hippocampal CA1 neurons. CX 3CL1 causes a reversible depression of excitatory postsynaptic current (EPSC), which is abolished by the A 3R antagonist MRS1523, but not by A 1R (DPCPX) or A 2AR (SCH58261) antagonists. Consistently, CX 3CL1-induced EPSC depression is absent in slices from A 3R −/− but not A 1R −/− or A 2AR −/− mice. Further, A 3R stimulation causes similar EPSC depression. In cultured neurons, CX 3CL1-induced depression of AMPA current shows A 1R–A 3R pharmacology. We conclude that glutamatergic depression induced by released adenosine requires the stimulation of different ARs.

Protracted downregulation of CX 3CR1 on microglia of aged mice after lipopolysaccharide challenge

Fractalkine (CX 3CL1) to fractalkine receptor (CX 3CR1) interactions in the brain are involved in the modulation of microglial activation. Our recent findings indicate that there is microglial hyperactivity in the aged brain during an inflammatory challenge. The underlying cause of this amplified microglial response in the aged brain is unknown. Therefore, the purpose of this study was to determine the degree to which age-associated impairments of CX 3CL1 and CX 3CR1 in the brain contribute to exaggerated microglial activation after intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). Here we show that CX 3CL1 protein was reduced in the brain of aged (18–22 mo) BALB/c mice compared to adult (3–6 mo) controls. CX 3CL1 protein, however, was unaltered by LPS injection. Next, CX 3CR1 levels were determined in microglia (CD11b +/CD45 low) isolated by Percoll density gradient separation at 4 and 24 h after LPS injection. Flow cytometric and mRNA analyses of these microglia showed that LPS injection caused a marked decrease of CX 3CR1 and a simultaneous increase of IL-1β at 4 h after LPS injection. While surface expression of CX 3CR1 was enhanced on microglia of adult mice by 24 h, it was still significantly downregulated on a subset of microglia from aged mice. This protracted reduction of CX 3CR1 corresponded with a delayed recovery from sickness behavior, prolonged IL-1β induction, and decreased TGFß expression in the aged brain. In the last set of studies BV2 microglia were used to determine effect of TGFß on CX 3CR1. These results showed that TGFβ enhanced CX 3CR1 expression and attenuated the LPS-induced increase in IL-1β expression.

LTP impairment by fractalkine/CX 3CL1 in mouse hippocampus is mediated through the activity of adenosine receptor type 3 (A 3R)

We have examined how the chemokine fractalkine/CX 3CL1 influences long-term potentiation (LTP) in CA1 mouse hippocampal slices. Field potentials (fEPSPs) were recorded upon electrical stimulation of Schaffer collaterals. It was found that application of CX 3CL1 inhibits LTP when present during the critical induction period. LTP impairment (i) failed to occur in CX 3CR1 deficient mice (CX 3CR1 GFP/GFP) and in the presence of okadaic acid (OA); (ii) required the activation of adenosine receptor 3 (A 3R), since it was prevented in A 3R-deficient mice or by MRS1523, a selective A 3R antagonist. Together, these findings indicate that CX 3CL1 inhibits hippocampal LTP through A 3R activity.

Mast cell degranulation is induced by A549 airway epithelial cell infected with respiratory syncytial virus

Respiratory syncytial virus (RSV), a major causative agent of respiratory tract infections, influences allergic diseases. Mast cells, important effector cells in allergic disease, also express chemokine (C-X 3-C motif) receptor 1 (CX 3CR1). The RSV attachment glycoprotein (G protein) is structurally similar to CX 3C ligand 1 (CX 3CL1), the CX 3CR1 ligand, suggesting that RSV directly interacts with and affects mast cell function, including degranulation. In this paper, the effect of RSV infection on mast cell function was studied using the human mast cell line (HMC-1). The results showed that RSV infection and replication was inefficient in HMC-1 cells than in human epithelial A549 cells. Additionally, HMC-1 degranulation occurred only in coculture with RSV-infected A549 cells, with up-regulation of TNFα secretion. However, direct RSV inoculation and incubation with RSV-infected A549 cell culture medium failed to induce HMC-1 degranulation, suggesting that virus-infected cells are critical for degranulation during RSV infection; however, degranulation does not occur by direct RSV infection into mast cells.

B cell lymphomas express CX 3CR1 a non-B cell lineage adhesion molecule

To study the differential expression of cell membrane-bound receptors and their potential role in growth and/or survival of the tumor cells, highly purified follicular lymphoma cells were analyzed, using gene expression analysis, and compared to non-malignant B cell populations. Filtering the genome for overexpressed genes coding for cell membrane-bound proteins/receptors resulted in a hit list of 27 identified genes. Among these, we have focused on the aberrant over expression of CX 3CR1 , in different types of B cell lymphoma, as compared to non-malignant B cells. We show that CX 3CR1 , which normally is not expressed on B cells, is expressed both at the mRNA and protein level in several subtypes of lymphoma. CX 3CR1 has also shown to be involved in the homing to specific tissues that express the ligand, CX 3CL1, in breast and prostate cancer and may thus be involved in dissemination of lymphoma.

Fractalkine/CX 3CL1 depresses central synaptic transmission in mouse hippocampal slices

This work reports the effect of chemokine fractalkine/CX 3CL1, an endogenous small peptide highly expressed in the central nervous system, on evoked synaptic responses investigated in mouse CA1 stratum radiatum using an electrophysiological approach. We report that in acute mouse hippocampal slices, superfusion of CX 3CL1 resulted in a reversible depression of the field excitatory postsynaptic potential (fEPSP) which developed within few seconds, increased for up to 10 min of application and disappeared within 30 min after the end of CX 3CL1 treatment. We also show that CX 3CL1-induced synaptic depression is (i) dose-dependent with IC 50 and n H values of 0.7 nM and 1, respectively, (ii) not associated with a change in paired-pulse facilitation, (iii) mediated through CX 3CL1 receptor (CX 3CR1), being absent in CX 3CR1 −/− mice and inhibited in wild-type mice by a specific blocking antibody, and (iv) occluded by the induction of homosynaptic long-term depression (LTD). We conclude that CX 3CL1 is a potent neuromodulator of the evoked excitatory synaptic transmission, sharing common mechanisms with LTD.

Chemokine CX 3CL1 protects rat hippocampal neurons against glutamate-mediated excitotoxicity

Excitotoxicity is a cell death caused by excessive exposure to glutamate (Glu), contributing to neuronal degeneration in many acute and chronic CNS diseases. We explored the role of fractalkine/CX 3CL1 on survival of hippocampal neurons exposed to excitotoxic doses of Glu. We found that: CX 3CL1 reduces excitotoxicity when co-applied with Glu, through the activation of the ERK1/2 and PI3K/Akt pathways, or administered up to 8 h after Glu insult; CX 3CL1 reduces the Glu-activated whole-cell current through mechanisms dependent on intracellular Ca 2+; CX 3CL1 is released from hippocampal cells after excitotoxic insult, likely providing an endogenous protective mechanism against excitotoxic cell death.

TNF-α and IL-4 regulate expression of fractalkine (CX 3CL1) as a membrane-anchored proadhesive protein and soluble chemotactic peptide on human fibroblasts

The CX 3C chemokine, fractalkine (FKN, CX 3CL1), has multiple functions and exists as two distinct forms, a membrane-anchored protein and a soluble chemotactic peptide that cleaves from the cell surface FKN. In this study, we first demonstrated the expression of FKN in tumor necrosis factor (TNF)-α- and interleukin (IL)-4-stimulated human fibroblasts. The induction of FKN was observed for both forms. We also demonstrated monocyte chemotactic activity in the culture supernatant from the fibroblasts stimulated with these cytokines. These results suggest that TNF-α- and IL-4-stimulated fibroblasts may play an important role in accumulation of monocytes at inflammatory sites.

The chemokine CX 3CL1 regulates NK cell activity in vivo

In vitro, chemokines can both activate and induce migration of NK cells. However, little is known about how chemokines influence NK cell activity in vivo. We studied the role of CX 3CL1 and its receptor, CX 3CR1, in modulating NK cell activity in an established in vivo model of tumour cell clearance. Radiolabelled YAC-1 target cells intravenously injected into C57BL/6 mice rapidly localize to the lungs and are cleared by NK cells. In mice pre-treated with blocking anti-CX 3CL1 or anti-CX 3CR1 Ab, target cell clearance decreased by four- to fivefold ( p<0.001). In vitro, we found no effect of anti-CX 3CL1 or anti-CX 3CR1 Ab on NK lysis of target cells. We further examined adhesion of NK cells to Py-4-1 endothelial cells. NK cell binding to activated endothelial monolayers was significantly inhibited by anti-CX 3CR1 Ab or soluble CX 3CL1 ( p<0.001). These studies identify a critical role for CX 3CL1 in modulating NK cell activity in vivo.

CSF and serum levels of soluble fractalkine (CX 3CL1) in inflammatory diseases of the nervous system

The new CX 3C–chemokine fractalkine (CX 3CL1) was measured by Western blot in the cerebrospinal fluid (CSF) and serum of patients with inflammatory diseases of the peripheral and central nervous system (Bell's palsy, BP; Guillain–Barré Syndrome, GBS; multiple sclerosis, MS; viral meningitis, VM; bacterial meningitis, BM) and patients with noninflammatory neurological diseases (controls). In controls, fractalkine was detectable at low concentrations in the CSF and, at much higher levels, in serum. In all inflammatory neurological diseases under study, CSF fractalkine levels were significantly ( p<0.01) increased vs. controls (BM≫GBS>VM>MS>BP>controls). In serum, fractalkine levels were significantly increased only in MS patients. The fractalkine CSF/serum ratios (a measure of the chemotactic gradient) were significantly elevated in BM, VM and GBS; furthermore, they tended to be increased in BP and to be decreased in MS. The elevated fractalkine CSF/serum ratios in diseases without CSF pleocytosis (GBS, BP) and a lack of correlation between fractalkine levels and CSF leukocyte counts suggested that soluble fractalkine is not a major chemokine in the CSF. There was no evidence of significant intrathecal production of fractalkine as the mean fractalkine indices (fractalkine CSF/serum ratio:albumin CSF/serum ratio) were <1 in all inflammatory diseases and not significantly elevated vs. controls.