Single-cell analysis of age-related changes in leukocytes of diabetic mouse hindpaws.

Complications associated with Type 1 and Type 2 diabetes, such as diabetic peripheral neuropathy and diabetic foot ulcers, are a growing health-care concern. In addition, this concern increases as diabetic patients age due to their increased susceptibility to complications. To address this growing problem, it is important to understand fluctuations in physiology which lead to pathological changes associated with the metabolic disturbances of diabetes. Our study explores dysregulation of immune cell populations in the hindpaws of healthy and diabetic mice at 12 and 21 weeks of age using single-cell RNA sequencing to provide insight into immune disruptions occurring in the distal limb during chronic diabetes. In 21-week-old Leprdb/db mice, increases were seen in mast cells/basophils, dermal γδ T cells, heterogeneous T cells, and Type 2 innate lymphoid cells. In addition, macrophages represented the largest cluster of immune cells and showed the greatest increase in genes associated with immune-specific pathways. Sub-clustering of macrophages revealed a bias toward angiogenic Lyve1+MHCIIlo macrophages in the hindpaws of 21-week-old diabetic mice, which corresponded to an increase in Lyve1+ macrophages in the hindpaws of 21-week-old diabetic mice on histology. Our results show that in Type 2 diabetes, the immunological function and phenotype of multiple immune cell types shift not only with metabolic disturbance, but also with duration of disease, which may explain the increased susceptibility to pathologies of the distal limb in patients with more chronic diabetes.

Sustained pain and macrophage infiltration in a mouse muscle contusion model.

INTRODUCTION/AIMS: Prior studies have emphasized the role of inflammation in the response to injury and muscle regeneration, but little emphasis has been placed on characterizing the relationship between innate inflammation, pain, and functional impairment. The aim of our study was to determine the contribution of innate immunity to prolonged pain following muscle contusion. METHODS: We developed a closed-impact mouse model of muscle contusion and a macrophage-targeted near-infrared fluorescent nanoemulsion. Closed-impact contusions were delivered to the lower left limb. Pain sensitivity, gait dysfunction, and inflammation were assessed in the days and weeks post-contusion. Macrophage accumulation was imaged in vivo by injecting i.v. near-infrared nanoemulsion. RESULTS: Despite hindpaw hypersensitivity persisting for several weeks, disruptions to gait and grip strength typically resolved within 10 days of injury. Using non-invasive imaging and immunohistochemistry, we show that macrophage density peaks in and around the affected muscle 3 day post-injury and quickly subsides. However, macrophage density in the ipsilateral sciatic nerve and dorsal root ganglia (DRG) increases more gradually and persists for at least 14 days. DISCUSSION: In this study, we demonstrate pain sensitivity is influenced by the degree of lower muscle contusion, without significant changes to gait and grip strength. This may be due to modulation of pain signaling by macrophage proliferation in the sciatic nerve, upstream from the site of injury. Our work suggests chronic pain developing from muscle contusion is driven by macrophage-derived neuroinflammation in the peripheral nervous system.

Age-Dependent Effects of Transgenic 2D2 Mice Used to Induce Passive Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice.

INTRODUCTION: Multiple sclerosis (MS) is a neurodegenerative autoimmune disease that worsens with age. Here, we examined the influence of age on passive experimental autoimmune encephalomyelitis (P-EAE), a model to study MS, using young and mature adult 2D2 transgenic donor mice to induce pathology in WT C57BL6/J mice. METHODS: Lymphocytes from young adult (i.e., 10-week-old) or mature adult (i.e., 6-month-old) transgenic donor mice were characterized by flow cytometry prior to injection of cultured leukocytes into adult female WT recipient mice, with a special focus on transgenic T cell phenotypes. RESULTS: Our findings show age-dependent changes in memory T cell phenotypes correlated with more severe clinical and histological disease when donor cells originated from young as compared to mature adult mice. CONCLUSION: Not only do these results demonstrate that the age of the 2D2 transgenic donor mice is critical in establishing P-EAE, but the differential effects might also identify age-dependent factors that contribute to EAE and perhaps MS.

Sensory neuron dysfunction in orthotopic mouse models of colon cancer.

Reports of neurological sequelae related to colon cancer are largely restricted to rare instances of paraneoplastic syndromes, due to autoimmune reactions. Systemic inflammation associated with tumor development influences sensory neuron function in other disease models, though the extent to which this occurs in colorectal cancer is unknown. We induced orthotopic colorectal cancer via orthotopic injection of two colorectal cancer cell lines (MC38 and CT26) in two different mouse strains (C57BL/6 and Balb/c, respectively). Behavioral tests of pain sensitivity and activity did not detect significant alterations in sensory sensitivity or diminished well-being throughout tumor development. However, immunohistochemistry revealed widespread reductions in intraepidermal nerve fiber density in the skin of tumor-bearing mice. Though loss of nerve fiber density was not associated with increased expression of cell injury markers in dorsal root ganglia, lumbar dorsal root ganglia neurons of tumor-bearing animals showed deficits in mitochondrial function. These neurons also had reduced cytosolic calcium levels in live-cell imaging and reduced spontaneous activity in multi-electrode array analysis. Bulk RNA sequencing of DRGs from tumor-bearing mice detected activation of gene expression pathways associated with elevated cytokine and chemokine signaling, including CXCL10. This is consistent with the detection of CXCL10 (and numerous other cytokines, chemokines and growth factors) in MC38 and CT26 cell-conditioned media, and the serum of tumor-bearing mice. Our study demonstrates in a pre-clinical setting that colon cancer is associated with latent sensory neuron dysfunction and implicates cytokine/chemokine signaling in this process. These findings may have implications for determining risk factors and treatment responsiveness related to neuropathy in colorectal cancer.

Tracking macrophages in diabetic neuropathy with two-color nanoemulsions for near-infrared fluorescent imaging and microscopy.

BACKGROUND: The incidence of diabetes and diabetic peripheral neuropathy continues to rise, and studies have shown that macrophages play an important role in their pathogenesis. To date, macrophage tracking has largely been achieved using genetically-encoded fluorescent proteins. Here we present a novel two-color fluorescently labeled perfluorocarbon nanoemulsion (PFC-NE) designed to monitor phagocytic macrophages in diabetic neuropathy in vitro and in vivo using non-invasive near-infrared fluorescent (NIRF) imaging and fluorescence microscopy. METHODS: Presented PFC-NEs were formulated with perfluorocarbon oil surrounded by hydrocarbon shell carrying two fluorescent dyes and stabilized with non-ionic surfactants. In vitro assessment of nanoemulsions was performed by measuring fluorescent signal stability, colloidal stability, and macrophage uptake and subsequent viability. The two-color PFC-NE was administered to Leprdb/db and wild-type mice by tail vein injection, and in vivo tracking of the nanoemulsion was performed using both NIRF imaging and confocal microscopy to assess its biodistribution within phagocytic macrophages along the peripheral sensory apparatus of the hindlimb. RESULTS: In vitro experiments show two-color PFC-NE demonstrated high fluorescent and colloidal stability, and that it was readily incorporated into RAW 264.7 macrophages. In vivo tracking revealed distribution of the two-color nanoemulsion to macrophages within most tissues of Leprdb/db and wild-type mice which persisted for several weeks, however it did not cross the blood brain barrier. Reduced fluorescence was seen in sciatic nerves of both Leprdb/db and wild-type mice, implying that the nanoemulsion may also have difficulty crossing an intact blood nerve barrier. Additionally, distribution of the nanoemulsion in Leprdb/db mice was reduced in several tissues as compared to wild-type mice. This reduction in biodistribution appears to be caused by the increased number of adipose tissue macrophages in Leprdb/db mice. CONCLUSIONS: The nanoemulsion in this study has the ability to identify phagocytic macrophages in the Leprdb/db model using both NIRF imaging and fluorescence microscopy. Presented nanoemulsions have the potential for carrying lipophilic drugs and/or fluorescent dyes, and target inflammatory macrophages in diabetes. Therefore, we foresee these agents becoming a useful tool in both imaging inflammation and providing potential treatment in diabetic peripheral neuropathy.

Neuroinflammation and B-Cell Phenotypes in Cervical and Lumbosacral Regions of the Spinal Cord in Experimental Autoimmune Encephalomyelitis in the Absence of Pertussis Toxin.

OBJECTIVES: The active experimental autoimmune encephalomyelitis (EAE) model is often initiated using myelin oligodendrocyte glycoprotein (MOG) immunization followed by pertussis toxin (PTX) to study multiple sclerosis. However, PTX inactivates G protein-coupled receptors, and with increasing knowledge of the role that various G protein-coupled receptors play in immune homeostasis, it is valuable to establish neuroimmune endpoints for active EAE without PTX. METHODS: Female C57BL/6 mice were immunized with MOG35-55 peptide in Complete Freund's Adjuvant and neuroinflammation, including central nervous system B-cell infiltration, was compared to saline-injected mice. Since it was anticipated that disease onset would be slower and less robust than EAE in the presence of PTX, both cervical and lumbosacral sections of the spinal cord were evaluated. RESULTS: Immunohistochemical analysis showed that EAE without PTX induced immune infiltration, CCL2 and VCAM-1 upregulation. Demyelination in the cervical region correlated with the infiltration of CD19+ B cells in the cervical region. There was upregulation of IgG, CD38, and PDL1 on B cells in cervical and lumbosacral regions of the spinal cord in EAE without PTX. Interestingly, IgG was expressed predominantly by CD19- cells. CONCLUSIONS: These data demonstrate that many neuroimmune endpoints are induced in EAE without PTX and although clinical disease is mild, this can be used as an autoimmune model when PTX inactivation of G protein-coupled receptors is not desired.

The CB1 Receptor Differentially Regulates IFN-γ Production In Vitro and in Experimental Autoimmune Encephalomyelitis.

Introduction: Activation of the peripheral immune system and the infiltration of immune cells into the central nervous system are both key features of the experimental autoimmune encephalomyelitis (EAE) model. By exploring how the endocannabinoid system works to modulate this response, we can better understand how exogenous cannabinoids, such as THC, might be used to modulate the immune responses of multiple sclerosis patients. Materials and Methods: In this study, we examined the role of the CB1 receptor in IFN-γ and IL-17A production in the EAE model and in vitro stimulations of naive splenocytes using Cnr1-/- mice and wild-type (WT) littermates. We also introduce a novel method of scoring spinal cord histological sections to show the differences in disease severity between Cnr1-/- and WT mice with EAE. Results: Clinical scores of Cnr1-/-/EAE and WT/EAE mice showed more severe disease progression in Cnr1-/- mice, which was confirmed using our new histological scoring method. In the peripheral immune system, IFN-γ production by restimulated splenocytes from Cnr1-/-/EAE mice, compared with WT/EAE mice, was increased and the primary source of IFN-γ was a CD3- cell population; however, IFN-γ production by Cnr1-/- splenocytes was decreased compared with WT splenocytes when the primary source of IFN-γ was CD3+ T cells in cultures from naive mice stimulated by either anti-CD3/anti-CD28 antibodies or Staphylococcal superantigens. Conclusion: These findings suggest a duality to the CB1 receptor's effects on the peripheral immune response, which varies based on the specific cell types stimulated. Knowledge of the complex nature of a receptor is an important part of determining its potential usefulness as a therapeutic target, and these findings further define the role of CB1 in IFN-γ responses.

CBD Suppression of EAE Is Correlated with Early Inhibition of Splenic IFN-γ + CD8+ T Cells and Modest Inhibition of Neuroinflammation.

In this study cannabidiol (CBD) was administered orally to determine its effects and mechanisms in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). We hypothesized that 75 mg/kg of oral CBD given for 5 days after initiation of disease would reduce EAE severity through suppression of either the early peripheral immune or late neuroimmune response. EAE was induced in C57BL/6 mice at two different magnitudes, and peripheral inflammatory and neuroinflammatory responses were measured at days 3, 10, and 18. Th1, Th17, Tc1, Tc17, Tregs, and myeloid derived suppressor cells (MDSC) were identified from the lymph nodes and spleens of each mouse to determine if CBD altered the suppressor cell or inflammatory cell populations in secondary lymphoid tissues. Additionally, neuroinflammation was identified in brain and spinal cord tissues using various immunohistochemical techniques and flow cytometry. Early treatment of EAE with oral CBD reduced clinical disease at the day 18 timepoint which correlated with a significant decrease in the percentage of MOG35-55 specific IFN-γ producing CD8+ T cells in the spleen at day 10. Analysis of both T cell infiltration and lesion size within the spinal cord also showed a moderate reduction in neuroinflammation within the central nervous system (CNS). These results provide evidence that oral CBD suppressed the peripheral immune response that precedes neuroinflammation; however, analysis of the neuroinflammatory endpoints also suggest that the modest reduction in neuroinflammation was only partially responsible for CBD's neuroprotective capability. Graphical Abstract CBD was administered orally for the first 5 days following initiation of EAE. CBD attenuated clinical disease, and we found that CBD suppressed IFN-γ producing CD8+ T cells in the spleen at day 10. There was also modest suppression of neuroinflammation. Together these data demonstrate that early, oral administration of CBD protected mice from disease, but the modest effects on neuroinflammation suggest other mechanisms participate in CBD's neuroprotective effect in EAE.

Immune Responses Regulated by Cannabidiol.

Introduction: Cannabidiol (CBD) as Epidiolex® (GW Pharmaceuticals) was recently approved by the U.S. Food and Drug Administration (FDA) to treat rare forms of epilepsy in patients 2 years of age and older. Together with the increased societal acceptance of recreational cannabis and CBD oil for putative medical use in many states, the exposure to CBD is increasing, even though all of its biological effects are not understood. Once such example is the ability of CBD to be anti-inflammatory and immune suppressive, so the purpose of this review is to summarize effects and mechanisms of CBD in the immune system. It includes a consideration of reports identifying receptors through which CBD acts, since the "CBD receptor," if a single one exists, has not been definitively identified for the myriad immune system effects. The review then provides a summary of in vivo and in vitro effects in the immune system, in autoimmune models, with a focus on experimental autoimmune encephalomyelitis, and ends with identification of knowledge gaps. Conclusion: Overall, the data overwhelmingly support the notion that CBD is immune suppressive and that the mechanisms involve direct suppression of activation of various immune cell types, induction of apoptosis, and promotion of regulatory cells, which, in turn, control other immune cell targets.