Non-classical monocytes scavenge the growth factor CSF1 from endothelial cells in the peripheral vascular tree to ensure survival and homeostasis.

Unlike sessile macrophages that occupy specialized tissue niches, non-classical monocytes (NCMs)-circulating phagocytes that patrol and cleanse the luminal surface of the vascular tree-are characterized by constant movement. Here, we examined the nature of the NCM's nurturing niche. Expression of the growth factor CSF1 on endothelial cells was required for survival of NCMs in the bloodstream. Lack of endothelial-derived CSF1 did not affect blood CSF1 concentration, suggesting that NCMs rely on scavenging CSF1 present on endothelial cells. Deletion of the transmembrane chemokine and adhesion factor CX3CL1 on endothelial cells impaired NCM survival. Mechanistically, endothelial-derived CX3CL1 and integrin subunit alpha L (ITGAL) facilitated the uptake of CSF1 by NCMs. CSF1 was produced by all tissular endothelial cells, and deletion of Csf1 in all endothelial cells except bone marrow sinusoids impaired NCM survival, arguing for a model where the full vascular tree acts as a niche for NCMs and where survival and patrolling function are connected.

The cellular microenvironment regulates CX3CR1 expression on CD8+ T cells and the maintenance of CX3CR1+ CD8+ T cells.

Expression levels of the chemokine receptor CX3CR1 serve as high-resolution marker delineating functionally distinct antigen-experienced T-cell states. The factors that influence CX3CR1 expression in T cells are, however, incompletely understood. Here, we show that in vitro priming of naïve CD8+ T cells failed to robustly induce CX3CR1, which highlights the shortcomings of in vitro priming settings in recapitulating in vivo T-cell differentiation. Nevertheless, in vivo generated memory CD8+ T cells maintained CX3CR1 expression during culture. This allowed us to investigate whether T-cell receptor ligation, cell death, and CX3CL1 binding influence CX3CR1 expression. T-cell receptor stimulation led to downregulation of CX3CR1. Without stimulation, CX3CR1+ CD8+ T cells had a selective survival disadvantage, which was enhanced by factors released from necrotic but not apoptotic cells. Exposure to CX3CL1 did not rescue their survival and resulted in a dose-dependent loss of CX3CR1 surface expression. At physiological concentrations of CX3CL1, CX3CR1 surface expression was only minimally reduced, which did not hamper the interpretability of T-cell differentiation states delineated by CX3CR1. Our data further support the broad utility of CX3CR1 surface levels as T-cell differentiation marker and identify factors that influence CX3CR1 expression and the maintenance of CX3CR1 expressing CD8+ T cells.

Validating Fractalkine receptor as a target and identifying candidates for drug discovery against type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is one of the most common chronic diseases employing abnormal levels of insulin. Enhancing the insulin production is greatly aided by the regulatory mechanisms of the Fractalkine receptor (CX3CR1) system in islet ß-cell function. However, elements including a high-fat diet, obesity, and ageing negatively impact the expression of CX3CR1 in islets. CX3CL1/CX3CR1 receptor-ligand complex is now recognized as a novel therapeutic target. It suggests that T2DM-related ß-cell dysfunction may result from lower amount of these proteins. We analyzed the differential expression of CX3CR1 gene samples taken from persons with T2DM using data obtained from the Gene Expression Omnibus database. Homology modeling enabled us to generate the three-dimensional structure of CX3CR1 and a possible binding pocket. The optimized CX3CR1 structure was subjected to rigorous screening against a massive library of 693 million drug-like molecules from the ZINC15 database. This screening process led to the identification of three compounds with strong binding affinity at the identified binding pocket of CX3CR1. To further evaluate the potential of these compounds, molecular dynamics simulations were conducted over a 50 ns time scale to assess the stability of the protein-ligand complexes. These simulations revealed that ZINC000032506419 emerged as the most promising drug-like compound among the three potent molecules. The discovery of ZINC000032506419 holds exciting promise as a potential therapeutic agent for T2D and other related metabolic disorders. These findings pave the way for the development of effective medications to address the complexities of T2DM and its associated metabolic diseases.

Laminar shear stress alleviates monocyte adhesion and atherosclerosis development via miR-29b-3p/CX3CL1 axis regulation.

Laminar shear stress (Lss) is an important anti-atherosclerosis (anti-AS) factor, but its mechanism network is not clear. Therefore, this study aimed to identify how Lss acts against AS formation from a new perspective. In this study, we analyzed high-throughput sequencing data from static and Lss-treated human aortic and human umbilical vein endothelial cells (HAECs and HUVECs, respectively) and found that the expression of CX3CL1, which is a target gene closely related to AS development, was lower in the Lss group. Lss alleviated the inflammatory response in TNF-α (also known as TNF)-activated HAECs by regulating the miR-29b-3p/CX3CL1 axis, and this was achieved by blocking nuclear factor (NF)-κB signaling. In complementary in vivo experiments, a high-fat diet (HFD) induced inflammatory infiltration and plaque formation in the aorta, both of which were significantly reduced after injection of agomir-miRNA-29b-3p via the tail vein into HFD-fed ApoE-/- mice. In conclusion, this study reveals that the Lss-sensitive miR-29b-3p/CX3CL1 axis is an important regulatory target that affects vascular endothelial inflammation and AS development. Our study provides new insights into the prevention and treatment of AS.

Blockage of the fractalkine pathway reduces hyperalgesia and prevents morphological glial alterations-Comparison between inflammatory and neuropathic orofacial pain in male rats.

This study aimed to evaluate the effects of inhibitors of the fractalkine pathway in hyperalgesia in inflammatory and neuropathic orofacial pain in male rats and the morphological changes in microglia and satellite glial cells (SGCs). Rats were submitted to zymosan-induced arthritis of the temporomandibular joint or infraorbital nerve constriction, and treated intrathecally with a P2 X7 antagonist, a cathepsin S inhibitor or a p-38 mitogen-activated protein kinase (MAPK) inhibitor. Mechanical hyperalgesia was evaluated 4 and 6 h following arthritis induction or 7 and 14 days following nerve ligation. The expression of the receptor CX3 CR1 , phospho-p-38 MAPK, ionized calcium-binding adapter molecule-1 (Iba-1), and glutamine synthetase and the morphological changes in microglia and SGCs were evaluated by confocal microscopy. In both inflammatory and neuropathic models, untreated animals presented a higher expression of CX3 CR1 and developed hyperalgesia and up-regulation of phospho-p-38 MAPK, which was prevented by all drugs (p < .05). The number of microglial processes endpoints and the total branch length were lower in the untreated animals, but the overall immunolabeling of Iba-1 was altered only in neuropathic rats (p < .05). The mean area of SGCs per neuron was significantly altered only in the inflammatory model (p < .05). All morphological alterations were reverted by modulating the fractalkine pathway (p < .05). In conclusion, the blockage of the fractalkine pathway seemed to be a possible therapeutic strategy for inflammatory and neuropathic orofacial pain, reducing mechanical hyperalgesia by impairing the phosphorylation of p-38 MAPK and reverting morphological alterations in microglia and SGCs.

CX3C chemokine: Hallmarks of fibrosis and ageing.

Fibrosis refers to the progressive tissue lesion process characterized by excessive secretion and deposition of extracellular matrix (ECM). Abnormal fibrous tissue deposition distorts tissue architecture and leads to the progressive loss of organ function. Notably, fibrosis is one of the primary pathological appearances of many end stage illnesses, and is considered as a lethal threat to human health, especially in the elderly with ageing-related diseases. CX3C ligand 1 (CX3CL1) is the only member of chemokine CX3C and binds specifically to CX3C receptor 1 (CX3CR1). Different from other chemokines, CX3CL1 possesses both chemotactic and adhesive activity. CX3CL1/CX3CR1 axis involves in various physiological and pathological processes, and exerts a critical role in cells from the immune system, vascular system, and nervous system etc. Notably, increasing evidence has demonstrated that CX3CL1/CX3CR1 signaling pathway is closely related to the pathological process of fibrosis in multiple tissue and organs. We reviewed the crucial role of CX3CL1/CX3CR1 axis in fibrosis and ageing and systematically summarized the underlying mechanism, which offers prospective strategies of targeting CX3C for the therapy of fibrosis and ageing-related diseases.

CX3CL1/Fractalkine as a biomarker for early pregnancy prediction of preterm premature rupture of membranes.

OBJECTIVES: The objective of our study was to evaluate serum CX3CL1/Fractalkine, a monocyte/macrophage chemoattractant expressed in cytotrophoblasts and decidual cells, as a predictive biomarker for the occurrence of preterm premature rupture of membranes (PPROM). METHODS: A case-control study of 438 pregnancies including 82 PPROM cases and 64 preterm labor with intact membranes cases with blood samples collected at first trimester, second trimester and delivery was conducted. The predictive ability of CX3CL1 and maternal risk factors for the occurrence of PPROM was assessed by receiver operating characteristic curve analysis. A second, independent cohort was prospectively constituted to confirm the case-control study results. RESULTS: First trimester CX3CL1 was significantly increased in PPROM cases when compared to matched controls. Multivariate regression analysis highlighted a significant difference for CX3CL1 measured during the first trimester (p<0.001). Alone, CX3CL1 predicts PPROM with a 90 % sensitivity and a specificity around 40 %. The area under the receiver operating characteristic curve for PPROM prediction were 0.64 (95% confidence interval: 0.57-0.71) for first trimester CX3CL1, and 0.61 (95% confidence interval: 0.54-0.68) for maternal risk factors (body mass index<18.5 kg/m2, nulliparity, tobacco use and the absence of high school diploma). The combination of CX3CL1 and maternal risk factors significantly improved the area under the curve: 0.72 (95% confidence interval: 0.66-0.79) (p<0.001). The results were confirmed on a second independent cohort. CONCLUSIONS: CX3CL1 is a promising blood biomarker in the early (first trimester) prediction of PPROM.

Neutralization of CX3CL1 Attenuates TGF-ß-Induced Fibroblast Differentiation Through NF-κB Activation and Mitochondrial Dysfunction in Airway Fibrosis.

BACKGROUND: Severe asthma, characterized by inflammation and airway remodeling, involves fibroblast differentiation into myofibroblasts expressing α-SMA. This process leads to the production of fibronectin and connective tissue growth factor (CTGF), driven by factors such as transforming growth factor (TGF)-ß. Furthermore, the persistent presence of myofibroblasts is associated with resistance to apoptosis and mitochondrial dysfunction. The chemokine (C-X3-C motif) ligand 1 (CX3CL1) plays a role in tissue fibrosis. However, it is currently unknown whether neutralization of CX3CL1 decreases TGF-ß-induced fibroblast differentiation and mitochondrial dysfunction in normal human lung fibroblasts (NHLFs). METHODS: CX3CL1/C-X3-C motif chemokine receptor 1 (CX3CR1), CX3CL1 was analyzed by immunofluorescence (IF) or immunohistochemical (IHC) staining of ovalbumin-challenged mice. CX3CL1 release was detected by ELISA. TGF-ß-induced CTGF, fibronectin, and α-SMA expression were evaluated in NHLFs following neutralization of CX3CL1 (TP213) treatment for the indicated times by Western blotting or IF staining. Mitochondrion function was detected by a JC-1 assay and seahorse assay. Cell apoptosis was observed by a terminal uridine nick-end labeling (TUNEL) assay. RESULTS: An increase in CX3CL1 expression was observed in lung tissues from mice with ovalbumin-induced asthma by IF staining. CX3CR1 was increased in the subepithelial layer of the airway by IHC staining. Moreover, CX3CR1 small interfering (si)RNA downregulated TGF-ß-induced CTGF and fibronectin expression in NHLFs. CX3CL1 induced CTGF and fibronectin expression in NHLFs. TGF-ß-induced CX3CL1 secretion from NHLFs. Furthermore, TP213 decreased TGF-ß-induced CTGF, fibronectin, and α-SMA expression in NHLFs. Mitochondrion-related differentially expressed genes (DEGs) were examined after CX3CL1 neutralization in TGF-ß-treated NHLFs. TP213 alleviated TGF-ß-induced mitochondrial dysfunction and apoptosis resistance in NHLFs. CX3CL1 induced p65, IκBα, and IKKα phosphorylation in a time-dependent manner. Furthermore, CX3CL1-induced fibronectin expression and JC-1 monomer were decreased by p65 siRNA. TP213 reduced TGF-ß-induced p65 and α-SMA expression in NHLFs. CONCLUSIONS: These findings suggest that neutralizing CX3CL1 attenuates lung fibroblast activation and mitochondrial dysfunction. Understanding the impacts of CX3CL1 neutralization on fibroblast mitochondrial function could contribute to the development of therapeutic strategies for managing airway remodeling in severe asthma.

Effects of Cerebrospinal Fluids from Alzheimer and Non-Alzheimer Patients on Neurons-Astrocytes-Microglia Co-Culture.

Alzheimer's disease (AD) is the most common form of dementia, characterized by the accumulation of ß-amyloid plaques, tau tangles, neuroinflammation, and synaptic/neuronal loss, the latter being the strongest correlating factor with memory and cognitive impairment. Through an in vitro study on a neurons-astrocytes-microglia (NAM) co-culture system, we analyzed the effects of cerebrospinal fluid (CSF) samples from AD and non-AD patients (other neurodegenerative pathologies). Treatment with CSF from AD patients showed a loss of neurofilaments and spheroids, suggesting the presence of elements including CX3CL1 (soluble form), destabilizing the neurofilaments, cellular adhesion processes, and intercellular contacts. The NAM co-cultures were analyzed in immunofluorescence assays for several markers related to AD, such as through zymography, where the expression of proteolytic enzymes was quantified both in cell extracts and the co-cultures' conditioned medium (CM). Through qRT-PCR assays, several genes involved in the formation of ß-amyloid plaque, in phosphorylation of tau, and in inflammation pathways and MMP expression were investigated.

Alterations in CX3CL1 Levels and Its Role in Viral Pathogenesis.

CX3CL1, also named fractalkine or neurotactin, is the only known member of the CX3C chemokine family that can chemoattract several immune cells. CX3CL1 exists in both membrane-anchored and soluble forms, with each mediating distinct biological activities. CX3CL1 signals are transmitted through its unique receptor, CX3CR1, primarily expressed in the microglia of the central nervous system (CNS). In the CNS, CX3CL1 acts as a regulator of microglia activation in response to brain disorders or inflammation. Recently, there has been a growing interest in the role of CX3CL1 in regulating cell adhesion, chemotaxis, and host immune response in viral infection. Here, we provide a comprehensive review of the changes and function of CX3CL1 in various viral infections, such as human immunodeficiency virus (HIV), SARS-CoV-2, influenza virus, and cytomegalovirus (CMV) infection, to highlight the emerging roles of CX3CL1 in viral infection and associated diseases.

Chemokine CX3CL1 (Fractalkine) Signaling and Diabetic Encephalopathy.

Diabetes mellitus (DM) is the most common metabolic disease in humans, and its prevalence is increasing worldwide in parallel with the obesity pandemic. A lack of insulin or insulin resistance, and consequently hyperglycemia, leads to many systemic disorders, among which diabetic encephalopathy (DE) is a long-term complication of the central nervous system (CNS), characterized by cognitive impairment and motor dysfunctions. The role of oxidative stress and neuroinflammation in the pathomechanism of DE has been proven. Fractalkine (CX3CL1) has unique properties as an adhesion molecule and chemoattractant, and by acting on its only receptor, CX3CR1, it regulates the activity of microglia in physiological states and neuroinflammation. Depending on the clinical context, CX3CL1-CX3CR1 signaling may have neuroprotective effects by inhibiting the inflammatory process in microglia or, conversely, maintaining/intensifying inflammation and neurotoxicity. This review discusses the evidence supporting that the CX3CL1-CX3CR1 pair is neuroprotective and other evidence that it is neurotoxic. Therefore, interrupting the vicious cycle within neuron-microglia interactions by promoting neuroprotective effects or inhibiting the neurotoxic effects of the CX3CL1-CX3CR1 signaling axis may be a therapeutic goal in DE by limiting the inflammatory response. However, the optimal approach to prevent DE is simply tight glycemic control, because the elimination of dysglycemic states in the CNS abolishes the fundamental mechanisms that induce this vicious cycle.

CX3CL1 (Fractalkine)-CX3CR1 Axis in Inflammation-Induced Angiogenesis and Tumorigenesis.

The chemotactic cytokine fractalkine (FKN, chemokine CX3CL1) has unique properties resulting from the combination of chemoattractants and adhesion molecules. The soluble form (sFKN) has chemotactic properties and strongly attracts T cells and monocytes. The membrane-bound form (mFKN) facilitates diapedesis and is responsible for cell-to-cell adhesion, especially by promoting the strong adhesion of leukocytes (monocytes) to activated endothelial cells with the subsequent formation of an extracellular matrix and angiogenesis. FKN signaling occurs via CX3CR1, which is the only known member of the CX3C chemokine receptor subfamily. Signaling within the FKN-CX3CR1 axis plays an important role in many processes related to inflammation and the immune response, which often occur simultaneously and overlap. FKN is strongly upregulated by hypoxia and/or inflammation-induced inflammatory cytokine release, and it may act locally as a key angiogenic factor in the highly hypoxic tumor microenvironment. The importance of the FKN/CX3CR1 signaling pathway in tumorigenesis and cancer metastasis results from its influence on cell adhesion, apoptosis, and cell migration. This review presents the role of the FKN signaling pathway in the context of angiogenesis in inflammation and cancer. The mechanisms determining the pro- or anti-tumor effects are presented, which are the cause of the seemingly contradictory results that create confusion regarding the therapeutic goals.

Chemokine Fractalkine and Non-Obstructive Coronary Artery Disease-Is There a Link?

Non-obstructive coronary artery disease (NO-CAD) constitutes a heterogeneous group of conditions collectively characterized by less than 50% narrowing in at least one major coronary artery with a fractional flow reserve (FFR) of ≤0.80 observed in coronary angiography. The pathogenesis and progression of NO-CAD are still not fully understood, however, inflammatory processes, particularly atherosclerosis and microvascular dysfunction are known to play a major role in it. Chemokine fractalkine (FKN/CX3CL1) is inherently linked to these processes. FKN/CX3CL1 functions predominantly as a chemoattractant for immune cells, facilitating their transmigration through the vessel wall and inhibiting their apoptosis. Its concentrations correlate positively with major cardiovascular risk factors. Moreover, promising preliminary results have shown that FKN/CX3CL1 receptor inhibitor (KAND567) administered in the population of patients with ST-elevation myocardial infarction (STEMI) undergoing percutaneous coronary intervention (PCI), inhibits the adverse reaction of the immune system that causes hyperinflammation. Whereas the link between FKN/CX3CL1 and NO-CAD appears evident, further studies are necessary to unveil this complex relationship. In this review, we critically overview the current data on FKN/CX3CL1 in the context of NO-CAD and present the novel clinical implications of the unique structure and function of FKN/CX3CL1 as a compound which distinctively contributes to the pathomechanism of this condition.

CX3CL1 induces cell migration and invasion through ICAM-1 expression in oral squamous cell carcinoma cells.

Human oral squamous cell carcinoma (OSCC) has been associated with a relatively low survival rate over the years and is characterized by a poor prognosis. C-X3-C motif chemokine ligand 1 (CX3CL1) has been involved in advanced migratory cells. Overexpressed CX3CL1 promotes several cellular responses related to cancer metastasis, including cell movement, migration and invasion in tumour cells. However, CX3CL1 controls the migration ability, and its molecular mechanism in OSCC remains unknown. The present study confirmed that CX3CL1 increased cell movement, migration and invasion. The CX3CL1-induced cell motility is upregulated through intercellular adhesion molecule-1 (ICAM-1) expression in OSCC cells. These effects were significantly suppressed when OSCC cells were pre-treated with CX3CR1 monoclonal antibody (mAb) and small-interfering RNA (siRNA). The CX3CL1-CX3CR1 axis activates promoted PLCß/PKCα/c-Src phosphorylation. Furthermore, CX3CL1 enhanced activator protein-1 (AP-1) activity. The CX3CR1 mAb and PLCß, PKCα, c-Src inhibitors reduced CX3CL1-induced c-Jun phosphorylation, c-Jun translocation into the nucleus and c-Jun binding to the ICAM-1 promoter. The present results reveal that CX3CL1 induces the migration of OSCC cells by promoting ICAM-1 expression through the CX3CR1 and the PLCß/PKCα/c-Src signal pathway, suggesting that CX3CL1-CX3CR1-mediated signalling is correlated with tumour motility and appealed to be a precursor for prognosis in human OSCC.

The CX3CL1 intracellular domain exhibits neuroprotection via insulin receptor/insulin-like growth factor receptor signaling.

CX3CL1, also known as fractalkine, is best known for its signaling activity through interactions with its cognate receptor CX3CR1. However, its intrinsic function that is independent of interaction with CX3CR1 remains to be fully understood. We demonstrate that the intracellular domain of CX3CL1 (CX3CL1-ICD), generated upon sequential cleavages by α-/ß-secretase and γ-secretase, initiates a back signaling activity, which mediates direct signal transmission to gene expression in the nucleus. To study this, we fused a synthetic peptide derived from CX3CL1-ICD, named Tet34, with a 13-amino acid tetanus sequence at the N terminus to facilitate translocation into neuronal cells. We show that treatment of mouse neuroblastoma Neuro-2A cells with Tet34, but not its scrambled control (Tet34s), induced cell proliferation, as manifested by changes in protein levels of transcription factors and progrowth molecules cyclin D1, PCNA, Sox5, and Cdk2. Further biochemical assays reveal elevation of phosphorylated insulin receptor ß subunit, insulin-like growth factor-1 receptor ß subunit, and insulin receptor substrates as well as activation of proliferation-linked kinase AKT. In addition, transgenic mice overexpressing membrane-anchored C-terminal CX3CL1 also exhibited activation of insulin/insulin-like growth factor-1 receptor signaling. Remarkably, we found that this Tet34 peptide, but not Tet34s, protected against endoplasmic reticulum stress and cellular apoptosis when Neuro-2A cells were challenged with toxic oligomers of ß-amyloid peptide or hydrogen peroxide. Taken together, our results suggest that CX3CL1-ICD may have translational potential for neuroprotection in Alzheimer's disease and for disorders resulting from insulin resistance.

Concentration and proteolysis of CX3CL1 may regulate the microglial response to CX3CL1.

Fractalkine (FKN) is a membrane-bound chemokine that can be cleaved by proteases such as ADAM 10, ADAM 17, and cathepsin S to generate soluble fragments. Studies using different forms of the soluble FKN yield conflicting results in vivo. These observations prompted us to investigate the function and pharmacology of two commonly used isoforms of FKN, a human full-length soluble FKN (sFKN), and a human chemokine domain only FKN (cdFKN). Both are prevalent in the literature and are often assumed to be functionally equivalent. We observed that recombinant sFKN and cdFKN exhibit similar potencies in a cell-based cAMP assay, but binding affinity for CX3CR1 was modestly different. There was a 10-fold difference in potency between sFKN and cdFKN when assessing their ability to stimulate ß-arrestin recruitment. Interestingly, high concentrations of FKN, regardless of cleavage variant, were ineffective at reducing pro-inflammatory microglial activation and may induce a pro-inflammatory response. This effect was observed in mouse and rat primary microglial cells as well as microglial cell lines. The inflammatory response was exacerbated in aged microglia, which is known to exhibit age-related inflammatory phenotypes. We observed the same effects in Cx3cr1-/- primary microglia and therefore speculate that an alternative FKN receptor may exist. Collectively, these data provide greater insights into the function and pharmacology of these common FKN reagents, which may clarify conflicting reports and urge greater caution in the selection of FKN peptides for use in in vitro and in vivo studies and the interpretation of results obtained using these differing peptides.

VPS13A knockdown impairs corticostriatal synaptic plasticity and locomotor behavior in a new mouse model of chorea-acanthocytosis.

Chorea-acanthocytosis (ChAc) is an inherited neurodegenerative movement disorder caused by VPS13A gene mutations leading to the absence of protein expression. The striatum is the most affected brain region in ChAc patients. However, the study of the VPS13A function in the brain has been poorly addressed. Here we generated a VPS13A knockdown (KD) model and aimed to elucidate the contribution of VPS13A to synaptic plasticity and neuronal communication in the corticostriatal circuit. First, we infected primary cortical neurons with miR30-shRNA against VPS13A and analyzed its effects on neuronal plasticity. VPS13A-KD neurons showed a higher degree of branching than controls, accompanied by decreased BDNF and PSD-95 levels, indicative of synaptic alterations. We then injected AAV-KD bilaterally in the frontal cortex and two different regions of the striatum of mice and analyzed the effects of VPS13A-KD on animal behavior and synaptic plasticity. VPS13A-KD mice showed modification of the locomotor behavior pattern, with increased exploratory behavior and hyperlocomotion. Corticostriatal dysfunction in VPS13A-KD mice was evidenced by impaired striatal long-term depression (LTD) after stimulation of cortical afferents, which was partially recovered by BDNF administration. VPS13A-KD did not lead to neuronal loss in the cortex or the striatum but induced a decrease in the neuronal release of CX3CL1 and triggered a microglial reaction, especially in the striatum. Notably, CX3CL1 administration partially restored the impaired corticostriatal LTD in VPS13A-KD mice. Our results unveil the involvement of VPS13A in neuronal connectivity modifying BDNF and CX3CL1 release. Moreover, the involvement of VPS13A in synaptic plasticity and motor behavior provides key information to further understand not only ChAc pathophysiology but also other neurological disorders.

Targeted inhibition of CX3CL1 limits podocytes ferroptosis to ameliorate cisplatin-induced acute kidney injury.

BACKGROUND: It is widely acknowledged that cisplatin-induced nephrotoxicity hinders its efficacy during clinical therapy. Effective pharmaceutical interventions for cisplatin-induced acute kidney injury (Cis-AKI) are currently lacking. Prior studies have implicated the chemokine CX3CL1 in the development of lipopolysaccharide-induced AKI; however, its specific role in Cis-AKI remains uncertain. This research aimed to comprehensively characterize the therapeutic impact and mechanism of CX3CL1 inhibition on Cis-AKI. METHODS: This study employed an in vivo Cis-AKI mouse model and in vitro cisplatin-treated podocytes. Kidney pathological changes were assessed using hematoxylin-eosin (HE) and Periodic-Schiff (PAS) staining. Transcriptome changes in mouse kidney tissue post-cisplatin treatment were analyzed through RNA sequencing (RNA-seq) datasets. Evaluation parameters included the expression of inflammatory markers, intracellular free iron levels, ferroptosis-related proteins-solute carrier family 7 member 11 (SLC7A11/XCT) and glutathione peroxidase 4 (GPX4)-as well as lipid peroxidation markers and mitochondrial function proteins. Mitochondrial morphological changes were visualized through transmission electron microscopy. The impact of CX3CL1 on the glucose-regulated protein 78/eukaryotic translation initiation factor 2A/CCAAT enhancer binding protein-homologous protein (GRP78/eIF2α/CHOP) and hypoxia-inducible factor 1-alpha/heme oxygenase-1 (HIF1A/HO-1) pathways in Cis-AKI was assessed via Western Blot and Immunofluorescence experiments, both in vivo and in vitro. RESULTS: Kidney CX3CL1 levels were elevated following cisplatin injection in wild-type (WT) mice. Cisplatin-treated CX3CL1-Knockout mice exhibited reduced renal histological changes, lowered blood creatinine (Cre) and blood urea nitrogen (BUN) levels, and decreased expression of inflammatory mediators compared to cisplatin-treated WT mice. RNA-seq analysis revealed the modulation of markers associated with oxidative stress and lipid metabolism related to ferroptosis in the kidneys of mice with Cis-AKI. Both the in vivo Cis-AKI mouse model and in vitro cisplatin-treated podocytes demonstrated that CX3CL1 inhibition could mitigate ferroptosis. This effect was characterized by alleviated intracellular iron overload, malondialdehyde (MDA) content, and reactive oxygen species (ROS) production, alongside increased glutathione/glutathione disulfide ratio, superoxide dismutase (SOD), XCT, and GPX4 activity. CX3CL1 inhibition also ameliorated mitochondrial dysfunction and upregulated expression of mitochondrial biogenesis proteins-uncoupling protein (UCP), mitofusin 2 (Mfn2), and peroxisome proliferators-activated receptor γ coactivator l-alpha (PGC1α)-both in vivo and in vitro. Furthermore, CX3CL1 inhibition attenuated cisplatin-induced endoplasmic reticulum (ER) stress in podocytes. Notably, CX3CL1 inhibition reduced cisplatin-induced expression of HIF-1α and HO-1 in vivo and in vitro. CONCLUSION: Our findings suggest that CX3CL1 inhibition exerts therapeutic effects against Cis-AKI by suppressing podocyte ferroptosis.

Microglia sustain anterior cingulate cortex neuronal hyperactivity in nicotine-induced pain.

BACKGROUND: Long-term smoking is a risk factor for chronic pain, and chronic nicotine exposure induces pain-like effects in rodents. The anterior cingulate cortex (ACC) has been demonstrated to be associated with pain and substance abuse. This study aims to investigate whether ACC microglia are altered in response to chronic nicotine exposure and their interaction with ACC neurons and subsequent nicotine-induced allodynia in mice. METHODS: We utilized a mouse model that was fed nicotine water for 28 days. Brain slices of the ACC were collected for morphological analysis to evaluate the impacts of chronic nicotine on microglia. In vivo calcium imaging and whole-cell patch clamp were used to record the excitability of ACC glutamatergic neurons. RESULTS: Compared to the vehicle control, the branch endpoints and the length of ACC microglial processes decreased in nicotine-treated mice, coinciding with the hyperactivity of glutamatergic neurons in the ACC. Inhibition of ACC glutamatergic neurons alleviated nicotine-induced allodynia and reduced microglial activation. On the other hand, reactive microglia sustain ACC neuronal excitability in response to chronic nicotine, and pharmacological inhibition of microglia by minocycline or liposome-clodronate reduces nicotine-induced allodynia. The neuron-microglia interaction in chronic nicotine-induced allodynia is mediated by increased expression of neuronal CX3CL1, which activates microglia by acting on CX3CR1 receptors on microglial cells. CONCLUSION: Together, these findings underlie a critical role of ACC microglia in the maintenance of ACC neuronal hyperactivity and resulting nociceptive hypersensitivity in chronic nicotine-treated mice.

CX3CL1 promotes M1 macrophage polarization and osteoclast differentiation through NF-κB signaling pathway in ankylosing spondylitis in vitro.

BACKGROUND: Ankylosing spondylitis (AS) is an autoimmune disease with a genetic correlation and is characterized by inflammation in the axial skeleton and sacroiliac joints. Many AS patients also have inflammatory bowel diseases (IBD), but the underlying causes of intestinal inflammation and osteoporosis in AS are not well understood. CX3CL1, a protein involved in inflammation, has been found to be up-regulated in AS patients and AS-model mice. METHODS: The authors investigated the effects of CX3CL1 on AS by studying its impact on macrophage polarization, inflammation factors, and osteoclast differentiation. Furthermore, the effects of inhibiting the NF-κB pathway and blocking CX3CL1 were assessed using BAY-117082 and anti-CX3CL1 mAb, respectively. AS model mice were used to evaluate the effects of anti-CX3CL1 mAb on limb thickness, spine rupture, and intestinal tissue damage. RESULTS: The authors found that CX3CL1 increased the expression of M1-type macrophage markers and inflammation factors, and promoted osteoclast differentiation. This effect was mediated through the NF-κB signaling pathway. Inhibition of the NF-κB pathway prevented M1-type macrophage polarization, reduced inflammation levels, and inhibited osteoclast differentiation. Injection of anti-CX3CL1 mAb alleviated limb thickness, spine rupture, and intestinal tissue damage in AS model mice by inhibiting M1-type macrophage polarization and reducing intestinal tissue inflammation. CONCLUSIONS: The study demonstrated that up-regulated CX3CL1 promotes M1-type macrophage polarization and osteoclast differentiation through the NF-κB signaling pathway. Inhibition of this pathway and blocking CX3CL1 can alleviate inflammation and bone destruction in AS. These findings contribute to a better understanding of the pathogenesis of AS and provide a basis for clinical diagnosis and treatment.