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CD200-CD200R1 interaction contributes to neuroprotective effects of anandamide on experimentally induced inflammation
Abstract
The endocannabinoid anandamide (AEA) is released by macrophages and microglia on pathological neuroinflammatory conditions such as multiple sclerosis (MS). CD200 is a membrane glycoprotein expressed in neurons that suppresses immune activity via its receptor (CD200R) mainly located in macrophages/microglia. CD200-CD200R interactions contribute to the brain immune privileged status. In this study, we show that AEA protects neurons from microglia-induced neurotoxicity via CD200-CD200R interaction. AEA increases the expression of CD200R1 in LPS/IFN-γ activated microglia through the activation of CB(2) receptors. The neuroprotective effect of AEA disappears when microglial cells derive from CD200R1(-/-) mice. We also show that engagement of CD200R1 by CD200Fc decreased the production of the proinflammatory cytokines IL-1β and IL-6, but increased IL-10 in activated microglia. In the chronic phases of Theiler's virus-induced demyelinating disease (TMEV-IDD) the expression of CD200 and CD200R1 was reduced in the spinal cord. AEA-treated animals up-regulated the expression of CD200 and CD200R1, restoring levels found in sham animals together with increased expression of IL-10 and reduced expression of IL-1β and IL-6. Treated animals also improved their motor behavior. Because AEA up-regulated the expression of CD200R1 in microglia, but failed to enhance CD200 in neurons we suggest that AEA-induced up-regulation of CD200 in TMEV-IDD is likely due to IL-10 as this cytokine increases CD200 in neurons. Our findings provide a new mechanism of action of AEA to limit immune response in the inflamed brain.
... Moreover, microglia in the resting state are continually monitoring neurons' activity and surveilling the brain microenvironment; ramified microglia are often called surveilling or homeostatic microglia [6,7]. Contextually, microglia are kept inhibited in the resting state, partly through their crosstalk with neurons and astrocytes [13,14]. However, this situation changes when a neuron is unhealthy or dysfunctional. ...
... Other protein complexes that facilitate cell-contact mediated microglia-neuron interaction include CD200/CD200R. In the CNS, CD200, a transmembrane glycoprotein, is largely expressed on the surface of neurons and astrocytes, while the expression of the CD200 receptor (CD200R) is confined to microglia [14,35]. It is thought that CD200/ CD200R signaling has a neuroprotective role since, upon microglianeuron interaction, CD200/ CD200R signaling operates to retain microglia in an inhibited resting state. ...
... It is thought that CD200/ CD200R signaling has a neuroprotective role since, upon microglianeuron interaction, CD200/ CD200R signaling operates to retain microglia in an inhibited resting state. The loss of this signaling can lead to microglia activation and the eventual elimination of neuronal synapses [14,35]. Consequently, it is not surprising that defects in CD200/CD200R signaling are involved in a handful of neuronal conditions, such as multiple sclerosis, AD, and neurotrauma [14,35,80]. ...
Microglia are the resident immune cells of the brain and play a crucial role in housekeeping and maintaining homeostasis of the brain microenvironment. Upon injury or disease, microglial cells become activated, at least partly, via signals initiated by injured neurons. Activated microglia, thereby, contribute to both neuroprotection and neuroinflammation. However, sustained microglial activation initiates a chronic neuroinflammatory response which can disturb neuronal health and disrupt communications between neurons and microglia. Thus, microglia-neuron crosstalk is critical in a healthy brain as well as during states of injury or disease. As most studies focus on how neurons and microglia act in isolation during neurotrauma, there is a need to understand the interplay between these cells in brain pathophysiology. This review highlights how neurons and microglia reciprocally communicate under physiological conditions and during brain injury and disease. Furthermore, the modes of microglia-neuron communication are exposed, focusing on cell-contact dependent signaling and communication by the secretion of soluble factors like cytokines and growth factors. In addition, how microglia-neuron interactions could exert either beneficial neurotrophic effects or pathologic proinflammatory responses are discussed. We further explore how aberrations in microglia-neuron crosstalk may be involved in central nervous system (CNS) anomalies, namely: traumatic brain injury (TBI), neurodegeneration, and ischemic stroke. A clear understanding of how the microglia-neuron crosstalk contributes to the pathogenesis of brain pathologies may offer novel therapeutic avenues of brain trauma treatment.
... These changes lead to allodynia and hyperalgesia, which are typical symptoms of neuropathic pain [16][17][18]. CB 2 receptors are expressed in immune cells including microglia, and activation by CB 2 , agonists can reduce pro-inflammatory cytokine production in those cells [19][20][21][22][23][24][25][26]. Previously, we have reported that a CB 2 subtype-selective agonist, ABK5, which is a compound with a distinct structure from the known CB 2 , agonists, has anti-inflammatory effects in T-cells. ...
... Activation of the CB 2 receptor is known to be associated with a decreased production of various pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. [26,[29][30][31]. To best characterize anti-inflammatory effects of our compound ABK5-1, we screened for changes in the levels of various cytokines and chemokines after LPS stimulation and LPS stimulation plus ABK5-1 treatment in BV-2 mouse microglia cell line. ...
... Although some studies showed an increase of antiinflammatory cytokines such as IL-4 and IL-10 after activation of CB 2 by various methods [26,29,[34][35][36], we did not observe increase of such cytokines in the current condition. This might be due to the difference between different cell lines and primary microglia and the difference of the stimulation method. ...
Background and objectives:
Neuroinflammation is closely associated with various diseases including neuropathic pain. Microglia are immune cells in the central nervous system which are the main players of immunity and inflammation. Since microglia are activated by nerve injury, and they produce proinflammatory mediators to cause neuropathic pain, targeting activated microglia is considered to be a strategy for treating neuropathic pain. Activation of the cannabinoid CB2 receptor is known to have anti-inflammatory effects in microglia. ABK5-1 is a CB2 subtype selective agonist which inhibits IL-1β and IL-6 production in the microglia cell line BV-2. The purpose of the current study is to further analyze anti-inflammatory effects of ABK5 in terms of different cytokines and the possible pathway involved in the effect in the BV-2 cell line.
Methods:
A cytokine array was performed to screen the effect of ABK5-1 on forty inflammatory mediators in BV-2 cells. Changes of the inflammatory mediators was further supported by mRNA analysis, and a possible signaling molecule that involved the observation was evaluated by western blot.
Results:
Stimulating BV-2 cells by lipopolysaccharide increased expression of eleven inflammatory mediators, and ABK5-1 treatment resulted in more than a 50% decrease of sICAM1, IL-6, and RANTES. Real-time PCR results showed a decrease of G-CSF, ICAM1, MCP-1, MIP-1α, and MIP-1β mRNA levels. Western blot analysis showed that ABK5-1 inhibited LPS-induced ERK phosphorylation, which can be a mechanism of ABK5-1-mediated anti-inflammatory effect.
Conclusions:
Our current results support the possibility that ABK5-1 is an anti-inflammatory drug for microglia.
... One of the primary methods of communication between microglia and other cells of the CNS is surface-surface binding of CD200. In a physiological state, CD200 is expressed most cells of the brain, with its receptor CD200R only found on microglia and other macrophages [ 209 ]. CD200-CD200R binding is a critical mediator of microglial activation. ...
... CD200-CD200R binding is a critical mediator of microglial activation. When bound, microglia are observed in a resting and surveillant state [ 209 ]. Importantly, expression of CD200 and CD200R are significantly reduced in areas of the brain affected by AD [ 104 , 210 ]. ...
... Cannabinoid receptor 2 in microglia appears to be involved in a variety of paradigms and diseases involving neuroinflammation, including Alzheimer's disease (AD), Parkinson's disease, multiple sclerosis (MS), as well as stress or addiction (8,31,32). Some studies showed that activation of CB2 receptors prevents the activation of microglia and the release of pro-inflammatory mediators (33)(34)(35). ...
... CD200R is expressed on microglia, whereas CD200 is expressed on neurons, and their interaction plays a crucial role in neuronal protection in inflammationmediated neurodegeneration. Furthermore, CD200R1-CD200 interaction was associated with decreased production of the proinflammatory cytokines IL-1b and IL-6 but increased IL-10 in activated microglia after anandamide treatment (32). Another possible mechanism related to microglia-neuron interaction and CB2r involves the up-regulation of microglial CX3CR1. ...
Emotional behavior, memory, and learning have been associated with alterations in the immune system in neuropsychiatric and neurodegenerative diseases. In recent years, several studies pointed out the involvement of the cannabinoid receptor 2 (CB2r) in the immune system and the regulation of inflammation. This receptor is widely distributed in different tissues and organs with higher expression in spleen and immune system cells. However, CB2r has also been detected in several brain areas and different brain cell types, such as neurons and glia. These findings suggest that CB2r may closely relate the immune system and the brain circuits regulating inflammation, mood, and cognitive functions. Therefore, we review the studies that may help elucidate the molecular bases of CB2r in regulating inflammation in different brain cells and its role in the pathophysiology of psychiatric and neurodegenerative disorders.
... To obtain all possible combinations of neuron-glia co-cultures (neuron-astrocyte, neuron-microglia and neuron-astrocytemicroglia), primary cortical cell cultures were prepared as stated above. Cells were plated at different ratios as it has been described (Hernangómez et al., 2012;Correa et al., 2013) to find the adequate conditions. The optimal conditions are described in Figure 1A. ...
... In order to establish an in vitro protocol where we could study neuron-glia communication and recapitulate neurodegeneration features such as synaptic loss, we optimized an easy and straightforward murine triple co-culture model with neurons, astrocytes and microglia in all possible combinations (Figures 1A,B). We adapted the protocols for culturing individual murine neurons (Alberdi et al., 2018), astrocytes (Wyssenbach et al., 2016) and microglia (Zabala et al., 2018) to establish the triple co-culture model by identifying the appropriate platting order and ratios for the three cell types and by confirming their survival (Hernangómez et al., 2012;Correa et al., 2013). Plating astrocytes on top of neurons compromised neuronal survival (data not shown). ...
Glial cells are essential to understand Alzheimer’s disease (AD) progression, given their role in neuroinflammation and neurodegeneration. There is a need for reliable and easy to manipulate models that allow studying the mechanisms behind neuron and glia communication. Currently available models such as co-cultures require complex methodologies and/or might not be affordable for all laboratories. With this in mind, we aimed to establish a straightforward in vitro setting with neurons and glial cells to study AD. We generated and optimized a 2D triple co-culture model with murine astrocytes, neurons and microglia, based on sequential seeding of each cell type. Immunofluorescence, western blot and ELISA techniques were used to characterize the effects of oligomeric Aβ (oAβ) in this model. We found that, in the triple co-culture, microglia increased the expression of anti-inflammatory marker Arginase I, and reduced pro-inflammatory iNOS and IL-1β, compared with microglia alone. Astrocytes reduced expression of pro-inflammatory A1 markers AMIGO2 and C3, and displayed a ramified morphology resembling physiological conditions. Anti-inflammatory marker TGF-β1 was also increased in the triple co-culture. Lastly, neurons increased post-synaptic markers, and developed more and longer branches than in individual primary cultures. Addition of oAβ in the triple co-culture reduced synaptic markers and increased CD11b in microglia, which are hallmarks of AD. Consequently, we developed a straightforward and reproducible triple co-cultured model, where cells resemble physiological conditions better than in individual primary cultures: microglia are less inflammatory, astrocytes are less reactive and neurons display a more mature morphology. Moreover, we are able to recapitulate Aβ-induced synaptic loss and CD11b increase. This model emerges as a powerful tool to study neurodegeneration and neuroinflammation in the context of AD and other neurodegenerative diseases.
... CB 2 is expressed in microglia, and the level of expression is further enhanced when the microglia are activated [39][40][41]. Both endocannabinoids and exogenous cannabinoid receptor agonists are reported to decrease production of pro-inflammatory cytokines in primary microglia and microglial cell lines [19,34,42,43]. In agreement with these studies, our results also showed inhibitory effects of a CB 2 agonist ABK5-1 on inhibiting production of IL-1β and IL-6, supporting the beneficial effects of CB 2 in inhibition of neuroinflammation. ...
... In addition, microglia are also known to be able to produce antiinflammatory cytokine IL-10 when they undergo alternative activation [44]. Cannabinoid receptor activation by endocannabinoids and some synthetic agonists are also reported to increase IL-10-producing anti-inflammatory microglia (commonly known as M2 microglia) [34,42,43,45,46]. In contrast, CB 2 activation is also reported to promote microglia activation to pro-inflammatory (known as M1 microglia) phases in cancer pain with morphine tolerance [47]. ...
Activation of the CB2 receptor has been shown to have anti-inflammatory and antinociceptive effects without causing psychoactive effects. Previously, we reported that the compound ethyl 2(2-(N-(2,3-dimethylphenyl) phenylsulfonamido)acetamido)benzoate (ABK5) is a CB2 subtype selective agonist with anti-inflammatory and antinociceptive effects. In the present study, we tested four ABK5 derivatives, ABK5-1, ABK5-2, ABK5-5, and ABK5-6, to analyze the structure of ABK5 to obtain CB2-selective agonists with higher affinity and efficacy. Affinity, subtype selectivity, and G-protein coupling were determined by radioligand binding assays. Selected compounds were then subjected to evaluation of anti-inflammatory effects using two different cell lines, Jurkat (ABK5-1 and 5-2) and BV-2 cells (ABK5-1), which are models of T cells and microglia, respectively. ABK5-1, ABK5-2, and ABK5-6 had comparable CB2 binding affinity with ABK5 (and stimulated G-protein coupling), while only ABK5-1 and ABK5-2 maintained CB2-subtype selectivity. ABK5-5 did not bind CB2 in the detectable range. RT-PCR and ELISA analysis showed that the two compounds also inhibit IL-2 and TNF-α production, and they were more efficacious than ABK5 in inhibiting TNF-α production. CXCL-12 mediated chemotaxis was also evaluated by the transwell migration assay, and both ABK5-1 and ABK5-2 inhibited chemotaxis with a stronger effect observed in ABK5-1. In the microglia cell line BV-2, ABK5-1 inhibited IL-1β and IL-6 production, which suggests this compound has anti-inflammatory effects through targeting multiple immune cells, and may be a candidate for treatment of inflammation.
... Notable single protein changes in PSP included downregulation of CD200, which plays a key role in protecting neurons from microglia-induced neurotoxicity [45], and of the synaptic protein neuronal pentraxin-2 (NPTX2), which has been found to be decreased in CSF of patients with PD, PSP, and MSA, and likely reflects synaptic dysfunction and loss in the prefrontal cortex [46]. This protein was also significantly decreased in our PD samples, but not in the MSA samples. ...
The molecular pathogenesis of degenerative parkinsonisms, including Parkinson’s disease (PD), progressive supranuclear palsy (PSP), and Multiple system atrophy (MSA), remains largely unknown. To gain novel insight into molecular processes associated with these diseases, we conducted a proteome-wide expression study in prefrontal cortex tissue from a cohort of 181 individuals, comprising PD (N = 73), PSP (N = 18), MSA (N = 17) and healthy control (N = 73). Using marker gene profiles, we first assess the cellular composition of the samples and, subsequently, identify distinct protein signatures for each disease, while correcting for cell composition. Our findings indicate that all three diseases are characterized by a structural and/or functional loss of deep cortical neurons, while PD exhibits an additional decrease in somatostatin-expressing interneurons, as well as in endothelial cells. Differential protein expression analysis identified multiple proteins and pathways with disease-specific expression, some of which have previously been associated with parkinsonism or neurodegeneration in general. Notably, we observed a strong mitochondrial signature which was present in both PD and PSP, albeit of a different composition and most pronounced in PSP, but not in MSA where immunological/inflammation-related pathways dominated. Additionally, we identified protein signatures associated with the severity of α-synuclein pathology in PD and showed that these are highly enriched in an upregulation of mitochondrial processes, specifically related to oxidative phosphorylation and in particular respiratory complexes I and IV. We identify multiple novel signatures of protein expression, associated with PD, PSP, and MSA, as well as with the severity of α-synuclein pathology in the PD brain.
... Briefly, primary embryonic hippocampal cultures containing neurons and astrocytes were first prepared as described above. Pure microglia were then isolated at 14 days in vitro (DIV) and seeded onto the neuron-astrocyte cultures at DIV 21 (Hernangómez et al., 2012). For this purpose, cerebral cortices from C57BL/6 J mice were harvested after the third to fifth postnatal day in cold 1X HBSS medium (Gibco,14, and centrifuged at 2000 rcf for 5 min at 4°C. ...
Neuroinflammation can be triggered by various stimuli, including viral infections. Viruses can directly invade the brain and infect neuronal cells or indirectly trigger a “cytokine storm” in the periphery that eventually leads to microglial activation in the brain. While this initial activation of microglial cells is important for viral clearance, chronic activation leads to excessive inflammation and oxidative stress, which can be neurotoxic. Remarkebly, recent studies have shown that certain viruses such as influenza A virus, coronavirus, herpes virus and Epstein–Barr virus may be involved in the development of neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis. Therefore, it is important to find therapeutic strategies against chronic neuroinflammation triggered by viral infections. Here, we investigated the effects of urolithin A (UA) on microglial activation in vitro induced by a viral mimetic, poly I:C, in a triple co-culture system of neurons, astrocytes and microglial cells. Immunocytochemistry was used to perform a comprehensive single-cell analysis of the morphological changes of microglia as an indicator of their reactive state. Treatment with UA significantly prevented the poly I:C-induced reactive state of microglia, which was characterized by increased expression of the microglial activation markers CD68 and IBA-1. UA restored the poly I:C-induced morphology by restoring microglial ramification. In addition, UA was able to reduce the release of the pro-inflammatory mediators CCL2, TNF-α, and IL-1β and showed a trend toward attenuation of cellular ROS production in poly I:C-treated cultures. Overall, this study suggests that UA as a component of a healthy diet may help prevent virus-induced neuroinflammation and may have therapeutic potential for future studies to prevent or treat neurodegenerative diseases by targeting the associated neuroinflammatory processes.
... In particular, microglial CB2R appears to be involved in various conditions and diseases related to neuroinflammation, including AD, PD, MS, depression, anxiety and addiction [23][24][25]. ...
Mental disorders account for one of the most prevalent categories of the burden of disease worldwide, with depression expected to be the largest contributor by 2030, closely followed by anxiety. The COVID-19 pandemic possibly exacerbated these challenges, especially amongst adolescents, who experienced isolation, disrupted routines, and limited healthcare access. Notably, the pandemic has been associated with long-term neurological effects known as “long-COVID”, characterized by both cognitive and psychopathological symptoms. In general, psychiatric disorders, including those related to long-COVID, are supposed to be due to widespread inflammation leading to neuroinflammation. Recently, the endocannabinoid system (ECS) emerged as a potential target for addressing depression and anxiety pathophysiology. Specifically, natural or synthetic cannabinoids, able to selectively interact with cannabinoid type-2 receptor (CB2R), recently revealed new therapeutic potential in neuropsychiatric disorders with limited or absent psychotropic activity. Among the most promising natural CB2R ligands, the bicyclic sesquiterpene β-caryophyllene (BCP) has emerged as an excellent anti-inflammatory and antioxidant therapeutic agent. This review underscores BCP’s immunomodulatory and anti-inflammatory properties, highlighting its therapeutic potential for the management of depression and anxiety.
... While microglial products can promote a neuroinflammatory response, other microglial-expressed proteins may promote an anti-inflammatory and neuroprotective environment. For example, the neuronallyexpressed proteins CD200 and fractalkine (Eyo and Wu, 2013) and CD200 receptor (CD200R) expressed on macrophages and microglia may induce CD200-CD200R signaling to maintain a resting or non-activated microglial state (Wright et al., 2001;Hernangómez et al., 2012). Defects in CD200 signaling promote microglial activation and are associated with neuro-inflammatory conditions such as multiple sclerosis (MS) and Alzheimer's disease (AD) (Walker et al., 2009). ...
Despite structural similarity with other tumor necrosis factor receptor superfamily (TNFRSF) members, the p75 neurotrophin receptor (p75 NTR , TNFR16) mediates pleiotropic biological functions not shared with other TNFRs. The high level of p75 NTR expression in the nervous system instead of immune cells, its utilization of co-receptors, and its interaction with soluble dimeric, rather than soluble or cell-tethered trimeric ligands are all characteristics which distinguish it from most other TNFRs. Here, we compare these attributes to other members of the TNFR superfamily. In addition, we describe the recent evolutionary adaptation in B7-1 (CD80), an immunoglobulin (Ig) superfamily member, which allows engagement to neuronally-expressed p75 NTR . B7-1-mediated binding to p75 NTR occurs in humans and other primates, but not lower mammals due to specific sequence changes that evolved recently in primate B7-1. This discovery highlights an additional mechanism by which p75 NTR can respond to inflammatory cues and trigger synaptic elimination in the brain through engagement of B7-1, which was considered to be immune-restricted. These observations suggest p75 NTR does share commonality with other immune co-modulatory TNFR family members, by responding to immunoregulatory cues. The evolution of primate B7-1 to bind and elicit p75 NTR -mediated effects on neuronal morphology and function are discussed in relationship to immune-driven modulation of synaptic actions during injury or inflammation.
... Moreover, it is well ascertained that the activation of CB 2 signaling in microglia polarizes these cells toward an anti-inflammatory/pro-homeostatic phenotype [80,204] that may favor the establishment of a neurogenic milieu in the niche. In particular, CB 2 agonism not only suppresses the expression of pro-inflammatory cytokines and enzymes in LPS/INFγactivated microglial cells but, at the same time, can promote an antiinflammatory switching of these cells by stimulating phagocytosis and increasing the expression of arginase 1, IL-10, and critical neurotrophic factors, such as BDNF and GDNF [202,[205][206][207][208][209]. ...
Dentate gyrus of the hippocampus continuously gives rise to new neurons, namely, adult-born granule cells, which contribute to conferring plasticity to the mature brain throughout life. Within this neurogenic region, the fate and behavior of neural stem cells (NSCs) and their progeny result from a complex balance and integration of a variety of cell-autonomous and cell-to-cell-interaction signals and underlying pathways. Among these structurally and functionally diverse signals, there are endocannabinoids (eCBs), the main brain retrograde messengers. These pleiotropic bioactive lipids can directly influence and/or indirectly adult hippocampal neurogenesis (AHN) by modulating, both positively and negatively, multiple molecular and cellular processes in the hippocampal niche, depending on the cell type or stage of differentiation. Firstly, eCBs act directly as cell-intrinsic factors, cell-autonomously produced by NSCs following their stimulation. Secondly, in many, if not all, niche-associated cells, including some local neuronal and nonneuronal elements, the eCB system indirectly modulates the neurogenesis, linking neuronal and glial activity to regulating distinct stages of AHN. Herein, we discuss the crosstalk of the eCB system with other neurogenesis-relevant signal pathways and speculate how the hippocampus-dependent neurobehavioral effects elicited by (endo)cannabinergic medications are interpretable in light of the key regulatory role that eCBs play on AHN.
... 55 Several studies support our hypothesis demonstrating that M2 polarization is potentially regulated by endocannabinoids. [56][57][58][59][60] In addition to activating microglia in M2, endocannabinoids can also be released from it. Mecha et al. 59 demonstrated that M2-polarized microglia promote endocannabinoid synthesis and have a lower amount of degradative enzymes associated with resting or M1 phenotypes. ...
Bacterial infections are often accompanied by fever and generalized muscle pain. However, the treatment of pain with an infectious etiology has been overlooked. Thus, we investigated the impact of cannabidiol (CBD) in bacterial lipopolysaccharide (LPS)-induced nociception. Male Swiss mice received intrathecal (i.t.) LPS injection, and the nociceptive threshold was measured by the von Frey filaments test. Spinal involvement of the cannabinoid CB2 receptor, toll-like receptor 4 (TLR4), microglia and astrocytes were evaluated by i.t. administration of their respectively antagonists or inhibitors. Western blot, immunofluorescence, ELISA and liquid chromatography-mass spectrometry were used to assess Cannabinoid CB2 receptors and TLR4 spinal expression, proinflammatory cytokines and endocannabinoid levels. CBD was administered intraperitoneally at 10 mg/kg. The pharmacological assay demonstrated TLR4 participation in LPS-induced nociception. In addition, spinal TLR4 expression and proinflammatory cytokine levels were increased in this process. CBD treatment prevented LPS-induced nociception and TLR4 expression. AM630 reversed antinociception and reduced CBD-induced endocannabinoids upregulation. Increased spinal expression of the cannabinoid CB2 receptor was also found in animals receiving LPS, which was accompanied by reduced TLR4 expression in CBD-treated mice. Taken together, our findings indicated that CBD is a potential treatment strategy to control LPS-induced pain by attenuating TLR4 activation via the endocannabinoid system.
... Upregulation of DLG4 within EBV + and CIS ABCs is also noteworthy, as this gene encodes a neuronal signaling regulator that was identified within MS patient leukocytes and exhibited significantly reduced expression during IFNb treatment 104 . ABCs even from healthy individuals basally express Src family kinases (HCK, FGR), which are essential to myeloid cell inflammatory responses 105 , but also inflammatory response inhibitors (e.g., CD200R1) 106 . EBV infection has previously been shown to induce FGR expression in an EBNA2-dependent fashion 78,79 . ...
Expansion and pathogenicity of CD19 ⁺ /CD20 ⁺ /CD11c ⁺ /T-bet ⁺ atypical B cells (ABCs) are hallmarks of numerous autoimmune disorders and chronic infections. In many such cases Epstein-Barr virus (EBV) is another associated or etiologic factor, though EBV involvement in these diseases remains poorly understood. Notably, the expansion of pro-inflammatory ABCs and a putative causal role for EBV have been identified independently in multiple sclerosis (MS). A common precipitating event in MS onset is Clinically Isolated Syndrome (CIS), a neuroinflammatory demyelinating condition of which 60-80% of cases progress to relapsing-remitting MS (RRMS). Here we report single-cell gene and surface protein expression (scRNA/CITE-seq) in peripheral B cells collected longitudinally from patients with CIS during the Immune Tolerance Network STAyCIS Trial. We focus on the transcriptomic signatures of ABCs from this cohort, publicly available scRNA-seq datasets from six other autoimmune and chronic infectious diseases, and in vitro EBV infection. Conservation of an expanded ABC expression profile across diseases establishes ABC dysregulation as a feature of CIS. Critically, we also observed transcriptomic features that distinguished CIS and de novo EBV-infected ABCs from those found in healthy controls and other disease contexts. Outcome stratification of CIS samples revealed a rare yet distinctive pro-inflammatory ABC subset that was significantly underrepresented in long-term non-progressor (LTNP) versus cases with RRMS activity (∼5-fold difference). Collectively, this study provides evidence for altered ABC regulation – possibly arising from niche-specific responses to EBV infection – preceding MS onset.
SUMMARY
Single-cell transcriptomics establishes an EBV-associated signature in T-bet ⁺ atypical B cells in CIS and a pro-inflammatory phenotype underrepresented in patients with no disease progression.
... Furthermore, the AMPK pathway is upregulated after CB2R activation, which can downregulate the synthesis of nitric oxide (NO) [196]. Actually, the activation of CB2R in microglial cells has been found to lead to spinal decreased iNOS, IL-6, BDNF, CCR2, and TNFα receptor expression during neuropathic pain [25,197], and increased release of anti-inflammatory cytokines, such as IL-10 [174,198,199]. Specifically, the activation of spinal CB2R by exercise-induced AEA release also reduces the production of IL-1β and TNFα in mice within a carrageenan-induced pain model [136]. ...
Pain, especially chronic pain, can strongly affect patients’ quality of life. Cannabinoids ponhave been reported to produce potent analgesic effects in different preclinical pain models, where they primarily function as agonists of Gi/o protein-coupled cannabinoid CB1 and CB2 receptors. The CB1 receptors are abundantly expressed in both the peripheral and central nervous systems. The central activation of CB1 receptors is strongly associated with psychotropic adverse effects, thus largely limiting its therapeutic potential. However, the CB2 receptors are promising targets for pain treatment without psychotropic adverse effects, as they are primarily expressed in immune cells. Additionally, as the resident immune cells in the central nervous system, microglia are increasingly recognized as critical players in chronic pain. Accumulating evidence has demonstrated that the expression of CB2 receptors is significantly increased in activated microglia in the spinal cord, which exerts protective consequences within the surrounding neural circuitry by regulating the activity and function of microglia. In this review, we focused on recent advances in understanding the role of microglial CB2 receptors in spinal nociceptive circuitry, highlighting the mechanism of CB2 receptors in modulating microglia function and its implications for CB2 receptor- selective agonist-mediated analgesia.
... Microglia, the resident immune cells of the brain, possess a self-contained ECS including receptors for cannabinoid-like molecules and the capacity to biosynthesize and metabolize AEA and 2-AG (Stella, 2010(Stella, , 2009). In the brain, endocannabinoids released by post-synaptic neurons neuroprotective in animal models of multiple sclerosis (Hernangómez et al., 2012;Lourbopoulos et al., 2011). However, 2-AG is also an intermediate metabolite in prostaglandin synthesis and excess quantities of 2-AG could promote neuroinflammation and subsequent neurodegeneration via prostaglandin-mediated microglial activity (Klawonn et al., 2021;Nomura et al., 2011). ...
Perturbation of the endocannabinoid system can have profound effects on immune function and synaptic plasticity. Microglia are one of few cell types with a self-contained endocannabinoid system and are positioned at the interface between the immune system and the central nervous system. Past work has produced conflicting results with respect to the effects of pro-inflammatory conditions on the microglial endocannabinoid system. Thus, we systematically investigated the relationship between the concentration of two distinct pro-inflammatory stimuli, lipopolysaccharide and interferon gamma, on the abundance of components of the endocannabinoid system within microglia. Here we show that lipopolysaccharide and interferon gamma influence messenger RNA abundances of the microglial endocannabinoid system in a concentration-dependent manner. Furthermore, we demonstrate that the efficacy of different synthetic cannabinoid treatments with respect to inhibition of microglia nitric oxide release is dependent on the concentration and type of pro-inflammatory stimuli presented to the microglia. This indicates that different pro-inflammatory stimuli influence the capacity of microglia to synthesize, degrade, and respond to cannabinoids which has implications for the development of cannabinoid-based treatments for neuroinflammation.
... The ligand/receptor complexes that regulate microglial/ neuronal interactions are incompletely understood; however, several candidate systems have been identified, including neuronally expressed proteins CD200 and fractalkine (48) and CD200 receptor (CD200R), expressed on macrophages and microglia. CD200/CD200R signaling maintains a resting or nonactivated microglial state (49,50), and defects in CD200 signaling, which result in increased microglial activation, are associated with neuroinflammatory conditions such as MS and AD. Impaired CD200 signaling is also associated with an increased aging phenotype in the brain (51,52). ...
Cell surface receptors, ligands and adhesion molecules underlie development, circuit formation and synaptic function of the central nervous system and represent important therapeutic targets for many neuropathologies. The functional contributions of interactions between cell surface proteins of neurons and non-neuronal cells have not been fully addressed. Using an unbiased protein-protein interaction screen, we showed that the human immunomodulatory ligand, B7-1 (hB7-1), interacts with the p75 neurotrophin receptor (p75NTR), and that the B7-1:p75NTR interaction is a recent evolutionary adaptation present in humans and other primates, but absent in mice, rats, and other lower mammals. The surface of hB7-1 that engages p75NTR overlapped with the hB7-1 surface involved in CTLA-4/CD28 recognition, and these molecules directly competed for binding to p75NTR. Soluble or membrane bound hB7-1 altered dendritic morphology of cultured hippocampal neurons, with loss of the postsynaptic protein PSD95 in a p75NTR-dependent manner. Abatacept, an FDA-approved therapeutic (CTLA-4-hFc fusion) inhibited these processes. In vivo injection of hB7-1 into the murine subiculum, a hippocampal region affected in Alzheimer's Disease, resulted in p75NTR-dependent pruning of dendritic spines. Collectively, we have reported the biochemical interaction between B7-1 and p75NTR, described biological effects on neuronal morphology, and identified a therapeutic opportunity for treatment of neuroinflammatory diseases.
... Previous work has demonstrated that the endocannabinoid system modulates the pro-inflammatory activity of microglia (Askari and Shafiee-Nick, 2019;Correa et al., 2010;Eljaschewitsch et al., 2006;Hernangómez et al., 2012;Ma et al., 2018;Malek et al., 2015;Tanaka et al., 2020). Both CB 2 receptor-selective agonists and nonselective agonists have demonstrated the potential to suppress the LPS-mediated induction of pro-inflammatory markers in immortalized and primary microglia (Askari and Shafiee-Nick, 2019;Ehrhart et al., 2005;Ma et al., 2018Ma et al., , 2015Mecha et al., 2015;Waksman et al., 1999). ...
Microglia are resident immune cells of the brain that survey the microenvironment, provide trophic support to neurons, and clear debris to maintain homeostasis and healthy brain function. Microglia are also drivers of neuroinflammation in several neurodegenerative diseases. Microglia produce endocannabinoids and express both cannabinoid receptor subtypes suggesting that this system is a target to suppress neuroinflammation. We tested whether cannabinoid type 1 (CB1) or type 2 (CB2) receptors could be targeted selectively or in combination to dampen the pro-inflammatory behavior of microglia, and whether this would have functional relevance to decrease secondary neuronal damage. We determined that components of the endocannabinoid system were altered when microglia are treated with lipopolysaccharide and interferon-gamma and shift to a pro-inflammatory phenotype. Furthermore, pro-inflammatory microglia released cytotoxic factors that induced cell death in cultured STHdhQ7/Q7 neurons. Treatment with synthetic cannabinoids that were selective for CB1 receptors (ACEA) or CB2 receptors (HU-308) dampened the release of nitric oxide (NO) and pro-inflammatory cytokines and decreased levels of mRNA for several pro-inflammatory markers. A nonselective agonist (CP 55,940) exhibited similar influence over NO release but to a lesser extent relative to ACEA or HU-308. All three classes of synthetic cannabinoids ultimately reduced the secondary damage to the cultured neurons. The mechanism for the observed neuroprotective effects appeared to be related to cannabinoid-mediated suppression of MAPK signaling in microglia. Taken together, the data indicate that activation of CB1 or CB2 receptors interfered with the pro-inflammatory activity of microglia in a manner that also reduced secondary damage to neurons.
... A previous study showed that PPAR-γ activation in microglia could attenuate axonal injury [100]. Furthermore, microglial PPAR-γ may be important for bidirectional microglia-neuron crosstalk, as PPAR-γ can regulate glycoprotein CD200 expression on neurons [101], which, in turn, interacts with the CD200 receptor found only on microglia [102,103]. In the case of SOD1, SOD1 released by microglia is known to mediate neuroprotection under neurotoxic environments [104]. ...
Although different regions of the brain are dedicated to specific functions, the intra- and inter-regional heterogeneity of astrocytes and microglia in these regions has not yet been fully understood. Recently, an advancement in various technologies, such as single-cell RNA sequencing, has allowed for the discovery of astrocytes and microglia with distinct molecular fingerprints and varying functions in the brain. In addition, the regional heterogeneity of astrocytes and microglia exhibits different functions in several situations, such as aging and neurodegenerative diseases. Therefore, investigating the region-specific astrocytes and microglia is important in understanding the overall function of the brain. In this review, we summarize up-to-date research on various intra- and inter-regional heterogeneities of astrocytes and microglia, and provide information on how they can be applied to aging and neurodegenerative diseases.
... These findings are consistent with many other studies carried out on primary cells and several immortalized cell lines. When microglia are experimentally activated in a reactive state, CB 2 activation could inhibit the release of pro-inflammatory and cytotoxic factors interfering with their switching to a neurotoxic phenotype [102][103][104][105][106]. ...
Chronic inflammation in Alzheimer’s disease (AD) has been recently identified as a major contributor to disease pathogenesis. Once activated, microglial cells, which are brain-resident immune cells, exert several key actions, including phagocytosis, chemotaxis, and the release of pro- or anti-inflammatory mediators, which could have opposite effects on brain homeostasis, depending on the stage of disease and the particular phenotype of microglial cells. The endocannabinoids (eCBs) are pleiotropic bioactive lipids increasingly recognized for their essential roles in regulating microglial activity both under normal and AD-driven pathological conditions. Here, we review the current literature regarding the involvement of this signalling system in modulating microglial phenotypes and activity in the context of homeostasis and AD-related neurodegeneration.
... The neuroprotective effect of AEA against microglial neurotoxicity was mediated by a mechanism between neurons (with reference to membrane glycoproteins) and microglia/macrophages that involves CD200 ligand-receptor interaction [227]. ...
Simple Summary
Neurodegenerative diseases represent an important cause of morbidity and mortality worldwide. Existing therapeutic options are limited and focus mostly on improving symptoms and reducing exacerbations. The endocannabinoid system is involved in the pathophysiology of such disorders, an idea which has been highlighted by recent scientific work. The current work focusses its attention on the importance and implications of this system and its synthetic and natural ligands in disorders such as Alzheimer’s, Parkinson’s, Huntington’s and multiple sclerosis.
Abstract
Neurodegenerative diseases are an increasing cause of global morbidity and mortality. They occur in the central nervous system (CNS) and lead to functional and mental impairment due to loss of neurons. Recent evidence highlights the link between neurodegenerative and inflammatory diseases of the CNS. These are typically associated with several neurological disorders. These diseases have fundamental differences regarding their underlying physiology and clinical manifestations, although there are aspects that overlap. The endocannabinoid system (ECS) is comprised of receptors (type-1 (CB1R) and type-2 (CB2R) cannabinoid-receptors, as well as transient receptor potential vanilloid 1 (TRPV1)), endogenous ligands and enzymes that synthesize and degrade endocannabinoids (ECBs). Recent studies revealed the involvement of the ECS in different pathological aspects of these neurodegenerative disorders. The present review will explore the roles of cannabinoid receptors (CBRs) and pharmacological agents that modulate CBRs or ECS activity with reference to Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Huntington’s Disease (HD) and multiple sclerosis (MS).
... Indeed, CB2R activation in a paclitaxel model of chemotherapyinduced neuropathy leads to decreased IL-6, BDNF, P2X4, and TNFα receptor expression, and increased release of the antiinflammatory cytokine IL-10 (Burgos et al., 2012;Wu et al., 2019). This cannabinoid-induced release of IL-10 by microglia is a phenomenon shared with other areas of the CNS (Correa et al., 2010;Hernangómez et al., 2012). Additionally, activation of CB2R by exercise-induced AEA release reduces levels of TNFα and IL-1β in the spinal cord of mice with carrageenaninduced pain hypersensitivity (dos Santos et al., 2019). ...
Chronic pain is a complex sensory, cognitive, and emotional experience that imposes a great personal, psychological, and socioeconomic burden on patients. An estimated 1.5 billion people worldwide are afflicted with chronic pain, which is often difficult to treat and may be resistant to the potent pain-relieving effects of opioid analgesics. Attention has therefore focused on advancing new pain therapies directed at the cannabinoid system because of its key role in pain modulation. Endocannabinoids and exogenous cannabinoids exert their actions primarily through G i/o -protein coupled cannabinoid CB1 and CB2 receptors expressed throughout the nervous system. CB1 receptors are found at key nodes along the pain pathway and their activity gates both the sensory and affective components of pain. CB2 receptors are typically expressed at low levels on microglia, astrocytes, and peripheral immune cells. In chronic pain states, there is a marked increase in CB2 expression which modulates the activity of these central and peripheral immune cells with important consequences for the surrounding pain circuitry. Growing evidence indicate that interventions targeting CB1 or CB2 receptors improve pain outcomes in a variety of preclinical pain models. In this mini-review, we will highlight recent advances in understanding how cannabinoids modulate microglia function and its implications for cannabinoid-mediated analgesia, focusing on microglia-neuron interactions within the spinal nociceptive circuitry.
... One of the inhibitory mechanisms to control the microglial in ammatory response is CD200/CD200R1 interaction. CD200 is a surface glycoprotein generally expressed by neurons, whereas CD200R1, the receptor of CD200R, is chie y located in microglia [3]. In the retina, microglia migration is promoted by the combination of CD200R and CD200, while preventing M1 phenotype activation of resident microglia [4]. ...
Objective
Based on recent reports, peroxisome proliferator-activated receptor-γ (PPAR-γ) could promote microglial M2 polarization to inhibit inflammation. However, the specific molecular mechanisms instigate the anti-inflammatory ability of PPAR-γ in microglia have not been expounded. In the present study, we explored the molecular mechanisms of the anti-inflammatory effects of PPAR-γ in hypoxia-stimulated rat microglial cells.
Methods
shRNA expressing lentivirus was used to knock down PPAR-γ and CD200 genes. The hypoxia-induced polarization markers release (M1: iNOS, IL-1β, IL-6 and TNF-α; M2: Arg-1, YM1, IL-4 and IL-10) was assessed by RT-PCR, while PPAR-γ-related signals (PPAR-γ, PPAR-γ in cytoplasm or nucleus, CD200 and CD200Rs) were monitored by western blot and RT-PCR.
Results
Hypoxia enhanced PPAR-γ and CD200 expressions in microglial cells. In addition, PPAR-γ agonist 15d-PGJ2 elevated CD200 and CD200R1 expressions, while sh-PPAR-γ (PPAR-γ knock-down) had just the opposite effect. Following hypoxia, expressions of M1 markers increased significantly, while those of M2 markers decreased, and the above effects were attenuated by 15d-PGJ2. Conversely, knocking down PPAR-γ or CD200 inhibited the polarization of microglial cells to M2 phenotype.
Conclusion
Results demonstrated that PPAR-γ performed an anti-inflammatory function in hypoxia-stimulated microglial cells by promoting their polarization to M2 phenotype via CD200-CD200R1 pathway.
... Conversely, the administration of recombinant CD200-His reverses the effect (19). CD200R1 is also associated with microglia priming (20), antigen presentation (18), and cytokine production (21). Activation of CD200R1 slows down microglial migration and phagocytosis (22), whereas microglia isolated from CD200 deficient mouse model shows augmented phagocytosis and lysosomal activity (23). ...
CD200R1 is an inhibitory surface receptor expressed in microglia and blood macrophages. Microglial CD200R1 is known to control neuroinflammation by keeping the microglia in resting state and therefore, tight regulation of its expression is important. CCAAT/enhancer binding protein β (CEBPβ) is the known regulator of CD200R1 transcription. In the present study our specific intention was to find a possible posttranscriptional regulatory mechanism of CD200R1 expression. Here we investigated a novel regulatory mechanism of CD200R1 expression following exposure to an environmental stressor, arsenic, combining in silico analysis, in vitro, and in vivo experiments, as well as validation in human samples. The in silico analysis and in vitro studies with primary neonatal microglia and BV2 microglia revealed that arsenic demethylates the promoter of a microRNA, miR-129-5p, thereby increasing its expression, which subsequently represses CD200R1 by binding to its 3´- untranslated region and shuttling the CD200R1 mRNA to the cytoplasmic-processing body in mouse microglia. The role of miR-129-5p was further validated in BALB/c mouse by stereotaxically injecting anti-miR-129. We found that anti-miR-129 reversed the expression of CD200R1, as well as levels of inflammatory molecules IL-6 and TNF-α. Experiments with a CD200R1 siRNA-induced loss-of-function mouse model confirmed a miR-129-5p→CD200R1→IL-6/TNF-α signaling axis. These main findings were replicated in a human cell line and validated in human samples. Taken together, our study revealed miR-129-5p as a novel post-transcriptional regulator of CD200R1 expression with potential implications in neuroinflammation and related complications.
... Studies have reported the dysregulation of CD200R1 expression in neurodegenerative diseases including PD. For instance, viral model of multiple sclerosis showed reduced CD200R1 expression in the spinal cord at the later chronic-phase [20]. Walker et al. demonstrated that the mRNA expression of CD200R1 was signi cantly reduced in hippocampal and infratemporal gyrus samples from patients with Alzheimer's disease (AD) [21]. ...
Background
Neuroinflammation is known to be involved in the pathogenesis of Parkinson's disease (PD). Abnormal activation of microglia plays a key role in this pathological process. CD200R1 is a membrane glycoprotein primarily expressed in microglia in central nervous system responsible for transducing signaling maintaining microglia in stationary status. Our previous studies have demonstrated the dysregulation of CD200R1 and its involvement in PD pathogenesis. The binding of transcription factors with promoter regions is the basic and essential step for the regulation of gene expression. However, little is known about the human CD200R1 promoter region and the mechanism of the dysregulated expression of CD200R1 in PD. Methods
Luciferase reporter system was initially employed to identify the core region of CD200R1 promoter and figure out its potential transcription factors. Subsequently, we investigated the interaction adopting electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The regulatory function of the detected transcription factors were further proved through its down-regulation and overexpression. We then collected the peripheral blood mononuclear cells from both PD patients and their healthy counterparts with matched age and sex to evaluate whether consistent results existed under clinical setting. Ultimately, the mouse model was established through knocking-out the identified transcription factor and its role in neuroinflammation and pathogenesis of PD was explored. ResultsWe defined that the core promoter region of CD200R was located within -482 to -146 bp upstream of the translation initiation site (TIS). In addition, we demonstrated that NFKB1 directly bound to the CD200R1 core promoter region and regulated its transcriptional activity. Besides, the expression of NFKB1 and CD200R1 was significantly correlated in human peripheral blood mononuclear cells and knocking down NFKB1 significantly reduced CD200R1 expression. Moreover, both NFKB1 and CD200R1 were significantly downregulated in samples from PD patients. Furthermore, NFKB1-/- mice exhibited exacerbated microglia activation and dopaminergic neuron loss after MPTP treatment. ConclusionOur study provided novel understanding of the transcriptional regulation of CD200R1 and its role in microglia homeostasis in the pathogenesis of PD.
... The interaction between neurons and microglia occurs in a ligand-dependent way, involving many ligand and receptors. Three main signaling pathways (CD200/ CD200R, CX3CL1/CX3CR1 and Cq1-C3/C3R pathways) have been well described [52]. In all cases, neurons express specific ligands that, by binding to their receptors localized on the microglia, act as a pro-survival stimulus and modulate microglia activation, keeping them in a quiescent state [53]. ...
... 2021, 11, 1098 4 of 15 superfamily of cell surface proteins [72], which is mainly expressed by neurons [73], and interacts with a receptor, OX2R (CD200R), found mainly in microglia in the CNS [74,75]. Previous studies have indicated that OX2R activation reduces the activity of myeloid cells such as microglia and macrophages [76,77]. The interaction of OX2 with its receptor acts to suppress inflammatory responses [76,[78][79][80]. ...
Microglia are the first line of defense at the level of the central nervous system (CNS). Phenotypic change in microglia can be regulated by various factors, including the orexin system. Neuroinflammation is an inflammatory process mediated by cytokines, by the lack of interaction between neurotransmitters and their specific receptors, caused by systemic tissue damage or, more often, associated with direct damage to the CNS. Chronic activation of microglia could lead to long-term neurodegenerative diseases. This review aims to explore how tocopherol (vitamin E) and the orexin system may play a role in the prevention and treatment of microglia inflammation and, consequently, in neurodegenerative diseases thanks to its antioxidant properties. The results of animal and in vitro studies provide evidence to support the use of tocopherol for a reduction in microglia inflammation as well as a greater activation of the orexinergic system. Although there is much in vivo and in vitro evidence of vitamin E antioxidant and protective abilities, there are still conflicting results for its use as a treatment for neurodegenerative diseases that speculate that vitamin E, under certain conditions or genetic predispositions, can be pro-oxidant and harmful.
... Additionally, AEA upregulated CD200 and IL-10 but downregulated pro-inflammatory cytokines IL-1β and IL-6 in a mixed culture of neurons and reactive microglia activated by LPS and IFNγ [87]. The CD200-CD200R axis enhanced by AEA is believed to shift microglial cells from an M1 to M2 phenotype, which provides neuroprotection [88]. Furthermore, CB1 agonist WIN55,212-2 induces astrocytes to release purines, which reduce tremors in an animal model of essential tremors [24]. ...
Chronic inflammation is considered to be a silent killer because it is the underlying cause of a wide range of clinical disorders, from cardiovascular to neurological diseases, and from cancer to obesity. In addition, there are over 80 different types of debilitating autoimmune diseases for which there are no cure. Currently, the drugs that are available to suppress chronic inflammation are either ineffective or overtly suppress the inflammation, thereby causing increased susceptibility to infections and cancer. Thus, the development of a new class of drugs that can suppress chronic inflammation is imperative. Cannabinoids are a group of compounds produced in the body (endocannabinoids) or found in cannabis (phytocannabinoids) that act through cannabinoid receptors and various other receptors expressed widely in the brain and immune system. In the last decade, cannabinoids have been well established experimentally to mediate anti-inflammatory properties. Research has shown that they suppress inflammation through multiple pathways, including apoptosis and inducing immunosuppressive T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Interestingly, cannabinoids also mediate epigenetic alterations in genes that regulate inflammation. In the current review, we highlight how the epigenetic modulations caused by cannabinoids lead to the suppression of inflammation and help identify novel pathways that can be used to target autoimmune diseases.
... Among their properties that could be interesting in the context of MS, NAEs are described to exert anti-inflammatory and neuroprotective effects [6][7][8][9]. Although N-arachidonoylethanolamine (anandamide, AEA) and N-oleoylethanolamine (OEA) have been shown to exert neuroprotective effects [6,10,11], the implication of N-palmitoylethanolamine (PEA) in (neuro)inflammation has been more thoroughly investigated. For instance, PEA administration was demonstrated to reduce inflammation and to exert neuroprotective effects in murine models of Alzheimer's disease [7,12] and Parkinson's disease [13,14]. ...
N-acylethanolamines (NAEs) are endogenous bioactive lipids reported to exert anti-inflammatory and neuroprotective effects mediated by cannabinoid receptors and peroxisome proliferator–activated receptors (PPARs), among others. Therefore, interfering with NAE signaling could be a promising strategy to decrease inflammation in neurological disorders such as multiple sclerosis (MS). Fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase (NAAA) are key modulators of NAE levels. This study aims to investigate and compare the effect of NAAA inhibition, FAAH inhibition, and dual inhibition of both enzymes in a mouse model of MS, namely the experimental autoimmune encephalomyelitis (EAE). Our data show that NAAA inhibition strongly decreased the hallmarks of the pathology. Interestingly, FAAH inhibition was less efficient in decreasing inflammatory hallmarks despite the increased NAE levels. Moreover, the inhibition of both NAAA and FAAH, using a dual-inhibitor or the co-administration of NAAA and FAAH inhibitors, did not show an added value compared to NAAA inhibition. Furthermore, our data suggest an important role of decreased activation of astrocytes and microglia in the effects of NAAA inhibition on EAE, while NAAA inhibition did not affect T cell recall. This work highlights the beneficial effects of NAAA inhibition in the context of central nervous system inflammation and suggests that the simultaneous inhibition of NAAA and FAAH has no additional beneficial effect in EAE.
... In addition, a beneficial role for CD200-CD200R1 signalling has been described in rodent models of brain ischemia using CD200Fc [69,88] or recombinant CD200 [85]. Several in vitro and in vivo studies showed that the CD200R1 agonist exerts its effect by suppressing the proinflammatory microglial activation [15,24,25,33,47,51,79]. In addition, Varnum et al. [75] demonstrated that a CD200R1 agonist stimulated the production of neurotrophic factors in primary microglial cell cultures. ...
Background
It is suggested that neuroinflammation, in which activated microglial cells play a relevant role, contributes to the development of Parkinson’s disease (PD). Consequently, the modulation of microglial activation is a potential therapeutic target to be taken into account to act against the dopaminergic neurodegeneration occurring in this neurological disorder. Several soluble and membrane-associated inhibitory mechanisms contribute to maintaining microglial cells in a quiescent/surveillant phenotype in physiological conditions. However, the presence of activated microglial cells in the brain in PD patients suggests that these mechanisms have been somehow overloaded. We focused our interest on one of the membrane-associated mechanisms, the CD200-CD200R1 ligand-receptor pair.
Methods
The acute MPTP experimental mouse model of PD was used to study the temporal pattern of mRNA expression of CD200 and CD200R1 in the context of MPTP-induced dopaminergic neurodegeneration and neuroinflammation. Dopaminergic damage was assessed by tyrosine hydroxylase (TH) immunoreactivity, and neuroinflammation was evaluated by the mRNA expression of inflammatory markers and IBA1 and GFAP immunohistochemistry. The effect of the modulation of the CD200-CD200R1 system on MPTP-induced damage was determined by using a CD200R1 agonist or CD200 KO mice.
Results
MPTP administration resulted in a progressive decrease in TH-positive fibres in the striatum and TH-positive neurons in the substantia nigra pars compacta, which were accompanied by transient astrogliosis, microgliosis and expression of pro- and anti-inflammatory markers. CD200 mRNA levels rapidly decreased in the ventral midbrain after MPTP treatment, while a transient decrease of CD200R1 mRNA expression was repeatedly observed in this brain area at earlier and later phases. By contrast, a transient increase in CD200R1 expression was observed in striatum. The administration of a CD200R1 agonist resulted in the inhibition of MPTP-induced dopaminergic neurodegeneration, while microglial cells showed signs of earlier activation in CD200-deficient mice.
Conclusions
Collectively, these findings provide evidence for a correlation between CD200-CD200R1 alterations, glial activation and neuronal loss. CD200R1 stimulation reduces MPTP-induced loss of dopaminergic neurons, and CD200 deficiency results in earlier microglial activation, suggesting that the potentiation of CD200R1 signalling is a possible approach to controlling neuroinflammation and neuronal death in PD.
... Endocannabinoid ligands in both the early and late phases of gliosis have been tested in rodent models (Schmidt et al., 2012) and the effects have been associated with prevention of neurodegenerative symptoms in disorders such as Huntington's Disease (Ullrich et al., 2007;Palazuelos et al., 2009). Specifically, endocannabinoids have a direct effect on the interactions between microglial CD200 and CD200R via anandamide that enhances CD200R expression and increases IL-10 levels to have a neuroprotective effect (Hernangomez et al., 2012). In reciprocal communication, microglia may transmit AEA through EVs to stimulate CB1 receptors, leading to the suppression GABA inhibitory transmission that may lead to neuron vulnerability, if prolonged (Gabrielli et al., 2015). ...
The aging brain seems to be characterized by neuronal loss leading to cognitive decline and progressively worsening symptoms related to neurodegeneration. Also, pro-inflammatory states, if prolonged, may increase neuronal vulnerability via excessive activation of microglia and their pro-inflammatory by-products, which is seen as individuals increase in age. Consequently, microglial activity is tightly regulated by neuron-microglia communications. The endocannabinoid system (ECS) is emerging as a regulator of microglia and the neuronal-microglia communication system. Recently, it has been demonstrated that cannabinoid 1 (CB1) receptor signaling on GABAergic interneurons plays a crucial role in regulating microglial activity. Interestingly, if endocannabinoid signaling on GABAergic neurons are disturbed, the phenotypes mimic central nervous system insult models by activating microglia and leading to accelerated brain aging. Investigating the endocannabinoid receptors, ligands, and genetic deletions yields the potential to understand the communication system and mechanism by which the ECS regulates glial cells and aspects of aging. While there remains much to discover with the ECS, the information gathered and identified already could lead to the development of cell-specific therapeutic interventions that help in reducing the effects of age-related pro-inflammatory states and neurodegeneration.
... CBD produces its anxiolytic effects via activation of the 5-HT1A receptor associated with 5-HT, affecting positively the amygdala. Studies show that CBD acts via the modulation of the 5-HT1A receptors [15]. ...
The endocannabinoid system (ECS) is a neuromodulator system with a crucial role in CNS and the reaction to endogenous and exogenous compounds and inflammation. Cannabidiol (CBD) is a basic part of the ECS which is the overwhelming causative and/or protective factor of major depressive disease (MDD). CBD interacts with brain-derived neurotropic factor (BDNF) that responds to inflammation, dysregulations of the hypothalamic-pituitary-adrenal (HPA) axis, and many more imbalances in MDD patients for which the ECS is a vital part to analyze, diagnose, and reflect the treatment. The ECS and MDD appear to have strong connections and interactions, so interest in ECS and CBD use in MDD patients is developing as a rescue resort.
... One of the main agents through which neurons affect glial activation is the CD200 glycoprotein, which inhibits microglial priming and holds microglia in a quiescent state (Shrivastava et al., 2012). Indeed, alterations in the distribution of the CD200 or its glial receptor CD200R can lead to overactivation of microglia and neuroinflammation (Hernangómez et al., 2012) that accompany neurodegeneration (Oria et al., 2018). Other neuronal agents that affect the course of neuroinflammation are chemokines, such as CX3CL1, CXCR4 and CCL2 which promotes microglial scavenging (Meucci et al., 2000;Hatori et al., 2002;Paolicelli et al., 2014), regulation of the BBB integrity (Rabinovich-Nikitin et al., 2016) and chemotaxis (Coughlan et al., 2000;Conductier et al., 2010) respectively. ...
Glia, a non-excitable cell type once considered merely as the connective tissue between neurons, is nowadays acknowledged for its essential contribution to multiple physiological processes including learning, memory formation, excitability, synaptic plasticity, ion homeostasis, and energy metabolism. Moreover, as glia are key players in the brain immune system and provide structural and nutritional support for neurons, they are intimately involved in multiple neurological disorders. Recent advances have demonstrated that glial cells, specifically microglia and astroglia, are involved in several neurodegenerative diseases including Amyotrophic lateral sclerosis (ALS), Epilepsy, Parkinson’s disease (PD), Alzheimer’s disease (AD), and frontotemporal dementia (FTD). While there is compelling evidence for glial modulation of synaptic formation and regulation that affect neuronal signal processing and activity, in this manuscript we will review recent findings on neuronal activity that affect glial function, specifically during neurodegenerative disorders. We will discuss the nature of each glial malfunction, its specificity to each disorder, overall contribution to the disease progression and assess its potential as a future therapeutic target.
... Possible mechanisms for this effect would be the switch in microglial phenotypes after CB2 receptor activation with a shift in the gene expression balance towards a neuroprotective phenotype that releases neurotrophic factors and a different pattern of pro-and anti-inflammatory cytokines [36,50]. In addition, endocannabinoids potentiate the expression of CX 3 CR1 [51] and CD200R [52] that interact with their corresponding ligands in the cell surface of neurons promoting the establishment of protective neuron-glia interactions. Modulation of CB1 receptors did not affect neuronal survival at any condition, suggesting a preferential role in other functions not necessarily related to degeneration or repair. ...
Monoacylglycerol lipase inhibition (MAGL) has emerged as an interesting therapeutic target for neurodegenerative disease treatment due to its ability to modulate the endocannabinoid system and to prevent the production of proinflammatory mediators. To obtain a beneficial response, it is necessary to understand how this inhibition affects the neuron–glia crosstalk and neuron viability. In this study, the effect of MAGL inhibition by KML29 was evaluated in two types of rat cortical primary cultures; mixed cultures, including neuron and glial cells, and neuron-enriched cultures. The risk of neuronal death was estimated by longitudinal survival analysis. The spontaneous neuronal risk of death in culture was higher in the absence of glial cells, a process that was enhanced by KML29 addition. In contrast, neuronal survival was not compromised by MAGL inhibition in the presence of glial cells. Blockade of cannabinoid type 2 (CB2) receptors expressed mainly by microglial cells did not affect the spontaneous neuronal death risk but decreased neuronal survival when KML29 was added. Modulation of cannabinoid type 1 (CB1) receptors did not affect neuronal survival. Our results show that neuron–glia interactions are essential for neuronal survival. CB2 receptors play a key role in these protective interactions when neurons are exposed to toxic conditions.
For decades, the interactions between the peripheral immune system and the brain have played a key role in disease. However, the molecular, biochemical, and cellular interactions that affect neuroimmune responses remain under investigation. Only in the last two decades has it become evident that the neuroimmune axis is reciprocal to maintaining health. When it becomes dysfunctional, it can underline disease pathobiology. This chapter focuses on what is known and what still remains to unravel the neuroimmune axis.
With the recent introduction of a number of highly effective disease-modifying treatments (DMTs) and the resulting almost complete prevention of acute relapses in many patients with multiple sclerosis (MS), the interest of MS clinicians has gradually shifted from relapse prevention to counteraction of disease progression and the treatment of residual symptoms. Targeting the cannabinoid system with nabiximols is an approved and effective strategy for the treatment of spasticity secondary to MS. Recently, the concept of spasticity plus syndrome (SPS) was introduced to account for the evidence that spasticity often appears in MS patients in clusters with other symptoms (such as pain, bladder dysfunction, sleep, and mood disorders), where cannabinoids can also be effective due to their broader action on many immune and neuronal functions. Interestingly, outside these symptomatic benefits, extensive pre-clinical and clinical research indicated how the modulation of the cannabinoid system results in significant anti-inflammatory and neuroprotective effects, all potentially relevant for MS disease control. This evidence makes nabiximols a potential disease modifying symptomatic treatment (DMST), a concept introduced in an attempt to overcome the often artificial distinction between DMTs and symptomatic therapies (STs).
Detection of merely apoptosis does not reveal the type of central nervous system (CNS) cells that are dying in the CNS diseases and injuries. In situ detection and estimation of amount of apoptosis specifically in neurons or glial cells (astrocytes, oligodendrocytes, and microglia) can unveil valuable information for designing therapeutics for protection of the CNS cells and functional recovery. A method was first developed and reported from our laboratory for in situ detection and estimation of amount of apoptosis precisely in neurons and glial cells using in vitro and in vivo models of CNS diseases and injuries. This is a combination of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and double immunofluorescent labeling (DIFL) or simply TUNEL-n-DIFL method for in situ detection and estimation of amount of apoptosis in a specific CNS cell type. An anti-digoxigenin (DIG) IgG antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) for blue fluorescence, fluorescein isothiocyanate (FITC) for green fluorescence, or Texas Red (TR) for red fluorescence can be used for in situ detection of apoptotic cell DNA, which is earlier labeled with TUNEL using alkali-stable DIG-11-dUTP. A primary anti-NeuN (neurons), anti-GFAP (astrocytes), anti-MBP (oligodendrocytes), or anti-OX-42 (microglia) IgG antibody and a secondary IgG antibody conjugated with one of the above fluorophores (other than that of ani-DIG antibody) are used for in situ detection of apoptosis in a specific CNS cell type in the mixed culture and animal models of the CNS diseases and injuries.
Microglia play key roles in the post‐ischemic inflammatory response and damaged tissue removal reacting rapidly to the disturbances caused by ischemia and working to restore the lost homeostasis. However, the modified environment, encompassing ionic imbalances, disruption of crucial neuron–microglia interactions, spreading depolarization, and generation of danger signals from necrotic neurons, induce morphological and phenotypic shifts in microglia. This leads them to adopt a proinflammatory profile and heighten their phagocytic activity. From day three post‐ischemia, macrophages infiltrate the necrotic core while microglia amass at the periphery. Further, inflammation prompts a metabolic shift favoring glycolysis, the pentose‐phosphate shunt, and lipid synthesis. These shifts, combined with phagocytic lipid intake, drive lipid droplet biogenesis, fuel anabolism, and enable microglia proliferation. Proliferating microglia release trophic factors contributing to protection and repair. However, some microglia accumulate lipids persistently and transform into dysfunctional and potentially harmful foam cells. Studies also showed microglia that either display impaired apoptotic cell clearance, or eliminate synapses, viable neurons, or endothelial cells. Yet, it will be essential to elucidate the viability of engulfed cells, the features of the local environment, the extent of tissue damage, and the temporal sequence. Ischemia provides a rich variety of region‐ and injury‐dependent stimuli for microglia, evolving with time and generating distinct microglia phenotypes including those exhibiting proinflammatory or dysfunctional traits and others showing pro‐repair features. Accurate profiling of microglia phenotypes, alongside with a more precise understanding of the associated post‐ischemic tissue conditions, is a necessary step to serve as the potential foundation for focused interventions in human stroke.
Background
Recent reports suggest that peroxisome proliferator-activated receptor-γ (PPAR-γ) could promote microglial M2 polarization to inhibit inflammation. However, the specific molecular mechanisms that trigger PPAR-γ’s anti-inflammatory ability in microglia are yet to be expounded. Thus, in this study, we aimed to explore the molecular mechanisms behind the anti-inflammatory effects of PPAR-γ in hypoxia-stimulated rat retinal microglial cells.
Methods and results
We used shRNA expressing lentivirus to knock down PPAR-γ and CD200 genes, and we assessed hypoxia-induced polarization markers release – M1 (iNOS, IL-1β, IL-6, and TNF-α) and M2 (Arg-1, YM1, IL-4, and IL-10) by RT-PCR. We also monitored PPAR-γ-related signals (PPAR-γ, PPAR-γ in cytoplasm or nucleus, CD200, and CD200Rs) by Western blot and RT-PCR. Our results showed that hypoxia enhanced PPAR-γ and CD200 expressions in microglial cells. Moreover, PPAR-γ agonist 15d-PGJ2 elevated CD200 and CD200R1 expressions, whereas sh-PPAR-γ had the opposite effect. Following hypoxia, expressions of M1 markers increased significantly, while those of M2 markers decreased, and the above effects were attenuated by 15d-PGJ2. Conversely, knocking down PPAR-γ or CD200 inhibited the polarization of microglial cells to M2 phenotype.
Conclusion
Our findings demonstrated that PPAR-γ performed an anti-inflammatory function in hypoxia-stimulated microglial cells by promoting their polarization to M2 phenotype via the CD200-CD200R1 pathway.
Chronic alcohol use induces innate immune genes, which activate the innate immune system. Neuroimmune regulatory proteins [e.g., Cluster of Differentiation 200 (CD200)] are immune response regulators and are involved in balancing the immune response. This study aimed to investigate the expression of CD200 on the surface of peripheral blood leucocytes in patients with alcohol use disorder and compare them with controls. Fifty male patients with alcohol use disorder were included in the study. A baseline assessment was done, and alcohol use history, craving, and withdrawal scores were collected. A two-milliliter venous blood sample was collected from cases and controls for immunophenotyping of CD200. The control group consisted of 50 participants with similar socio-economic backgrounds. The cellular expression of CD200 on total leucocytes (median ± IQR) (39.94 (28.85, 50.01) in cases was significantly lower compared to controls (45.07 (37.70, 51.69) [U = 896, P = 0.015]. Expression of CD200 on lymphocytes in cases was negatively correlated with years since heavy drinking and this was statistically significant (r = -0.321, P = 0.023). The study indicates that cellular expression of CD200 on the surface of peripheral blood leucocytes is reduced in alcohol-dependent patients. This reduction can contribute to exaggerated immune activity, release of pro-inflammatory cytokines, chronic microglial activation, neuroinflammation, and neurodegeneration in alcohol dependence.
Sleep deprivation alters orexinergic neuronal activity in the lateral hypothalamus (LH), which is the main regulator of sleep-wake, arousal, appetite, and energy regulation processes. Cannabinoid receptor (CBR) expression in this area is involved in modulating the function of orexin neurons. In this study, we investigated the effects of endocannabinoid anandamide (AEA) administration on improving food intake and appetite by modulating the activity of orexin neurons and CB1R expression after chronic sleep deprivation. Adult male Wistar rats (200-250 g) were randomly divided into three groups: control + vehicle (Control), chronic sleep deprivation + vehicle (SD), and chronic sleep deprivation +20 mg/kg AEA (SD + A). For SD induction, the rats were kept in a sleep deprivation device for 18 h (7 a.m. to 1 a.m.) daily for 21 days. Weight gain, food intake, the electrical power of orexin neurons, CB1R mRNA expression in hypothalamus, CB1R protein expression in the LH, TNF-α, IL-6, IL-4 levels and antioxidant activity in hypothalamus were measured after SD induction. Our results showed that AEA administration significantly improved food intake (p < 0.01), Electrical activity of orexin neurons (p < 0.05), CB1R expression in the hypothalamus (p < 0.05), and IL-4 levels (p < 0.05). AEA also reduced mRNA expression of OX1R and OX2R (p < 0.01 and p < 0.05 respectively), also IL-6 and TNF-α (p < 0.01) and MDA level (p < 0.05) in hypothalamic tissue. As a consequence, AEA modulates orexinergic system function and improves food intake by regulating the expression of the CB1 receptor in the LH in sleep deprived rats.
Depressive disorders can occur in up to 50% of people with multiple sclerosis in their lifetime. If left untreated, comorbid major depressive disorders may not spontaneously remit and is associated with an increased morbidity and mortality. Conversely, epidemiological evidence supports increased psychiatric visit as a significant prodromal event prior to diagnosis of MS. Are there common molecular pathways that contribute to the co-development of MS and psychiatric illnesses? We discuss immune cells that are dysregulated in MS and how such dysregulation can induce or protect against depressive symptoms. This is not meant to be a comprehensive review of all molecular pathways but rather a framework to guide future investigations of immune responses in depressed versus euthymic people with MS. Currently, there is weak evidence supporting the use of antidepressant medication in comorbid MS patients. It is our hope that by better understanding the neuroimmune crosstalk in the context of depression in MS, we can enhance the potential for future therapeutic options.
The spine has essential roles in supporting body weight, and passaging the neural elements between the body and the brain. In this study, we used integrated single-cell RNA sequencing and single-cell transposase-accessible chromatin sequencing analyses to reveal the cellular heterogeneity, lineage, and transcriptional regulatory network of the developing human spine. We found that EPYC + HAPLN1+ fibroblasts with stem cell characteristics could differentiate into chondrocytes by highly expressing the chondrogenic markers SOX9 and MATN4. Neurons could originate from neuroendocrine cells, and MEIS2 may be an essential transcription factor that promotes spinal neural progenitor cells to selectively differentiate into neurons during early gestation. Furthermore, the interaction of NRP2_SEMA3C and CD74_APP between macrophages and neurons may be essential for spinal cord development. Our integrated map provides a blueprint for understanding human spine development in the early and midgestational stages at single-cell resolution and offers a tool for investigating related diseases.
During the mid-1980s, in vitro rosette formation between macrophages and sheep erythrocytes was reported; however, sialidase treatment on erythrocytes abolished the rosettes [1]. Rosette formation is based on sialic acid-binding receptors expressed by macrophages. Later, these receptors were identified to be sialoadhesin that binds to sialic acids as ligands [2]. The SA-recognizing property of the innate immune system of vertebrates led to the discovery of SA-bearing glycans as the ligands for lectins. The binding of SA ligands to Siglecs with immune inhibitory properties leads to suppressed immune functions. The SA-to-Siglec recognition implicates immune activation to limit self-recognition and to destroy the defense mechanism in hosts. Siglecs attenuate ‘self’-inflammatory triggers called DAMPs. During the early 1990s, the first sialyl carbohydrate-binding receptor protein called Siglecs was discovered, for example, Siglec-2 (or CD22) present on B cells. Siglec-1, known as sialoadhesin present on macrophage surfaces, was found. The Ig-like domains of such lectins are different from those of other known C-type lectins or Ca2+-dependent lectins. Among them, I-type lectins are named I-type because they belong to the varied immunoglobulin superfamily of proteins. As I-type lectins share similar characteristics with the immunoglobulin superfamily of proteins, they contain Ig folds, which consist of antiparallel β-sheets. I-type lectins can be broken down into many different regions: V-set, C1 and C2 sets, and ITIM and ITIM-like domains. The V-set domain is the primary site of ligand binding and recognition. The C1 and C2 sets act as spacers and are believed to control the entire length of lectins. ITIM and ITIM-like domains (in some cases, ITAM domain) are essentially tyrosine-based signaling motifs that inhibit (or, in the case of the ITAM domain, activate) downstream signaling and thereby modulate cell activities. More than 16 I-type lectins, such as Siglecs, recognize diverse sialoglycans by immune cells. Even among I-type lectins, there are many different subtypes and the best characterized subtype is called Siglecs. Siglecs are SA-binding lectins and they are the most well-studied I-type lectins. Like any other lectins, Siglecs are composed of many different domains, namely, the V-set, C1 and C2 sets, and mostly the ITIM domains.
Background and Purpose
CD200 (cluster of differentiation 200), a highly glycosylated protein primarily expressed on neurons in the central nervous system, binds with its receptor CD200R to form an endogenous inhibitory signal against immune responses. However, little is known about the effect of neuronal CD200 signaling in cerebral ischemia. The aim of this study was to investigate how neuronal CD200 signaling impacts poststroke inflammation and the ischemic injury.
Methods
CD200 tma1 lf/fl :Thy1CreER mice were treated with tamoxifen to induce conditional gene knockout (ICKO) of neuronal CD200. The mice were subjected to a 60-minute transient middle cerebral artery occlusion. Stroke outcomes, apoptotic cell death, immune cell infiltration, microglia activation, and other inflammatory profiles were evaluated at 3 and 7 days after stroke.
Results
Infarct volumes were significantly larger, and behavioral deficits more severe in ICKO versus control mice at 3 days after middle cerebral artery occlusion. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay also revealed a significant increase in apoptotic neuronal death in CD200 ICKO mice. An enhancement in lymphocytic infiltration and microglial proinflammatory responses were revealed by flow cytometry at 3 and 7 days after stroke in ICKO mice, accompanied by an increased microglial phagocytosis activity. Plasma proinflammatory cytokine (TNFα [tumor necrosis factor alpha] and IL [interleukin]-1β) levels significantly increased at 3 days, and IL-1β/IL-6 levels increased at 7 days in ICKO versus control animals. ICKO led to significantly lower baseline level of CD200 both in brain and plasma.
Conclusions
Neuronal CD200 inhibits proinflammatory responses and is protective against stroke injury.
Increasing evidence suggests that SARS-CoV-2, the virus responsible for the COVID-19 pandemic, is associated with increased risk of developing neurological or psychiatric conditions such as depression, anxiety or dementia. While the precise mechanism underlying this association is unknown, aberrant activation of toll-like receptor (TLR)3, a viral recognizing pattern recognition receptor, may play a key role. Synthetic cannabinoids and enhancing cannabinoid tone via inhibition of fatty acid amide hydrolase (FAAH) has been demonstrated to modulate TLR3-induced neuroimmune responses and associated sickness behaviour. However, the role of individual FAAH substrates, and the receptor mechanisms mediating these effects, are unknown. The present study examined the effects of intracerebral or systemic administration of the FAAH substrates N-oleoylethanolamide (OEA), N-palmitoylethanolamide (PEA) or the anandamide (AEA) analogue meth-AEA on hyperthermia and hypothalamic inflammatory gene expression following administration of the TLR3 agonist, and viral mimetic, poly I:C. The data demonstrate that meth-AEA does not alter TLR3-induced hyperthermia or hypothalamic inflammatory gene expression. In comparison, OEA and PEA attenuated the TLR3-induced hyperthermia, although only OEA attenuated the expression of hyperthermia-related genes (IL-1β, iNOS, COX2 and m-PGES) in the hypothalamus. OEA, but not PEA, attenuated TLR3-induced increases in the expression of all IRF- and NFκB-related genes examined in the hypothalamus, but not in the spleen. Antagonism of PPARα prevented the OEA-induced attenuation of IRF- and NFκB-related genes in the hypothalamus following TLR3 activation but did not significantly alter temperature. PPARα agonism did not alter TLR3-induced hyperthermia or hypothalamic inflammatory gene expression. These data indicate that OEA may be the primary FAAH substrate that modulates TLR3-induced neuroinflammation and hyperthermia, effects partially mediated by PPARα.
Decades after identifying cannabinoids and their beneficial effects on Parkinson's disease (PD), many gaps are still missing. Although, CB2-dependent actions have been shown as underlying positive effects of cannabinoid treatment, in recent years, another receptor of cannabinoids, CB1, emerged as a valuable player in cannabinoid-induced neuroprotection. Remarkably, the effects of CB1 are mainly related to immune cells in the CNS, microglia, and astrocytes. However, oxidative stress, α-syn accumulation, and immune disbalance are essential aspects of both neurons and glial cells. Therefore, in this study, we investigated the effects of the CB1 on both α-syn and rotenone-treated SH-SY5Y and C8-D1A cells. ACEA and AM-251 were used as CB1 agonists and antagonists. Cell viability, IL-1β, IL-6, TNF-α levels, and CD200 expressions were determined in culture mediums. Our results demonstrated that preformed fibril form (pFF) of α-syn did not cause any significant change in SH-SY5Y cells compared to C8-D1A cells. Rotenone significantly increased the expression of IL-1β, IL-6, and TNF-α levels in both cells. pFF α-syn and rotenone treatment caused a decrease in CD200 expression. Surprisingly both ACEA and AM-251 alleviated rotenone-induced increase in cytokine levels in both cell lines. Although ACEA prevented pFF α-syn induced increase in cytokine levels and decrease in CD200 expression in C8-D1A cells, AM-251 failed to affect CD200 expression levels. Additionally, ACEA + AM-251 abolished the protective effects of both ACEA and AM-251 against rotenone and α-syn insults in both cell lines. The current study suggests that cannabinoid receptor agonism alleviates rotenone and α-syn-dependent inflammation in neurons and astrocytes.
Stressful experiences evoke, among others, a rapid increase of brain (nor)epinephrine (NE) levels and a slower increase in glucocorticoid hormones (GCs) in the brain. Microglia are key regulators of neuronal function and contain receptors for NE and GCs. These brain cells may therefore potentially be involved in modulating stress‐effects on neuronal function and learning and memory. In this review, we discuss that stress induces (1) an increase in microglial numbers as well as (2) a shift towards a proinflammatory profile. These microglia have (3) impaired crosstalk with neurons, and (4) disrupted glutamate signaling. Moreover, microglial immune responses after stress (5) alter the kynurenine pathway through metabolites that impair glutamatergic transmission. All these effects could be involved in the impairment in memory and memory synapses caused by (prolonged) stress, implicating this cell type as a potential target in stress‐related memory impairments.
Objective
To study how the CD200‐CD200R1 signaling pathway modulates poststroke inflammation and advances our knowledge of immune responses to ischemia insults in stroke.
Methods
Focal middle cerebral artery occlusion (MCAO) was induced in mice for 90 min, and mice were sacrificed at 1, 3, and 7 days of reperfusion. CD200, CD200R1, iNOS, and Arg‐1 expression in ischemic brains was assessed by Western blotting (WB), and immunohistochemical (IHC) staining was performed to examine the expression of CD200 on neurons and CD200R1 on infiltrating lymphocytes. The severity of neurobehavioral deficits was evaluated by neurological deficit scores (NDS) and infarction volume estimated by TTC staining. To study the relationship between CD200/CD200R1 expression and the diversity of the neuroinflammatory response in stroke, CD200Fc (CD200R1 agonist) was subcutaneously injected at onset, at 1 day and 2 days after MCAO operation, and the brains were collected for detection at 3 days after MCAO/R (reperfusion).
Results
CD200 expression on neurons increased at 1 day and then decreased at 3 days after MCAO/R, and the expression of CD200R1 on lymphocytes showed an opposite temporal pattern as tested by IHC. The WB results showed that CD200/CD200R1 variance exhibited a similar pattern of IHC results, and the level of iNOS peaked at 1 day and then decreased gradually, but Arg‐1 increased with time after MCAO/R in ischemic brains. After CD200Fc injection, CD200R1 expression significantly increased, and CD200Fc promoted Arg‐1 but inhibited iNOS expression. The infarct volume and NDS of the group treated with CD200Fc were significantly smaller than those of the IgG2a‐treated group.
Conclusions
The CD200‐CD200R1 signaling pathway regulates neuroinflammation after stroke. Stimulation of CD200R1 by CD200Fc promotes the anti‐inflammatory response and alleviates ischemic injury.
Aging is a complex biological process that increases the risk of age-related cognitive degenerative diseases such as dementia, including Alzheimer’s disease (AD), Lewy Body Dementia (LBD), and mild cognitive impairment (MCI). Even non-pathological aging of the brain can involve chronic oxidative and inflammatory stress, which disrupts the communication and balance between the brain and the immune system. There has been an increasingly strong connection found between chronic neuroinflammation and impaired memory, especially in AD. While microglia and astrocytes, the resident immune cells of the central nervous system (CNS), exerting beneficial effects during the acute inflammatory phase, during chronic neuroinflammation they can become more detrimental. Central cholinergic circuits are involved in maintaining normal cognitive function and regulating signaling within the entire cerebral cortex. While neuronal-glial cholinergic signaling is anti-inflammatory and anti-oxidative, central cholinergic neuronal degeneration is implicated in impaired learning, memory sleep regulation, and attention. Although there is evidence of cholinergic involvement in memory, fewer studies have linked the cholinergic anti-inflammatory and anti-oxidant pathways to memory processes during development, normal aging, and disease states. This review will summarize the current knowledge of cholinergic effects on microglia and astroglia, and their role in both anti-inflammatory and anti-oxidant mechanisms, concerning normal aging and chronic neuroinflammation.
Although significant advances have taken place in recent years on our understanding of the molecular mechanisms of different neurodegenerative diseases, its translation into effective therapeutic treatments has not been as successful as could be expected. There is still a dramatic lack of curative treatments for the most frequent disorders and only symptomatic relief for many others. Under this perspective, the search for novel therapeutic approaches is demanding and significant attention and efforts have been directed to studying additional neurotransmission systems including the endocannabinoid system (ECS). The neuroprotective properties of exogenous as well as endogenous cannabinoids have been known for years and the underlying molecular mechanisms have been recently unveiled. As discussed later, antioxidative, antiglutamatergic and antiinflammatory effects are now recognized as derived from cannabinoid action and are known to be of common interest for many neurodegenerative processes. Thus, these characteristics make cannabinoids attractive candidates for the development of novel therapeutic strategies [1]. The present review will focus on the existing data regarding the possible usefulness of cannabinoid agents for the treatment of relevant neurological pathologies for our society such as Alzheimer's disease, multiple sclerosis, Huntington's disease and amyotrophic lateral sclerosis.
OX2 (CD200) is a broadly expressed membrane glycoprotein, shown here to be important for regulation of the macrophage lineage. In mice lacking CD200, macrophage lineage cells, including brain microglia, exhibited an activated phenotype and were more numerous. Upon facial nerve transection, damaged CD200-deficient neurons elicited an accelerated microglial response. Lack of CD200 resulted in a more rapid onset of experimental autoimmune encephalomyelitis (EAE). Outside the brain, disruption of CD200-CD200 receptor interaction precipitated susceptibility to collagen-induced arthritis (CIA) in mice normally resistant to this disease. Thus, in diverse tissues OX2 delivers an inhibitory signal for the macrophage lineage.
The medicinal properties of marijuana have been recognized for centuries, but clinical and societal acceptance of this drug of abuse as a potential therapeutic agent remains fiercely debated. An attractive alternative to marijuana-based therapeutics would be to target the molecular pathways that mediate the effects of this drug. To date, these neural signaling pathways have been shown to comprise a cannabinoid receptor (CB(1)) that binds the active constituent of marijuana, tetrahydrocannabinol (THC), and a postulated endogenous CB(1) ligand anandamide. Although anandamide binds and activates the CB(1) receptor in vitro, this compound induces only weak and transient cannabinoid behavioral effects in vivo, possibly a result of its rapid catabolism. Here we show that mice lacking the enzyme fatty acid amide hydrolase (FAAH(-/-)) are severely impaired in their ability to degrade anandamide and when treated with this compound, exhibit an array of intense CB(1)-dependent behavioral responses, including hypomotility, analgesia, catalepsy, and hypothermia. FAAH(-/-)-mice possess 15-fold augmented endogenous brain levels of anandamide and display reduced pain sensation that is reversed by the CB(1) antagonist SR141716A. Collectively, these results indicate that FAAH is a key regulator of anandamide signaling in vivo, setting an endogenous cannabinoid tone that modulates pain perception. FAAH may therefore represent an attractive pharmaceutical target for the treatment of pain and neuropsychiatric disorders.
Cannabinoids exert pleiotropic actions in the CNS, including the inhibition of inflammatory responses and the enhancement of neuronal survival after injury. Although cannabinoid receptors are distributed widely in brain, their presence has not been investigated previously in oligodendrocytes. This study examined the expression of cannabinoid type 1 (CB1) receptors in rat oligodendrocytes in vivo and in culture and explored their biological function. Expression of CB1 receptors by oligodendrocytes was demonstrated immunocytochemically in postnatal and in adult white matter as well as in oligodendrocyte cultures. Reverse transcription-PCR and Western blotting further confirmed the presence of CB1 receptors. Oligodendrocyte progenitors undergo apoptosis with the withdrawal of trophic support, as determined by TUNEL assay and caspase-3 activation, and both the selective CB1 agonist arachidonyl-2'-chloroethylamide/(all Z)-N-(2-cycloethyl)-5,8,11,14-eicosatetraenamide (ACEA) and the nonselective cannabinoid agonists HU210 and (+)-Win-55212-2 enhanced cell survival. To investigate intracellular signaling involved in cannabinoid protection, we focused on the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. HU210, (+)-Win-55212-2, and ACEA elicited a time-dependent phosphorylation of Akt. Pertussis toxin abolished Akt activation, indicating the involvement of G(i)/G(o)-protein-coupled receptors. The CB1 receptor antagonist SR141716A partially inhibited Akt phosphorylation in response to HU210 and (+)-Win-55212-2 and abolished the effects of ACEA. Trophic support deprivation downregulated Akt activity, and cannabinoids recovered phospho-Akt levels. Inhibition of PI3K abrogated the survival action and the recovery of Akt activity in response to cannabinoids. SR141716A prevented only the protection conferred by ACEA. Nevertheless, SR141716A and the selective CB2 receptor antagonist SR144528 in combination inhibited the prosurvival action of HU210, which is in accordance with the finding of CB2 receptor expression by oligodendroglial cells. These data identify oligodendrocytes as potential targets of cannabinoid action in the CNS.
Theiler murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is a mouse model of chronic-progressive multiple sclerosis (MS) characterized by Th1-mediated CNS demyelination and spastic hindlimb paralysis. Existing MS therapies reduce relapse rates in 30% of relapsing-remitting MS patients, but are ineffective in chronic-progressive disease, and their effects on disability progression are unclear. Experimental studies demonstrate cannabinoids are useful for symptomatic treatment of spasticity and tremor in chronic-relapsing experimental autoimmune encephalomyelitis. Cannabinoids, however, have reported immunosuppressive properties. We show that the cannabinoid receptor agonist, R+WIN55,212, ameliorates progression of clinical disease symptoms in mice with preexisting TMEV-IDD. Amelioration of clinical disease is associated with downregulation of both virus and myelin epitope-specific Th1 effector functions (delayed-type hypersensitivity and IFN-gamma production) and the inhibition of CNS mRNA expression coding for the proinflammatory cytokines, TNF-alpha, IL1-beta, and IL-6. Clinical trials investigating the therapeutic potential of cannabinoids for the symptomatic treatment of MS are ongoing, and this study demonstrates that they may also have potent immunoregulatory properties.
CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed on rodent myeloid cells and is involved in regulation of macrophage function. We report the first characterization of human CD200R (hCD200R) and define its binding characteristics to hCD200. We also report the identification of a closely related gene to hCD200R, designated hCD200RLa, and four mouse CD200R-related genes (termed mCD200RLa-d). CD200, CD200R, and CD200R-related genes were closely linked in humans and mice, suggesting that these genes arose by gene duplication. The distributions of the receptor genes were determined by quantitative RT-PCR, and protein expression was confirmed by a set of novel mAbs. The distribution of mouse and human CD200R was similar, with strongest labeling of macrophages and neutrophils, but also other leukocytes, including monocytes, mast cells, and T lymphocytes. Two mCD200 receptor-like family members, designated mCD200RLa and mCD200RLb, were shown to pair with the activatory adaptor protein, DAP12, suggesting that these receptors would transmit strong activating signals in contrast to the apparent inhibitory signal delivered by triggering the CD200R. Despite substantial sequence homology with mCD200R, mCD200RLa and mCD200RLb did not bind mCD200, and presently have unknown ligands. The CD200 receptor gene family resembles the signal regulatory proteins and killer Ig-related receptors in having receptor family members with potential activatory and inhibitory functions that may play important roles in immune regulation and balance. Because manipulation of the CD200-CD200R interaction affects the outcome of rodent disease models, targeting of this pathway may have therapeutic utility.
Chronic progression of two T cell-mediated central nervous system (CNS) demyelinating models of multiple sclerosis, relapsing EAE (R-EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is dependent on the activation of T cells to endogenous myelin epitopes (epitope spreading). Using transfer of carboxyfluorescein succinyl ester (CFSE)-labeled T-cell receptor (TCR)-transgenic T cells and mixed bone marrow chimeras, we show that activation of naive proteolipid protein (PLP)139-151-specific T cells in SJL mice undergoing PLP178-191-induced R-EAE or TMEV-IDD occurs directly in the CNS and not in the cervical lymph nodes or other peripheral lymphoid organs. Examination of the antigen-presentation capacity of antigen-presenting cell (APC) populations purified from the CNS of mice with PLP178-191-induced R-EAE shows that only F4/80-CD11c+CD45hi dendritic cells (DCs) efficiently present endogenous antigen to activate naive PLP139-151-specific T cells in vitro. In contrast, DCs as well as F4/80+CD45hi macrophages and F4/80+CD45lo microglia activate a PLP139-151-specific helper T cell line. The data suggest that naive T cells enter the inflamed CNS and are activated by local APCs, possibly DCs, to initiate epitope spreading.
Theiler's virus infection of the CNS induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). Cannabinoids may act as immunosuppressive compounds that have shown therapeutic potential in chronic inflammatory disorders. Using the Theiler's murine encephalomyelitis virus model, we report here that treatment with the synthetic cannabinoids WIN 55,212-2, ACEA, and JWH-015 during established disease significantly improved the neurological deficits in a long-lasting way. At a histological level, cannabinoids reduced microglial activation, abrogated major histocompatibility complex class II antigen expression, and decreased the number of CD4+ infiltrating T cells in the spinal cord. Both recovery of motor function and diminution of inflammation paralleled extensive remyelination. Overall, the data presented may have potential therapeutic implications in demyelinating pathologies such as MS; in particular, the possible involvement of cannabinoid receptor CB2 would enable nonpsychoactive therapy suitable for long-term use.
Myeloid cells play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remodeling capacities. CD200 is widely expressed on a variety of cell types, while the recently identified CD200R is expressed on myeloid cells and T cells. CD200 deletion in vivo results in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation, suggesting that the CD200-CD200R interaction is involved in immune suppression. We demonstrate in this study that CD200R agonists suppress mouse and human myeloid cell function in vitro, and also define a dose relationship between receptor expression and cellular inhibition. IFN-gamma- and IL-17-stimulated cytokine secretion from mouse peritoneal macrophages was inhibited by CD200R engagement. Inhibitory effects were not universal, as LPS-stimulated responses were unaffected. Inhibition of U937 cell cytokine production correlated with CD200R expression levels, and inhibition was only observed in low CD200R expressing cells, if the CD200R agonists were further cross-linked. Tetanus toxoid-induced human PBMC IL-5 and IL-13 secretion was inhibited by CD200R agonists. This inhibition was dependent upon cross-linking the CD200R on monocytes, but not on cross-linking the CD200R on CD4+ T cells. In all, we provide direct evidence that the CD200-CD200R interaction controls monocyte/macrophage function in both murine and human systems, further supporting the potential clinical application of CD200R agonists for the treatment of chronic inflammatory diseases.
The ability of cannabinoids to modulate both inflammatory and degenerative neuronal damage prompted investigations on the potential benefits of such compounds in multiple sclerosis (MS) and in animal models of this disorder. Here we measured endocannabinoid levels, metabolism and binding, and physiological activities in 26 patients with MS (17 females, aged 19-43 years), 25 healthy controls and in mice with experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS. Our results show that MS and EAE are associated with significant alterations of the endocannabinoid system. We found that anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), was increased in the CSF of relapsing MS patients. AEA concentrations were also higher in peripheral lymphocytes of these patients, an effect associated with increased synthesis and reduced degradation of this endocannabinoid. Increased synthesis, reduced degradation, and increased levels of AEA were also detected in the brains of EAE mice in the acute phase of the disease, possibly accounting for its anti-excitotoxic action in this disorder. Accordingly, neurophysiological recordings from single neurons confirmed that excitatory transmission in EAE slices is inhibited by CB1 receptor activation, while inhibitory transmission is not. Our study suggests that targeting the endocannabinoid system might be useful for the treatment of MS.
Deficits in cognitive function are associated with neuroinflammatory changes, typified by activation of glial cells and an alteration of the pro- and anti-inflammatory cytokine balance in the brain. Although there is evidence to suggest that activation of microglia is regulated by interaction with other cell types in the brain, the mechanism(s) involved is poorly understood. Here, we provide evidence that interaction between CD200 and its receptor plays a role in modulating microglial activation under conditions of chronic and acute inflammation of the brain. We report that interleukin-4 (IL-4) plays a central role in modulating expression of CD200 and identify a mechanism by which IL-4 directly controls microglial cell activation. Our findings provide the first demonstration of a role for IL-4 in modulating CD200 expression and suggest a mechanism for regulation of microglial activation in the intact CNS under inflammatory conditions.
In previous studies we reported that while interaction between the relatively ubiquitously expressed molecule CD200 and one of its receptors, CD200R1, resulted in direct suppression of alloreactivity, engagement of alternate receptors led instead to altered differentiation of dendritic cells (DCs) from marrow precursors, which could in turn foster development of Foxp3(+) regulatory T cells. We have explored this effect of engagement of alternate receptors by using a monoclonal agonist Ab to CD200R2 and investigating expression of TLRs on DCs induced in vivo and in vitro after CD200 stimulation in mice in which the gene encoding CD200R1 was deleted. CD200 stimulation was achieved by using either a soluble form of CD200 (CD200Fc) or overexpression of CD200 as a doxycycline-inducible transgene. Although broadly similar effects were seen, consistent with the hypothesis that triggering of CD200R2 does produce DCs with a characteristic TLR repertoire, there are subtle differences in suppression of alloreactivity achieved by CD200 delivered in these two manners, which is consistent with a complexity of CD200:CD200R engagement not previously appreciated.
The aim of this study was to evaluate how endocannabinoids interact with excitotoxic processes both in vitro, using primary neural cell cultures, and in vivo, in the TMEV-IDD model of multiple sclerosis. First, we observed that neuronal cells respond to excitotoxic challenges by the production of endocannabinoid molecules which in turn exerted neuroprotective effects against excitotoxicity. The inhibitor of endocannabinoid uptake, UCM707, protected specifically against AMPA-induced excitotoxicity, by activating CB1 and CB2 cannabinoid receptors, as well as the nuclear factor, PPARγ. This neuroprotective effect was reverted by blocking the glial glutamate transporter, GLT-1. Mice subjected to the model of multiple sclerosis showed a decrease in the expression of GLT-1. UCM707 reversed this loss of GLT-1 and induced a therapeutic effect. Our data indicate that the enhancement of the endocannabinoid tone leads to neuroprotection against AMPA-induced excitotoxicity and provides therapeutic effects in this model of multiple sclerosis.
DBA/1 mice immunized with 100 μg bovine collagen type II emulsified in Freund's adjuvant, followed by booster injection in incomplete adjuvant at 18 days, develop profound arthritis (>50% of animals) by 30 days postinjection. The molecule CD200 (previously called OX2), associated with, among others, follicular dendritic cells, is implicated in delivery of immunosuppressive signals to the immune system, and an immunoadhesin in which the extracellular domains of CD200 were linked to a mouse IgG2a Fc region has been shown to promote renal allograft survival. DBA/1 mice receiving 15 μg/mouse CD200Fc at 3-day intervals following immunization with collagen did not develop arthritis in this model. Lymphocytes taken from CD200Fc-treated, collagen-immunized mice produced significantly lower levels of TNFα and IFN-γ in culture supernatants after restimulation in vitro with collagen, in contrast to cells taken from control mice treated with PBS or normal mouse Ig. Serum from CD200Fc-treated mice contained less anti-collagen IgG (∼50% reduction), with relatively more IgG2b and IgG3, and lower levels of TNFα and IFN-γ, than control mice. These data indicate that this immunoadhesin may have a potent role to play in the regulation of autoimmune disorders.
Cannabinoids have recently been approved as a treatment for pain in multiple sclerosis (MS). Increasing evidence from animal studies suggests that this class of compounds could also prove efficient to fight neurodegeneration, demyelination, inflammation and autoimmune processes occurring in this pathology. However, the use of cannabinoids is limited by their psychoactive effects. In this context, potentiation of the endogenous cannabinoid signalling could represent a substitute to the use of exogenously administrated cannabinoid ligands. Here, we studied the expression of different elements of the endocannabinoid system in a chronic model of MS in mice. We first studied the expression of the two cannabinoid receptors, CB(1) and CB(2), as well as the putative intracellular cannabinoid receptor peroxisome proliferator-activated receptor-alpha. We observed an upregulation of CB(2), correlated to the production of proinflammatory cytokines, at 60 days after the onset of the MS model. At this time, the levels of the endocannabinoid, 2-arachidonoylglycerol, and of the anti-inflammatory anandamide congener, palmithoylethanolamide, were enhanced, without changes in the levels of anandamide. These changes were not due to differences in the expression of the degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase, or of biosynthetic enzymes, diacylglycerol lipase-alpha and N-acylphosphatidylethanolamine phospholipase-D at this time (60 days). Finally, the exogenous administration of palmitoylethanolamide resulted in a reduction of motor disability in the animals subjected to this model of MS, accompanied by an anti-inflammatory effect. This study overall highlights the potential therapeutic effects of endocannabinoids in MS.
Theiler's virus (TMEV) infection of the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). The endocannabinoid system represents a novel therapeutic target for autoimmune and chronic inflammatory diseases due to its anti-inflammatory properties by regulating cytokine network. IL-12p70 and IL-23 are functionally related heterodimeric cytokines that play a crucial role in the pathogenesis of MS. In the present study we showed that the endocannabinoid anandamide (AEA) downregulated the gene expression of IL-12p70 and IL-23 forming subunits mRNAs in the spinal cord of TMEV-infected mice and ameliorated motor disturbances. This was accompanied by significant decreases on the serological levels of IL-12p70/IL-23 and more interestingly, of IL-17A. In contrast, serum levels of IL-10 resulted elevated. In addition, we studied the signalling pathways involved in the regulation of IL-12p70/IL-23 and IL-10 expression in TMEV-infected microglia and addressed the possible interactions of AEA with these pathways. AEA acted through the ERK1/2 and JNK pathways to downregulate IL-12p70 and IL-23 while upregulating IL-10. These effects were partially mediated by CB2 receptor activation. We also described an autocrine circuit of cross-talk between IL-12p70/IL-23 and IL-10, since endogenously produced IL-10 negatively regulates IL-12p70 and IL-23 cytokines in TMEV-infected microglia. This suggests that by altering the cytokine network, AEA could indirectly modify the type of immune responses within the CNS. Accordingly, pharmacological modulation of endocannabinoids might be a useful tool for treating neuroinflammatory diseases.
The Cannabis sativa plant has been exploited for medicinal, agricultural and spiritual purposes in diverse cultures over thousands of years. Cannabis has been used recreationally for its psychotropic properties, while effects such as stimulation of appetite, analgesia and anti‐emesis have lead to the medicinal application of cannabis. Indeed, reports of medicinal efficacy of cannabis can been traced back as far as 2700 BC, and even at that time reports also suggested a neuroprotective effect of the cultivar. The discovery of the psychoactive component of cannabis resin, Δ ⁹ ‐tetrahydrocannabinol (Δ ⁹ ‐THC) occurred long before the serendipitous identification of a G‐protein coupled receptor at which Δ ⁹ ‐THC is active in the brain. The subsequent finding of endogenous cannabinoid compounds, the synthesis of which is directed by neuronal excitability and which in turn served to regulate that excitability, further widened the range of potential drug targets through which the endocannabinoid system can be manipulated. As a result of this, alterations in the endocannabinoid system have been extensively investigated in a range of neurodegenerative disorders. In this review we examine the evidence implicating the endocannabinoid system in the cause, symptomatology or treatment of neurodegenerative disease. We examine data from human patients and compare and contrast this with evidence from animal models of these diseases. On the basis of this evidence we discuss the likely efficacy of endocannabinoid‐based therapies in each disease context.
This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476‐5381.2010.00831.x
Cannabinoids have been proposed as clinically promising neuroprotective molecules, based on their capability to normalize glutamate homeostasis, reducing excitotoxicity, to inhibit calcium influx, lowering intracellular levels and the subsequent activation of calcium-dependent destructive pathways, and to reduce the generation of reactive oxygen intermediates or to limit their toxicity, decreasing oxidative injury. Cannabinoids are also able to decrease local inflammatory events by acting on glial processes that regulate neuronal survival, and to restore blood supply by reducing vasocontriction produced by several endothelium-derived factors.
Current literature supporting these neuroprotective effects, particularly evidence generated during the last ten years, concentrating on targets within the cannabinoid signaling system that facilitate these effects. Acute or chronic neurodegenerative disorders where cannabinoids have shown neuroprotective effect.
Most of the information reviewed here relates to preclinical studies. However, these molecules may progress from the present preclinical evidence to clinical applications.
Treatment of neurodegenerative disorders is a challenge for neuroscientists and neurologists. Unhappily, the efficacy of available medicines is still poor and there is an urgent need for novel neuroprotective agents. Cannabinoids can serve this purpose given their recognized antiexcitotoxic, antioxidant and anti-inflammatory properties.
LPS activates microglia, which are normally maintained in a quiescent state by CD200-CD200 receptor (CD200R) interaction. MAC-1 (a microglia marker) mRNA expression was increased in mice brains up to 1 year post LPS administration (i.p.). Minocycline treatment did not prevent LPS (5 mg/kg)-induced increase in MAC-1 mRNA but reduced that induced by 0.1 mg/kg LPS. CD200R mRNA decreased starting at 4 h, whereas CD200 mRNA increased at 4 h and decreased at 1 year post LPS inoculation. Thus, LPS-induced changes in CD200-CD200R equilibrium might keep microglia chronically activated. Minocycline does not effectively inhibit microglia activation induced by high-dose LPS.
The endocannabinoid system exhibits anti-inflammatory properties by regulating cytokine production. Anandamide (AEA) down-regulates proinflammatory cytokines in a viral model of multiple sclerosis (MS). However, little is known about the mechanisms by which AEA exerts these effects. Microglial cells are the main source of cytokines within the brain and the first barrier of defense against pathogens by acting as antigen presenting cells. IL-10 is a key physiological negative regulator of microglial activation. In this study we show that AEA enhances LPS/IFNgamma-induced IL-10 production in microglia by targeting CB(2) receptors through the activation of ERK1/2 and JNK MAPKs. AEA also inhibits NF-kappaB activation by interfering with the phosphorylation of IkappaBalpha, which may result in an increase of IL-10 production. Moreover, endogenously produced IL-10 negatively regulates IL-12 and IL-23 cytokines, which in its turn modify the pattern of expression of transcription factors involved in Th commitment of splenocytes. This suggests that by altering the cytokine network, AEA could indirectly modify the type of immune responses within the central nervous system (CNS). Accordingly, pharmacological modulation of AEA uptake and degradation might be a useful tool for treating neuroinflammatory diseases.
CD200 is a membrane glycoprotein that suppresses immune activity via its receptor, CD200R. CD200-CD200R interactions have recently been considered to contribute to the "immune privileged" status of the central nervous system (CNS). The mechanisms by which these interactions take place are not well understood in part because there is limited detailed information on the distribution of CD200 and CD200R in the CNS. Here, we used immunohistochemistry to characterize the distinct anatomical and cellular distribution of these molecules in multiple sclerosis (MS) lesions and controls. CD200 was robustly expressed in gray matter areas including the cerebral cortex, hippocampus, striatum, cerebellum, and spinal cord, where neurons appeared immunopositive. CD200 expression was also detected in oligodendrocytes, but not in astrocytes or microglia. In CNS samples from MS patients, CD200 expression was additionally observed on reactive astrocytes in chronic active plaque centers, despite our previous finding of an overall decrease ofCD200 expression in MS lesions. In contrast to CD200, the immunolocalization pattern of CD200R was very distinct, showing high expression on perivascular macrophages in both gray and white matter. Using flow cytometry, we also found that human primary microglia express low levels of CD200R. These data suggest that CD200-mediated immune suppression may occur not only via neuron-microglia interactions, but also via glia-glia interactions, especially in inflammatory conditions in which an immune-suppressive environment needs to be restored; this may occur as a result of increased CD200 expression on reactive astrocytes.
Inflammatory activation of microglia in response to neurodegenerative changes in diseases such as Alzheimer's disease (AD) and Parkinson's disease has been extensively described. These observations have suggested that inflammation could be contributing to disease progression. In this paper, the potential role of CD200 and CD200 receptor (CD200R), whose known functions are to activate anti-inflammatory pathways and induce immune tolerance through binding of CD200 to CD200 receptor (CD200R), was studied in AD. Quantitative studies showed a significant decrease in CD200 protein and mRNA in AD hippocampus and inferior temporal gyrus, but not cerebellum. Immunohistochemistry of brain tissue sections of hippocampus, superior frontal gyrus, inferior temporal gyrus and cerebellum from AD and non-demented cases demonstrated a predominant, though heterogeneous, neuronal localization for CD200. Decreased neuronal expression was apparent in brain regions affected by AD pathology. There was also a significant decrease in CD200R mRNA expression in AD hippocampus and inferior temporal gyrus, but not cerebellum. Low expression of CD200R by microglia was confirmed at the mRNA and protein level using cultured human microglia compared to blood-derived macrophages. Treatment of microglia and macrophages with interleukin-4 and interleukin-13 significantly increased expression of CD200R. Expression of these cytokines was not generally detectable in brain. These data indicate that the anti-inflammatory CD200/CD200R system may be deficient in AD brains. Mechanisms aimed at increasing levels of CD200 and CD200R could have therapeutic potential for controlling inflammation in human neurodegenerative diseases.
Endocannabinoids are lipid mediators with protective effects in many diseases of the nervous system. We have studied the modulation of the endocannabinoid system after a spinal cord contusion in rats. In early stages, lesion induced increases of anandamide and palmitoylethanolamide (PEA) levels, an upregulation of the synthesizing enzyme NAPE-phospholipase D and a downregulation of the degradative enzyme FAAH. In delayed stages, lesion induced increases in 2-arachidonoylglycerol and a strong upregulation of the synthesizing enzyme DAGL-alpha, that is expressed by neurons, astrocytes and immune infiltrates. The degradative enzyme MAGL was also moderately increased but only 7 days after the lesion. We have studied the cellular targets for the newly formed endocannabinoids using RT-PCR and immunohistochemistry against CB(1) and CB(2) receptors. We observed that CB(1) was constitutively expressed by neurons and oligodendrocytes and induced in reactive astrocytes. CB(2) receptor was strongly upregulated after lesion, and mostly expressed by immune infiltrates and astrocytes. The endocannabinoid system may represent an interesting target for new therapeutical approaches to spinal cord injury.
The endocannabinoid signaling system (eCBSS) is composed of cannabinoid (CB) receptors, their endogenous ligands (the endocannabinoids, eCB) and the enzymes that produce and inactivate these ligands. Neurons use this signaling system to communicate with each other and Delta9-tetrahydrocannabinol (THC), the main psychotropic ingredient of Cannabis sativa, induces profound behavioral effects by impinging on this communication. Evidence now shows that microglia, the macrophages of the brain, also express a functional eCBSS and that activation of CB receptors expressed by activated microglia controls their immune-related functions. This review summarizes this evidence, discusses how microglia might use the eCBSS to communicate with each other and neighboring cells, and argues that compounds selectively targeting the eCBSS expressed by microglia constitute valuable therapeutics to manage acute and chronic neuroinflammation, without inducing the psychotropic effects and underlying addictive properties commonly associated with THC.
Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis of bioactive fatty acid amides and esters such as the endogenous cannabinoid receptor ligands, anandamide (N-arachidonoyl-ethanolamine) and 2-arachidonoylglycerol, and the putative sleep inducing factor cis-9-octadecenoamide (oleamide). Most FAAH blockers developed to date also inhibit cytosolic phospholipase A2 (cPLA2) and/or bind to the CB1 cannabinoid receptor subtype. Here we report the finding of four novel FAAH inhibitors, two of which, malhamensilipin A and grenadadiene, were screened out of a series of thirty-two different algal natural products, and two others, arachidonoylethylene glycol (AEG) and arachidonoyl-serotonin (AA-5-HT) were selected out of five artificially functionalized polyunsaturated fatty acids. When using FAAH preparations from mouse neuroblastoma N18TG2 cells and [14C]anandamide as a substrate, the IC50s for these compounds ranged from 12.0 to 26 microM, the most active compound being AA-5-HT. This substance was also active on FAAH from rat basophilic leukaemia (RBL-2H3) cells (IC50 = 5.6 microM), and inhibited [14C]anandamide hydrolysis by both N18TG2 and RBL-2H3 intact cells without affecting [14C]anandamide uptake. While AEG behaved as a competitive inhibitor and was hydrolyzed to arachidonic acid (AA) by FAAH preparations, AA-5-HT was resistant to FAAH-catalyzed hydrolysis and behaved as a tight-binding, albeit non-covalent, mixed inhibitor. AA-5-HT did not interfere with cPLA2-mediated, ionomycin or antigen-induced release of [3H]AA from RBL-2H3 cells, nor with cPLA2 activity in cell-free experiments. Finally, AA-5-HT did not activate CB1 cannabinoid receptors since it acted as a very weak ligand in in vitro binding assays, and, at 10-15 mg/kg body weight, it was not active in the 'open field', 'hot plate' and rectal hypothermia tests carried out in mice. Conversely AEG behaved as a cannabimimetic substance in these tests as well as in the 'ring' immobility test where AA-5-HT was also active. AA-5-HT is the first FAAH inhibitor reported to date which is inactive both against cPLA2 and at CB1 receptors, whereas AEG represents a new type of cannabinoid receptor agonist.
The innate immune system and notably the complement (C) system play important roles in host defense to recognise and kill deleterious invaders or toxic entities, but activation at inappropriate sites or to an excessive degree can cause severe tissue damage. C has been implicated as a factor in the exacerbation and propagation of tissue injury in numerous diseases including neurodegenerative disorders. In this article, we review the evidence indicating that brain cells can synthesise a full lytic C system and also express specific C inhibitors (to protect from C activation and C lysis) and C receptors (involved in cell activation, chemotaxis and phagocytosis). We also summarise the mechanisms involved in the antibody-independent activation of the classical pathway of C in Alzheimer's disease, Huntington's disease and Pick's disease. Although the primary role of C activation on a target cell is to induce cell lysis (particularly of neurons), we present evidence indicating that C (C3a, C5a, sublytic level of C5b-9) may also be involved in pro- as well as anti-inflammatory activities. Moreover, we discuss evidence suggesting that local C activation may contribute to tissue remodelling activities during repair in the CNS.
The OX2 membrane glycoprotein (CD200) is expressed on a broad range of tissues including lymphoid cells, neurons, and endothelium. We report the characterization of an OX2 receptor (OX2R) that is a novel protein restricted to cells of the myeloid lineage. OX2 and its receptor are both cell surface glycoproteins containing two immunoglobulin-like domains and interact with a dissociation constant of 2.5 microM and koff 0.8 s(-1), typical of many leukocyte protein membrane interactions. Pervanandate treatment of macrophages showed that OX2R could be phosphorylated on tyrosine residues. Blockade of the OX2-OX2R interaction with an OX2R mAb exacerbated the disease model experimental allergic encephalomyelitis. These data, together with data from an OX2-deficient mouse (R. M. Hoek et al., submitted), suggest that myeloid function can be controlled in a tissue-specific manner by the OX2-OX2R interaction.
Spasticity is a complicating sign in multiple sclerosis that also develops in a model of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice. In areas associated with nerve damage, increased levels of the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and of the AEA congener, palmitoylethanolamide (PEA), were detected here, whereas comparable levels of these compounds were found in normal and non-spastic CREAE mice. While exogenously administered endocannabinoids and PEA ameliorate spasticity, selective inhibitors of endocannabinoid re-uptake and hydrolysis-probably through the enhancement of endogenous levels of AEA, and, possibly, 2-arachidonoyl glycerol-significantly ameliorated spasticity to an extent comparable with that observed previously with potent cannabinoid receptor agonists. These studies provide definitive evidence for the tonic control of spasticity by the endocannabinoid system and open new horizons to therapy of multiple sclerosis, and other neuromuscular diseases, based on agents modulating endocannabinoid levels and action, which exhibit little psychotropic activity.
DBA/1 mice immunized with 100 microg bovine collagen type II emulsified in Freund's adjuvant, followed by booster injection in incomplete adjuvant at 18 days, develop profound arthritis (>50% of animals) by 30 days postinjection. The molecule CD200 (previously called OX2), associated with, among others, follicular dendritic cells, is implicated in delivery of immunosuppressive signals to the immune system, and an immunoadhesin in which the extracellular domains of CD200 were linked to a mouse IgG2a Fc region has been shown to promote renal allograft survival. DBA/1 mice receiving 15 microg/mouse CD200Fc at 3-day intervals following immunization with collagen did not develop arthritis in this model. Lymphocytes taken from CD200Fc-treated, collagen-immunized mice produced significantly lower levels of TNFalpha and IFN-gamma in culture supernatants after restimulation in vitro with collagen, in contrast to cells taken from control mice treated with PBS or normal mouse Ig. Serum from CD200Fc-treated mice contained less anti-collagen IgG (approximately 50% reduction), with relatively more IgG2b and IgG3, and lower levels of TNFalpha and IFN-gamma, than control mice. These data indicate that this immunoadhesin may have a potent role to play in the regulation of autoimmune disorders.
Cannabinoid receptors and their endogenous ligands, the endocannabinoids, have been detected in several blood immune cells, including monocytes/macrophages, basophils and lymphocytes. However, their presence in dendritic cells, which play a key role in the initiation and development of the immune response, has never been investigated. Here we have analyzed human dendritic cells for the presence of the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), the cannabinoid CB1 and CB2 receptors, and one of the enzymes mostly responsible for endocannabinoid hydrolysis, the fatty acid amide hydrolase (FAAH). By using a very sensitive liquid chromatography-atmospheric pressure chemical ionization-mass spectrometric (LC-APCI-MS) method, lipids extracted from immature dendritic cells were shown to contain 2-AG, anandamide and the anti-inflammatory anandamide congener, N-palmitoylethanolamine (PalEtn) (2.1 +/- 1.0, 0.14 +/- 0.02 and 8.2 +/- 3.9 pmol x 10(-7) cells, respectively). The amounts of 2-AG, but not anandamide or PalEtn, were significantly increased following cell maturation induced by bacterial lipopolysaccharide (LPS) or the allergen Der p 1 (2.8- and 1.9-fold, respectively). By using both RT-PCR and Western immunoblotting, dendritic cells were also found to express measurable amounts of CB1 and CB2 receptors and of FAAH. Cell maturation did not consistently modify the expression of these proteins, although in some cell preparations a decrease of the levels of both CB1 and CB2 mRNA transcripts was observed after LPS stimulation. These findings demonstrate for the first time that the endogenous cannabinoid system is present in human dendritic cells and can be regulated by cell activation.
Immunization of DBA/1 with 100 microg bovine collagen type II emulsified in Freund's adjuvant, followed by booster injection in incomplete adjuvant at 18 days, leads to development of arthritis in more than 70% of mice by 28 days postinjection. We have previously shown that the novel immunosuppressant molecule CD200Fc (linking an extracellular domain of CD200 with a murine IgG2a Fc region) can suppress induction of disease when given to mice from the time of collagen injection. This occurs in concert with a decrease in the serum levels of anti-collagen IgG ( approximately 50% reduction), with relatively more IgG2b and IgG3, decreased serum levels of TNFalpha and IFN-gamma, and decreased production of those same cytokines after restimulation of lymphocytes in vitro with collagen. Since CD200 induces suppression following engagement of a receptor (CD200R), known to be expressed on, among other cells, macrophages, we investigated whether infusion of anti-CD200R and/or CD200Fc would ameliorate established disease in DBA mice, when injections were begun following collagen immunization. Our data indicate an arrest of disease following either treatment, with modification of a number of immune parameters (serum and lymphocyte cytokine production) consistent with a general role for CD200:CD200R interactions in the regulation of induction and/or expression of autoimmune disorders. When a higher dose (250 microg/mouse) of anti-CD200R was infused into a group of overtly arthritic mice, a significant ( approximately 50%) decrease in arthritic joint score occurred over the 4-week treatment period.
Microglial cells, the macrophages of the brain, express low, yet detectable levels of cannabinoid CB(1) receptors, which are known to modulate cell migration. To determine if cannabinoid CB(1) receptors expressed by microglial cells modulate their migration, we assessed whether arachidonylcyclopropylamide (ACPA, an agonist shown to selectively activate CB(1) receptors) affects the migration of BV-2 cells, a mouse microglial cell line. We found that ACPA induced a dose-dependent increase in BV-2 cell migration (EC(50)=2.2 nM). This ACPA response was blocked by pertussis toxin pretreatment, suggesting the involvement of G(i/o) protein-coupled receptors. However, the cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A) did not prevent ACPA-induced BV-2 cell migration. Two antagonists of cannabinoid CB(2) receptors N-(1,S)-endo-1,3,3-trimethyl bicyclo(2,2,1)heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) and cannabinol, as well as two antagonists of the newly identified "abnormal-cannabidiol-sensitive" (abn-CBD) receptors (O-1918 and cannabidiol) prevented this response. Our results suggest that cannabinoid CB(2) receptors and abn-CBD receptors, rather than cannabinoid CB(1) receptors, regulate microglial cell migration, and that ACPA is a broad cannabinoid receptor agonist.
Theiler's virus infection of the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). Cannabinoids have been shown to exert beneficial effects on animal models of MS and evidence suggests that the endocannabinoid system plays a role in the tonic control of spasticity. In this study we show that OMDM1 [(R)-N-oleoyl-(1'-hydroxybenzyl)-2'-ethanolamine] and OMDM2 [(S)-N-oleoyl-(1'-hydroxybenzyl)-2'-ethanolamine], two selective inhibitors of the putative endocannabinoid transporter and hence of endocannabinoid inactivation, provide an effective therapy for Theiler murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Treatment of TMEV-infected mice with OMDM1 and OMDM2 enhanced anandamide levels in the spinal cord and ameliorated motor symptoms. This was associated with a down-regulation of inflammatory responses in the spinal cord. In addition we show that OMDM1 and OMDM2 down-regulate macrophage function by (i) decreasing the surface expression of major histocompatibility complex (MHC) class II molecules, (ii) inhibiting nitric oxide synthase-2 (NOS-2) expression and (iii) reducing the production of the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and interleukin-12 (IL-12p40). Taken together, these results point to the manipulation of the endocannabinoid system as a possible strategy to develop future MS therapeutic drugs.
In normal brain aging, CNS resident macrophages exhibit increased expression of major histocompatibility complex (MHC) II expression. However, the transcriptional basis for this observation has not been clarified nor have age-related alterations in pivotal pro-inflammatory genes been characterized. Age-related mRNA alterations in MHC II, MHC II accessory molecules and several pro-inflammatory mediators were measured in older (24 months) and younger (3 months) male F344xBN F1 rats. Real time RT-PCR was utilized to measure steady state mRNA levels in hippocampus. Older as compared to younger animals exhibited increased mRNA levels of MHC II, CD86, CIITA and IFN-gamma. Furthermore, IL-10 and CD200 mRNA, molecules that down-regulate macrophage activation, was decreased in older animals. The present results indicate that normal brain aging is characterized by a shift towards a pro-inflammatory microenvironment in the CNS.
Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is a well-characterized murine model of human multiple sclerosis (MS) that closely resembles the chronic and progressive clinical form of the disease. Recent studies have described the involvement of the cannabinoid system in the progression of the disease and the benefits associated with the administration of cannabinoid agonists. With the objective to study whether "indirect" agonists, that is, compounds able to reinforce the physiological endocannabinoid transmission and, therefore, devoid of the psychotropic effects of "direct" agonists, could be suitable agents for the amelioration of MS neurological deficits, we administered the potent and selective anandamide uptake inhibitor UCM707 to TMEV-infected mice. Our results indicate that treatment during established disease significantly improves the motor function of the diseased mice. At the histological level, UCM707 is able to reduce microglial activation, diminish major histocompatibility complex class II antigen expression, and decrease cellular infiltrates in the spinal cord. Additionally, in microglial cells, UCM707 decreases the production of the proinflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6; reduces nitric oxide levels and inducible nitric oxide synthase expression; and is able to potentiate the action of a subeffective dose of the endocannabinoid anandamide. Overall, these results suggest that agents able to activate the endocannabinoid system could constitute a new series of drugs for the treatment of MS.
For Abstract see ChemInform Abstract in Full Text.
Recent studies have addressed the changes in endocannabinoid ligands and receptors that occur in multiple sclerosis, as a way to explain the efficacy of cannabinoid compounds to alleviate spasticity, pain, tremor, and other signs of this autoimmune disease. Using Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, we recently found a decrease in cannabinoid CB1 receptors mainly circumscribed to the basal ganglia, which could be related to the motor disturbances characteristic of these rats. In the present study, using the same model, we explored the potential changes in several neurotransmitters in the basal ganglia that might be associated with the motor disturbances described in these rats, but we only found a small increase in glutamate contents in the globus pallidus. We also examined whether the motor disturbances and the changes of CB1 receptors found in the basal ganglia of EAE rats disappear after the treatment with rolipram, an inhibitor of type IV phosphodiesterase able to supress EAE in different species. Rolipram attenuated clinical decline, reduced motor inhibition, and normalized CB1 receptor gene expression in the basal ganglia. As a third objective, we examined whether EAE rats also exhibited changes in endocannabinoid levels as shown for CB1 receptors. Anandamide and 2-arachidonoylglycerol levels decreased in motor related regions (striatum, midbrain) but also in other brain regions, although the pattern of changes for each endocannabinoid was different. Finally, we hypothesized that the elevation of the endocannabinoid activity, following inhibition of endocannabinoid uptake, might be beneficial in EAE rats. AM404, arvanil, and OMDM2 were effective to reduce the magnitude of the neurological impairment in EAE rats, whereas VDM11 did not produce any effect. The beneficial effects of AM404 were reversed by blocking TRPV1 receptors with capsazepine, but not by blocking CB1 receptors with SR141716, thus indicating the involvement of endovanilloid mechanisms in these effects. However, a role for CB1 receptors is supported by additional data showing that CP55,940 delayed EAE progression. In summary, our data suggest that reduction of endocannabinoid signaling is associated with the development of EAE in rats. We have also proved that the reduction of CB1 receptors observed in these rats is corrected following treatment with a compound used in EAE such as rolipram. In addition, the direct or indirect activation of vanilloid or cannabinoid receptors may reduce the neurological impairment experienced by EAE rats, although the efficacy of the different compounds examined seems to be determined by their particular pharmacodynamic and pharmacokinetic characteristics.
We previously reported that the compound O‐2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re‐examined the activity of O‐2093 in vivo and synthesized four structural analogs (O‐2247, O‐2248, O‐3246, and O‐3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB 1 and CB 2 receptors; (b) assays of transient receptor potential of the vanilloid type‐1 (TRPV1) channel functional activity (measurement of [Ca ²⁺ ] i ); (c) [ ¹⁴ C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL‐2H3) cells; (d) the mouse ‘tetrad’ tests (analgesia on a hot plate, immobility on a ‘ring’, rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS).
O‐2093, either synthesized by us or commercially available, was inactive in the ‘tetrad’ up to a 20 mg kg ⁻¹ dose (i.v.). Like O‐2093, the other four compounds exhibited low affinity in CB 1 ( K i from 1.3 to >10 μ M ) and CB 2 binding assays (1.3< K i < 8 μ M ), low potency and efficacy in a TRPV1 functional assay (EC 50 >10 μ M ), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC 50 >25 μ M ) and were inactive in the ‘tetrad’ up to a 30 mg kg ⁻¹ dose (i.v.).
While O‐2247 and O‐2248 were poor inhibitors of [ ¹⁴ C]AEA cellular uptake (IC 50 >40 μ M ), O‐3246 and O‐3262 were quite potent in this assay. O‐3246, which exhibits only a very subtle structural difference with O‐2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC 50 =1.4 μ M ) and is 12‐fold more potent than O‐2093.
When injected intravenously O‐3246 and O‐3262, again like O‐2093 and unlike O‐2247 and O‐2248, significantly inhibited limb spasticity in mice with CREAE.
These data confirm the potential utility of selective AEA uptake inhibitors as anti‐spasticity drugs in MS and, given the very subtle chemical differences between potent and weak inhibitors of uptake, support further the existence of a specific mechanism for this process.
British Journal of Pharmacology (2006) 147 , 83–91. doi: 10.1038/sj.bjp.0706418
Endocannabinoids are released after brain injury and believed to attenuate neuronal damage by binding to CB(1) receptors and protecting against excitotoxicity. Such excitotoxic brain lesions initially result in primary destruction of brain parenchyma, which attracts macrophages and microglia. These inflammatory cells release toxic cytokines and free radicals, resulting in secondary neuronal damage. In this study, we show that the endocannabinoid system is highly activated during CNS inflammation and that the endocannabinoid anandamide (AEA) protects neurons from inflammatory damage by CB(1/2) receptor-mediated rapid induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) in microglial cells associated with histone H3 phoshorylation of the mkp-1 gene sequence. As a result, AEA-induced rapid MKP-1 expression switches off MAPK signal transduction in microglial cells activated by stimulation of pattern recognition receptors. The release of AEA in injured CNS tissue might therefore represent a new mechanism of neuro-immune communication during CNS injury, which controls and limits immune response after primary CNS damage.
Axonal damage secondary to inflammation is likely the substrate of chronic disability in multiple sclerosis and is found in the animal model of experimental autoimmune encephalomyelitis (EAE). Wld(s) mice have a triplication of the fusion gene Ube4b/Nmnat and a phenotype of axon protection. Wld(s) mice develop an attenuated disease course of EAE, with decreased demyelination, reduced axonal pathology, and decreased central nervous system (CNS) macrophage and microglial accumulation. We show that attenuated disease in Wld(s) mice was associated with robust constitutive expression of the nonsignaling CD200 molecule on neurons in the CNS compared with control mice. CD200 interacts with its signaling receptor CD200R, which we found to be expressed on microglia, astrocytes, and oligodendrocytes at similar levels in control and Wld(s) mice. Administration of blocking anti-CD200 antibody to Wld(s) mice abrogated disease attenuation and was associated with increased CNS inflammation and neurodegeneration. In vitro, Wld(s) neuronal cultures were protected from microglial-induced neurotoxicity compared with control cultures, but protection was abrogated by anti-CD200 antibody. The CD200-CD200R pathway plays a critical role in attenuating EAE and reducing inflammation-mediated damage in the CNS. Strategies that up-regulate the expression of CD200 in the CNS or molecules that ligate the CD200R may be relevant as neuroprotective strategies in multiple sclerosis.
To ensure an adequate response against pathogens and prevent unwanted self-reactivity, immune cells need to functionally express both activating and inhibitory receptors. CD200R is an inhibitory receptor mainly expressed on myeloid cells that down-modulates cellular activation both in vivo and in vitro. Although previously mainly studied as a regulator of myeloid function, we now show that CD200R is differentially expressed on human and mouse T-cell subsets. In both species, CD4+ T cells express higher amounts of CD200R than CD8+ T cells, and memory cells express higher amounts of CD200R than naïve or effector cells. CD200R expression is up-regulated on both CD4+ and CD8+ T cells after stimulation in vitro. Furthermore, we show CD200R expression on human and mouse B cells. In human tonsils, CD200R is differentially expressed on B cells, with high expression on memory cells and plasmablasts. Mice lacking the ligand for CD200R, CD200-/- mice, do not show abnormal composition of the lymphocyte compartment and have normal B cell responses to antigenic challenge. Although the functional implications remain to be elucidated, the expression of CD200R on lymphocytes suggests a much broader role for CD200R-mediated immune regulation than previously anticipated.
Inflammatory and demyelinating activity of activated resident macrophages (microglia) and recruited blood-borne macrophages are considered crucial in multiple sclerosis (MS) lesion development. The membrane glycoproteins CD200 and CD47, highly expressed on neurons, are mediators of macrophage inhibition via their receptors CD200R and signal-regulatory protein alpha, respectively, on myeloid cells. We determined the expression pattern of immune inhibitory molecules in relation to genes involved in macrophage activation and MS lesion pathology.
Laser dissection microscopy was combined with real-time polymerase chain reaction to quantitatively study these gene expression patterns in specific subareas (rim, center, and normal-appearing white matter) of chronic active and inactive MS lesions.
Hallmarks of MS pathology were confirmed by messenger RNA expression patterns of glial fibrillary acidic protein, neurofilament (NF), myelin basic protein, growth factors, chemokines and receptors, and macrophage activation markers, although expression of osteopontin and alphaB-crystallin was decreased. CD200 and CD47 were downregulated in the center of chronic active and inactive MS lesions. CD47 expression was also decreased in the rim of chronic active lesions, where complement expression was increased. This expression profile was also found in normal-appearing white matter surrounding these lesions, but not surrounding inactive lesions. Expression of CD200R and signal-regulatory protein alpha was not altered.
These data suggest that diminished immune inhibition via decreased CD200 and CD47 expression contributes to a disturbed equilibrium in macrophage and microglia activation in MS lesions. Furthermore, this may result in a proinflammatory predisposition in the area surrounding chronic active lesions, thereby contributing to axonal injury, demyelination, and possible lesion expansion.
Amongst the various demyelinating diseases that affect the central nervous system, those induced by an inflammatory response stand out because of their epidemiological relevance. The best known inflammatory-induced demyelinating disease is multiple sclerosis, but the immune response is a common pathogenic mechanism in many other less common pathologies (e.g., acute disseminated encephalomyelitis and acute necrotizing haemorrhagic encephalomyelitis). In all such cases, modulation of the immune response seems to be a logical therapeutic approach. Cannabinoids are well known immunomodulatory molecules that act through CB1 and CB2 receptors. While activation of CB1 receptors has a psychotropic effect, activation of CB2 receptors alone does not. Therefore, to bypass the ethical problems that could result from the treatment of inflammation with psychotropic molecules, considerable effort is being made to study the potential therapeutic value of activating CB2 receptors. In this review we examine the current knowledge and understanding of the utility of cannabinoids as therapeutic molecules for inflammatory-mediated demyelinating pathologies. Moreover, we discuss how CB2 receptor activation is related to the modulation of immunopathogenic states.
British Journal of Pharmacology (2008) 153, 216–225; doi:10.1038/sj.bjp.0707466; published online 24 September 2007
Microglial cells constitute the resident macrophage population of the CNS. Recent in vivo studies have shown that microglia carry out active tissue scanning, which challenges the traditional notion of 'resting' microglia in the normal brain. Transformation of microglia to reactive states in response to pathology has been known for decades as microglial activation, but seems to be more diverse and dynamic than ever anticipated--in both transcriptional and nontranscriptional features and functional consequences. This may help to explain why engagement of microglia can be either neuroprotective or neurotoxic, resulting in containment or aggravation of disease progression. Moreover, little is known about the heterogeneity of microglial responses in different pathologic contexts that results from regional adaptations or from the progression of a disease. In this review, we focus on several key observations that illustrate the multi-faceted activities of microglia in the normal and pathologic brain.
Recently, it has been recognized that the cannabinoid receptor CB2 may play a functionally relevant role in the central nervous system (CNS). This role is mediated primarily through microglia, a resident population of cells in the CNS that is morphologically, phenotypically, and functionally related to macrophages. These cells also express the cannabinoid receptor CB1. The CB1 receptor (CB1R) is constitutively expressed at low levels while the CB2 receptor (CB2R) is expressed at higher levels and is modulated in relation to cell activation state. The relatively high levels of the CB2R correspond with microglia being in ‘responsive’ and ‘primed’ states, suggesting the existence of a ‘window’ of functional relevance during which activation of the CB2R modulates microglial activities. Signature activities of ‘responsive’ and ‘primed’ microglia are chemotaxis and antigen processing, respectively. The endocannabinoid 2-arachidonylglycerol has been reported to stimulate a chemotactic response from these cells through the CB2R. In contrast, we have shown in vivo and in vitro that the exogenous cannabinoids delta-9-tetrahydrocannabinol and CP55940 inhibit the chemotactic response of microglia to Acanthamoeba culbertsoni, an opportunistic pathogen that is the causative agent of Granulomatous Amoebic Encephalitis, through activation of the CB2R. It is postulated that these exogenous cannabinoids superimpose an inhibitory effect on pro-chemotactic endocannabinoids that are elicited in response to Acanthamoeba. Furthermore, the collective results suggest that the CB2R plays a critical immune functional role in the CNS.
British Journal of Pharmacology (2008) 153, 240–251; doi:10.1038/sj.bjp.0707584; published online 26 November 2007
Multiple sclerosis is a chronic disabling CNS disorder, characterized by autoimmune inflammatory demyelination and neurodegeneration. CD200, broadly expressed on neurons and endothelial cells, mediates inhibitory signals through its receptor, CD200R, on cells of myeloid origin. Antibody-mediated blockade of CD200R leads to an aggravated clinical course of rodent experimental autoimmune encephalomyelitis in vivo, accompanied by profoundly augmented cellular infiltrates consisting of T cells and activated iNOS(+) macrophages in inflammatory spinal cord lesions. In vitro blockade of CD200R on macrophages leads to enhanced IFN-gamma-induced release of IL6 and neuronal cell death in co-cultures with hippocampal neurons expressing CD200. CD200 and its receptor could also be detected on neurons and macrophages in human MS plaques. Therefore the CD200-CD200R pathway seems of critical relevance for macrophage-mediated damage in autoimmune inflammation of the CNS.
Cannabinoids have been shown to exert beneficial actions in different animal models of multiple sclerosis (MS). However, the use of cannabinoids compounds in human therapy is greatly limited by their psychoactivity. Thus, new hopes in MS therapy have arisen from the evidence for a cannabinoid receptor, termed CB2, which is devoid of psychoactive effects in animal models.
This review discusses the different mechanisms by which CB2 activation could induce therapeutic actions in MS.
Particular focus is given to the potential effects on inflammation/autoimmunity, remyelination and neuroprotection.
This review discusses the importance of glial cells in sustaining these effects, as well as the putative secondary effects that would limit the use of CB2 agonists in the treatment of MS.
The CD200 receptor (CD200R) is an inhibitory receptor expressed by myeloid cells that is postulated to play an important role in regulation of the immune system. The purpose of this study was to evaluate the efficacy of a soluble ligand of CD200R in established collagen-induced arthritis (CIA) in mice and to analyze changes in cytokine expression following therapy in order to understand its primary mechanism of action.
Arthritis was induced in DBA/1 mice, and CD200-Fc fusion protein, an isotype control monoclonal antibody, or TNFR-Fc fusion protein was administered over a period of 10 days (total of 4 doses). Cytokine expression in the joint was assessed by flow cytometry, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction.
CD200-Fc significantly reduced the severity of established arthritis at the clinical and histologic levels. The therapeutic effect of CD200-Fc at 1 mg/kg was comparable with that of TNFR-Fc at 4 mg/kg. CD200R was found to be expressed in arthritic synovia and in lymph nodes, yet no changes in T cell cytokine levels (interferon-gamma, interleukin-5 [IL-5], IL-10, IL-17) were detected after CD200-Fc therapy. There was no evidence of an expansion of forkhead box P3-positive regulatory T cells or a change in serum anticollagen IgG1 and IgG2a levels. However, administration of CD200-Fc markedly decreased the expression of messenger RNA for tumor necrosis factor alpha, IL-1beta, IL-10, and matrix metalloproteinase 13 in the joint to the same extent as administration of TNFR-Fc.
CD200-Fc is an effective therapeutic agent in established CIA that targets proinflammatory cytokine expression in the joint without any obvious systemic immunosuppressive effects. Our findings indicate that CD200-Fc has considerable potential as a novel therapeutic agent in rheumatoid arthritis in humans.