Fractalkine Expression Research Articles

Fractalkine/CX3CR1 axis is critical for neuroprotection induced by hypoxic postconditioning against cerebral ischemic injury

Microglial activation-mediated neuroinflammation is a major contributor to neuronal damage after cerebral ischemia. The Fractalkine (FKN)/CX3C chemokine receptor 1 (CX3CR1) axis plays a critical role in regulating microglial activation and neuroinflammation. The aim of this study is to ascertain the role and mechanism of FKN/CX3CR1 axis in hypoxic postconditioning (HPC)-induced anti-inflammatory and neuroprotective effects on transient global cerebral ischemia (tGCI). We found that HPC suppressed microglial activation and alleviated neuroinflammation in hippocampal CA1 after tGCI. Meanwhile, HPC upregulated the expression of FKN and CX3CR1 in neurons, but it downregulated the expression of CX3CR1 in glial cells after tGCI. In addition, the overexpression of FKN induced by the administration of FKN-carried lentivirus reduced microglial activation and inhibited neuroinflammation in CA1 after tGCI. Furthermore, silencing CX3CR1 with CX3CRi-carried lentivirus in CA1 after tGCI suppressed microglial activation and neuroinflammation and exerted neuroprotective effects. Finally, the overexpression of FKN caused a marked increase of neuronal CX3CR1 receptors, upregulated the phosphorylation of Akt, and reduced neuronal loss of rats in CA1 after tGCI. These findings demonstrated that HPC protected against neuronal damage in CA1 of tGCI rats through inhibiting microglial activation and activating Akt signaling pathway via FKN/CX3CR1 axis.

Phagocytosis of aggrecan-positive perineuronal nets surrounding motor neurons by reactive microglia expressing MMP-9 in TDP-43Q331K ALS model mice

Perineuronal nets (PNNs) are extracellular matrix structures that surround excitable neurons and their proximal dendrites. PNNs play an important role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can act as a trigger for neuronal death, and this has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We therefore characterised PNNs around alpha motor neurons and the possible contributing cellular factors in the mutant TDP-43Q331K transgenic mouse, a slow onset ALS mouse model. PNNs around alpha motor neurons showed significant loss at mid-stage disease in TDP-43Q331K mice compared to wild type strain control mice. PNN loss coincided with an increased expression of matrix metallopeptidase-9 (MMP-9), an endopeptidase known to cleave PNNs, within the ventral horn. During mid-stage disease, increased numbers of microglia and astrocytes expressing MMP-9 were present in the ventral horn of TDP-43Q331K mice. In addition, TDP-43Q331K mice showed increased levels of aggrecan, a PNN component, in the ventral horn by microglia and astrocytes during this period. Elevated aggrecan levels within glia were accompanied by an increase in fractalkine expression, a chemotaxic protein responsible for the recruitment of microglia, in alpha motor neurons of onset and mid-stage TDP-43Q331K mice. Following PNN loss, alpha motor neurons in mid-stage TDP-43Q331K mice showed increased 3-nitrotyrosine expression, an indicator of protein oxidation. Together, our observations along with previous PNN research provide suggests a possible model whereby microglia and astrocytes expressing MMP-9 degrade PNNs surrounding alpha motor neurons in the TDP-43Q331K mouse. This loss of nets may expose alpha-motor neurons to oxidative damage leading to degeneration of the alpha motor neurons in the TDP-43Q331K ALS mouse model.

Expression and significance of Fractalkine/CX3CL1 in MPO-AAV-associated glomerulonephritis rats

ObjectiveTo investigate the expression and significance of Fractalkine (CX3CL1, FKN) in serum and renal tissue of myeloperoxidase and anti-neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV) rats.MethodsThirty Wistar-Kyoto (WKY) rats were randomly divided into: Control group, MPO-AAV group (400 µg/kg MPO mixed with Freund’s complete adjuvant i.p), MPO-AAV + Anti-FKN group (400 µg/kg MPO mixed with Freund’s complete adjuvant i.p), anti-FKN group (1 µg/ rat /day, i.p) after 6 weeks. MPO-AAV associated glomerulonephritis model was established by intraperitoneal injection of MPO + Freund’s complete adjuvant with 10 mice in each group. The concentration of MPO-ANCA and FKN in serum was detected by Enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was used to detect pathological changes of kidney tissue. Western blot and immunofluorescence staining were used to detect the expression and localization of FKN protein in kidney tissue. Renal function test indicators: 24-hour urinary protein (UAER), blood urea nitrogen (BUN), serum creatinine (Scr). The expression levels of p65NF-κB and IL-6 was detected by Immunohistochemical assays.ResultsCompared with the control group, the serum MPO-ANCA antibody expression level in the MPO-AAV group was significantly increased (P < 0.01), and the contents of UAER, BUN and Scr were significantly up-regulated at 24 h (P < 0.01). Compared with the control group, the glomeruli in the MPO-AAV group had different degrees of damage, infiltration of inflammatory cell, and membrane cell hyperplasia and renal tubule edema. Compared with the control group, rats in the MPO-AAV group had significantly higher levels of FKN in serum and renal tissues (P < 0.01), and high expression of p65NF-κB and IL-6 in renal tissues (P < 0.01) (P < 0.05), whereas anti-FKN reversed the expression of the above factors. In MPO-AAV renal tissue, FKN was mainly expressed in the cytoplasm of renal tubular epithelial cells and glomerular podocytes. In addition, the contents of 24 h UAER, BUN and Scr of renal function in MPO-AAV rats were significantly decreased (P < 0.01) and the damage of renal tissue was significantly ameliorated after the administration of antagonistic FKN.ConclusionFKN may play a key role in the pathogenesis of MPO-AAV associated glomerulonephritis.

Diagnostic value of galectin-3, fractalkine, IL-6, miR-21 and cardiac troponin I in human ischemic cardiomyopathy.

The primary objective of this study was to assess the diagnostic potential of galectin-3 (Gal-3), fractalkine (FKN), interleukin (IL)-6, microRNA(miR)-21, and cardiac troponin I (cTnI) in patients with ischemic cardiomyopathy (ICM). A total of 78 ICM patients (Case group) and 80 healthy volunteers (Control group) admitted to our hospital for treatment or physical examination from Aug. 2018 to Feb. 2020 were included in the current study. The serum concentration of Gal-3, FKN, IL-6, miR-21, and plasma expression of cTnI of both groups were determined. The severity of ICM was classified using New York Heart Association (NYHA) scale. When compared with the control group, the case group had a significantly high blood concentration of Gal-3, FKN, IL-6, miR-21, and cTnI (P < 0.001). NYHA class II patients had lower blood levels of Gal-3, FKN, IL-6, miR-21, and cTnI than that in patients of NYHA class III and IV without statistical significance (P > 0.05). However, statistical significance could be achieved when comparing the above-analyzed markers in patients classified between class III and IV. Correlation analysis also revealed that serum levels of Gal-3, FKN, IL-6, miR-21, and cTnI were positively correlated with NYHA classification (R = 0.564, 0.621, 0.792, 0.981, P < 0.05). Our study revealed that up-regulated serum Gal-3, FKN, IL-6, miR-21, and cTnI levels were closely related to the progression of ICM. This association implies that these biomarkers have diagnostic potential, offering a promising avenue for early detection and monitoring of ICM progression.

Perineuronal nets are phagocytosed by MMP-9 expressing microglia and astrocytes in the SOD1G93A ALS mouse model.

Perineuronal nets (PNNs) are an extracellular matrix structure that encases excitable neurons. PNNs play a role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can trigger neuronal death, which has been implicated in amyotrophic lateral sclerosis (ALS). We investigated the spatio-temporal timeline of PNN breakdown and the contributing cellular factors in the SOD1G93A strain, a fast-onset ALS mouse model. This was conducted at the presymptomatic (P30), onset (P70), mid-stage (P130), and end-stage disease (P150) using immunofluorescent microscopy, as this characterisation has not been conducted in the SOD1G93A strain. We observed a significant breakdown of PNNs around α-motor neurons in the ventral horn of onset and mid-stage disease SOD1G93A mice compared with wild-type controls. This was observed with increased numbers of microglia expressing matrix metallopeptidase-9 (MMP-9), an endopeptidase that degrades PNNs. Microglia also engulfed PNN components in the SOD1G93A mouse. Further increases in microglia and astrocyte number, MMP-9 expression, and engulfment of PNN components by glia were observed in mid-stage SOD1G93A mice. This was observed with increased expression of fractalkine, a signal for microglia engulfment, within α-motor neurons of SOD1G93A mice. Following PNN breakdown, α-motor neurons of onset and mid-stage SOD1G93A mice showed increased expression of 3-nitrotyrosine, a marker for protein oxidation, which could render them vulnerable to death. Our observations suggest that increased numbers of MMP-9 expressing glia and their subsequent engulfment of PNNs around α-motor neurons render these neurons sensitive to oxidative damage and eventual death in the SOD1G93A ALS model mouse.

Human bocavirus 1 coinfection is associated with decreased cytokine expression in the rhinovirus-induced first wheezing episode in children.

Rhinovirus (RV)-induced first wheezing episodes in children are associated with a markedly increased risk of asthma. Previous studies have suggested that human bocavirus 1 (HBoV1) may modify RV-induced immune responses in young children. We investigated cytokine profiles of sole RV- and dual RV-HBoV1-induced first wheezing episodes, and their association with severity and prognosis. Fifty-two children infected with only RV and nine children infected with dual RV-HBoV1, aged 3-23months, with severe first wheezing episodes were recruited. At acute illness and 2 weeks later, peripheral blood mononuclear cells were isolated, and stimulated with anti-CD3/anti-CD28 in vitro. Multiplex ELISA was used to quantitatively identify 56 different cytokines at both study points. Patients were prospectively followed for 4years. The mean age of the children was 14.3months, and 30% were sensitized. During the acute illness, the adjusted analyses revealed a decrease in the expression of IL-1b, MIP-1b, Regulated upon activation, normal T cell expressed and presumably secreted (CCL5), TNF-a, TARC, and ENA-78 in the RV-HBoV1 group compared with the RV group. In the convalescence phase, the RV-HBoV1 group was characterized by decreased expression of Fractalkine, MCP-3, and IL-8 compared to the RV group. Furthermore, the hospitalization time was associated with the virus group and cytokine response (interaction p<0.05), signifying that increased levels of epidermal growth factor and MIP-1b were related with a shorter duration of hospitalization in the RV-HBoV1 coinfection group but not in the RV group. Different cytokine response profiles were detected between the RV and the RV-HBoV1 groups. Our results show the idea that RV-induced immune responses may be suppressed by HBoV1.

Hydralazine alleviates noise-induced hearing loss by scavenging acrolein

Noise-induced hearing loss (NIHL) is the most common cause of hearing loss. This study investigates the therapeutic efficacy of the acrolein scavenger hydralazine for NIHL in rats. NIHL was induced by exposure to a continuous pure tone of 10 kHz. Auditory function was evaluated by auditory brainstem response (ABR) testing and scanning electron microscopy. The expression of acrolein and glial cell markers GFAP and OX42 was assessed by immunofluorescence staining. The protein and mRNA expression of GFAP, OX42, interleukin-18 (IL-18), IL-1β, and fractalkine (FTK) was measured by qRT-PCR and western blotting. A rat model of NIHL was successfully developed, as evidenced by increased ABR thresholds. The results showed that noise exposure increased the expression of acrolein, GFAP, OX42, FTK, IL-1β, and IL-18 in the rat cochlear nucleus. Furthermore, hydralazine alleviates NIHL by reversing the effects of acrolein. These results demonstrate that acrolein is involved in glial cell activation and NIHL, it’s also a therapeutic potential target for NIHL.

FKN secreted by kidney epithelial cells regulates macrophage activation in lupus nephritis via the Hippo signaling pathway.

Lupus nephritis (LN) is a serious complication of systemic lupus erythematosus (SLE), and its pathogenesis is not fully understood. Previously, we showed that fractalkine (FKN) expression was positively correlated with the severity of LN. Here, we aimed to study the role of the Hippo signaling pathway (HSP) and its interaction with FKN in LN in an attempt to provide novel strategies for LN treatment. In this study, lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-stimulated THP-1 cells were co-cultured with FKN up-regulated or down-regulated kidney epithelial cells Hkb20. FKN-knockout (KO-FKN) mice were used to construct LN model. Flow cytometric analysis, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), pathological staining, Western blot, and immunofluorescence (IF) staining were employed to investigate the role of FKN and its interaction with the Hippo signaling pathway (HSP) in LN. Up-regulation of FKN in kidney epithelial cells was associated with increased macrophage activation. FKN overexpression in kidney epithelial cells suppressed apoptosis, inflammation levels, and M1 polarization of THP-1 cells and inhibited the HSP. Oppositely, FKN knockdown in kidney epithelial cells increased apoptosis, inflammation, and M1 polarization and activated the HSP. HSP inhibitor reversed the effect of FKN knockdown on THP-1 cells. In LN mice, FKN knockout and YAP inhibitor decreased the levels of renal function markers, alleviated kidney injury induced by LN, and inhibited macrophage activation in LN mice. FKN down-regulation reduced the activation of macrophages in renal tissue and alleviated kidney damage by activating HSP. The regulatory effect of FKN on HSP should be confirmed in patients with LN, and the mechanism of FKN in LN should be further explored.

Spinal Cathepsin S promotes visceral hypersensitivity via FKN/CX3CR1/p38 MAPK signaling pathways.

Irritable bowel syndrome (IBS) is one of the typical representatives of chronic functional visceral pain that lacks effective treatment. Recently, attention has been given to the role of microglia in IBS, particularly the activation of spinal microglia and the subsequent release of Cathepsin S (Cat S), a proteolytic enzyme. However, the specific role of spinal Cat S in IBS remains to be elucidated. The purpose of this study is to investigate the mechanisms underlying the regulation of visceral hypersensitivity in IBS-like rats by Cat S. An IBS-like rat model was developed, and visceral sensitivity was tested via the electromyographic (EMG) response to colorectal distention (CRD) and pain threshold. Western blot and immunofluorescence were used to examine the expressions of proteins. The effects of inhibitors or neutralizing antibodies on visceral pain and the downstream molecular expressions were detected. The open-field test was performed to evaluate locomotor activity and anxiety-like behaviors in rats. We discovered that spinal Cat S was upregulated and colocalized with microglia in IBS-like rats. Treatment with LY3000328, a selective inhibitor of Cat S, dose-dependently down-regulated EMG amplitude and Fractalkine (FKN) expression, indicating that Cat S regulated visceral hypersensitivity via activating FKN in IBS-like rats. Furthermore, the expressions of FKN, CX3CR1, and p-p38 MAPK were elevated in IBS-like rats whereas inhibition of these molecules could alleviate visceral pain. Moreover, pharmacological inhibitor experiments suggested the activation of CX3CR1 by FKN facilitated p38 MAPK phosphorylation, which in turn promoted Cat S expression in IBS-like rats. Neonatal adverse stimulation might enhance the expression of spinal microglial Cat S, thereby activating the FKN/CX3CR1/p38 MAPK pathway and lead to visceral hypersensitivity in IBS-like rats. As a selective inhibitor of Cat S, LY3000328 could become a potential therapeutic option for IBS.

997 Nucleic acid induced fractalkine expression in human keratinocytes

997 Nucleic acid induced fractalkine expression in human keratinocytes

Human-specific duplicate CHRFAM7A gene is associated with more severe osteoarthritis and amplifies pain behaviours

ObjectivesCHRFAM7A is a uniquely human fusion gene that functions as a dominant negative regulator of alpha 7 acetylcholine nicotinic receptor (α7nAChR) in vitro. This study determined the impact of CHRFAM7A...

Cytokine expression in rhinovirus- vs. respiratory syncytial virus-induced first wheezing episode and its relation to clinical course.

Rhinovirus (RV) and respiratory syncytial virus (RSV) are common causes of bronchiolitis. Unlike an RSV etiology, an RV etiology is associated with a markedly increased risk of asthma. We investigated the cytokine profiles of RV- and RSV-induced first wheezing episode and their correlation with prognosis. We recruited 52 sole RV- and 11 sole RSV-affected children with a severe first wheezing episode. Peripheral blood mononuclear cells (PBMCs) were isolated during acute illness and 2 weeks later and stimulated in vitro with anti-CD3/anti-CD28. Culture medium samples were analyzed for 56 different cytokines by multiplex ELISA. Recurrences were prospectively followed for 4 years. In adjusted analyses, the cytokine response from PBMCs in the RV group was characterized by decreased expression of interleukin 1 receptor antagonist (IL-1RA), interleukin 1 beta (IL-1β), and monocyte chemoattractant protein-1 (MCP-1) and increased expression of eosinophil chemotactic protein 2 (eotaxin-2), thymus- and activation-regulated chemokine (TARC), and epithelial-derived neutrophil-activating peptide 78 (ENA-78) in the acute phase and increased expression of fractalkine in the convalescent phase compared to those in the RSV group. An analysis of the change in cytokine expression between study points revealed an increased expression of fractalkine and IL-1β and decreased expression of I-309 (CCL1) and TARC in the RV group compared to those in the RSV group.. Considering hospitalization time, a significant non-adjusted group × cytokine interaction was observed in the levels of interferon gamma (IFN-γ), macrophage-derived chemokine (MDC), IL-1RA, and vascular endothelial growth factor (VEGF), indicating that a higher expression of cytokine was associated with shorter hospitalization time in the RSV group but not in the RV group. A significant interaction was also found in interleukin 6 (IL-6), but the cytokine response was not associated with hospitalization time in the RSV or RV group. In the RV group, increased expression of I-309 (CCL1) and TARC was associated with fewer relapses within 2 months, and decreased expression of interleukin 13 (IL-13) and increased expression of I-309 (CCL1) were associated with less relapses within 12 months. Differences in cytokine response from PBMCs were observed between RV- and RSV-induced first severe wheezing episode. Our findings also reveal new biomarkers for short- and medium-term prognosis in first-time wheezing children infected with RV or RSV.

Alterations of the expression levels of glucose, inflammation, and iron metabolism related miRNAs and their target genes in the hypothalamus of STZ-induced rat diabetes model

BackgroundThe hypothalamus of the central nervous system is implicated in the development of diabetes due to its glucose-sensing function. Dysregulation of the hypothalamic glucose-sensing neurons leads to abnormal glucose metabolism. It has been described that fractalkine (FKN) is involved in the development of hypothalamic inflammation, which may be one of the underlying causes of a diabetic condition. Moreover, iron may play a role in the pathogenesis of diabetes via the regulation of hepcidin, the iron regulatory hormone synthesis. MicroRNAs (miRNAs) are short non-coding molecules working as key regulators of gene expression, usually by inhibiting translation. Hypothalamic miRNAs are supposed to have a role in the control of energy balance by acting as regulators of hypothalamic glucose metabolism via influencing translation.MethodsUsing a miRNA array, we analysed the expression of diabetes, inflammation, and iron metabolism related miRNAs in the hypothalamus of a streptozotocin-induced rat type 1 diabetes model. Determination of the effect of miRNAs altered by STZ treatment on the target genes was carried out at protein level.ResultsWe found 18 miRNAs with altered expression levels in the hypothalamus of the STZ-treated animals, which act as the regulators of mRNAs involved in glucose metabolism, pro-inflammatory cytokine synthesis, and iron homeostasis suggesting a link between these processes in diabetes. The alterations in the expression level of these miRNAs could modify hypothalamic glucose sensing, tolerance, uptake, and phosphorylation by affecting the stability of hexokinase-2, insulin receptor, leptin receptor, glucokinase, GLUT4, insulin-like growth factor receptor 1, and phosphoenolpyruvate carboxykinase mRNA molecules. Additional miRNAs were found to be altered resulting in the elevation of FKN protein. The miRNA, mRNA, and protein analyses of the diabetic hypothalamus revealed that the iron import, export, and iron storage were all influenced by miRNAs suggesting the disturbance of hypothalamic iron homeostasis.ConclusionIt can be supposed that glucose metabolism, inflammation, and iron homeostasis of the hypothalamus are linked via the altered expression of common miRNAs as well as the increased expression of FKN, which contribute to the imbalance of energy homeostasis, the synthesis of pro-inflammatory cytokines, and the iron accumulation of the hypothalamus. The results raise the possibility that FKN could be a potential target of new therapies targeting both inflammation and iron disturbances in diabetic conditions.Graphical

Knock-out of 5-lipoxygenase in overexpressing tumor cells—consequences on gene expression and cellular function

5-Lipoxygenase (5-LO), the central enzyme in the biosynthesis of leukotrienes, is frequently expressed in human solid malignancies even though the enzyme is not present in the corresponding healthy tissues. There is little knowledge on the consequences of this expression for the tumor cells regarding gene expression and cellular function. We established a knockout (KO) of 5-LO in different cancer cell lines (HCT-116, HT-29, U-2 OS) and studied the consequences on global gene expression using next generation sequencing. Furthermore, cell viability, proliferation, migration and multicellular tumor spheroid (MCTS) formation were studied in these cells. Our results show that 5-LO influences the gene expression and cancer cell function in a cell type-dependent manner. The enzyme affected genes involved in cell adhesion, extracellular matrix formation, G protein signaling and cytoskeleton organization. Furthermore, absence of 5-LO elevated TGFβ2 expression in HCT-116 cells while MCP-1, fractalkine and platelet-derived growth factor expression was attenuated in U-2 OS cells suggesting that tumor cell-derived 5-LO shapes the tumor microenvironment. In line with the gene expression data, KO of 5-LO had an impact on cell proliferation, motility and MCTS formation. Interestingly, pharmacological inhibition of 5-LO only partly mimicked the KO suggesting that also noncanonical functions are involved.

Artificial Microvesicles: New Perspective on Healing Tendon Wounds

Tendons have a limited capacity to repair both naturally and following clinical interventions. Damaged tissue often presents with structural and functional differences, adversely affecting animal performance, mobility, health, and welfare. Advances in cell therapies have started to overcome some of these issues, however complications such as the formation of ectopic bone remain a complication of this technique. Regenerative medicine is therefore looking toward future therapies such as the introduction of microvesicles (MVs) derived from stem cells (SCs). The aim of the present study was to assess the characteristics of artificially derived MVs, from equine mesenchymal stem cells (MSCs), when delivered to rat tendon cells in vitro and damaged tendons in vivo. The initial stages of extracting MVs from equine MSCs and identifying and characterizing the cultured tendon stem/progenitor cells (TSCs) from rat Achilles tendons were undertaken successfully. The horse MSCs and the rat tendon cells were both capable of differentiating in 3 directions: adipogenic, osteogenic, and chondrogenic pathways. The artificially derived equine MVs successfully fused with the TSC membranes, and no cytotoxic or cytostimulating effects were observed. In addition, co-cultivation of TSCs with MVs led to stimulation of cell proliferation and migration, and cytokine VEGF and fractalkine expression levels were significantly increased. These experiments are the first to show that artificially derived MVs exhibited regeneration-stimulating effects in vitro, and that fusion of cytoplasmic membranes from diploid cell lines originating from different species was possible. The experiment in vivo demonstrated the influence of MVs on synthesis of collagen I and III types in damaged tendons of rats. Explorations in vivo showed accelerated regeneration of injured tendons after introduction of the MVs into damaged areas. The results from the studies performed indicated obvious positive modifying effects following the administration of MVs. This represents the initial successful step required prior to translating this regenerative medicine technique into clinical trials, such as for tendon repair in injured horses.

Dapagliflozin Protects H9c2 Cells Against Injury Induced by Lipopolysaccharide via Suppression of CX3CL1/CX3CR1 Axis and NF-κB Activity.

Dapagliflozin, a selective Sodium-glucose cotransporter-2 (SGLT2) inhibitor, has been shown to play a key role in the control and management of metabolic and cardiac diseases. The current study aims to address the effects of dapagliflozin on the expression of fractalkine (FKN), known as CX3CL1, and its receptors CX3CR1, Nuclear factor-kappa B(NF-κB) p65 activity, Reactive oxygen species (ROS), and inflammation in LPS-treated H9c2 cell line. H9c2 cells were cultured with lipopolysaccharide (LPS) to establish a model of LPS-induced damage, and then, subsequently were treated with dapagliflozin for 72 h. Our work included measurement of cell viability (MTT), Malondialdehyde (MDA), intracellular ROS, tumor necrosis factor-α (TNF-α), NF-κB activity, and expression of CX3CL1/CX3CR1. The results showed that LPS-induced reduction of cell viability was successfully rescued by dapagliflozin treatment. The cellular levels of MDA, ROS, and TNF-α, as an indication of cellular oxidative stress and inflammation, were significantly elevated in H9c2 cells compared to the control group. Furthermore, dapagliflozin ameliorated inflammation and oxidative stress through the modulation of the levels of MDA, TNF-α, and ROS. Correspondingly, dapagliflozin reduced the expression of CX3CL1/CX3CR1, NF-κB p65 DNA binding activity, and it also attenuated nuclear acetylated NF-κB p65 in LPS-induced injury in H9c2 cells compared to untreated cells. These findings shed light on the novel pharmacological potential of dapagliflozin in the alleviation of LPS-induced CX3CL1/CX3CR1-mediated injury in inflammatory conditions such as sepsis-induced cardiomyopathy.

Phrenic Motor Neuron Fractalkine Expression After Intermittent Hypoxia and Spinal Cord Injury

Acute intermittent hypoxia (AIH) elicits spinal respiratory motor plasticity, and has emerged as a promising therapeutic approach to restore lost respiratory (and non‐respiratory) motor function following cervical spinal cord injury (cSCI). Conversely, most individuals with cSCI experience sleep disordered breathing, generating chronic intermittent hypoxia (CIH) at intensities sufficient to trigger pathology. Since glia play a critical role in regulating neuroplasticity, they may play a key role in differentiating responses to therapeutic (AIH) versus pathogenic (CIH) intermittent hypoxia. Motor neuron‐microglial interactions regulate AIH‐induced phrenic motor plasticity. Specifically, phrenic motor neuron fractalkine, a chemokine unique to neurons released during hypoxia activates its high affinity receptor, CX3CR1—which is unique to microglia in the CNS. In response, microglia release ATP/adenosine, inhibiting AIH‐induced phrenic motor plasticity. Thus, if intermittent hypoxia or cSCI impact phrenic motor neuron fractalkine expression, they may directly impact plasticity and the potential of therapeutic AIH. In this study, we tested the hypothesis that phrenic motor neuron fractalkine expression is attenuated by daily AIH (but not CIH) in spinally intact rats, but not in rats with cSCI. Fractalkine was assessed in phrenic motor neurons of male rats with/without C2 hemisection (12 weeks post‐injury) exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5‐min episodes per day, 10.5% O2; 5‐min intervals); and 3) moderate CIH (2‐min 10.5% O2 episodes; 2‐min normoxic intervals; 8 hr/day). Fourteen days before hemisection, intrapleural Cholera toxin B injections were made to retrogradely label phrenic motor neurons. Fractalkine expression was assessed in 40 mm sections using a custom MATLAB algorithm to assess immunofluorescence optical density within labeled phrenic motor neurons. Spinally intact and injured rats have differential responses to daily AIH vs CIH. Whereas injured rats had no change in phrenic motor neuron fractalkine expression after daily AIH or CIH, fractalkine expression was marginally reduced in intact rats following daily AIH (p = 0.07; cohen’s d= 132%), but not CIH (p = 0.88; cohen’s d= 23%). Thus, daily (therapeutic) AIH may decrease motor neuron fractalkine and microglial adenosine release, thereby enhancing moderate AIH‐induced phrenic motor plasticity.

Neuron derived fractalkine promotes microglia to absorb hematoma via CD163/HO-1 after intracerebral hemorrhage.

Hematoma leads to progressive neurological deficits and poor outcomes after intracerebral hemorrhage (ICH). Early clearance of hematoma is widely recognized as an essential treatment to limit the damage and improve the clinical prognosis. CD163, alias hemoglobin (Hb) scavenger receptor on microglia, plays a pivotal role in hematoma absorption, but CD163 on neurons permits Hb uptake and results in neurotoxicity. In this study, we focus on how to specially promote microglial but not neuronal CD163 mediated-Hb uptake and hematoma absorption. RNA sequencing was used to explore the potential molecules involved in ICH progression, and hematoma was detected by magnetic resonance imaging (MRI). Western blot and immunofluorescence were used to evaluate the expression and location of fractalkine (FKN) after ICH. Erythrophagocytosis assay was performed to study the specific mechanism of action of FKN in hematoma clearance. Small interfering RNA (siRNA) transfection was used to explore the effect of peroxisome proliferator-activated receptor-γ (PPAR-γ) on hematoma absorption. Enzyme-linked immunosorbent assay (ELISA) was used to determine the serum FKN concentration in ICH patients. FKN was found to be significantly increased around the hematoma in a mouse model after ICH. With its unique receptor CX3CR1 in microglia, FKN significantly decreased the hematoma size and Hb content, and improved neurological deficits in vivo. Further, FKN could enhance erythrophagocytosis of microglia in vitro via the CD163/ hemeoxygenase-1 (HO-1) axis, while AZD8797 (a specific CX3CR1 inhibitor) reversed this effect. Moreover, PPAR-γ was found to mediate the increase in the CD163/HO-1 axis expression and erythrophagocytosis induced by FKN in microglia. Of note, a higher serum FKN level was found to be associated with better hematoma resolution in ICH patients. We systematically identified that FKN may be a potential therapeutic target to improve hematoma absorption and we shed light on ICH treatment.

Role of Fractalkine-CX3CR1 Axis in Acute Rejection of Mouse Heart Allografts Subjected to Ischemia Reperfusion Injury.

Transplantation outcomes are affected by the increase in rejection associated with ischemia reperfusion injury (IRI). Fractalkine (FKN), a chemokine for recruitment of CX3CR1+ leukocytes, contributes to the pathogenesis of various inflammatory diseases. Herein, we evaluated the importance of the FKN-CX3CR1 axis during IRI-related rejections using a mouse heterotopic heart transplantation model. FKN expression and graft survival was compared between wild-type C57BL/6 recipients transplanted with BALB/c hearts preserved for 8 (WT-IRI) and 0.5 h (WT-control) at 4°C. Graft survival of WT-IRI was shorter than that of WT-control. FKN was expressed on the vascular endothelium in WT-IRI allografts, but minimally in WT-control. The role of the FKN-CX3CR1 axis in IRI-related rejection was directly investigated using the transplant model with CX3CR1-deficient recipients (CX3CR1 KO-IRI) or treatment with anti-mouse FKN monoclonal antibodies. Graft survival of CX3CR1 KO-IRI was longer than that of WT-IRI; antibody treatment prolonged graft survival. The contribution of CX3CR1+ monocytes to IRI-related rejection was evaluated by adoptive transfer to CX3CR1 KO-IRI. Adoptive transfer of CX3CR1+ monocytes attenuated the effect of prolonged graft survival in CX3CR1 KO-IRI. Overall, the FKN-CX3CR1 axis plays a major role during IRI-related rejection; its blockade has the potential to improve the outcomes of deceased donor transplantation.

Enhancing fractalkine/CX3CR1 signalling pathway can reduce neuroinflammation by attenuating microglia activation in experimental diabetic retinopathy.

The concept of diabetic retinopathy (DR) has been extended from microvascular disease to neurovascular disease in which microglia activation plays a remarkable role. Fractalkine (FKN)/CX3CR1 is reported to regulate microglia activation in central nervous system diseases. To characterize the effect of FKN on microglia activation in DR, we employed streptozotocin‐induced diabetic rats, glyoxal‐treated R28 cells and hypoxia‐treated BV2 cells to mimic diabetic conditions and explored retinal neuronal apoptosis, reactive oxygen species (ROS), as well as the expressions of FKN, Iba‐1, TSPO, NF‐κB, Nrf2 and inflammation‐related cytokines. The results showed that FKN expression declined with diabetes progression and in glyoxal‐treated R28 cells. Compared with normal control, retinal microglia activation and inflammatory factors surged in both diabetic rat retinas and hypoxia‐treated microglia, which was largely dampened by FKN. The NF‐κB and Nrf2 expressions and intracellular ROS were up‐regulated in hypoxia‐treated microglia compared with that in normoxia control, and FKN significantly inhibited NF‐κB activation, activated Nrf2 pathway and decreased intracellular ROS. In conclusion, the results demonstrated that FKN deactivated microglia via inhibiting NF‐κB pathway and activating Nrf2 pathway, thus to reduce the production of inflammation‐related cytokines and ROS, and protect the retina from diabetes insult.