Fractalkine isoforms differentially regulate microglia-mediated inflammation and enhance visual function in the diabetic retina.

Diabetic retinopathy (DR) affects about 200 million people worldwide, causing leakage of blood components into retinal tissues, leading to activation of microglia, the resident phagocytes of the retina, promoting neuronal and vascular damage. The microglial receptor, CX3CR1, binds to fractalkine (FKN), an anti-inflammatory chemokine that is expressed on neuronal membranes (mFKN), and undergoes constitutive cleavage to release a soluble domain (sFKN). Deficiencies in CX3CR1 or FKN showed increased microglial activation, inflammation, vascular damage, and neuronal loss in experimental mouse models. To understand the mechanism that regulates microglia function, recombinant adeno-associated viral vectors (rAAV) expressing mFKN or sFKN were delivered to intact retinas prior to diabetes. High-resolution confocal imaging and mRNA-seq were used to analyze microglia morphology and markers of expression, neuronal and vascular health, and inflammatory mediators. We confirmed that prophylactic intra-vitreal administration of rAAV expressing sFKN (rAAV-sFKN), but not mFKN (rAAV-mFKN), in FKNKO retinas provided vasculo- and neuro-protection, reduced microgliosis, mitigated inflammation, improved overall optic nerve health by regulating microglia-mediated inflammation, and prevented fibrin(ogen) leakage at 4 weeks and 10 weeks of diabetes induction. Moreover, administration of sFKN improved visual acuity. Our results elucidated a novel intervention via sFKN gene therapy that provides an alternative pathway to implement translational and therapeutic approaches, preventing diabetes-associated blindness.

Therapeutic Delivery of Soluble Fractalkine Ameliorates Vascular Dysfunction in the Diabetic Retina.

Diabetic retinopathy (DR)-associated vision loss is a devastating disease affecting the working-age population. Retinal pathology is due to leakage of serum components into retinal tissues, activation of resident phagocytes (microglia), and vascular and neuronal damage. While short-term interventions are available, they do not revert visual function or halt disease progression. The impact of microglial inflammatory responses on the neurovascular unit remains unknown. In this study, we characterized microglia-vascular interactions in an experimental model of DR. Early diabetes presents activated retinal microglia, vascular permeability, and vascular abnormalities coupled with vascular tortuosity and diminished astrocyte and endothelial cell-associated tight-junction (TJ) and gap-junction (GJ) proteins. Microglia exclusively bind to the neuronal-derived chemokine fractalkine (FKN) via the CX3CR1 receptor to ameliorate microglial activation. Using neuron-specific recombinant adeno-associated viruses (rAAVs), we therapeutically overexpressed soluble (sFKN) or membrane-bound (mFKN) FKN using intra-vitreal delivery at the onset of diabetes. This study highlights the neuroprotective role of rAAV-sFKN, reducing microglial activation, vascular tortuosity, fibrin(ogen) deposition, and astrogliosis and supporting the maintenance of the GJ connexin-43 (Cx43) and TJ zonula occludens-1 (ZO-1) molecules. The results also show that microglia-vascular interactions influence the vascular width upon administration of rAAV-sFKN and rAAV-mFKN. Administration of rAAV-sFKN improved visual function without affecting peripheral immune responses. These findings suggest that overexpression of rAAV-sFKN can mitigate vascular abnormalities by promoting glia-neural signaling. sFKN gene therapy is a promising translational approach to reverse vision loss driven by vascular dysfunction.

Defective fractalkine-CX3CR1 signaling aggravates neuroinflammation and affects recovery from cuprizone-induced demyelination.

Microglia have been implicated in multiple sclerosis (MS) pathogenesis. The fractalkine receptor CX3CR1 limits the activation of pathogenic microglia and the human polymorphic CX3CR1I249/M280 (hCX3CR1I249/M280 ) variant increases disease progression in models of MS. However, the role of hCX3CR1I249/M280 variant on microglial activation and central nervous system repair mechanisms remains unknown. Therefore, using transgenic mice expressing the hCX3CR1I249/M280 variant, we aimed to determine the contribution of defective CX3CR1 signaling to neuroinflammation and remyelination in the cuprizone model of focal demyelination. Here, we report that mice expressing hCX3CR1I249/M280 exhibit marked demyelination and microgliosis following acute cuprizone treatment. Nanostring gene expression analysis in demyelinated lesions showed that hCX3CR1I249/M280 but not CX3CR1-deficient mice up-regulated the cuprizone-induced gene profile linked to inflammatory, oxidative stress, and phagocytic pathways. Although CX3CR1-deficient (CX3CR1-KO) and fractalkine-deficient (FKN-KO) mice displayed a comparable demyelination and microglial activation phenotype to hCX3CR1I249/M280 mice, only CX3CR1-deficient and CX3CR1-WT mice showed significant myelin recovery 1 week from cuprizone withdrawal. Confocal microscopy showed that hCX3CR1I249/M280 variant inhibits the generation of cells involved in myelin repair. Our results show that defective fractalkine signaling contributes to regional differences in demyelination, and suggest that the CX3CR1 pathway activity may be a key mechanism for limiting toxic gene responses in neuroinflammation. Cover Image for this issue: https://doi.org/10.1111/jnc.15416.

Models of microglia depletion and replenishment elicit protective effects to alleviate vascular and neuronal damage in the diabetic murine retina.

Microglia, the resident phagocytes of the retina, are believed to influence the development of retinopathy, but their exact contributions to vascular integrity and neuronal loss are unknown. Therefore, utilizing two models of microglia depletion, we aimed to deplete and repopulate microglia to clarify the contribution of microglia to neuronal loss and vascular damage in the diabetic retina in an STZ-induced model of hyperglycemia. Here, we report that 2 weeks exposure to diphtheria toxin (DTx) in diabetic CX3CR1CreER:R26iDTR transgenic mice induced a 62% increase in Iba1+ microglia associated with an increase in TUJ1+ axonal density and prevention of NeuN+RBPMS+ neuronal loss. Conversely, diabetic PBS controls exhibited robust TUJ1+ axonal and NeuN+RBPMS+ neuronal loss compared to non-diabetic controls. A 2-week recovery period from DTx was associated with a 40% reduction in angiogenesis and an 85% reduction in fibrinogen deposition into the diabetic retina in comparison to diabetic PBS-treated controls. Analysis of microglia morphology and marker expression revealed that following a 2-week recovery period microglia displayed a P2RY12+Ly6C- phenotype and high transformation index (TI) values complimented by a ramified-surveillant morphology closely resembling non-diabetic controls. In contrast, diabetic PBS-treated control mice displayed P2RY12+Ly6C+ microglia, with a 50% reduction in TI values with an amoeboid morphology. To validate these observations were due to microglia depletion, we used PLX-5622 to assess vascular and neuronal damage in the retinas of diabetic mice. Confocal microscopy revealed that PLX-5622 also induced an increase in TUJ1+ axonal density and prevented fibrinogen extravasation into the diabetic retina. mRNAseq gene expression analysis in retinal isolates revealed that PLX-5622-induced microglia depletion and repopulation induced a downregulation in genes associated with microglial activation and phagocytosis, B2m, Cx3cr1, and Trem2, and complement-associated synaptic pruning, C1qa, C1qb, and C1qc. Although the levels of microglia depletion induced with DTx in the CX3CR1CreER:R26iDTR model and those induced with the CSF-1R antagonists are distinct, our results suggest that microglia depletion and replenishment is neuroprotective by inducing the proliferation of a homeostatic microglia pool that supports neuronal and vascular integrity.

Elimination of intravascular thrombi prevents early mortality and reduces gliosis in hyper-inflammatory experimental cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is the most lethal outcome of Plasmodium infection. There are clear correlations between expression of inflammatory cytokines, severe coagulopathies, and mortality in human CM. However, the mechanisms intertwining the coagulation and inflammation pathways, and their roles in CM, are only beginning to be understood. In mice with T cells deficient in the regulatory cytokine IL-10 (IL-10 KO), infection with Plasmodium chabaudi leads to a hyper-inflammatory response and lethal outcome that can be prevented by anti-TNF treatment. However, inflammatory T cells are adherent within the vasculature and not present in the brain parenchyma, suggesting a novel form of cerebral inflammation. We have previously documented behavioral dysfunction and microglial activation in infected IL-10 KO animals suggestive of neurological involvement driven by inflammation. In order to understand the relationship of intravascular inflammation to parenchymal dysfunction, we studied the congestion of vessels with leukocytes and fibrin(ogen) and the relationship of glial cell activation to congested vessels in the brains of P. chabaudi-infected IL-10 KO mice. METHODS: Using immunofluorescence microscopy, we describe severe thrombotic congestion in these animals. We stained for immune cell surface markers (CD45, CD11b, CD4), fibrin(ogen), microglia (Iba-1), and astrocytes (GFAP) in the brain at the peak of behavioral symptoms. Finally, we investigated the roles of inflammatory cytokine tumor necrosis factor (TNF) and coagulation on the pathology observed using neutralizing antibodies and low-molecular weight heparin to inhibit both inflammation and coagulation, respectively. RESULTS: Many blood vessels in the brain were congested with thrombi containing adherent leukocytes, including CD4 T cells and monocytes. Despite containment of the pathogen and leukocytes within the vasculature, activated microglia and astrocytes were prevalent in the parenchyma, particularly clustered near vessels with thrombi. Neutralization of TNF, or the coagulation cascade, significantly reduced both thrombus formation and gliosis in P. chabaudi-infected IL-10 KO mice. CONCLUSIONS: These findings support the contribution of cytokines, coagulation, and leukocytes within the brain vasculature to neuropathology in malaria infection. Strikingly, localization of inflammatory leukocytes within intravascular clots suggests a mechanism for interaction between the two cascades by which cytokines drive local inflammation without considerable cellular infiltration into the brain parenchyma.

Genetically enhancing the expression of chemokine domain of CX3CL1 fails to prevent tau pathology in mouse models of tauopathy.

BACKGROUND: Fractalkine (CX3CL1) and its receptor (CX3CR1) play an important role in regulating microglial function. We have previously shown that Cx3cr1 deficiency exacerbated tau pathology and led to cognitive impairment. However, it is still unclear if the chemokine domain of the ligand CX3CL1 is essential in regulating neuronal tau pathology. METHODS: We used transgenic mice lacking endogenous Cx3cl1 (Cx3cl1-/-) and expressing only obligatory soluble form (with only chemokine domain) and lacking the mucin stalk of CX3CL1 (referred to as Cx3cl1105Δ mice) to assess tau pathology and behavioral function in both lipopolysaccharide (LPS) and genetic (hTau) mouse models of tauopathy. RESULTS: First, increased basal tau levels accompanied microglial activation in Cx3cl1105Δ mice compared to control groups. Second, increased CD45+ and F4/80+ neuroinflammation and tau phosphorylation were observed in LPS, hTau/Cx3cl1-/-, and hTau/Cx3cl1105Δ mouse models of tau pathology, which correlated with impaired spatial learning. Finally, microglial cell surface expression of CX3CR1 was reduced in Cx3cl1105Δ mice, suggesting enhanced fractalkine receptor internalization (mimicking Cx3cr1 deletion), which likely contributes to the elevated tau pathology. CONCLUSIONS: Collectively, our data suggest that overexpression of only chemokine domain of CX3CL1 does not protect against tau pathology.

The IL-1ß phenomena in neuroinflammatory diseases.

It is becoming increasingly clear that neuroinflammation has a causal role in the pathogenesis of central nervous system (CNS)-related diseases, and therefore therapeutic strategies targeting the regulation or availability of inflammatory mediators can be used to prevent or mitigate pathology. Interestingly, the proinflammatory cytokine, interleukin-1 beta (IL-1ß), has been implicated in perpetuating immune responses and contributing to disease severity in a variety of CNS diseases ranging from multiple sclerosis, neurodegenerative diseases, traumatic brain injury, and diabetic retinopathy. Moreover, pharmacological blockade of IL-1 signaling has shown to be beneficial in some autoimmune and autoinflammatory diseases, making IL-1ß a promising therapeutic target in neuroinflammatory conditions. This review highlights recent advances of our understanding on the multifaceted roles of IL-1ß in neuroinflammatory diseases.

CX3CR1-dependent recruitment of mature NK cells into the central nervous system contributes to control autoimmune neuroinflammation.

Fractalkine receptor (CX3CR1)-deficient mice develop very severe experimental autoimmune encephalomyelitis (EAE), associated with impaired NK cell recruitment into the CNS. Yet, the precise implications of NK cells in autoimmune neuroinflammation remain elusive. Here, we investigated the pattern of NK cell mobilization and the contribution of CX3CR1 to NK cell dynamics in the EAE. We show that in both wild-type and CX3CR1-deficient EAE mice, NK cells are mobilized from the periphery and accumulate in the inflamed CNS. However, in CX3CR1-deficient mice, the infiltrated NK cells displayed an immature phenotype contrasting with the mature infiltrates in WT mice. This shift in the immature/mature CNS ratio contributes to EAE exacerbation in CX3CR1-deficient mice, since transfer of mature WT NK cells prior to immunization exerted a protective effect and normalized the CNS NK cell ratio. Moreover, mature CD11b(+) NK cells show higher degranulation in the presence of autoreactive 2D2 transgenic CD4(+) T cells and kill these autoreactive cells more efficiently than the immature CD11b(-) fraction. Together, these data suggest a protective role of mature NK cells in EAE, possibly through direct modulation of T cells inside the CNS, and demonstrate that mature and immature NK cells are recruited into the CNS by distinct chemotactic signals.

The myeloid cells of the central nervous system parenchyma.

A microglial cell is both a glial cell of the central nervous system and a mononuclear phagocyte, which belongs to the haematopoietic system and is involved in inflammatory and immune responses. As such, microglia face a challenging task. The neurons of the central nervous system cannot divide and be replenished, and therefore need to be protected against pathogens, which is a key role of the immune system, but without collateral damage. In addition, after physical injury, neural cells need restorative support, which is provided by inflammatory responses. Excessive or chronic inflammatory responses can, however, be harmful. How microglia balance these demands, and how their behaviour can be modified to ameliorate disorders of the central nervous system, is becoming clear.

Time-dependent effects of CX3CR1 in a mouse model of mild traumatic brain injury.

BACKGROUND: Neuroinflammation is an important secondary mechanism that is a key mediator of the long-term consequences of neuronal injury that occur in traumatic brain injury (TBI). Microglia are highly plastic cells with dual roles in neuronal injury and recovery. Recent studies suggest that the chemokine fractalkine (CX3CL1, FKN) mediates neural/microglial interactions via its sole receptor CX3CR1. CX3CL1/CX3CR1 signaling modulates microglia activation, and depending upon the type and time of injury, either protects or exacerbates neurological diseases. METHODS: In this study, mice deficient in CX3CR1 were subjected to mild controlled cortical impact injury (CCI), a model of TBI. We evaluated the effects of genetic deletion of CX3CR1 on histopathology, cell death/survival, microglia activation, and cognitive function for 30 days post-injury. RESULTS: During the acute post-injury period (24 h-15 days), motor deficits, cell death, and neuronal cell loss were more profound in injured wild-type than in CX3CR1(-/-) mice. In contrast, during the chronic period of 30 days post-TBI, injured CX3CR1(-/-) mice exhibited greater cognitive dysfunction and increased neuronal death than wild-type mice. The protective and deleterious effects of CX3CR1 were associated with changes in microglia phenotypes; during the acute phase CX3CR1(-/-) mice showed a predominant anti-inflammatory M2 microglial response, with increased expression of Ym1, CD206, and TGFß. In contrast, increased M1 phenotypic microglia markers, Marco, and CD68 were predominant at 30 days post-TBI. CONCLUSION: Collectively, these novel data demonstrate a time-dependent role for CX3CL1/CX3CR1 signaling after TBI and suggest that the acute and chronic responses to mild TBI are modulated in part by distinct microglia phenotypes.

The kinetics of myelin antigen uptake by myeloid cells in the central nervous system during experimental autoimmune encephalomyelitis.

Induction of experimental autoimmune encephalomyelitis (EAE) in susceptible animals requires reactivation of encephalitogenic CD4(+) T cells by APCs in the CNS. However, it has remained unresolved from where APCs in the CNS acquire myelin Ag for T cell activation and under which conditions, that is, whether only during EAE or also in the naive CNS. In this study, we investigated the kinetics of myelin Ag uptake by CNS APCs during EAE and in the naive CNS. Our results show that during EAE CX3CR1(+)CD11b(+) microglia were the first APCs in the CNS to contain myelin Ag upon induction of disease, albeit in very small numbers. Dendritic cells (DCs) arrived in the CNS in sizable numbers significantly later (day 5 postimmunization), without detectable myelin Ag, but acquired it by day 7 postimmunization. Furthermore, a sharp increase in neuroantigen-containing DCs coincided with the onset of EAE symptoms. Importantly, in naive mice a low but consistent number of microglia contained myelin Ag, suggesting release by oligodendrocytes under steady state conditions. Although microglia isolated from naive brain and spinal cord did not elicit a strong CD4(+) T cell response in vitro, myelin Ag-containing microglia may still play a local role in modulating encephalitogenic CD4(+) T cell responses in early EAE prior to the arrival of other professional APCs, such as DCs. Finally, newly arriving DCs in the CNS not yet loaded with myelin Ag before the onset of EAE may be a potential therapeutic target.

Regulation of adaptive immunity by the fractalkine receptor during autoimmune inflammation.

Fractalkine, a chemokine anchored to neurons or peripheral endothelial cells, serves as an adhesion molecule or as a soluble chemoattractant. Fractalkine binds CX3CR1 on microglia and circulating monocytes, dendritic cells, and NK cells. The aim of this study is to determine the role of CX3CR1 in the trafficking and function of myeloid cells to the CNS during experimental autoimmune encephalomyelitis (EAE). Our results show that, in models of active EAE, Cx3cr1(-/-) mice exhibited more severe neurologic deficiencies. Bone marrow chimeric mice confirmed that CX3CR1 deficiency in bone marrow enhanced EAE severity. Notably, CX3CR1 deficiency was associated with an increased accumulation of CD115(+)Ly6C(-)CD11c(+) dendritic cells into EAE-affected brains that correlated with enhanced demyelination and neuronal damage. Furthermore, higher IFN-γ and IL-17 levels were detected in cerebellar and spinal cord tissues of CX3CR1-deficient mice. Analyses of peripheral responses during disease initiation revealed a higher frequency of IFN-γ- and IL-17-producing T cells in lymphoid tissues of CX3CR1-deficient as well as enhanced T cell proliferation induced by CX3CR1-deficient dendritic cells. In addition, adoptive transfer of myelin oligodendrocyte glycoprotein35-55-reactive wild-type T cells induced substantially more severe EAE in CX3CR1-deficient recipients when compared with wild-type recipients. Collectively, the data demonstrate that besides its role in chemoattraction, CX3CR1 is a key regulator of myeloid cell activation contributing to the establishment of adaptive immune responses.

The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.

Microglial cells are difficult to track during development because of the lack of specific reagents for myeloid subpopulations. To further understand how myeloid lineages differentiate during development to create microglial cells, we investigated CX3CR1 and CCR2 transcription unit activation in Cx3cr1(+/GFP)CCR2(+/RFP) knockin fluorescent protein reporter mice. The principal findings include: 1) CX3CR1(+) cells localized to the aorta-gonad-mesonephros region, and visualized at embryonic day (E)9.0 in the yolk sac and neuroectoderm; 2) at E10.5, CX3CR1 single-positive microglial cells were visualized penetrating the neuroepithelium; and 3) CX3CR1 and CCR2 distinguished infiltrating macrophages from resident surveillant or activated microglia within tissue sections and by flow cytometric analyses. Our results support the contribution of the yolk sac as a source of microglial precursors. We provide a novel model to monitor chemokine receptor expression changes in microglia and myeloid cells early (E8.0-E10.5) in development and during inflammatory conditions, which have been challenging to visualize in mammalian tissues.

Astrogliosis in the GFAP-CreERT2:Rosa26iDTR Mouse Model Does Not Exacerbate Retinal Microglia Activation or Müller Cell Gliosis under Hypoxic Conditions.

Diabetic retinopathy (DR) affects over 140 million people globally. The mechanisms that lead to blindness are still enigmatic but there is evidence that sustained inflammation and hypoxia contribute to vascular damage. Despite efforts to understand the role of inflammation and microglia in DR's pathology, the contribution of astrocytes to hypoxic responses is less clear. To investigate the role of astrocytes in hypoxia-induced retinopathy, we utilized a 7-day systemic hypoxia model using the GFAP-CreERT2:Rosa26iDTR transgenic mouse line. This allows for the induction of inflammatory reactive astrogliosis following tamoxifen and diphtheria toxin administration. We hypothesize that DTx-induced astrogliosis is neuroprotective during hypoxia-induced retinopathy. Glial, neuronal, and vascular responses were quantified using immunostaining, with antibodies against GFAP, vimentin, IBA-1, NeuN, fibrinogen, and CD31. Cytokine responses were measured in both the brain and serum. We report that while both DTx and hypoxia induced a phenotype of reduced microglia morphological activation, DTx, but not hypoxia, induced an increase in the Müller glia marker vimentin. We did not observe that the combination of DTx and hypoxic treatments exacerbated the signs of reactive glial cells, nor did we observe a significant change in the expression immunomodulatory mediators IL-1ß, IL2, IL-4, IL-5, IL-6, IL-10, IL-18, CCL17, TGF-ß1, GM-CSF, TNF-α, and IFN-γ. Overall, our results suggest that, in this hypoxia model, reactive astrogliosis does not alter the inflammatory responses or cause vascular damage in the retina.

Increased accumulation of regulatory granulocytic myeloid cells in mannose receptor C type 1-deficient mice correlates with protection in a mouse model of neurocysticercosis.

Neurocysticercosis (NCC) is a central nervous system (CNS) infection caused by the metacestode stage of the parasite Taenia solium. During NCC, the parasites release immunodominant glycan antigens in the CNS environment, invoking immune responses. The majority of the associated pathogenesis is attributed to the immune response against the parasites. Glycans from a number of pathogens, including helminths, act as pathogen-associated molecular pattern molecules (PAMPs), which are recognized by pattern recognition receptors (PRRs) known as C-type lectin receptors (CLRs). Using a mouse model of NCC by infection with the related parasite Mesocestoides corti, we have investigated the role of mannose receptor C type 1 (MRC1), a CLR which recognizes high-mannose-containing glycan antigens. Here we show that MRC1(-/-) mice exhibit increased survival times after infection compared with their wild-type (WT) counterparts. The decreased disease severity correlates with reduced levels of expression of markers implicated in NCC pathology, such as interleukin-1ß (IL-1ß), IL-6, CCL5, and matrix metalloproteinase 9 (MMP9), in addition to induction of an important repair marker, fibroblast growth factor 2 (FGF2). Furthermore, the immune cell subsets that infiltrate the brain of MRC1(-/-) mice are dramatically altered and characterized by reduced numbers of T cells and the accumulation of granulocytic cells with an immune phenotype resembling granulocytic myeloid-dependent suppressor cells (gMDSCs). The results suggest that MRC1 plays a critical role in myeloid plasticity, which in turn affects the adaptive immune response and immunopathogenesis during murine NCC.

Dynamic intravital imaging reveals reactive vessel-associated microglia play a protective role in cerebral malaria coagulopathy.

Vascular congestion and coagulopathy have been shown to play a role in human and experimental cerebral malaria (eCM), but little is known about the role of microglia, or microglia-vascular interactions and hypercoagulation during disease progression in this fatal infection. Recent studies show microglia bind to fibrinogen, a glycoprotein involved in thrombosis. An eCM model of Plasmodium chabaudi infection in mice deficient in the regulatory cytokine IL-10 manifests neuropathology, including hypercoagulation with extensive fibrin(ogen) deposition and neuroinflammation. Intravital microscopy and immunofluorescence are applied to elucidate the role of microglia in eCM. Results show microgliosis and coagulopathy occur early in disease at 3 dpi (day post-infection), and both are exacerbated as disease progresses to 7dpi. Vessel associated microglia increase significantly at 7 dpi, and the expression of the microglial chemoattractant CCL5 (RANTES) is increased versus uninfected and localized with fibrin(ogen) in vessels. PLX3397 microglia depletion resulted in rapid behavioral decline, severe hypothermia, and greater increase in vascular coagulopathy. This study suggests that microglia play a prominent role in controlling infection-initiated coagulopathy and supports a model in which microglia play a protective role in cerebral malaria by migrating to and patrolling the cerebral vasculature, potentially regulating degree of coagulation during systemic inflammation.

Pharmacological depletion of microglia alleviates neuronal and vascular damage in the diabetic CX3CR1-WT retina but not in CX3CR1-KO or hCX3CR1I249/M280-expressing retina.

Diabetic retinopathy, a microvascular disease characterized by irreparable vascular damage, neurodegeneration and neuroinflammation, is a leading complication of diabetes mellitus. There is no cure for DR, and medical interventions marginally slow the progression of disease. Microglia-mediated inflammation in the diabetic retina is regulated via CX3CR1-FKN signaling, where FKN serves as a calming signal for microglial activation in several neuroinflammatory models. Polymorphic variants of CX3CR1, hCX3CR1I249/M280 , found in 25% of the human population, result in a receptor with lower binding affinity for FKN. Furthermore, disrupted CX3CR1-FKN signaling in CX3CR1-KO and FKN-KO mice leads to exacerbated microglial activation, robust neuronal cell loss and substantial vascular damage in the diabetic retina. Thus, studies to characterize the effects of hCX3CR1I249/M280 -expression in microglia-mediated inflammation in the diseased retina are relevant to identify mechanisms by which microglia contribute to disease progression. Our results show that hCX3CR1I249/M280 mice are significantly more susceptible to microgliosis and production of Cxcl10 and TNFα under acute inflammatory conditions. Inflammation is exacerbated under diabetic conditions and coincides with robust neuronal loss in comparison to CX3CR1-WT mice. Therefore, to further investigate the role of hCX3CR1I249/M280 -expression in microglial responses, we pharmacologically depleted microglia using PLX-5622, a CSF-1R antagonist. PLX-5622 treatment led to a robust (~70%) reduction in Iba1+ microglia in all non-diabetic and diabetic mice. CSF-1R antagonism in diabetic CX3CR1-WT prevented TUJ1+ axonal loss, angiogenesis and fibrinogen deposition. In contrast, PLX-5622 microglia depletion in CX3CR1-KO and hCX3CR1I249/M280 mice did not alleviate TUJ1+ axonal loss or angiogenesis. Interestingly, PLX-5622 treatment reduced fibrinogen deposition in CX3CR1-KO mice but not in hCX3CR1I249/M280 mice, suggesting that hCX3CR1I249/M280 expressing microglia influences vascular pathology differently compared to CX3CR1-KO microglia. Currently CX3CR1-KO mice are the most commonly used strain to investigate CX3CR1-FKN signaling effects on microglia-mediated inflammation and the results in this study indicate that hCX3CR1I249/M280 receptor variants may serve as a complementary model to study dysregulated CX3CR1-FKN signaling. In summary, the protective effects of microglia depletion is CX3CR1-dependent as microglia depletion in CX3CR1-KO and hCX3CR1I249/M280 mice did not alleviate retinal degeneration nor microglial morphological activation as observed in CX3CR1-WT mice.

Defibrinogenation Ameliorates Retinal Microgliosis and Inflammation in A CX3CR1-Independent Manner.

SUMMARY STATEMENT: Diabetic human and murine retinas revealed pronounced microglial morphological activation and vascular abnormalities associated with inflammation. Pharmacological fibrinogen depletion using ancrod dampened microglial morphology alterations, resolved fibrinogen accumulation, rescued axonal integrity, and reduced inflammation in the diabetic murine retina.

Analyses of phenotypic and functional characteristics of CX3CR1-expressing natural killer cells.

We previously demonstrated a correlation between the frequency of CX3CR1-expressing human natural killer (NK) cells and disease activity in multiple sclerosis and showed that CX3CR1(high) NK cells were more cytotoxic than their CX3CR1(neg/low) counterparts. Here we aimed to determine whether human NK cell fractions defined by CX3CR1 represent distinct subtypes. Phenotypic and functional NK cell analyses revealed that, distinct from CX3CR1(high), CX3CR1(neg/low) NK cells expressed high amounts of type 2 cytokines, proliferated robustly in response to interleukin-2 and promoted a strong up-regulation of the key co-stimulatory molecule CD40 on monocytes. Co-expression analyses of CX3CR1 and CD56 demonstrated the existence of different NK cell fractions based on the surface expression of these two surface markers, the CX3CR1(neg) CD56(bright), CX3CR1(neg) CD56(dim) and CX3CR1(high) CD56(dim) fractions. Additional investigations on the expression of NK cell receptors (KIR, NKG2A, NKp30 and NKp46) and the maturation markers CD27, CD62L and CD57 indicated that CX3CR1 expression of CD56(dim) discriminated between an intermediary CX3CR1(neg) CD56(dim) and fully mature CX3CR1(high) CD56(dim) NK cell fractions. Hence, CX3CR1 emerges as an additional differentiation marker that may link NK cell maturation with the ability to migrate to different organs including the central nervous system.

CX3CR1 deficiency alters microglial activation and reduces beta-amyloid deposition in two Alzheimer's disease mouse models.

Microglia, the primary immune effector cells in the brain, continually monitor the tissue parenchyma for pathological alterations and become activated in Alzheimer's disease. Loss of signaling between neurons and microglia via deletion of the microglial receptor, CX3CR1, worsens phenotypes in various models of neurodegenerative diseases. In contrast, CX3CR1 deficiency ameliorates pathology in murine stroke models. To examine the role of CX3CR1 in Alzheimer's disease-related ß-amyloid pathology, we generated APPPS1 and R1.40 transgenic mouse models of Alzheimer's disease deficient for CX3CR1. Surprisingly, CX3CR1 deficiency resulted in a gene dose-dependent reduction in ß-amyloid deposition in both the APPPS1 and R1.40 mouse models of AD. Immunohistochemical analysis revealed reduced staining for CD68, a marker of microglial activation. Furthermore, quantitative immunohistochemical analysis revealed reduced numbers of microglia surrounding ß-amyloid deposits in the CX3CR1-deficient APPPS1 animals. The reduced ß-amyloid pathology correlated with reduced levels of TNFα and CCL2 mRNAs, but elevated IL1ß mRNA levels, suggesting an altered neuroinflammatory milieu. Finally, to account for these seemingly disparate results, both in vitro and in vivo studies provided evidence that CX3CL1/CX3CR1 signaling alters the phagocytic capacity of microglia, including the uptake of Aß fibrils. Taken together, these results demonstrate that loss of neuron-microglial fractalkine signaling leads to reduced ß-amyloid deposition in mouse models of AD that is potentially mediated by altered activation and phagocytic capability of CX3CR1-deficient microglia.