Microglial CX3CR1I249/M280 variant limits neurogenesis and remyelination in cuprizone-induced multiple sclerosis model

Microglia have been implicated in multiple sclerosis (MS) pathogenesis. The fractalkine receptor CX3CR1 regulates the activation of pathogenic microglia in models of MS and the human polymorphic CX3CR1I249/M280 (hCX3CR1I249/M280) variant increases MS disease progression. However, the role of hCX3CR1I249/M280 on microglial activation and central nervous system repair and regenerative mechanisms remain unknown. Therefore, using transgenic mice expressing the hCX3CR1I249/M280 variant, we aimed to determine the contribution of defective CX3CR1 signaling to remyelination and neurogenesis in the cuprizone model of focal demyelination. Here, we report that mice expressing hCX3CR1I249/M280 exhibit marked demyelination and microgliosis follow acute cuprizone treatment. Cuprizone-treated CX3CR1-deficient and fractalkine-deficient mice displayed a comparable phenotype. Nanostring gene expression analysis in demyelinated lesions showed that hCX3CR1I249/M280 upregulates genes associated with inflammation, oxidative stress and disease-associated microglia. In addition, gene expression analysis in the subgranular zone (SGZ) of the hippocampus in hCX3CR1I249/M280 mice was associated with a significant downregulation of gene networks linked to neurogenesis following acute demyelination. Confocal microscopy showed that hCX3CR1I249/M280 or loss of CX3CR1 signaling inhibits the generation of progeny from the neurogenic niche, including cells involved in myelin repair. These results provide evidence for the pathogenic capacity of hCX3CR1I249/M280 on microglia dysfunction and therapeutic targeting of CX3CR1 to promote CNS repair in MS.

Anti Human CX3CR1 VHH Molecule Attenuates Venous Neointimal Hyperplasia of Arteriovenous Fistula in Mouse Model

BackgroundFractalkine receptor 1 (CX3CR1) mediates macrophage infiltration and accumulation, causing venous neointimal hyperplasia (VNH)/venous stenosis (VS) in arteriovenous fistula (AVF). The effect of blocking CX3CR1 using an anti–human variable VHH molecule (hCX3CR1 VHH, BI 655088) on VNH/VS was determined using a humanized mouse in which the human CX3CR1 (hCX3CR1) gene was knocked in (KI).MethodsWhole-transcriptomic RNA sequencing with bioinformatics analysis was used on human stenotic AVF samples, C57BL/6J, hCX3CR1 KI mice with AVF and CKD, and in in vitro experiments to identify the pathways involved in preventing VNH/VS formation after hCX3CR1 VHH administration.ResultsAccumulation of CX3CR1 and CD68 was significantly increased in stenotic human AVFs. In C57BL/6J mice with AVF, there was increased Cx3cr1, Cx3cl1, Cd68, and Tnf-α gene expression, and increased immunostaining of CX3CR1 and CD68. In hCX3CR1-KI mice treated with hCX3CR1 VHH molecule (KI-A), compared with vehicle controls (KI-V), there was increased lumen vessel area and patency, and decreased neointima in the AVF outflow veins. RNA-seq analysis identified TNF-α and NF-κB as potential targets of CX3CR1 inhibition. In KI-A–treated vessels compared with KI-V, there was decreased gene expression of Tnf-α, Mcp-1, and Il-1β; with reduction of Cx3cl1, NF-κB, and Cd68; decreased M1, Ly6C, smooth muscle cells, fibroblast-activated protein, fibronectin, and proliferation; and increased TUNEL and M2 staining. In cell culture, monocytes stimulated with PMA and treated with hCX3CR1 VHH had decreased TNF-α, CD68, proliferation, and migration.ConclusionsCX3CR1 blockade reduces VNH/VS formation by decreasing proinflammatory cues.

Hdac1 and Hdac2 are essential for physiological maturation of a Cx3cr1 expressing subset of T-lymphocytes

Objective Histone acetylation is an important mechanism in the regulation of gene expression and plays a crucial role in both cellular development and cellular response to external or internal stimuli. One key aspect of this form of regulation is that acetylation marks can be added and removed from sites of regulation very quickly through the activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The activity of both HATs and HDACs has been shown to be important for both physiological hematopoiesis as well as during development of hematological neoplasia, such as lymphomas. In the present study we analyzed the effect of knockout of the two HDACs, Hdac1 and Hdac2 in cells expressing the fractalkine receptor (Cx3cr1) on lymphocyte development. Results We report data showing a maturation defect in mice harboring a Cx3cr1 dependent knockout of Hdac1 and 2. Furthermore, we report that these mice develop a T-cell neoplasia at about 4–5 months of age, suggesting that a Cx3cr1 expressing subpopulation of immature T-cells gives rise to T-cell lymphomas in the combined absence of Hdac1 and Hdac2.

Targeting CX3CR1 Suppresses the Fanconi Anemia DNA Repair Pathway and Synergizes with Platinum

The C-X3-C motif chemokine receptor 1 (CX3CR1, fractalkine receptor) is associated with neoplastic transformation, inflammation, neurodegenerative diseases and aging, and the small molecule inhibitor KAND567 targeting CX3CR1 (CX3CR1i) is evaluated in clinical trials for acute systemic inflammation upon SARS-CoV-2 infections. Here we identify a hitherto unknown role of CX3CR1 in Fanconi anemia (FA) pathway mediated repair of DNA interstrand crosslinks (ICLs) in replicating cells. FA pathway activation triggers CX3CR1 nuclear localization which facilitates assembly of the key FA protein FANCD2 into foci. Interfering with CX3CR1 function upon ICL-induction results in inability of replicating cells to progress from S phase, replication fork stalling and impaired chromatin recruitment of key FA pathway factors. Consistent with defective FA repair, CX3CR1i results in increased levels of residual cisplatin-DNA adducts and decreased cell survival. Importantly, CX3CR1i synergizes with platinum agents in a nonreversible manner in proliferation assays including platinum resistant models. Taken together, our results reveal an unanticipated interplay between CX3CR1 and the FA pathway and show for the first time that a clinical-phase small molecule inhibitor targeting CX3CR1 might show benefit in improving responses to DNA crosslinking chemotherapeutics.

Association of CX3CR1 Gene Polymorphisms with Fractalkine, Fractalkine Receptor, and C-Reactive Protein Levels in Patients with Kidney Failure

Fractalkine (CX3CL1) is a chemokine that plays a significant role in inflammation, one of the pathophysiological processes underlying end-stage renal disease (ESRD). Genetic factors are significantly involved in cytokine expression and have been studied as potential risk factors for chronic kidney disease (CKD). Objectives: We aimed to elucidate the association of CX3CR1 gene polymorphisms rs3732378 and rs3732379 with the levels of CX3CL1, CX3CL1 receptor (CX3CR1), as well as C-reactive protein (CRP). Patients and methods: We enrolled 198 participants, including 106 patients with ESRD and 92 controls. Peripheral blood samples were collected from each patient for genetic (rs3732378 and rs3732379 polymorphisms) and immunoenzymatic (fractalkine, CX3CR1, CRP) tests. Results: CX3CR1 and CRP levels were higher in patients with ESRD than in controls (p < 0.05). Fractalkine levels were significantly higher in ESRD patients who were homozygous for the G allele of the rs3732378 polymorphism and for the C allele of the rs3732379 polymorphism than in homozygous controls. Moreover, carriers of these alleles among patients with ESRD had significantly higher CX3CR1 levels than controls. Conclusions: The G allele of the rs3732378 polymorphism and the C allele of the rs3732379 polymorphism of the CX3CR1 gene are associated with higher CX3CL1 and CX3CR1 levels. Our study suggests that CX3CR1 gene polymorphisms could be potentially involved in the pathogenesis of ESRD, but the study needs to be replicated in a larger population with a longitudinal follow-up study. Identification of genetic factors associated with inflammation in ESRD may contribute to the development of targeted gene therapies in the future.

A developmental analysis of juxtavascular microglia dynamics and interactions with the vasculature

ABSTRACTMicroglia, the resident macrophages of the central nervous system (CNS), are dynamic cells, constantly extending and retracting their processes as they contact and functionally regulate neurons and other glial cells. There is far less known about microglia-vascular interactions, particularly under healthy steady-state conditions. Here, we use the male and female mouse cerebral cortex to show that a higher percentage of microglia associate with the vasculature during the first week of postnatal development compared to older ages and the timing of these associations are dependent on the fractalkine receptor (CX3CR1). Similar developmental microglia-vascular associations were detected in the prenatal human brain. Using live imaging in mice, we found that juxtavascular microglia migrated when microglia are actively colonizing the cortex and became stationary by adulthood to occupy the same vascular space for nearly 2 months. Further, juxtavascular microglia at all ages contact vascular areas void of astrocyte endfeet and the developmental shift in microglial migratory behavior along vessels corresponded to when astrocyte endfeet more fully ensheath vessels. Together, our data provide a comprehensive assessment of microglia-vascular interactions. They support a mechanism by which microglia use the vasculature to migrate within the developing brain parenchyma. This migration becomes restricted upon the arrival of astrocyte endfeet when juxtavascular microglia then establish a long-term, stable contact with the vasculature.SIGNIFICANCE STATEMENTWe report the first extensive analysis of juxtavascular microglia in the healthy, developing and adult brain. Live imaging revealed that juxtavascular microglia within the cortex are highly motile and migrate along vessels as they are colonizing cortical regions. Using confocal, expansion, super-resolution, and electron microscopy, we determined that microglia associate with the vasculature at all ages in areas lacking full coverage astrocyte endfoot coverage and motility of juxtavascular microglia ceases as astrocyte endfeet more fully ensheath the vasculature. Our data lay the fundamental groundwork to investigate microglia-astrocyte crosstalk and juxtavascular microglial function in the healthy and diseased brain. They further provide a potential vascular-dependent mechanism by which microglia colonize the brain to later regulate neural circuit development.

Microglia depletion fails to abrogate inflammation-induced sickness in mice and rats

Background: Production of inflammatory mediators by reactive microglial cells in the brain is generally considered the primary mechanism underlying the development of symptoms of sickness in response to systemic inflammation.Methods: Depletion of microglia was achieved in C57BL/6 mice by chronic oral administration of PLX5622, a specific antagonist of colony stimulating factor-1 receptor, and in rats by a knock-in model in which the diphtheria toxin receptor was expressed under the control of the endogenous fractalkine receptor (CX3CR1) promoter sequence. After successful microglia depletion, mice and rats were injected with a sickness-inducing dose of lipopolysaccharide according to a 2 (depletion versus control) x 2 (LPS versus saline) factorial design. Sickness was measured by body weight loss and decreased locomotor activity in rats and mice, and reduced voluntary wheel running in mice. Results: Chronic administration of PLX5622 in mice and administration of diphtheria toxin to knock-in rats depleted microglia and peripheral tissue macrophages. However, it did not abrogate the inducible expression of proinflammatory cytokines in the brain in response to LPS and even exacerbated it for some of the cytokines. In accordance with these neuroimmune effects, LPS-induced sickness was not abrogated, rather it was exacerbated when measured by running wheel activity in mice. Conclusions: These findings reveal that the sickness-inducing effects of acute inflammation can develop independently of microglia activation.

Microglia depletion fails to abrogate inflammation-induced sicknessin mice and rats

Background: Production of inflammatory mediators by reactive microglial cells in the brain is generally considered the primary mechanism underlying the development of symptoms of sickness in response to systemic inflammation.Methods: Depletion of microglia was achieved in C57BL/6 mice by chronic oral administration of PLX5622, a specific antagonist of colony stimulating factor-1 receptor, and in rats by a knock-in model in which the diphtheria toxin receptor was expressed under the control of the endogenous fractalkine receptor (Cx3cr1) promoter sequence. After successful microglia depletion, mice and rats were injected with a sickness-inducing dose of lipopolysaccharide according to a 2 (depletion versus control) x 2 (LPS versus saline) factorial design. Sickness was measured by body weight loss and decreased locomotor activity in rats and mice, and reduced voluntary wheel running in mice. Results: Chronic administration of PLX5622 in mice and administration of diphtheria toxin to knock-in rats depleted microglia and peripheral tissue macrophages. However, it did not abrogate the inducible expression of proinflammatory cytokines in the brain in response to LPS and even exacerbated it for some of the cytokines. In accordance with these neuroimmune effects, LPS-induced sickness was not abrogated, rather it was exacerbated when measured by running wheel activity in mice. Conclusions: These findings reveal that the sickness-inducing effects of acute inflammation can develop independently of microglia activation.

Murine cytomegalovirus disseminates independently of CX3CR1, CCL2 or its m131/m129 chemokine homologue

Cytomegaloviruses (CMVs) use myeloid cells to move within their hosts. Murine CMV (MCMV) colonizes the salivary glands for long-term shedding, and reaches them via CD11c+ infected cells. A need to recruit patrolling monocytes for systemic spread has been proposed, based on poor salivary gland infection in fractalkine receptor (CX3CR1)-deficient mice. We found no significant CX3CR1 dependence of salivary gland infection. CCL2 and the viral m131/m129 chemokine homologue were also redundant for acute MCMV spread, arguing against a need for inflammation or infection to recruit additional monocytes to the entry site. M131/m129 promoted salivary gland infection, but only after the initial seeding of infected cells to this site. Our data support the idea that MCMV disseminates by infecting and mobilizing tissue-resident dendritic cells.