Single-cell RNA sequencing reveals peripheral blood leukocyte responses to spinal cord injury in mice with humanised immune systems.

Next-generation humanised mouse models and single-cell RNA sequencing (scRNAseq) approaches enable in-depth studies into human immune cell biology. Here we used NSG-SGM3 mice engrafted with human umbilical cord haematopoietic stem cells to investigate how human immune cells respond to and/or are changed by traumatic spinal cord injury (SCI). We hypothesised that the use of such mice could help advance our understanding of spinal cord injury-induced immune depression syndrome (SCI-IDS), and also how human leukocytes change as they migrate from the circulation into the lesion site. Our scRNAseq experiments, supplemented by flow cytometry, demonstrate the existence of up to 11 human immune cell (sub-) types and/or states across the blood and injured spinal cord (7 days post-SCI) of humanised NSG-SGM3 mice. Further comparisons of human immune cell transcriptomes between naïve, sham-operated and SCI mice identified a total of 579 differentially expressed genes, 190 of which were 'SCI-specific' (that is, genes regulated only in response to SCI but not sham surgery). Gene ontology analysis showed a prominent downregulation of immune cell function under SCI conditions, including for T cell receptor signalling and antigen presentation, confirming the presence of SCI-IDS and the transcriptional signature of human leukocytes in association with this phenomenon. We also highlight the activating influence of the local spinal cord lesion microenvironment by comparing the transcriptomes of circulating versus infiltrated human immune cells; those isolated from the lesion site were enriched for genes relating to both immune cell activity and function (e.g., oxidative phosphorylation, T cell proliferation and antigen presentation). We lastly applied an integrated bioinformatics approach to determine where immune responses in humanised NSG-SGM3 mice appear congruent to the native responses of human SCI patients, and where they diverge. Collectively, our study provides a valuable resource and methodological framework for the use of these mice in translational research.

CSF1R inhibition promotes neuroinflammation and behavioral deficits during graft-versus-host disease in mice.

ABSTRACT: Chronic graft-versus-host disease (cGVHD) remains a significant complication of allogeneic hematopoietic stem cell transplantation. Central nervous system (CNS) involvement is becoming increasingly recognized, in which brain-infiltrating donor major histocompatibility complex (MHC) class II+ bone marrow-derived macrophages (BMDM) drive pathology. BMDM are also mediators of cutaneous and pulmonary cGVHD, and clinical trials assessing the efficacy of antibody blockade of colony-stimulating factor 1 receptor (CSF1R) to deplete macrophages are promising. We hypothesized that CSF1R antibody blockade may also be a useful strategy to prevent/treat CNS cGVHD. Increased blood-brain barrier permeability during acute GVHD (aGVHD) facilitated CNS antibody access and microglia depletion by anti-CSF1R treatment. However, CSF1R blockade early after transplant unexpectedly exacerbated aGVHD neuroinflammation. In established cGVHD, vascular changes and anti-CSF1R efficacy were more limited. Anti-CSF1R-treated mice retained donor BMDM, activated microglia, CD8+ and CD4+ T cells, and local cytokine expression in the brain. These findings were recapitulated in GVHD recipients, in which CSF1R was conditionally depleted in donor CX3CR1+ BMDM. Notably, inhibition of CSF1R signaling after transplant failed to reverse GVHD-induced behavioral changes. Moreover, we observed aberrant behavior in non-GVHD control recipients administered anti-CSF1R blocking antibody and naïve mice lacking CSF1R in CX3CR1+ cells, revealing a novel role for homeostatic microglia and indicating that ongoing clinical trials of CSF1R inhibition should assess neurological adverse events in patients. In contrast, transfer of Ifngr-/- grafts could reduce MHC class II+ BMDM infiltration, resulting in improved neurocognitive function. Our findings highlight unexpected neurological immune toxicity during CSF1R blockade and provide alternative targets for the treatment of cGVHD within the CNS.

Robust mapping of spatiotemporal trajectories and cell-cell interactions in healthy and diseased tissues.

Spatial transcriptomics (ST) technologies generate multiple data types from biological samples, namely gene expression, physical distance between data points, and/or tissue morphology. Here we developed three computational-statistical algorithms that integrate all three data types to advance understanding of cellular processes. First, we present a spatial graph-based method, pseudo-time-space (PSTS), to model and uncover relationships between transcriptional states of cells across tissues undergoing dynamic change (e.g. neurodevelopment, brain injury and/or microglia activation, and cancer progression). We further developed a spatially-constrained two-level permutation (SCTP) test to study cell-cell interaction, finding highly interactive tissue regions across thousands of ligand-receptor pairs with markedly reduced false discovery rates. Finally, we present a spatial graph-based imputation method with neural network (stSME), to correct for technical noise/dropout and increase ST data coverage. Together, the algorithms that we developed, implemented in the comprehensive and fast stLearn software, allow for robust interrogation of biological processes within healthy and diseased tissues.

The neuroprotective effects of estrogen and estrogenic compounds in spinal cord injury.

Spinal cord injury (SCI) occurs when the spinal cord is damaged from either a traumatic event or disease. SCI is characterised by multiple injury phases that affect the transmission of sensory and motor signals and lead to temporary or long-term functional deficits. There are few treatments for SCI. Estrogens and estrogenic compounds, however, may effectively mitigate the effects of SCI and therefore represent viable treatment options. This review systematically examines the pre-clinical literature on estrogen and estrogenic compound neuroprotection after SCI. Several estrogens were examined by the included studies: estrogen, estradiol benzoate, Premarin, isopsoralen, genistein, and selective estrogen receptor modulators. Across these pharmacotherapies, we find significant evidence that estrogens indeed offer protection against myriad pathophysiological effects of SCI and lead to improvements in functional outcomes, including locomotion. A STRING functional network analysis of proteins modulated by estrogen after SCI demonstrated that estrogen simultaneously upregulates known neuroprotective pathways, such as HIF-1, and downregulates pro-inflammatory pathways, including IL-17. These findings highlight the strong therapeutic potential of estrogen and estrogenic compounds after SCI.

Acute IL-1RA treatment suppresses the peripheral and central inflammatory response to spinal cord injury.

BACKGROUND: The acute phase response (APR) to CNS insults contributes to the overall magnitude and nature of the systemic inflammatory response. Aspects of this response are thought to drive secondary inflammatory pathology at the lesion site, and suppression of the APR can therefore afford some neuroprotection. In this study, we examined the APR in a mouse model of traumatic spinal cord injury (SCI), along with its relationship to neutrophil recruitment during the immediate aftermath of the insult. We specifically investigated the effect of IL-1 receptor antagonist (IL-1RA) administration on the APR and leukocyte recruitment to the injured spinal cord. METHODS: Adult female C57BL/6 mice underwent either a 70kD contusive SCI, or sham surgery, and tissue was collected at 2, 6, 12, and 24 hours post-operation. For IL-1RA experiments, SCI mice received two intraperitoneal injections of human IL-1RA (100mg/kg), or saline as control, immediately following, and 5 hours after impact, and animals were sacrificed 6 hours later. Blood, spleen, liver and spinal cord were collected to study markers of central and peripheral inflammation by flow cytometry, immunohistochemistry and qPCR. Results were analysed by two-way ANOVA or student's t-test, as appropriate. RESULTS: SCI induced a robust APR, hallmarked by elevated hepatic expression of pro-inflammatory marker genes and a significantly increased neutrophil presence in the blood, liver and spleen of these animals, as early as 2 hours after injury. This peripheral response preceded significant neutrophil infiltration of the spinal cord, which peaked 24 hours post-SCI. Although expression of IL-1RA was also induced in the liver following SCI, its response was delayed compared to IL-1ß. Exogenous administration of IL-1RA during this putative therapeutic window was able to suppress the hepatic APR, as evidenced by a reduction in CXCL1 and SAA-2 expression as well as a significant decrease in neutrophil infiltration in both the liver and the injured spinal cord itself. CONCLUSIONS: Our data indicate that peripheral administration of IL-1RA can attenuate the APR which in turn reduces immune cell infiltration at the spinal cord lesion site. We propose IL-1RA treatment as a viable therapeutic strategy to minimise the harmful effects of SCI-induced inflammation.

Repopulating Microglia Promote Brain Repair in an IL-6-Dependent Manner.

Cognitive dysfunction and reactive microglia are hallmarks of traumatic brain injury (TBI), yet whether these cells contribute to cognitive deficits and secondary inflammatory pathology remains poorly understood. Here, we show that removal of microglia from the mouse brain has little effect on the outcome of TBI, but inducing the turnover of these cells through either pharmacologic or genetic approaches can yield a neuroprotective microglial phenotype that profoundly aids recovery. The beneficial effects of these repopulating microglia are critically dependent on interleukin-6 (IL-6) trans-signaling via the soluble IL-6 receptor (IL-6R) and robustly support adult neurogenesis, specifically by augmenting the survival of newborn neurons that directly support cognitive function. We conclude that microglia in the mammalian brain can be manipulated to adopt a neuroprotective and pro-regenerative phenotype that can aid repair and alleviate the cognitive deficits arising from brain injury.

p110δ PI3-Kinase Inhibition Perturbs APP and TNFα Trafficking, Reduces Plaque Burden, Dampens Neuroinflammation, and Prevents Cognitive Decline in an Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is associated with the cleavage of the amyloid precursor protein (APP) to produce the toxic amyloid-ß (Aß) peptide. Accumulation of Aß, together with the concomitant inflammatory response, ultimately leads to neuronal death and cognitive decline. Despite AD progression being underpinned by both neuronal and immunological components, therapeutic strategies based on dual targeting of these systems remains unexplored. Here, we report that inactivation of the p110δ isoform of phosphoinositide 3-kinase (PI3K) reduces anterograde axonal trafficking of APP in hippocampal neurons and dampens secretion of the inflammatory cytokine tumor necrosis factor-alpha by microglial cells in the familial AD APPswe/PS1ΔE9 (APP/PS1) mouse model. Moreover, APP/PS1 mice with kinase-inactive PI3Kδ (δD910A) had reduced Aß peptides levels and plaques in the brain and an abrogated inflammatory response compared with APP/PS1 littermates. Mechanistic investigations reveal that PI3Kδ inhibition decreases the axonal transport of APP by eliciting the formation of highly elongated tubular-shaped APP-containing carriers, reducing the levels of secreted Aß peptide. Importantly, APP/PS1/δD910A mice exhibited no spatial learning or memory deficits. Our data highlight inhibition of PI3Kδ as a new approach to protect against AD pathology due to its dual action of dampening microglial-dependent neuroinflammation and reducing plaque burden by inhibition of neuronal APP trafficking and processing.SIGNIFICANCE STATEMENT During Alzheimer's disease (AD), the accumulation of the toxic amyloid-ß (Aß) peptide in plaques is associated with a chronic excessive inflammatory response. Uncovering new drug targets that simultaneously reduce both Aß plaque load and neuroinflammation holds therapeutic promise. Using a combination of genetic and pharmacological approaches, we found that the p110δ isoform of phosphoinositide 3-kinase (PI3K) is involved in anterograde trafficking of the amyloid precursor protein in neurons and in the secretion of tumor necrosis factor-alpha from microglial cells. Genetic inactivation of PI3Kδ reduces Aß plaque deposition and abrogates the inflammatory response, resulting in a complete rescue of the life span and spatial memory performance. We conclude that inhibiting PI3Kδ represents a novel therapeutic approach to ameliorate AD pathology by dampening plaque accumulation and microglial-dependent neuroinflammation.

Complement receptor C3aR1 controls neutrophil mobilization following spinal cord injury through physiological antagonism of CXCR2.

Traumatic spinal cord injury (SCI) triggers an acute-phase response that leads to systemic inflammation and rapid mobilization of bone marrow (BM) neutrophils into the blood. These mobilized neutrophils then accumulate in visceral organs and the injured spinal cord where they cause inflammatory tissue damage. The receptor for complement activation product 3a, C3aR1, has been implicated in negatively regulating the BM neutrophil response to tissue injury. However, the mechanism via which C3aR1 controls BM neutrophil mobilization, and also its influence over SCI outcomes, are unknown. Here, we show that the C3a/C3aR1 axis exerts neuroprotection in SCI by acting as a physiological antagonist against neutrophil chemotactic signals. We show that C3aR1 engages phosphatase and tensin homolog (PTEN), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, to restrain C-X-C chemokine receptor type 2-driven BM neutrophil mobilization following trauma. These findings are of direct clinical significance as lower circulating neutrophil numbers at presentation were identified as a marker for improved recovery in human SCI. Our work thus identifies C3aR1 and its downstream intermediary, PTEN, as therapeutic targets to broadly inhibit neutrophil mobilization/recruitment following tissue injury and reduce inflammatory pathology.

The Alternative Receptor for Complement Component 5a, C5aR2, Conveys Neuroprotection in Traumatic Spinal Cord Injury.

This study investigated the role of the alternative receptor for complement activation fragment C5a, C5aR2, in secondary inflammatory pathology after contusive spinal cord injury (SCI) in mice. C5ar2-/- mice exhibited decreased intraparenchymal tumor necrosis factor alpha and interleukin-6 acutely post-injury, but these reductions did not translate into improved outcomes. We show that loss of C5aR2 leads to increased lesion volumes, reduced myelin sparing, and significantly worsened recovery from SCI in C5ar2-/- animals compared to wild-type (WT) controls. Loss of C5aR2 did not alter leukocyte mobilization from the bone marrow in response to SCI, and neutrophil recruitment/presence at the lesion site was also not different between genotypes. Acute treatment of SCI mice with the selective C5aR1 antagonist, PMX205, improved SCI outcomes, compared to vehicle controls, and, importantly, fully alleviated the worsened recovery of C5ar2-/- mice compared to their WT counterparts. Collectively, these findings indicate that C5aR2 is neuroprotective and a novel target to restrain injurious C5a signaling after a major neurotraumatic event.

It takes nerve to fight back: The significance of neural innervation of the bone marrow and spleen for immune function.

Inflammation is a natural part of wound healing but it can also cause secondary (bystander) damage and/or negatively interfere with endogenous repair mechanisms if non-resolving. Regulation of inflammation is traditionally looked at from the perspective of danger signals, cytokines and chemokines, and their respective receptors. A neuronal contribution to the regulation of inflammation is, however, increasingly appreciated, and this has important implications for the bodily response under conditions where the nervous system itself may be damaged. In this review article, we provide an up-to-date overview of the current literature on neural innervation of primary and secondary lymphoid organs, focusing in particular on the bone marrow and spleen, its significance in relation to immune function and, lastly, also briefly discussing how a major neurotraumatic event like spinal cord injury (SCI) may impact on this.

A case of mistaken identity: CD11c-eYFP(+) cells in the normal mouse brain parenchyma and neural retina display the phenotype of microglia, not dendritic cells.

Under steady-state conditions the central nervous system (CNS) is traditionally thought to be devoid of antigen presenting cells; however, putative dendritic cells (DCs) expressing enhanced yellow fluorescent protein (eYFP) are present in the retina and brain parenchyma of CD11c-eYFP mice. We previously showed that these mice carry the Crb1(rd8) mutation, which causes retinal dystrophic lesions; therefore we hypothesized that the presence of CD11c-eYFP(+) cells within the CNS may be due to pathology associated with the Crb1(rd8) mutation. We generated CD11c-eYFP Crb1(wt/wt) mice and compared the distribution and immunophenotype of CD11c-eYFP(+) cells in CD11c-eYFP mice with and without the Crb1(rd8) mutation. The number and distribution of CD11c-eYFP(+) cells in the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice. CD11c-eYFP(+) cells were distributed throughout the inner retina, and clustered in brain regions that receive input from the external environment or lack a blood-brain barrier. CD11c-eYFP(+) cells within the retina and cerebral cortex of CD11c-eYFP Crb1(wt/wt) mice expressed CD11b, F4/80, CD115 and Iba-1, but not DC or antigen presentation markers, whereas CD11c-eYFP(+) cells within the choroid plexus and pia mater expressed CD11c, I-A/I-E, CD80, CD86, CD103, DEC205, CD8α and CD135. The immunophenotype of CD11c-eYFP(+) cells and microglia within the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice; however, CD11c and I-A/I-E expression was significantly increased in CD11c-eYFP Crb1(rd8/rd8) mice. This study demonstrates that the overwhelming majority of CNS CD11c-eYFP(+) cells do not display the phenotype of DCs or their precursors and are most likely a subpopulation of microglia. GLIA 2016. GLIA 2016;64:1331-1349.

PI3Kδ inhibition reduces TNF secretion and neuroinflammation in a mouse cerebral stroke model.

Stroke is a major cause of death worldwide and the leading cause of permanent disability. Although reperfusion is currently used as treatment, the restoration of blood flow following ischaemia elicits a profound inflammatory response mediated by proinflammatory cytokines such as tumour necrosis factor (TNF), exacerbating tissue damage and worsening the outcomes for stroke patients. Phosphoinositide 3-kinase delta (PI3Kδ) controls intracellular TNF trafficking in macrophages and therefore represents a prospective target to limit neuroinflammation. Here we show that PI3Kδ inhibition confers protection in ischaemia/reperfusion models of stroke. In vitro, restoration of glucose supply following an episode of glucose deprivation potentiates TNF secretion from primary microglia-an effect that is sensitive to PI3Kδ inhibition. In vivo, transient middle cerebral artery occlusion and reperfusion in kinase-dead PI3Kδ (p110δ(D910A/D910A)) or wild-type mice pre- or post-treated with the PI3Kδ inhibitor CAL-101, leads to reduced TNF levels, decreased leukocyte infiltration, reduced infarct size and improved functional outcome. These data identify PI3Kδ as a potential therapeutic target in ischaemic stroke.

Mobilisation of the splenic monocyte reservoir and peripheral CX3CR1 deficiency adversely affects recovery from spinal cord injury.

Macrophages in the injured spinal cord originate from resident microglia and blood monocytes. Whether this diversity in origins contributes to their seemingly dual role in immunopathology and repair processes has remained poorly understood. Here we took advantage of Cx3cr1(gfp) mice to visualise monocyte-derived macrophages in the injured spinal cord via adoptive cell transfer and bone marrow (BM) chimera approaches. We show that the majority of infiltrating monocytes at 7 days post-injury originate from the spleen and only to a lesser extent from the BM. Prevention of early monocyte infiltration via splenectomy was associated with improved recovery at 42 days post-SCI. In addition, an increased early presence of infiltrating monocytes/macrophages, as a result of CX3CR1 deficiency within the peripheral immune compartment, correlated with worsened injury outcomes. Adoptive transfer of identified Cx3cr1(gfp/+) monocytes confirmed peak infiltration at 7 days post-injury, with inflammatory (Ly6C(high)) monocytes being most efficiently recruited. Focal SCI also changed the composition of the two major monocyte subsets in the blood, with more Ly6C(high) cells present during peak recruitment. Adoptive transfer experiments further suggested high turnover of inflammatory monocytes in the spinal cord at 7 days post-injury. Consistent with this, only a small proportion of infiltrating cells unequivocally expressed polarisation markers for pro-inflammatory (M1) or alternatively activated (M2) macrophages at this time point. Our findings offer new insights into the origins of monocyte-derived macrophages after SCI and their contribution to functional recovery, providing a basis for further scrutiny and selective targeting of Ly6C(high) monocytes to improve outcomes from neurotraumatic events.

Detection of endogenous iron deposits in the injured mouse spinal cord through high-resolution ex vivo and in vivo MRI.

The main aim of this study was to employ high-resolution MRI to investigate the spatiotemporal development of pathological features associated with contusive spinal cord injury (SCI) in mice. Experimental mice were subjected to either sham surgery or moderate contusive SCI. A 16.4-T small-animal MR system was employed for nondestructive imaging of post-mortem, fixed spinal cord specimens at the subacute (7 days) and more chronic (28-35 days) stages post-injury. Routine histological techniques were used for subsequent investigation of the observed neuropathology at the microscopic level. The central core of the lesion appeared as a dark hypo-intense area on MR images at all time points investigated. Small focal hypo-intense spots were also observed spreading through the dorsal funiculi proximal and distal to the site of impact, an area that is known to undergo gliosis and Wallerian degeneration in response to injury. Histological examination revealed these hypo-intense spots to be high in iron content as determined by Prussian blue staining. Quantitative image analysis confirmed the increased presence of iron deposits at all post-injury time points investigated (p<0.05). Distant iron deposits were also detectable through live imaging without the use of contrast-enhancing agents, enabling the longitudinal investigation of this pathology in individual animals. Further immunohistochemical evaluation showed that intracellular iron deposits localised to macrophages/microglia, astrocytes and oligodendrocytes in the subacute phase of SCI, but predominantly to glial fibrillary acidic protein-positive, CC-1-positive astrocytes at later stages of recovery. Progressive, widespread intracellular iron accumulation is thus a normal feature of SCI in mice, and high-resolution MRI can be effectively used to detect and monitor these neuropathological changes with time.

Vertebral landmarks for the identification of spinal cord segments in the mouse.

Accurate identification of spinal cord segments in relation to vertebral landmarks is essential to surgery aimed at experimental spinal cord injury. We have analyzed a complete series of high-resolution magnetic resonance (MR) images from the mouse spine in order to delineate the boundaries of spinal cord segments in relation to vertebral landmarks. The resulting atlas can be used to plan experimental approaches that require the accurate identification of a target spinal cord segment.

Accumulation of murine subretinal macrophages: effects of age, pigmentation and CX3CR1.

Macrophages or activated microglia in the subretinal space are considered a hallmark of some retinal pathologies. We investigated the effects of age, pigmentation and CX(3)CR1 deficiency on the accumulation of macrophages/activated microglia in the outer retina of young and old Cx(3)cr1(gfp/gfp) (CX(3)CR1-deficient) or Cx(3)cr1(gfp/+) mice on either a pigmented (C57BL/6) or albino (BALB/c) background. Quantitative analysis of immunostained retinal-choroidal whole mounts revealed an increase in subretinal macrophage (SRMΦ) numbers in young Cx(3)cr1(gfp/gfp) mice compared with Cx(3)cr1(gfp/+) mice, however the increase was more marked in albino Cx(3)cr1(gfp/gfp) mice. In aged mice, large numbers of SRMΦ/activated microglia replete with autofluorescent debris were noted in both old pigmented Cx(3)cr1(gfp/gfp) and Cx(3)cr1(gfp/+) mice proving this accumulation was not CX(3)CR1-dependent. While CX(3)CR1 deficiency leads to an early onset of SRMΦ accumulation, our data reveal that this change occurs in both aged Cx(3)cr1(gfp/+) and Cx(3)cr1(gfp/gfp) pigmented mice in the absence of marked retinal degeneration and is likely a normal response to aging.

CX3CR1 deficiency exacerbates neuronal loss and impairs early regenerative responses in the target-ablated olfactory epithelium.

The olfactory epithelium is a site of sustained adult neurogenesis where olfactory sensory neurons are continuously replaced from endogenous stem/progenitor cells. Epithelial macrophages have been implicated in the phagocytosis of degenerating cells but the molecular mechanisms allowing for their recruitment and activation while maintaining a neurogenic microenvironment are poorly understood. We have previously shown that the chemokine fractalkine (CX3CL1) is expressed by olfactory sensory neurons and ensheathing cells in the olfactory epithelium. In turn, the fractalkine receptor, CX3CR1, is expressed on macrophages and dendritic cells within the olfactory epithelium. We report that a selective cell death of olfactory sensory neurons in the epithelium of CX3CR1-deficient mice via target ablation (i.e. olfactory bulbectomy) results in an exacerbated loss of olfactory sensory neurons compared to wild-type mice. In addition, reduced proliferation of intraepithelial stem/progenitor cells was observed in lesioned CX3CR1-deficient mice, suggesting an impaired regenerative response. Importantly, a lack of CX3CL1-signaling caused increased recruitment of macrophages into the olfactory epithelium, which in turn contained higher levels of pro-inflammatory cytokines (e.g. TNF-α and IL-6) as determined by qPCR. We also present novel data showing that, relative to wild-type, CX3CR1-deficient macrophages have diminished phagocytic activity following stimulation with CX3CL1. Collectively, our data indicate that signaling through the CX3CR1 receptor modulates macrophage activity, resulting in an environment conducive to olfactory sensory neuron clearance and targeted replacement from endogenous stem/progenitor cells.

Novel characterization of monocyte-derived cell populations in the meninges and choroid plexus and their rates of replenishment in bone marrow chimeric mice.

The mouse dura mater, pia mater, and choroid plexus contain resident macrophages and dendritic cells (DCs). These cells participate in immune surveillance, phagocytosis of cellular debris, uptake of antigens from the surrounding cerebrospinal fluid and immune regulation in many pathologic processes. We used Cx3cr1 knock-in, CD11c-eYFP transgenic and bone marrow chimeric mice to characterize the phenotype, density and replenishment rate of monocyte-derived cells in the meninges and choroid plexus and to assess the role of the chemokine receptor CX3CR1 on their number and tissue distribution. Iba-1 major histocompatibility complex (MHC) Class II CD169 CD68 macrophages and CD11c putative DCs were identified in meningeal and choroid plexus whole mounts. Comparison of homozygous and heterozygous Cx3cr1 mice did not reveal CX3CR1-dependancy on density, distribution or phenotype of monocyte-derived cells. In turnover studies, wild type lethally irradiated mice were reconstituted with Cx3cr1/-positive bone marrow and were analyzed at 3 days, 1, 2, 4 and 8 weeks after transplantation. There was a rapid replenishment of CX3CR1-positive cells in the dura mater (at 4 weeks) and the choroid plexus was fully reconstituted by 8 weeks. These data provide the foundation for future studies on the role of resident macrophages and DCs in conditions such as meningitis, autoimmune inflammatory disease and in therapies involving irradiation and hematopoietic or stem cell transplantation.

The chemokine receptor CX3CR1 mediates homing of MHC class II-positive cells to the normal mouse corneal epithelium.

PURPOSE: Recent investigations have revealed that populations of macrophages and dendritic cells (DCs) are present in the stroma and epithelium of the cornea, although the precise phenotype and distribution are still controversial. CX(3)CR1, the sole receptor for the chemokine fractalkine, is expressed by these monocyte-derived cells. Transgenic CX(3)CR1(GFP) mice, in which either one (heterozygous) or both (homozygous) copies of the CX(3)CR1 gene were replaced by enhanced green fluorescent protein (eGFP), were used to characterize monocyte-derived cells in the mouse cornea and to determine whether the expression of this receptor influences the recruitment of these cells into the normal cornea. METHODS: Wholemount corneas were immunostained with anti-leukocyte antibodies to the phenotypic markers major histocompatibility complex (MHC) class II, CD169, CD68, CD11b, and CD45 and analyzed by epifluorescence and confocal microscopy. The density of intraepithelial MHC class II(+) cells was quantified in wild-type, CX(3)CR1(+/GFP) heterozygous, CX(3)CR1(GFP/GFP) homozygous, and CX(3)CR1-knockout mice. RESULTS: There was a significant reduction in the number of MHC class II(+) cells (putative DCs) in the corneal epithelium of CX(3)CR1-deficient mice (P < 0.009) compared with wild-type mice, and the few cells that were present did not possess classic dendriform morphology. No GFP(+) MHC class II(-) cells were noted in the epithelium. Dual immunostaining of corneas in both heterozygous and homozygous (CX(3)CR1-deficient) mice revealed GFP(+) cells with a more pleomorphic morphology throughout the entire corneal stroma that were CD11b(+) CD169(+), and had variable degrees of expression of CD68 andMHC class II. The immunophenotype and morphology of these intrastromal cells is strongly indicative of a macrophage phenotype. CONCLUSIONS: This study has identified a role for CX(3)CR1 in the normal recruitment of MHC class II(+) putative DCs into the corneal epithelium and establishes a model for investigating monocyte-derived cells and fractalkine/CX(3)CR1 interactions during corneal disease.

Olfactory ensheathing cells: characteristics, genetic engineering, and therapeutic potential.

Injured neurons in the mammalian central nervous system (CNS) do not normally regenerate their axons after injury. Neurotrauma to the CNS usually results in axonal damage and subsequent loss of communication between neuronal networks, causing long-term functional deficits. For CNS regeneration, repair strategies need to be developed that promote regrowth of lesioned axon projections and restoration of neuronal connectivity. After spinal cord injury (SCI), cystic cavitations are often found, particularly in the later stages, due to the loss of neural tissue at the original impact site. Ultimately, for the promotion of axonal regrowth in these situations, some form of transplantation will be required to provide lesioned axons with a supportive substrate along which they can extend. Here, we review the use of olfactory ensheathing cells: their location and role in the olfactory system, their use as cellular transplants in SCI paradigms, alone or in combination with gene therapy, and the unique properties of these cells that may give them a potential advantage over other cellular transplants.